EP1297131A2 - Cyk-4 polypeptides, dna molecules encoding them and their use in screening methods - Google Patents

Cyk-4 polypeptides, dna molecules encoding them and their use in screening methods

Info

Publication number
EP1297131A2
EP1297131A2 EP01947374A EP01947374A EP1297131A2 EP 1297131 A2 EP1297131 A2 EP 1297131A2 EP 01947374 A EP01947374 A EP 01947374A EP 01947374 A EP01947374 A EP 01947374A EP 1297131 A2 EP1297131 A2 EP 1297131A2
Authority
EP
European Patent Office
Prior art keywords
cyk
zen
cytokinesis
embryos
polynucleotide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01947374A
Other languages
German (de)
French (fr)
Inventor
Michael Glotzer
Verena Jantsch-Plunger
Alper Romano
Masanori Mishima
Susanne Kaitna
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Boehringer Ingelheim International GmbH
Original Assignee
Boehringer Ingelheim International GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from EP00112880A external-priority patent/EP1167525A1/en
Priority claimed from EP01110554A external-priority patent/EP1254912A1/en
Application filed by Boehringer Ingelheim International GmbH filed Critical Boehringer Ingelheim International GmbH
Priority to EP01947374A priority Critical patent/EP1297131A2/en
Publication of EP1297131A2 publication Critical patent/EP1297131A2/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Cyk-4 polypeptides DNA molecules encoding them and their use in screening methods
  • the present invention relates to a new protein Cyk-4, which is involved in cytokinesis, to its use in screening assays, and to therapies interfering with cell division, in particular tumor therapy.
  • Cytokinesis is one of the few processes in biology known to require coordination between microtubules and actin filaments. Indeed, in animal cells there are two steps in cytokinesis that rely on interactions between microtubules and the actin-based contractile ring (for review see (Field et al., 1999; Glotzer, 1997). In the first instance, the microtubule-based mitotic spindle specifies the position of the contractile ring. This allows the division plane to be positioned so that the separated chromosomes are partitioned equally into the two daughter cells.
  • the central spindle is also required for completion of cytokinesis (Wheatley and Wang, 1996) .
  • cytokinesis Wheatley and Wang, 1996) .
  • C. elegans embryos as in invertebrate embryos, only the later stages of cytokinesis appear to depend on the central spindle.
  • Embryos depleted of the kinesin-like protein ZEN-4/CeMKlpl fail to assemble the central spindle, yet cleavage furrows form and ingress, but cytokinesis does not proceed to completion (Powers et al . , 1998; Raich et al., 1998).
  • cytokinesis the initiation of cytokinesis depends on the central spindle in some but not in all organisms, whereas there appears to be a general requirement for the central spindle for the completion of cytokinesis in animal cells. While it is clear that the central spindle plays an important role in cytokinesis the underlying mechanism remains elusive.
  • RhoA GTPase RhoA GTPases
  • GEFs guanine nucleotide exchange factors
  • GAPs GTPase activating proteins
  • RhoA Inactivation of RhoA by the exoenzyme C3 (Aktories and Hall, 1989) , inhibits cytokinesis in a wide variety of experimental settings by causing disassembly of cortical actin structures and the contractile ring (Drechsel et al., 1997; Kishi et al., 1993; Mabuchi et al., 1993; Moorman et al., 1996; O'Connell et al., 1999). Further, a Rho GEF is essential for cytokinesis (Prokopenko et al., 1999; Tatsunaoto et al., 1999).
  • RhoA interacts with a number of putative effectors including formins, Rho Kinase, Citron kinase, and a regulatory subunit of myosin phosphatase (for review see (Van Aelst and D' Souza-Schorey, 1997).
  • the requirement for RhoA in 'cytokinesis may reflect its ability to regulate formins since members of the formin gene family are required for cytokinesis in budding yeast (BNI1/BNR1) (Imamura et al., 1997), fission yeast (Cdcl2) (Chang et al., 1997), Drosophila (dia) (Castrillon and Wasserman, 1994) and C. elegans ( cyk-1 ) (Swan et al., 1998).
  • microtubule-based central spindle arid the actin-based cleavage furrow are both essential for cytokinesis.
  • an interaction between the microtubule and actin cytoskeletal systems have been characterized. Examples include nuclear positioning in budding yeast (Carminati and Stearns, 1997; Fujiwara et al., 1999; Miller et al., 1999), spindle orientation in epithelial cells (Busson et al., 1998) and in certain asymmetrically dividing cells, such as the posterior blastomere of the two cell C.
  • cyk-4 a novel gene designated cyk-4 was identified and cloned and its role in the early divisions of the C. elegans embryo was characterized. It was shown that Cyk-4 is required for the late stages of cytokinesis. Interestingly, cyk-4 mutant embryos fail to assemble the central spindle. Positional cloning and localization studies revealed that the cyk- 4 gene encodes a novel GTPase activating protein (GAP) for the Rho family of GTPases that localizes to the central spindle.
  • GAP GTPase activating protein
  • the division of a cell into two daughters requires dynamic interactions between the microtubule-based mitotic spindle and the actin-based contractile ring.
  • the mutation responsible for the cyk-4 phenotype was found to be a missense mutation in a gene encoding a Rho family GAP that, in vitro, stimulates GTP hydrolysis by Rho, Rac, and Cdc42.
  • CYK-4 localizes to the central spindle and to cell division remnants.
  • CYK-4 colocalizes with the ZEN-4/CeMKLPl kinesin-like protein.
  • CYK-4 and ZEN-4/CeMKLPl are interdependent for their localization. It is concluded that the CYK-4 GAP and the ZEN-4/CeMKLPl kinesin-like protein cooperate to assemble the central spindle.
  • the spindle develops into two mitotic asters separated by a few overlapping, disorganized, microtubules.
  • a similar phenotype is observed in zen-4 mutant embryos (Powers et'al., 1998; Raich et al., 1998) .
  • both the ZEN-4/CeMKLPl kinesin-like protein and the CYK-4 GAP are essential for this microtubule bundling.
  • the Drosophila orthologue of ZEN-4/CeMKLPl is also required for cytokinesis, though it seems to be required for all aspects of furrow ingression (Adams et al., 1998).
  • a complex containing multiple motor proteins could specifically localize to overlapping, antiparallel microtubules (figure 10) . If such a motor complex transits along a microtubule it might continue to an end and dissociate. However, if such a motor complex transited along a microtubule in the vicinity of an antiparallel microtubule, the complex might bind simultaneously to both microtubules and attempt to move alternately in opposite directions, the net result being that the complex would concentrate in the region of microtubule overlap.
  • CYK-4 does not have a microtubule motor domain, yet it is essential for the formation of the central spindle, it is proposed that CYK-4 forms a complex with multiple ZEN-4 homodimers that localizes to and stabilizes overlapping antiparallel microtubules (figure 10) .
  • CYK-4 function in central spindle assembly is independent of the Rho GTPase, it was of interest to determine the function of the CYK-4 GAP domain.
  • CYK-4 • is likely bifunctional, one function being to promote the assembly of the central spindle, the second function being to promote GTP hydrolysis by Rho family members. These two functions might be related in that the first function would serve to concentrate CYK-4 at a site where GAP activity is required. It may be assumed that CYK-4 GAP activity is required late in cytokinesis, to promote GTP hydrolysis by a Rho family GTPase whose downregulation causes disassembly of the contractile ring and cell separation (fig. 10) .
  • the CYK-4 GAP domain has all the hallmarks of a Rho family GAP and it may therefore be expected that it will act on this subfamily of the GTPase superfamily.
  • the GAP domain of CYK-4 is promiscuous in its ability to promote GTP hydrolysis on Rho, Rac, and Cdc42 (Lamarche and Hall, 1994) .
  • Rho is the only one that is clearly essential for cytokinesis. The requirement for Rho in cell division is well documented in a variety of experimental systems.
  • RhoA is the key substrate for the CYK-4 GAP activity.
  • CYK-4 needs to act when the contractile ring is in close proximity to the central spindle. Since CYK-4 is concentrated on the central spindle at this time, its high local concentration might overcome its lower activity towards RhoA.
  • the central spindle was shown to be at the center of cytokinesis:
  • RhoA RhoA-independent manner in Xenopus embryos. It has been shown here that a Rho GAP is required for the late stages of cytokinesis, suggesting that the second process, completion of cytokinesis, does involve RhoA. It is therefore assumed that the two microtubule- dependent steps in cytokinesis are distinct.
  • CYK-4 and ZEN-4 are not the only components of the central spindle, a number of other components, some of which are required for cytokinesis, are also present at this site.
  • Polo kinase is known to associate with MKLPl and to concentrate in the central spindle (Adams et al., 1998; Lee et al., 1995), and this kinase is essential for cytokinesis. Rho associated kinase also localizes to this site (Kosako et al., 1999). The AIR-2 aurora-like kinase localizes to the central spindle (Schumacher et al., 1998).
  • Rho GEF that is required for cytokinesis, ECT2
  • ECT2 also accumulates on the central spindle (Tatsumoto et al., 1999), however the Drosophila ortholog, Pebble, does not localize in this manner (Prokopenko et al., 1999).
  • CYK-4/ZEN-4 complex which was named centralspindlin, consists of a two molecules each of CYK-4 and ZEN-4.
  • CYK-4 binds to the neck region of the ZEN-4 kinesin/ raising the possibility that the motor activity of the ZEN-4 is directly regulated by the CYK-4 RhoGAP.
  • HsCYK-4 and MKLPl are in a tetrameric complex.
  • immunopurification of HsCYK-4 and MKLPl reveals equal amounts of the two proteins.
  • these two proteins co-migrate on sucrose density gradients with a similar S value as observed for the C. ' elegans proteins.
  • CYK-4 binds to ZEN-4 in a particularly interesting region of this kinesin family member.
  • Rice et al., 1999 have established that a critical element of the kinesin molecule lies just C-terminal to the catalytic core; the neck linker region.
  • ATP binding to one catalytic core induces a large scale conformational change in the neck linker region that causes the other catalytic core to extend towards the adjacent tubulin dimer situated on the plus side of the initial microtubule contact (Rice et al., 1999).
  • CYK-4 binds to a region of ZEN-4 that includes the neck linker region.
  • the neck linker region corresponds to a region 15 amino acids long that connects the catalytic core of kinesin to the coiled coil stalk domain.
  • the MKLPl subfamily has a distinctly divergent neck linker region, it lacks several nearly invariant residues and the linker between the catalytic core and the coiled coil region is about 5 times longer than in other members on the
  • MKLPl may exist as a tetramer containing 4 motor domains (Chui et al., 2000; Kuriyama et al., 1994). If the two dimers were arranged in an antiparallel orientation, this arrangement could easily explain how this complex could crosslink antiparallel microtubules. However, the data obtained in the present invention are not consistent with this proposed structure.
  • the complex that has been characterized appears to contain two kinesin motors and two RhoGAP molecules . Since most kinesin motors are dimers in which both catalytic cores interact with a single microtubule protofilament, the architecture that is described here can not easily explain how microtubule bundling is achieved. If the two kinesin subunits of centralspindlin bind to the same microtubule protofilament, then how might microtubule crosslinking occur? At this point, at least three possible mechanisms can be envisaged (fig. 18) .
  • the first possibility is that there is an additional microtubule binding site elsewhere in the CYK-4/ZEN-4 complex. No additional binding site has been identified yet in CYK-4 nor has MKLPl, ZEN-4, or Pav been found to have an additional microtubule binding site.
  • MKLPl interacts differently with microtubules than most kinesin-like proteins. Specifically, ATP is usually sufficient to elute most kinesins from microtubules, but in the case of MKLPl, both ATP and high salt are required (Kuriyama et al., 1994; Nislow et al . , 1992). Thus it is possible that
  • MKLPl interacts with two microtubules, one by the motor domain and another via a different interaction surface. Consistent with this possibility is the finding that Rab6KIFL kinesin, which is quite similar to MKLPl in primary structure as well as in its localization and proposed function (Fontijn et al., 2001; Hill et al., 2000) , has been reported to contain a second microtubule binding activity in the C-terminal half of the molecule (Echard et al., 1998). However this possibility does not explain why CYK-4 is required for central spindle assembly.
  • MKLPl forms higher order structures and that the tetramer that has been characterized in the present invention is merely a building block.
  • This possibility gains some support from localization studies.
  • ring-like structures are found that are termed division remnants that persist in the cell cortex after division. These remnants appear to be large aggregates of centralspindlin which are not in direct association with microtubules. Higher order oligomers could potentially form in early anaphase and promote microtubule bundling.
  • the third possibility is that, unlike most N-terminal kinesins, the two catalytic cores of the kinesin subunits in the centralspindlin complex could bind to different microtubules. This would not be without precedent in that the KIN-N KIF1A moves processively along a microtubule using a single head (Okada and Hirokawa, 2000) .
  • the association of the two catalytic cores of MKLPl with different microtubules is made feasible by the fact that the linker region between the catalytic core and the coiled coil domain is much longer than that present in most N-terminal kinesins.
  • CYK-4 ensures that the two motor domains are oriented in such a way to bind to antiparallel microtubules (fig 18) . Structural analysis and biochemical reconstitution of centralspindlin from purified components will allow further insight into mechanism of antiparallel microtubule bundling.
  • RhoGAP domain of CYK-4 is highly conserved at the primary sequence level.
  • CYK-4 is significantly less active at inducing GTP hydrolysis by RhoA 1 as compared to Rac and Cdc42 (Jantsch-Plunger et al., 2000), and this feature of CYK-4 is conserved in HsCYK-4 (Toure et al., 1998).
  • CYK-4 has been proposed by the inventors that one function of the central spindle is to concentrate CYK-4 to a position in the cell where it could promote inactivation of RhoA at a late stage in cytokinesis. Consistent with this model, inactivation of RhoA by RNAi does not prevent CYK-4 localization nor does it impair central spindle assembly. In addition, the RhoGAP domain seems to be required at some stage of cytokinesis, since overexpression of a HsCYK- 4/MgcRacGAP bearing a mutation in the RhoGAP domain causes cytokinesis defects (Hirose et al., 2001). Further structure-function analysis of CYK-4 is necessary to test this model further. However such analyses must take into consideration that CYK-4 is able to dimerize or oligomerize and therefore such analysis is best done in the absence of endogenous CYK-4.
  • Aurora-B/Incenp complex is also required for the stable localization of centralspindlin in C. elegans embryos (Kaitna et al., 2000; Schumacher et al., 1998; Severson et al., 2000) and an in vitro biochemical interaction has been detected between Aurora-B (AIR-2) and ZEN-4 (Severson et al., 2000).
  • AIR-2 Aurora-B
  • ZEN-4 severeson et al., 2000
  • a stable association between Polo kinase or Aurora-B kinase with centralspindlin has not been observed. Reconstitution of central spindle assembly in vitro will allow the role of these and other regulators of centralspindlin to be dissected.
  • the experiments of the present invention comprise an initial phenotypic, molecular, biochemical, and cell biological analysis of the cyk-4 gene. Furthermore, they comprise an analysis of the CYK-4/ZEN-4 complex (centralspindlin) that consists of two molecules of each CYK-4 and ZEN-4.
  • CYK-4/ZEN-4 complex centralspindlin
  • Cyk-4 is an active GTPase activating protein that is required for cytokinesis, likely by its ability to regulate the RhoA GTPase. Quite surprisingly, one additional function of this protein is to promote assembly of the central spindle. Cyk-4 is a key molecule required for cytokinesis that regulates both the structure of the late mitotic spindle and the function of the contractile ring.
  • the present invention generally relates to Cyk-4 polypeptides, which have been shown to be essential for this second microtubule dependent ' step and thus define a functional link between the central spindle and the contractile ring.
  • the invention relates to a mammalian Cyk-4 polypeptide, in particular murine and human Cyk-4.
  • the invention relates to the urine Cyk-4 polypeptide with the amino acid sequence as set forth in SEQ ID NO: 4 or with the amino acid sequence encoded by a polynucleotide which hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO: 3.
  • the invention relates to the human Cyk-4 polypeptide with the amino acid sequence as set forth in SEQ ID NO: 2 or with the amino acid sequence encoded by a polynucleotide which hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO:l.
  • the present invention relates to ah isolated DNA molecule comprising a polynucleotide with the nucleotide sequence as set forth in SEQ ID NO:l encoding human Cyk-4 polypeptide, or an isolated DNA molecule encoding human Cyk-4 polypeptide comprising a polynucleotide which hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO:l.
  • the present invention relates to an isolated DNA molecule comprising a polynucleotide with the nucleotide sequence as set forth in SEQ ID NO: 3 encoding murine Cyk-4 polypeptide, or an isolated DNA molecule encoding murine Cyk-4 polypeptide comprising a polynucleotide which hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO: 3.
  • IX SSC 150 mM NaCl, 15mM trisodium citrate
  • 50 mM sodium phosphate pH 7.6
  • 5x Denhardt's solution 10% dextran sulfate
  • 20 ⁇ g/ml denatured, sheared salmon sperm DNA followed by washing the filters in O.lx SSC at about 65°C, or equivalent conditions.
  • Cyk-4" refers to both the murine and the human Cyk-4.
  • Cyk-4 polypeptides also include versions of the polypeptide which are resistant to post-translation modification or which alter the activity of the protein.
  • Cyk-4 polypeptides may comprise a full length protein, such as represented in SEQ ID NO: 2 or 4, or they may comprise fragments or variants therof.
  • the present invention relates to nucleic acid molecules in the form of RNA, such as mRNA.
  • the DNA molecules include cDNA and genomic DNA obtained by cloning or produced synthetically.
  • the DNA may be double-stranded or single-stranded.
  • Single-stranded DNA or RNA may be the coding strand, also known as the sense (or plus) strand, or it may be the non-coding strand, also referred to as the antisense (or minus) strand.
  • the present invention also relates to preparations of double stranded Cyk-4 RNA or derivatives thereof that can be used to interfere with gene expression by ds-RNA mediated gene interference as described by Fire et al. 1998 and reviewed by Fire, 1999; Bosher and Labouesse, 2000; Sharp, 1999.
  • isolated nucleic acid molecule is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment.
  • Recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells, and those DNA molecules purified (partially or substantially) from a solution whether produced by recombinant DNA or synthetic chemistry techniques.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. However, it is intended that "isolated” as used herein does not include the Cyk-4 cDNA present in a cDNA library or in a preparation of purified or isolated genomic DNA containing the Cyk-4 gene or a portion thereof in admixture with one or more other cDNA molecules or DNA fragments.
  • the nucleic acid molecules of the present invention further include genetic constructs comprising one or more Cyk-4 DNA sequences operably linked to regulatory DNA sequences (which may be heterologous regulatory sequences) , such as promoters or enhancers, wherein upon expression of these DNA sequences in host cells, preferably in bacterial, fungal (including yeast), -. * plant or animal (including insect or mammalian) cells, one or more Cyk-4 polypeptides are produced.
  • regulatory DNA sequences which may be heterologous regulatory sequences
  • promoters or enhancers such as promoters or enhancers
  • the regulatory sequences may be operably linked to a Cyk-4 polynucleotide encoding mature Cyk-4 polypeptide or any of its variants, precursors, fragments or derivatives described herein, which may include one or more polynucleotides having a nucleic acid sequence that is complementary to substantially all or a portion of a nucleic acid molecule having a nucleic acid sequence as shown in SEQ ID NO:l or 3.
  • a portion or "a fragment” of a nucleic acid molecule or a polypeptide means a segment of a polynucleotide or a polypeptide comprising at least 15, and more preferably at least 20, contiguous nucleotides or amino acids of a reference polynucleotide or polypeptide (for example, the polynucleotide and polypeptide shown in SEQ ID NOs: 1 or 3, respectively, unless otherwise specifically defined below.)
  • the invention also relates to DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the Cyk-4 mouse or human polypeptides. Since the genetic code is well known in the art, it is routine for one of ordinary skill in the art to produce the degenerate variants described above without undue experimentation.
  • the invention relates /to Cyk-4 polypeptides which have deviations' from the sequence shown in SEQ ID NO: 2 or 4, caused by the conservative exchange of amino acids, if they are Cyk-4 derivatives or fragments or peptides with the properties which are desirable for their use in therapy or in screening assays, or isolated DNA molecules encoding such derivatitives or fragments with a polynucleotide sequence varying in their sequence from SEQ ID NO:l or 3.
  • Nucleic acid molecules of the present invention which encode a Cyk-4 polypeptide or a derivative or fragment thereof may include, but are not limited to, those encoding the amino acid sequence of the polypeptide by itself, together with additional, non-coding sequences, including for example introns and non-coding 5 ' and 3 ' sequences, such as the transcribed, untranslated regions (UTRs) or other 5' flanking sequences that may play a role in transcription (e.g., via providing ribosome- or transcription factor-binding sites) , mRNA processing (e . g. splicing and polyadenylation signals) and stability of mRNA; the coding sequence for the
  • Cyk-4 polypeptide operably linked to a regulatory DNA sequence, particularly a heterologous regulatory DNA sequence such as a promoter or enhancer; and the coding sequence for the Cyk-4 polypeptide linked to one or more coding sequences which code for amino acids that provide additional functionalities.
  • the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide.
  • the marker amino /acid sequence may be a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • the "HA" tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al . , 2984.
  • Yet another useful marker peptide for facilitation of purification of Cyk-4 is glutathione S-transferase (GST) encoded by the pGEX fusion vector (see, e . g. , Winnacker, From Genes to Clones, New York: VCH Publishers, pp. 451-481 (1987)).
  • GST glutathione S-transferase
  • other such fusion proteins include the Cyk-4 fused to immunoglobulin Fc at the N- or C-terminus.
  • a still further aspect of the present invention relates to antibodies and antibody preparations specifically reactive with an epitope of the Cyk-4 polypeptide.
  • Polyclonal " antibodies are conventionally obtained by immunising animals, particularly rabbits, by injecting the antigen or fragments thereof and subsequently purifying the immunoglobulin.
  • Monoclonal anti-Cyk-4-antibodies may be obtained by standard procedures following the principle described by Kohler and Milstein, 1975, by immunising animals, particularly mice, then immortalising antibody- producing cells from the immunised animals, e.g. by fusion with myeloma cells, and screening the ' supernatant of the hybridomas obtained by immunological ' ' standard assays for monoclonal anti- Cyk-4 -antibodies.
  • these animal antibodies may optionally be chimerised in the conventional way (Neuberger et al., 1984, Boulianne et al., 1984) or humanised (Riechmann et al., 1988, Graziano et al., 1995). Cyk-4 specific antibodies can be used for both diagnostic and screening applications.
  • Cyk-4 specific antibodies are useful in clinical situations, where determining overexpression or underexpression of CYK-4 has predictive value.
  • Anti- CYK-4 antibodies can be used on tissue of cell specimens to evaluate the level of expression or changes in the subcellular localization in various disease states.
  • anti-CYK-4 antibodies can be used to detect the presence of CYK-4 in solid-phase binding assays as described in the subsequent subsection related to finding compounds that affect the interaction of CYK-4 with other proteins.
  • the present invention provides screening methods for identifying compounds capable of modulating, in particular inhibiting-, cytokinesis by modulating, in particular inhibiting, the function of CYK-4.
  • the screening method of the invention is used to identify compounds that modulate the function CYK-4 to promote GTP hydrolysis by Rho family GTPases. (Due to the evolutionary conservation of the GTPase domain and the requirement for Rho in cytokinesis, it can be concluded from the findings of the invention that the ability of CYK-4 to promote GTP hydrolysis by Rho is crucial for CYK-4 function.)
  • a compound's ability to promote GTP hydrolysis by a Rho family GTPase is determined by incubating a substrate selected from the members of the Rho family GTPases with GTP for a period of time sufficient to allow saturation of the substrate's GTP binding sites, adding Cyk-4 and allowing it to react in the presence or absence of the test compound, and determining the amount of hydrolyzed GTP.
  • GTP hydrolysis assay is a biochemical assay carried out according to standard protocols, as described, inter alia, by Self et al., 1995, or by Settleman and Foster, 1995.
  • this assay may be carried out as follows: In a first step, the substrate is incubated with GTP that carries a radioactive label (e.g. like the commercially available ⁇ - 32 P-GTP, or an otherwise measurable, e.g. a fluorescent label, as described, inter alia , by Hazlett et al., 1993, under conditions and for a period of time sufficient to allow saturation of GTP binding sites.
  • a radioactive label e.g. like the commercially available ⁇ - 32 P-GTP, or an otherwise measurable, e.g. a fluorescent label, as described, inter alia , by Hazlett et al., 1993, under conditions and for a period of time sufficient to allow saturation of GTP binding sites.
  • CYK-4 is added, optionally at various concentrations of from 0 to eg 1 uM; in the presence of absence of test compounds. After the appropriate time (e.g. 5 min.) the amount of GTP that has been hydrolysed is determined.
  • the reaction solution may be filtered through a protein binding matrix, e.g. nitrocellulose, that is arranged in the same geometrical pattern as the original microtiter plate where the reaction took place, and the radioactivity retained in the filters, or the amount of liberated free phosphate (or other label as appropriate) is quantified.
  • a protein binding matrix e.g. nitrocellulose
  • the Rh ⁇ protein may be immobilized on a solid matrix, either via a tag that allows for binding to a suitably modified solid support, e.g.
  • the solid matrix may also be in the form of beads.
  • the Rho protein is preferably human.
  • the assay may be conducted as follows: A member of the Rho family GTPases is immobilized on a solid support, e.g. a SPA (proximity scintillation assay bead) using either an epitope tag such as His6 or other similar tags.
  • the Rho protein is loaded with radioactive 32 P-gamma-GTP in the presence of EDTA.
  • Magnesium and Cyk-4 are added in the absence or presence of test compounds, incubated for various times at which point the amount of radioactivity or other suitable label remaining associated with the beads is measured.
  • Other embodiments of the assay include using alternative labels such as fluorescent GTP or other radioisotopes and using alternative measures of GTP hydrolysis such as measuring the amount of free 32 P released, as described above.
  • Rho protein a truncated version may be used, as long the GTPase
  • / activity is maintained, e.g. a fragment that lacks the critical cysteine residue in the CaaX box at the C-terminus that is required for membrane targetting.
  • Rho proteins are available that are suitable for use in a GTP hydrolysis assay as a substrate, they have been cloned from various species. Preferably, the are employed as recombinant proteins.
  • Rho substrates examples include human RhoA (Swiss Prot Primary Accession Number P06749) , human RhoB (Swiss Prot Primary Accession Number P01121) , human RhoC (Swiss Prot Primary Accession Number P08134), human RAC1 (Swiss Prot Primary Accession Number P15154), human RAC2 (Swiss Prot Primary Accession Number P15153) , human RAC3 (Swiss Prot Primary Accession Number 014658) and human GB25 (Swiss Prot Primary Accession Number P21181.
  • the Rho substrates and the potentially present co-factor (s) can be produced recombinantly in suitable host, cells, e.g. in E. coli or by means of baculovirus in insect cells, according to standard methods.
  • Rho family GTPases Compounds that have the ability to prevent CYK-4 stimulated GTP hydrolysis by Rho family GTPases are then tested for specificity in a similar assay format using other proteins that have Rho family GAP domains, e.g. pl90 or RhoGAP (Lamarche and Hall, 1994).
  • the compound' s ability to inhibit Cyk-4 function is determined by determining the compound' s ability to interfere with the biochemical interaction of CYK-4
  • CYK-4 in cytokinesis is intimately tied to its ability to interact with MKLPl, a kinesin like protein that is also required for central spindle assembly. Therefore compounds that inhibit this interaction are expected to prevent cytokinesis and to inhibit cell proliferation.
  • HsMKLPl human; GenBank ID X67155; SwissProt Q02241 .
  • HsMKLPl is employed.
  • CYK-4 and CeMKLPl are present in a protein complex and can be co-immunoprecipitated and co-purified.
  • the nature of this protein interaction can be further characterized, in particular by determining whether additional proteins also interact with this complex.
  • a compound's ability to modulate Cyk-4 function is determined by determining its effect on the interaction of said protein with the Cyk-4/MKLPl complex.
  • the domain of Cyk-4 that interacts with MKLPl i.e. the N terminal Cyk-4 region containing at least amino acids 1-120
  • the whole Cyk-4 protein is immobilized on a solid support, as described below.
  • the domain of MKLPl that interacts with CYK-4 i.e. the central MKLPl domain containing at least amino acids 503-603
  • the whole MLKPl protein that is modified with a suitable label to allow for rapid detection i.e. radiolabelled, fluorescently labeled, hapten labelled etc., as described below
  • a suitable label to allow for rapid detection i.e. radiolabelled, fluorescently labeled, hapten labelled etc., as described below
  • the amount of MKLPl bound to the immobilized CYK-4 is measured by use of the label outlined above or by the use of suitable antibodies in an ELISA type assay.
  • the assay may also be setup in the reverse, e.g. with Cyk-4 being labeled or with MKLPl immobilized, or by performing the binding reaction in solution and then capturing one of the components on a solid support and measuring the amount of the other component that is co-immobilized.
  • the invention relates to a screening method that takes advantage of the biochemical multimerization of CYK-4 on the one hand and the biochemical multimerization of the MKLPl subfamily of the kinesin-like protein superfamily on the other hand.
  • This approach is based on the finding that the domain of CYK-4 that is required for interaction with MKLPl contains a coiled-coil domain that mediates self association.
  • the domain of MKLPl that is required to interact with CYK-4 contains a coiled-coil domain that mediates self association. Since the ability of these proteins to ' self association is expected to be required for their function in vivo, compounds that inhibit the self assocation of either one or both of the interaction partners Cyk-4 and MKLPl are candidates for perturbing cytokinesis.
  • the N- terminal domain of CYK-4 (containing at least amino acids 1-120) , or the whole Cyk-4 protein, is immobilized on a solid support, either directly or through a tag.
  • Suitable tags are commercially available, e.g. the FLAG, HA, MYC, HIS tag, etc.).
  • solid supports useful in the present invention are commercially available immunobeads or immunoplates, e.g. 96- well immunoplates, or microchips, which are coated with an antibody directed against one of the above-listed tags fused to the interacting protein.
  • a second CYK-4 fragment consisting of or containing the N terminal domain of CYK-4, i.e. amino acids 1-120, or the whole Cyk-4 protein is modified with a suitable label that allows for rapid detection.
  • the immobilized and the labeled Cyk-4 protein (fragments) may be identical or different.
  • Suitable labels are commercially available radioactive or fluorescence labels, hapten labels, or peptide labels, e.g. Europium or the Green Fluorescent Protein (GFP), an enzyme label, e.g. luciferase, alkaline phosphatase etc.)
  • GFP Green Fluorescent Protein
  • an enzyme label e.g. luciferase, alkaline phosphatase etc.
  • the amount of CYK-4 bound to the immobilized CYK-4 is determined by measuring the signal of the label or by the use of suitable antibodies in an ELISA type assay.
  • the screening method to identify modulators of Cyk-4 function is based on determining whether a test compound has the ability to interfere with the self association of MKLPl.
  • the central domain of MKLPl containing at least amino acids 503-603 or the whole MKLPl protein, is immobilized on a solid support.
  • a second MKLPl fragment consisting of or containing the above-defined central domain of MKLPl, or the whole MKLPl protein is modified with a suitable label that allows for rapid detection.
  • the immobilized and the labeled MKLPl protein (fragments) may be identical or different) .
  • the assay for identifying inhibitors of MKLPl self assembly may be conducted in the same way as described above for identifying inhibitors of Cyk-4 self assembly.
  • All embodiments of the screening methods of the invention may be performed in the high throughput format by automation of the reaction steps.
  • a great number of compounds e.g. from compound or natural product libraries, are applied to microtiter plates containing the components necessary for the reaction.
  • the candidate compounds can be assayed for their effect on cytokinesis and other cellular processes in tissue culture of normal or transformed cells.
  • cytokinesis cytokinesis and other cellular processes in tissue culture of normal or transformed cells.
  • primary human tumor cells are incubated with the compound identified in the screen and the inhibition of tumor cell proliferation is tested by conventional methods, e.g. bromo-desoxy-uridine or 3 H thymidine incorporation.
  • Compounds that exhibit an anti-proliferative effect in these assays may be further tested in tumor animal models and used for the therapy of tumors.
  • the invention relates to compounds identified in the above screens for the therapy of tumors and any other situation in which cell overproliferation is observed.
  • Toxicity and therapeutic efficacy of the compounds identified as drug candidates by the screening methods of the invention can be determined by standard pharmaceutical procedures, which include conducting cell culture and animal experiments to determine the IC50, LD5O, the EDso- The data obtained are used for determining the human dose range, which will also depend on the dosage form (tablets, capsules, aerosol sprays, ampules, etc.) and the administration route (oral, buccal, nasal, paterental or rectal) .
  • a pharmaceutical composition containing the compound as the active ingredient can be formulated in conventional manner using one or more physologically active carriers and excipients. Methods for making such formulations can be found in manuals, e.g. Remington Pharmaceutical Sciences, 1980; Remington: The Science and Practice of Pharmacy, 2000.
  • cyk-4 (tl 689ts) mutant embryos fail to complete cytokinesis .
  • cyk-4 mutant embryos produce deeply ingressing cleavage furrows, but do not form a prominent central spindle.
  • RNAi cyk-4
  • CYK-4 enhances GTP hydrolysis by Rho, Rac, and Cdc42.
  • RNAi cytokinesis defects
  • cdc42 RNAi
  • CYK-4 and ZEN-4/CeMKLPl colocalize and are interdependent for their localization.
  • RhoA does not have a role in the formation of central spindle
  • CYK-4 and ZEN-4 associate in vivo and in vitro
  • ZEN-4 The central region of ZEN-4 is necessary and sufficient to bind to CYK-4
  • a complex of CYK-4 and ZEN-4 is required for function in vivo
  • the cyk-4 (tl 689ts) allele was identified in a search for maternal effect lethal mutations on chromosome III (see (G ⁇ nczy et al., 1999b) for details).
  • the strains DR104, BW1535, BW1369, and RW7000 were obtained from the CGC (C. elegans Genetics Center) .
  • the strain EU699 containing the zen-4 (orl53ts) allele was described by Severson, et al., 2000.
  • the end points of the deficiency tDflO Heinke Schnabel, unpublished data) are not molecularly defined, but it uncovers cyk-4, lit-1 , and bli-5 and it does not uncover unc-64.
  • tDflO Heinke Schnabel, unpublished data
  • Cyk-4 specific antisera were produced in rabbits using a His6-Cyk-4 fusion protein as immunogen (containing amino acids 407-613 of cyk-4) .
  • a GST-Cyk-4 fusion containing amino acids 407-681 of Cyk-4 was coupled to a Hi-Trap NHS resin (Amersham-Phamacia) and used to affinity purify anti-Cyk-4 antibodies which were used at a final concentration of 1:300.
  • the antibodies used for the studies are specific for Cyk-4 since the staining can be blocked with antigen, a similar pattern is observed when anti-GFP antibodies are used to detect a Cyk-4: GFP fusion construct, and the staining pattern is disrupted in cyk-4 mutant embryos.
  • the rat monoclonal YOL 1/34 anti-tubulin antibody was used at a dilution of 1:200-500.
  • Anti-GFP antibodies (Roche) were used at a dilution of 1:500.
  • Antisera specific for Zen-4/MKlpl was generously provided by Bill Saxton and Susan Strome (Univ. of Indiana) and used at a dilution of 1:4000.
  • Antisera specific for Air-2 was generously provided by Andy Golden (NIH) and used at a dilution of 1:1000.
  • the cyk-4 locus map's under the deficiency tDf6 which deletes a large fraction of the distal right arm of LGIII. Recombination mapping using unc-32 (el 89) cyk-4 (tl 689ts) /dpy-18 (e364) unc-25 (el56) placed cyk-4 distal to (or very close to) unc-25 (23/23 Dpy non Unc's carried the cyk-4 mutation).
  • Time lapse Nomarski imaging was performed as described previously (Jantsch-Plunger and Glotzer, 1999) .
  • Time lapse imaging of Cyk-4 GFP was performed on a Zeiss Axiovert microscope using a 100X/1.3 neofluar objective.
  • the illumination source an Atto-arc HBO-103
  • An intensified cooled CCD camera (GenlV pentamax, Princeton Instruments) was used for image acquisition.
  • the camera and other electronics were controlled with MetaMorph software (Universal Imaging) .
  • Typical acquisition times were 40-80 msec. Each 10 seconds, 4-5 fluorescent images were acquired at different focal planes and a nomarski image was acquired.
  • the fluorescent images 1 were projected onto a single frame using the maximum intensity from the stack of images. Under these j conditions, embryos could be filmed for more than one hour without affecting the cell cycle timing or pattern of cell divisions.
  • cosmid DNA from stocks kindly provided by Alan Coulson, Sanger Center
  • rol-6 sulOO ⁇
  • FI hermaphrodites that carried the rol-6 dominant marker were cloned to individual plates at 25°C and the presence of Unc progeny, indicating zygotic rescue of the cyk-4 mutation, was assessed.
  • Individual unc-32, cyk-4 worms carrying the extrachromosomal arrays were cloned to individual plates to assess the extent of germline rescue.
  • the cyk-4 genomic rescue construct MP17 contains a 4.9 kb genomic Xbal fragment excised from K08E3 and inserted into pBS-KS+.
  • the GFP cassette from vector pPD119.16 was excised with BspLUIII and inserted into the unique Ncol site of MP17 (see above) .
  • This construct was linearized with Xbal, and complex arrays containing linearized genomic DNA and linearized rol-6 (sulOO ⁇ ) DNA were mixed in a ratio of 1:100:1 and injected into unc-32 (el89) cyk-4 (tl 689 ts) /qCl hermaphrodites. Rolling FI heterozygotes were singled out at 25°C and rolling Unc F2 animals were picked.
  • a line MG110 was obtained that gave stable rescue of the cyk-4 (tl 689ts) mutation.
  • the structure of the cyk-4 gene was established by analysis of a large number of EST sequences available in the sequence databases at the Sanger Center and the National Institute of Genetics and by sequencing the clones yk63D6 and ykl04gl2 (kindly provided by Yuji Kohara) .
  • the structure of the gene is identical to the structure predicted by the C. elegans genome consortium.
  • Rho, Rac, and Cdc42 were PCR amplified and cloned into pET28b with an C-terminal polyhistidine tag.
  • the GTPases were expressed at 25°C and purified using Ni 2+ -NTA-agarose (QIAGEN) . Proteins were dialyzed into 50 mM Tris pH 7.5, 50 mM NaCl, 5 mM MgCl 2 and quick frozen. The GAP domain of Cyk-4 (amino acids 407-681) was cloned into pGEX4T-l. Proteins were expressed at 25°C and purified using GSH-agarose (Sigma) .
  • Proteins were dialyzed into 50 mM Tris pH 7.5, 50 mM NaCl, 5 mM MgCl 2 , 1 mM DTT and quick frozen.
  • 15 pmol of the GTPases were loaded with 1 pmol 32 P- -GTP in 20 mM Tris pH 7.6, 4 mM EDTA, 25 mM NaCl, 1 mM DTT, 1 mM ATP, 0.1 mg/ml BSA at room temperature. The sample was place on ice and MgCl 2 was added to 17 mM.
  • GST-Cyk-4-GAP was added at the indicated concentrations and, at intervals, samples were taken by dilution into 2% SDS, 20 mM EDTA. Aliquots were spotted onto TLC plates (PEI-cellulose, Machery-Nagel) and developed in 1M LiCl. The plates were dried and exposed using a Storm Phosphoimager: (Molecular Dynamics) and the data analysis was performed using the public domain NIH Image program (developed at the U.S. National Institutes of Health; http : //rsb . info . nih. gov/nih-image/) .
  • Example 1 Example 1
  • cyk-4 mutants initiate, but do not complete, cytokinesis
  • the cyk-4 (tl 689ts) allele was isolated in a screen for 5 maternal effect embryonic lethal mutations on chromosome III (G ⁇ nczy et al., 1999b).
  • the cyk-4 locus is defined by a single, temperature sensitive (ts) , allele.
  • cyk-4 mutant embryos 10 temperature (hereafter referred to as cyk-4 mutant embryos) during the first division is shown in figure IB.
  • cyk-4 mutant embryos appear normal until cytokinesis, except that defects in polar body extrusion are frequently observed (data not shown) .
  • Wildtype embryos (A) and embryos from cyk-4 mutant hermaphrodites (B) were dissected from young adults, mounted on agarose pads and observed by time-lapse nomarski microscopy. The ingressing cleavage furrow is indicated (arrows).10 ⁇ m
  • cyk-4 is required postembryonically, perhaps due to its role in cell division.
  • animals shifted at the L2/L3 stages become highly uncoordinated adults, even though most motor neurons would be expected to have completed all their divisions at the time of the shift. This may suggest that CYK-4 has additional roles besides its role in cell division. However, not out the possibility cannot be ruled out that some of the cells in the ventral nerve cord divide later in cyk-4 mutants.
  • FIG. 2A,B shows that cyk-4 mutant embryos produce deeply ingressing cleavage furrows, but do not form a prominent central spindle. Wild type (A, C) and cyk-4 mutant embryos (B, D) were fixed and stained for actin (green) and DNA (blue) (A,B); tubulin (green) and DNA (blue) (C,D). 10 ⁇ m scale bars.
  • the cyk-4 gene maps distal to unc-64 on the extreme right arm of LG III.
  • Figure 3 shows the positional cloning of the cyk-4 locus.
  • A A schematic of LGIII showing the positions of various loci and the extent of the deficiencies, cyk-4 is uncovered by tDflO, tDf ⁇ , and ctDf2, but not ctDf3.
  • B An enlargement of the physical map from the unc-64 locus until the end of LGIII.
  • the cyk-4 gene was then identified by functional rescue of the zygotic requirement for cyk-4 using pools of cosmids.
  • a pool of three cosmids (ZK520, W06F12, and K08E3) allowed cyk-4 homozygotes to hatch and develop to adulthood at 25°C. These cosmids were injected individually and cosmid K08E3 contained rescuing activity. Further subcloning revealed that a 4.9 kb genomic fragment, predicted to contain the complete K08E3.6 gene and no other intact gene, could rescue the cyk-4 zygotic and germline phenotypes.
  • the coding region was amplified from DNA derived from cyk-4 (tl 689) homozygotes and sequenced and a single point mutation was identified that differed from the, sequence provided by the genome project; this mutation was not observed in another line derived from the same parental strain.
  • Adjacent to the C-terminal GAP domain is a 5 Cl domain that is predicted to bind to diacylglycerol or phorbol esters.
  • Adjacent to the C-terminal GAP domain is a 5 Cl domain that is predicted to bind to diacylglycerol or phorbol esters.
  • At the amino terminus of the protein is a 90 amino acid region predicted to form a coiled-coil domain.
  • the S15L point mutation found in cyk-4 (tl 689ts) is located just amino terminal to the
  • the human and mouse cDNA's were identified by searching the DNA databases 25 with the C. elegans CYK-4 protein sequence.
  • RNA-mediated interference was used to deplete embryos of CYK-4 protein.
  • FIG. 4 shows that cyk-4 (RNAi) causes disorganization 30 of the proximal gonad and affects formation of the central spindle. Young adults were injected with cyk-4 dsRNA and the injected animals were analyzed 30 hours after injection (B) . The gonad of an uninjected worm is shown for comparison (A) . Wild-type embryos (C) and embryos from cyk-4 (RNAi) injected animals (D) were fixed and stained for tubulin. 10 ⁇ m scale bars.
  • RNAi of the predicted open reading frame K08E3.6 generated multinucleate embryos which exhibited a similar phenotype to that of the cyk-4 mutant, including loss of the central spindle (figure 4 D) and incomplete cytokinesis (not shown) .
  • the gonads of cyk-4 (RNAi) animals become disorganized 30 hours post-injection (figure 4 B) and irregularly sized embryos are produced, suggesting that CYK-4 acts not only during embryonic and zygotic development, but also in the female germline.
  • Rho-family GAP domain suggests that
  • CYK-4 may regulate one or more GTPases of Rho branch of the GTPase superfamil ⁇ . To determine whether CYK-4 is active as a GAP and,whether its GAP activity is
  • CYK-4 GAP domain was prepared and GTP hydrolysis assays were performed with recombinant C. elegans Rho, Rac, and Cdc42.
  • the GAP domain of CYK-4 promotes GTP hydrolysis by all three tested GTPases.
  • Figure 5 shows that CYK-4 enhances GTP hydrolysis by Rho, Rac, and Cdc42.
  • the GTPases were preloaded with 32 P- ⁇ -GTP and then GST-CYK-4-GAP or GST was added at the indicated concentration. Samples were taken at two minute intervals, the labeled nucleotide was resolved by thin layer chromatography, the fraction of GTP and GDP was quantitated. This graph shows the fraction of GTP hydrolyzed at the 2 minute time point as a function of CYK-4 concentration.
  • RNAi was used to determine which, if any, of the Rho family GTPases are required for cytokinesis in the C. elegans embryo. RNAi experiments were performed with RhoA, Racl, Cdc42 and three additional GTPases found in the genome that fall into the Rho subfamily. Approximately 90% of Rho (RNAi) embryos exhibit cytokinesis defects in the first and/or second cell cycle (table 2 and figure 6) .
  • Figure 6 shows that rho (RNAi) causes cytokinesis defects and cdc42 (RNAi) causes defects in spindle positioning. Young adults were injected with the indicated dsRNAs (see table 2) and the embryos produced by the injected worms were analyzed by time lapse nomarski microscopy. Images from a wild-type (A) , rho f- N ⁇ i) (B) and a cdc42 (RNAi) (C) embryo are shown. 10 ⁇ m scale bars.
  • RNAi RhoA embryos
  • RNAi central spindles assemble in RhoA embryos (data not shown) .
  • 88% of Cdc42 (RNAi) embryos complete cytokinesis normally.
  • a distinct defect in the early embryo is observed in 54% of Cdc42 (RNAi) embryos; defects in spindle positioning are observed in P0 and/or PI.
  • a minority (12%) of Cdc42 (RNAi) embryos fail to initiate cytokinesis; in most cases, these embryos appear osmotically swollen even when provided with osmotic support.
  • Rac (RNAi) embryos hatch with high efficiency and did not exhibit a detectable phenotype in the early embryo.
  • RhoA is the only member of the Rho family that is clearly required for cytokinesis and is therefore likely to be the critical target for the CYK-4 GAP domain.
  • CYK-4 localization is cell cycle dependent Figure 7 shows that CYK-4 localizes to the central spindle and division remnants.
  • A-F Wild-type embryos were fixed and stained for CYK-4 (green) , tubulin (red) and DNA (blue) .
  • the localization of CYK-4 to the central spindle (arrow) in a one cell embryo (D) and a two cell embryo (F) is shown.
  • the localization of CYK-4 to the division remnant from the polar body (A) , and between the AB and Pi blastomeres is indicated with arrows.
  • E The localization of CYK-4 to the division remnant from the polar body
  • G An embryo from a line expressing CYK-4: GFP is stained with anti-GFP antibodies. The same structures are seen as with CYK-4 antibodies (arrow) .
  • H The intrinsic fluorescence of the .gonad of a worm expressing CYK-4: GFP. CYK-4 is seen at the incomplete membranes of the syncytial gonad (arrow) and in oocyte nuclei. 10 ⁇ m scale bars.
  • CYK-4 In interphase cells, CYK-4 is present in the cytoplasm and slightly concentrated in the nucleus. CYK-4 is also highly concentrated in a spot at the anterior of the embryo, DNA labeling reveals that this localization corresponds to the site of polar body extrusion. As embryos enter mitosis, CYK-4 protein concentrates around the mitotic spindle. In early anaphase, CYK-4 concentrates to the central spindle. As the cleavage furrow ingresses, CYK-4 becomes highly concentrated on the central spindle ' into a structure that often appears ring shaped (not shown) . Upon completion of cytokinesis, CYK-4 staining persists at division remnants.
  • CYK-4 organized in ring-like structures averaging 1.2 ⁇ m in diameter are occasionally observed in the cytoplasm (not shown) .
  • the dynamics of CYK-4 localization in live embryos was investigated.
  • a transgenic line expressing a CYK-4: GFP fusion was generated and its localization followed by time lapse microscopy.
  • the CYK-4: GFP fusion is partially functional since cyk-4 xsExl [cyk-4 : GFP] animals are viable and fertile at 25°C whereas the parental cyk-4 strain is inviable at 25°C.
  • the fusion construct does not fully rescue the mutation, since about 40% of embryos produced by this line fail to hatch (table 1) .
  • CYK-4 GFP in living embryos. Embryos were imaged using a multi-mode imaging system whereby a series of z-sections and a nomarski image were recorded every 10 seconds. The fluorescent images from each time point were projected to form a single image.
  • Figure 8 shows the time lapse analysis of CYK-4: GFP.
  • An embryo from a line expressing CYK-4: GFP was imaged using low light level microscopy. The central spindle localization of CYK-4 is observed prior to furrow ingression. 10 ⁇ m scale bars.
  • Embryos expressing CYK-4: GFP were fixed and CYK-4 and ZEN-4 were localized simultaneously (using an anti-GFP antibody to detect CYK-4: GFP).
  • the two proteins co- localize both on central spindle structures and on division remnants ( Figure 9A-C) .
  • ZEN-4 staining of cyk-4 mutant embryos reveals that ZEN-4 localization to the central spindle is absent (figure 9E) , although staining of some microtubule bundles in the spindle midzone could be detected using an AIR-2 antibody (figure 9G) .
  • recruitment of ZEN-4 to the central spindle is CYK-4 dependent .
  • Figure 9 shows an embryo expressing CYK-4: GFP stained for GFP (A) , ZEN-4/CeMKLPl (B) and the merged image (C) .
  • CYK-4 and ZEN-4/CeMKLPl colocalize at division remnants (arrow) and a central spindle (arrowheads) structures.
  • ZEN-4 localization to the central spindle is CYK-4 dependent (arrow) .
  • Wild-type (D) and cyk-4 (tl 689ts) embryos (E) were fixed and stained for ZEN-4/CeMKLPl (green) , tubulin (red) and DNA (blue) .
  • AIR-2 localization to the central spindle is CYK-4 independent (arrow).
  • Wild-type (F) and cyk-4 (tl 689ts) embryos (G) were fixed and stained for AIR-2 (green) , tubulin (red) and DNA (blue) .
  • CYK-4 maintenance to division remnants (arrowheads) is ZEN-4/CeMKLPl dependent.
  • zen-4 (orl53ts) worms were maintained at 16°C and either fixed immediately (H) or shifted to 25°C for 18 minutes (I) .
  • Embryos were fixed and stained for CYK-4 (green), tubulin (red) and DNA (blue). 10 ⁇ m scale bars.
  • Suppressors of the cyk-4 (tl 689ts) mutation were obtained by mutagenizing cyk-4 (tl 689ts) animals with 30-40 mM EMS or 0.5 mM ENU. P0 animals were allowed to self fertilize for two generations at the permissive temperature. When F2 animals reached early adulthood, the population was shifted 20°C and fertile animals were selected. Approximately 110,000 FI genomes were screened and 18 suppressor mutations, all but 2 being unambiguously independent, were isolated. While all of the suppressor strains were viable and fertile at 20 °C, none were able to grow at 25°C, suggesting that none of them precisely reverted the original mutation. Several intragenic suppressor mutations were identified.
  • the ZEN-4 coding region of the remaining mutants were sequenced and 7 strains were found to contain substitutions within the CYK-4 binding region of ZEN-4 and one contained a substitution in the catalytic domain.
  • One suppressor strain, xs82 was characterized in detail. Suppressor activity was mapped using single nucleotide polymorphisms (SNPs) to the central region of chromosome IV (see methods) .
  • SNPs single nucleotide polymorphisms
  • the ZEN-4 coding region was sequenced in this strain and a point mutation was found that causes a substitution of glutamic acid for a lysine at position 502 (fig 15A) .
  • HeLa cells were routinely grown in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin.
  • DMEM Dulbecco's modified Eagle's medium
  • CYK-4 and ZEN-4 antibodies were affinity-purified with His6-CYK-4 (407-613) or His6-ZEN-4 (578-775) immobilized on NHS-Sepharose (Pharmacia) .
  • MKLP-1 specific antisera and HsCYK-4 specific antisera were produced in rabbits and mice (Gramsch
  • a cDNA clone for ZEN-4 in pBluescript (yk391b3) which lacks 13 nts at 5' of the coding region which were introduced by PCR.
  • the pCBD-TEV vector was constructed by inserting the sequences encoding the chitin-binding domain (CBD) and a TEV protease site into pET28a (Novagen) .
  • ZEN-4 fragments were amplified by PCR and cloned into pCBD-TEV.
  • CYK-4 and ZEN-4 were expressed by in vitro transcription and translation system with reticulocyte lysates using the TNT® Coupled Reticulocyte Lysate Systems (Promega) or the PROTEINscriptTM II (Ambion) kits, typically in 20 ⁇ l reactions.
  • Full length proteins without tag were expressed from the T3 promoter of pBluescript SK(-).
  • CYK-4 and ZEN-4 tagged with chitin-binding domain (CBD) at N-terminus were expressed from the T7 promoter of pET-CBD.
  • CBD chitin-binding domain
  • Triton X-100, ZEN-4 or CYK-4 proteins were immunoprecipitated with specific antibodies, or affinity-purified by chitin beads.
  • C. elegans embryos were prepared by bleaching a synchronous culture of adult worms grown on "egg plates" as described (Lewis and Fleming, 1995) . The embryos were washed with buffer A without Triton-X 100 and frozen with liquid nitrogen and kept at -80°C until use. Embryos were crushed by grinding in a mortar and pestle in liquid nitrogen.
  • HeLa cells were synchronised by treatment with 0.1 ⁇ g/ml nocodazole for 16 h. Cells were washed twice with cold PBS, frozen by liquid nitrogen and kept -80 °C until use. Cells were lysed and thawed by suspending into 10 volumes of buffer A with 0.5% (w/v) Triton X-100.
  • Frozen pellet (0.5 ml) of nocodazole arrested M-phase cells was thawed and lysed in 5 ml ice-cold BRB80 (80 mM K-Pipes, 1 mM MgCl 2 , 1 mM EGTA, pH 6.8) with 0.1% (w/v) Triton X-100.
  • the lysate was clarified by centrifuging at 10,000 x g for 15 min.
  • the resulting supernatant was centrifuged at 25,000 x g for 20 min at 4°C in S100AT6 rotor (Hitachi) .
  • Microtubules polymerized with glycerol and taxol was added to the supernatant. After incubating at 20°C for 20 min, microtubules were pelleted by centrifuging at
  • Sedimentation coefficient was estimated by ultracentrifugation through 2 ml linear gradient of 5 to 20% (w/v) sucrose in buffer A without Triton X-100 using S55S rotor (Hitachi) . 150 ⁇ l cell lysate or microtubule binding fraction was applied. Chicken ovalbumin (3.4 S) , bovine gamma globulin (7.1 S) , bovine catalase (11 S) and bovine thyroglobulin (19 S) were used as standards. The diffusion coefficient was estimated by gel filtration using Superdex 200 column (30 ml) in FPLC system (Pharmacia) .
  • Lysates (500 ⁇ l) were precleared with 50 ⁇ l protein A-Sepharose beads.
  • 1 ⁇ g affinity-purified anitibody was added to the lysate.
  • immunocomplex was recovered by incubation with 5 ⁇ l protein A-Sepharase (Pharmacia) at 4°C for 1 hr.
  • antibodies were covalently immobilized on protein A-beads (1 ⁇ g per 1 ⁇ l beads) with dimethyl pimelimidate.
  • the precleared lysate was incubated antibody-beads at 4°C for 1 to 4 h.
  • the beads were washed briefly with buffer A plus 0.5 % Triton X-100 three times.
  • Immunolocalization studies in worm embryos were performed as previously described ref. In brief, gravid hermaphrodites were placed on aminopropyl-silane treated slides, a coverslip was added, and sufficient pressure to extrude the embryos was applied (Jantsch- Plunger et al., 2000). The slide was placed into liquid nitrogen. The coverslip was removed while the sample was still frozen, the preparation was fixed with -20°C methanol, and antibody staining was performed according to standard procedures. Immunolocalization studies using HeLa cells were performed according to standard procedures following fixation in -20 °C methanol.
  • RhoGAP protein CYK-4 The process of central spindle assembly is dependent on the RhoGAP protein CYK-4. In the previous Examples, it was found that RhoA is essential for cytokinesis while Rac and Cdc42 are not (Jantsch-Plunger et al., 2000). Since RhoA is required for cytokinesis, it was tested if this GTPases is also required for central spindle assembly. To inactivate RhoA worms were grown on bacteria expressing dsRNA from the coding region of the gene (Timmons and (Fire, 1998) .
  • Embryos were fixed and immunoflorescence-stained for CYK-4, microtubules and DNA with anti-CYK-4 antibody, anti- ⁇ -tubulin antibody and Hoechst 33342) .
  • RNAi rhoA
  • RhoA dsRNAi causes a fully penetrant inhibition of cytokinesis
  • central spindle assembly occurs normally, and CYK-4 localizes to the central spindle (figure 11) .
  • RhoA in cytokinesis can not be accounted for by a requirement for RhoA in central spindle assembly.
  • CYK-4 and ZEN-4 form a complex in vivo and in vitro
  • CYK-4 and ZEN-4 are competent to interact in the absence of other nematode proteins
  • the two proteins were produced by in vitro translation and subjected to immunoprecipitation.
  • CYK-4 and ZEN-4 fused to the chitin binding domain (CBD)
  • CBD chitin binding domain
  • both proteins could be recovered in high yield by chitin beads (fig. 12B) .
  • the co-precipitation of CYK-4 was dependent on ZEN-4; CYK-4 was not recovered on chitin beads when ZEN-4 was substituted by CBD alone.
  • luciferase was substituted for CYK-4, it did not co-precipitate with ZEN-4.
  • the association between CYK-4 and ZEN-4 could also be detected when anti-CYK-4 antibodies were used to retrieve the complex (fig. 12C) .
  • Figure 12 shows that CYK-4 and ZEN-4 bind to each other in vivo (A) and in vitro (B and C) .
  • CYK-4 and ZEN-4 were immunoprecipitated from worm embryo extracts with anti-CYK-4 antibody, anti-ZEN-4 antibody, or with non specific rabbit IgG as a control. The immunoprecipitates were resolved on SDS-PAGE gels followed by western blotting with an anti-ZEN-4 antibody. Mock immunoprecipitation without extract was included to control for cross reactivity with the antibodies.
  • ZEN-4 specifically co-immunoprecipitated with CYK-4.
  • CYK-4 (or luciferase) and CBD-ZEN-4 (or CBD alone) were co-expressed as 35 S-labeled proteins in the indicated combinations (left panel) .
  • CYK-4 was immunoprecipitated with an anti-CYK-4 antibody (right panel).
  • ZEN-4 co-immunoprecipitated with CYK-4.
  • the C-terminal 250 amino acids contains a RhoGAP domain that is active in vitro against RhoA, Rac, and Cdc42 (Jantsch-Plunger et al., 2000).
  • the RhoGAP domain is preceded by a Cl domain, a cysteine-rich domain that mediates interactions with diacylglycerol (Hurley and Meyer, 2001) other ref.
  • Truncations of the C-terminus of CYK-4 revealed that the ZEN-4 binding region is contained within the N-terminal 232 amino acids (fig. 13A) .
  • Figure 13 shows the results of the in vitro binding assay that was used to define the ZEN-4 binding region of CYK-4.
  • A Full-length ZEN-4 and C-terminal deletion fragments of CYK-4 (1-232, 1-373 and 1-468) were separately expressed as 35 S-labeled proteins by in vitro translation (left two panels) . The reaction products were mixed and after incubation ZEN-4 was immunoprecipitated with an anti-ZEN-4 antibody (right panel) . The three fragments of CYK-4 co- immunoprecipitated with ZEN-4.
  • B Full length CYK-4 (1-681) and CYK-4 with N-terminal deletion (35-681) were expressed as 35 S-labeled proteins and mixed with unlabeled CBD-ZEN-4 (left panel) .
  • CBD-ZEN-4 was affinity purified on chitin beads (right panel) .
  • CYK-4 (35-681) did not copurify with CBD-ZEN- 4, but full length CYK-4 did.
  • CBD-CYK-4 fragments (1-232, 35-232, (121-232, 1-120 and 35-120) and full length ZEN-4 (o'r luciferase as a control) were co- expressed as 35 S-labeled proteins by in vitro translation (left panel) .
  • CBD-CYK-4 fragments were affinity purified (right panel) .
  • ZEN-4 copurified with CYK-4 (1-232) and (1-120), but not with CYK-4 (35-232), (121-232) or (35-120) .
  • D A schematic summary of the CYK-4 derivatives tested.
  • the ZEN-4 binding region was defined as residues 1-120 of CYK-4. This region contains a coiled coil region and a N-terminal extension.
  • the in vitro assay was used to define the region of ZEN-4 that binds to the N-terminal binding of CYK-4.
  • the CYK-4 binding activity of full length ZEN-4 was compared to that of three C-terminal deletion fragments of ZEN-4: the N-terminal catalytic core alone (1-434), the catalytic core domain and the linker region (1-507), and a longer derivative that also includes the coiled coil domain (1-603) .
  • the full length protein bound CYK-4 with high affinity, as did the next longer fragment (1-603), but the two smaller fragments did not interact with CYK-4 (fig. 14A) .
  • a minimal binding domain consisting of 169 amino acids was defined (fig. 14B) . This region appears to consist of two elements, the linker region and the coiled'coil; neither of these individual elements had detectable binding activity on their own.
  • Figure 14 shows the results of the in vitro binding assay that was used to show that the central region of ZEN-4 is necessary and sufficient to bind to CYK-4.
  • Full length ZEN-4 (1-775) and C-terminal deletion fragments of ZEN-4 were 35 S ⁇ labeled by in vitro translation and incubated with unlabeled CYK-4 (or luciferase as a control) (left panel) .
  • CYK-4 was precipitated with anti-CYK-4 antibody (right panel) .
  • Full length ZEN-4 and ZEN-4 (1-603) co-precipitated with CYK-4, while shorter fragments (1-507, 1-434) did not.
  • ZEN-4 migrates as a symmetrical peak with a sedimentation value of 6 S, significantly less dense than ZEN-4 sediments in wild type extracts (fig. 15C) .
  • this substitution maps directly to the region of ZEN-4 that was found to mediate the interaction with CYK-4 in vitro.
  • the position of the suppressor was mapped to less than 1 cM surrounding the zen-4 locus. This mapping data combined with the fact that the sequence polymorphism maps to the CYK-4 binding region suggests strongly that these point mutations are responsible for the suppressing activity.
  • CYK-4 S15L is unable to interact with ZEN-4 in the in vitro binding assay. If the immediate consequence of the S15L substitution in the N-terminus of CYK-4 is defective ZEN-4 binding, then an extragenic suppressor mutation in zen-4 that rescues cyk-4 (tl 689) might be expected to restore binding to CYK-4 S15L. To test this possibility a N terminal fragment of CYK-4 carrying the S15L point mutation was translated and this product was mixed with a C terminal fragment of ZEN-4 carrying either the wild-type glutamic acid at position 502 or the lysine substitute allele. The wild-type CYK-4 fragment bound equally well to wild-type and the E502K derivative of ZEN-4.
  • CYK-4 S15L is unable to bind to wild-type ZEN-4.
  • CYK-4 S15L was able to bind to ZEN-4 E502K, though this interaction was weaker than that seen with the wild-type proteins (fig. 15A) .
  • Figure 15 shows that a complex of CYK-4 and ZEN-4 is required for function' in vivo
  • a screen for suppressors of cyk-4 identified a point mutation of zen-4 (xs82) . This allele has a G to A point mutation, resulting in substitution of glutamate at residue 502 to lysine.
  • B The E502K mutation partially restores binding to CYK-4 S15L. CYK-4 fragments (1-232, 1-232 with a point mutation S15L, 35-232) were expressed as 35 S-labeled protein and incubated with unlabeled ZEN-4 fragments (435-775, 435-775 with the E502K mutation, 609-775) . ZEN-4 fragments were precipitated with anti-ZEN-4 antibody.
  • CYK-4 (1-232) co-precipitated with ZEN-4 (435-775) but not with ZEN-4 (609-775) as shown in Figure 13.
  • the S15L mutation in CYK-4 abolishes its ability to bind to ZEN-4 (CYK-4 1-232 S15L and ZEN-4 435-775 wt) .
  • the suppressor mutation in ZEN-4 (E502K) partially recovered this interaction ( (CYK-4 1-232 S15L and ZEN-4 435-775 E502K) .
  • the ZEN-4 E502K mutation does not affect the binding to wild-type CYK-4.
  • C The majority of ZEN-4 is in a complex with CYK-4 in vivo.
  • Extracts were made from wild type (N2) embryos or cyk-4 (tl 689ts) embryos. Sedimentation of ZEN-4 was analyzed by sucrose density gradient centrifugation followed by western blotting. The samples were spiked with the indicated standards and they ran identically in the two gradients. ZEN-4 from wild type embryos sediments at 8 S, whereas ZEN-4 from cyk-4 mutant embryos sediments at 7S.
  • central spindle assembly is dependent on an interaction between CYK-4 and ZEN-4 and have defined the regions of both proteins that are required for this interaction.
  • both binding domains contain coiled coil regions; these regions are required for binding activity. Since coiled coils are often dimerization motifs, and since kinesin motors are often composed of two motor domains tethered together through a coiled coil, it was examined whether CYK-4 and ZEN-4 are individually able self-associate.
  • ZEN-4 The capacity of ZEN-4 to self-associate was examined using a similar strategy.
  • Full length ZEN-4 was co- expressed with affinity-tagged deletion derivatives of ZEN-4.
  • ZEN-4 (503-775; 435-603) did bind to full length ZEN-4 (figure 16B) .
  • the region common to these two fragments consists of residues 503-603 which is the region predicted to form a coiled coil domain.
  • the ability of CYK-4 and ZEN-4 to self associate in vitro raise the possibility that in vivo, the CYK-4/ZEN-4 complex may contain, at a minimum, two molecules each of CYK-4 and ZEN-4.
  • Figure 16 shows the self-association of both CYK-4 and ZEN-4
  • CYK-4 (1-232) which does not contain the epitope recognized' by the anti-CYK-4 antibody, was co-expressed as a 35 S-labeled protein with CYK-4 (1-681) or (121-681) , which do contain the epitope for this antibody (left panel) .
  • CYK-4 (1-681) and (121-681) were immunoprecipitated.
  • CYK-4 (1-232) co- immunoprecipitated with CYK-4 (1-681) , but not with CYK-4 (121-681) .
  • B CBD-tagged fragments of ZEN-4 (1-507, 503-775, 435-603 or 604-775) were co-expressed with untagged ZEN-4 (1-775) (left panel) .
  • CBD-tagged fragments were purified by affinity chromatography with chitin beads (right panel) . Untagged ZEN-4 (1-775) copurified with CBD-ZEN-4 (503-775) and (435-603), but not with ZEN-4 (1-507) or (604-775) . The low extent of labelling of 604-775 as compared to 435-603 is attributable to low cysteine and methionine content of the C-terminal fragment.
  • CYK-4 and ZEN-4 associate in vivo and in vitro and this complex is important for central spindle assembly and cytokinesis.
  • HsCYK-4/MgcRacGAP Hirose et al., 2001; Toure et al., 1998; Wooltorton et al., 1999
  • MKLPl MKLPl
  • MKLPl could be co-immunoprecipitated with anti-HsCYK-4 antibodies and vice versa (fig. 17a) .
  • the immunoblots reveal that small amounts of HsCYK-4 and MKLPl migrate with reduced mobility, likely due to phosphorylation.
  • HsCYK-4 and MKLPl were examined in cultured (HeLa) human cells.
  • HeLa cultured
  • the two proteins precisely co-localize (insets fig 17b) .
  • the two proteins localize to a discrete, central region of the central spindle.
  • Three dimensional reconstruction of optically sectioned anaphase cells reveals that each bundle of microtubules in the central spindle is labeled at its distal end by MKLPl antibodies.
  • This region largely corresponds to the region that appears deficient in tubulin staining, but this is a known artifact caused by epitope masking (Saxton and Mclntosh, 1987) .
  • the extent of staining is somewhat narrower than previous ultrastructural determinations of the extent of microtubule overlap (> 2 ⁇ M) (Mastronarde et al., 1993). Later in cleavage, both proteins localize to a discrete, central portion of the midbody. During interphase, some cells contain HsCYK-4 and MKLPl in the nucleus, whereas others are do not.
  • HsCYK-4 and MKLPl were immunopurified from a mitotic MAP (microtubule- associated protein) fraction that was eluted with ATP and 500 mM NaCl.
  • Antibodies specific for HsCYK-4 and MKLPl were used in parallel for the immunoprecipitation.
  • Coomassie blue staining of both immunoprecipitates revealed two major bands and one minor band (fig. 17C) .
  • Mass spectrometry analysis revealed that the slower migrating major band and the minor band are MKLPl and the faster migrating major band is HsCYK-4.
  • centralspindlin has a native molecular mass of 300 kD and that it dotes not appear to contain any proteins in addition to/HsCYK-4 (70 kD) and MKLPl (100 kD) and given that both components are capable of self- association, it is concluded that centralspindlin is a tetramer containing two molecules each of HsCYK-4 and MKLPl.
  • Figure 17 shows that there exists a complex of MKLP-1 and HsCYK-4 in HeLa cells
  • HsCYK-4/MgcRacGAP and MKLP-1 the human orthologs of CYK-4 and ZEN-4, respectively, exist as a complex in mitotic HeLa cells.
  • HsCYK-4 and MKLP-1 were immunoprecipitated from HeLa cell lysate with specific rabbit antibodies (IP:HsCYK-4 or MKLP-1) and blotted with specific mouse antibodies (blot: HsCYK-4 or MKLP-1) .
  • HsCYK-4 co-immunoprecipitated with MKLP-1 and vice versa.
  • HsCYK-4 and MKLPl co-localize to the central spindle and the midbody.
  • Hela cells were fixed and immunostained with anti-CYK-4, anti-MKLPl, anti- tubulin antibodies and DNA was stained with Hoechst. Shown are cells in interphase (a) and various stages of cytokinesis (b-f) . In main panels, staining of CYK-4 (green) , microtubules (red) and DNA (blue) was shown. In inset, staining of CYK-4 (green) and MKLPl (red) was shown. (C) HeLa cell lysates were analyzed by sucrose density gradient centrifugation followed by blotting with anti-MKLP-1 antibody or anti-HsCYK-4 antibody.
  • MKLP-1 and HsCYK-4 complex were immunoprecipitated from a microtubule- binding fraction (MTfr.) of mitotic HeLa cells with specific antibodies.
  • Major 110 kDa and 75 kDa bands were identified by mass spectrometry as MKLP-1 and HsCYK-4, respectively.
  • the minor band at 115 kDa also corresponds to MKLPl. Broad bands around 50 kDa and bands near gel bottom are heavy (IgGH) and light (IgGL) chains of immunoglobulin leaked from antibody beads.
  • genotype stage at percent phenotype of N time of viable surviving shift to progeny
  • RhoA Y51H4A B 95% ( 175 ) 15 15 (100%)
  • dsRNAs that induced embryonic lethality were further characterized by dissecting embryos and scoring for multinucleate embryos and by performing time lapse recordings and evaluating cytokinesis, spindle orientation in the one-cell embryo (symmetric or asymmetric first cleavage) , and spindle positioning in the PI and AB blastomeres. Table 3
  • genot-ype temperature total total average nutribe. number of number of number of etribrj stained cells remnants/ remnants cell
  • cdcl2p a protein required for cytokinesis in fission yeast, •is a component of the cell division ring and interacts with profilin. J Cell Biol 137, 169-82. Chui, K. K. , Rogers, G. C, Kashina, A. M., Wedaman, K. P., Sharp, D.-J., Nguyen, D. T., Wilt, F. , and
  • Cytokinesis in eukaryotes a mechanistic comparison. Curr Opin Cell Biol 11, 68-80. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A.,
  • Cytoplasmic dynein is required for distinct aspects of MTOC positioning, including centrosome separation, in the one cell stage
  • Rab6- binding kinesin Rab6-KIFL
  • cytokinesis Embo J 19, 5711-9.
  • Centrosome movement in the early divisions of Caenorhabditis elegans a cortical site determining centrosome position. J Cell Biol 109, 1185-93. Imamura, H., Tanaka, K., Hihara, T., Umikawa, M.,
  • Bnilp and Bnrlp downstream targets of the Rho family small G-proteins which interact with profilin and regulate actin cytoskeleton in Saccharomyces cerevisiae. Embo J 16, 2745-55.
  • Rho-associated kinase cleavage furrow-specific phosphorylation of intermediate filaments. Oncogene 18, 2783-8.
  • Plk is an M-phase-specific protein kinase and interacts with a kinesin- like protein, CHOl/MKLP-1. Mol Cell Biol 15, 7143-51.
  • a plus-end-directed motor enzyme that moves antiparallel microtubules in vitro localizes to the interzone of mitotic spindles. Nature 359, 543-7.
  • Cytokinesis and midzone microtubule organization in Caenorhabditis elegans require the kinesin-like protein ZEN-4. Mol Biol Cell 9, 2037-49. 'Rappaport, R. (1985) . Repeated furrow formation from a single mitotic apparatus in cylindrical sand dollar eggs. J Exp Zool 234, 167-71.
  • AIR-2 An Aurora/Ipll-related protein kinase associated with chromosomes and midbody microtubules is required for polar body extrusion and cytokinesis in Caenorhabditis elegans embryos. J Cell Biol 143, 1635-46. Settleman, J. , and Foster, R. , (1995), Methods Enzymol; 256:105-13
  • cyk-1 a C. elegans FH gene required for a late step in embryonic cytokinesis. J Cell Sci 111, 2017-27. Tatsumoto, T., Xie, X., Blumenthal, R., Okamoto, I., and Miki, T. (1999) . Human ECT2 is an exchange factor for rho GTPases, phosphorylated in G2/M phases, and involved in cytokinesis [In Process Citation]. J Cell Biol 147, 921-8.
  • Timmons L., and Fire, A. (1998). Specific interference by ingested dsRNA. Nature 395, 854. Toure, A., Dorseuil, 0., Morin, L., Timmons, P., Jegou,
  • MgcRacGAP a new human GTPase-activating protein for Rac and Cdc42 similar to Drosophila rotundRacGAP gene product, is expressed in male germ cells.
  • pl40mDia a mammalian homolog of Drosophila diaphanous, is a target protein for Rho small
  • GTPase and is a ligand for profilin.
  • Stu-7/air-2 is a C. elegans aurora homologue essential- for chromosome segregation during embryonic and post- embryonic development. Mech Dev 82, 95-108.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Cell Biology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Mammalian CYK-4 polypeptides, which are key molecules required for cytokinesis, and DNA molecules encoding them. Screening methods based on biochemical reactions relevant for the function of CYK-4 can be employed to identify compounds interfering with cell division. Such compounds are useful in tumour therapy.

Description

Cyk-4 polypeptides, DNA molecules encoding them and their use in screening methods
The present invention relates to a new protein Cyk-4, which is involved in cytokinesis, to its use in screening assays, and to therapies interfering with cell division, in particular tumor therapy.
The process of cytokinesis produces two daughter cells from a single parental cell and permanently segregates •the products of the cell division cycle. Cytokinesis is one of the few processes in biology known to require coordination between microtubules and actin filaments. Indeed, in animal cells there are two steps in cytokinesis that rely on interactions between microtubules and the actin-based contractile ring (for review see (Field et al., 1999; Glotzer, 1997). In the first instance, the microtubule-based mitotic spindle specifies the position of the contractile ring. This allows the division plane to be positioned so that the separated chromosomes are partitioned equally into the two daughter cells. Subsequently, after ingression of the cleavage furrow, there is a second step that depends on both microtubules and the contractile ring. Completion of cytokinesis requires the central spindle, which contains bundled, antiparallel microtubules. The molecular mechanisms underlying these two microtubule dependent steps in cytokinesis are not known. The degree to which the progression of cytokinesis depends on the central spindle varies somewhat in different experimental organisms. In invertebrate embryos, a transient interaction between astral microtubules of the mitotic spindle and the cell cortex is sufficient to position the cleavage furrow (Rappaport, 1985) . Furrows specified in this manner ingress, but if the spindle is removed, these furrows do not usually complete cytokinesis (Rappaport, 1985) . In contrast, in Drosophila spermatocytes, contractile ring formation requires the central spindle (Giansanti et al., 1998). Similarly, in cultured mammalian cells, astral microtubules appear to be insufficient to induce furrow ingression, instead the presence or absence of a central spindle determines whether or not a cleavage furrow forms (Cao and Wang, 1996; Eckley et al., 1997; Rieder et al., 1997; Savoian et al., 1999; Wheatley and Wang, 1996) . Moreover, in cultured cells, the central spindle is also required for completion of cytokinesis (Wheatley and Wang, 1996) . In C. elegans embryos, as in invertebrate embryos, only the later stages of cytokinesis appear to depend on the central spindle. Embryos depleted of the kinesin-like protein ZEN-4/CeMKlpl fail to assemble the central spindle, yet cleavage furrows form and ingress, but cytokinesis does not proceed to completion (Powers et al . , 1998; Raich et al., 1998). In summary, the initiation of cytokinesis depends on the central spindle in some but not in all organisms, whereas there appears to be a general requirement for the central spindle for the completion of cytokinesis in animal cells. While it is clear that the central spindle plays an important role in cytokinesis the underlying mechanism remains elusive.
Cleavage furrow ingression is driven by the actin-based contractile ring. Like many actin-based structures, the contractile ring requires the RhoA GTPase for its assembly. Rho family GTPases are thought to act as molecular switches that cycle between inactive GDP-bound forms and active GTP-bound forms; their ability to exchange and hydrolyze GTP is regulated by additional factors, the so-called guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) . Inactivation of RhoA by the exoenzyme C3 (Aktories and Hall, 1989) , inhibits cytokinesis in a wide variety of experimental settings by causing disassembly of cortical actin structures and the contractile ring (Drechsel et al., 1997; Kishi et al., 1993; Mabuchi et al., 1993; Moorman et al., 1996; O'Connell et al., 1999). Further, a Rho GEF is essential for cytokinesis (Prokopenko et al., 1999; Tatsunaoto et al., 1999). GTP-bound RhoA interacts with a number of putative effectors including formins, Rho Kinase, Citron kinase, and a regulatory subunit of myosin phosphatase (for review see (Van Aelst and D' Souza-Schorey, 1997). The requirement for RhoA in 'cytokinesis may reflect its ability to regulate formins since members of the formin gene family are required for cytokinesis in budding yeast (BNI1/BNR1) (Imamura et al., 1997), fission yeast (Cdcl2) (Chang et al., 1997), Drosophila (dia) (Castrillon and Wasserman, 1994) and C. elegans ( cyk-1 ) (Swan et al., 1998).
Several formins also bind to profilin (Chang et al., 1997 ; Evangelista et al., 1997; Imamura et al., 1997; Watanabe et al., 1997), a key regulator of actin polymerization. It is conceivable that GTP-bound RhoA promotes contractile ring assembly by activating actin polymerization via the formins and by activating myosin motor activity.
There is compelling evidence that the microtubule-based central spindle arid the actin-based cleavage furrow are both essential for cytokinesis. Nest, the question was asked how these two cytoskeletal polymers interact. There are a few cases in which an interaction between the microtubule and actin cytoskeletal systems have been characterized. Examples include nuclear positioning in budding yeast (Carminati and Stearns, 1997; Fujiwara et al., 1999; Miller et al., 1999), spindle orientation in epithelial cells (Busson et al., 1998) and in certain asymmetrically dividing cells, such as the posterior blastomere of the two cell C. elegans embryo (Gδnczy et al., 1999a; Hyman and White, 1987; Skop and White, 1998; Waddle et al., 1994). In these examples there is evidence that the dynein/dynactin microtubule motor complex may mediate the interaction of microtubules with the cell cortex.
It was an object of the invention to elucidate the microtubule dependent steps in cytokinesis. In particular, it was sought to define the role of the central spindle in this process, in order to be able to interfere with this process and thus with cytokinesis, which provides a new approach for therapy, in particular cancer therapy. Therefore, to solve the problem underlying the invention, it was investigated how the central spindle assembles and how it functions in cytokinesis.
In the present invention, a novel gene designated cyk-4 was identified and cloned and its role in the early divisions of the C. elegans embryo was characterized. It was shown that Cyk-4 is required for the late stages of cytokinesis. Interestingly, cyk-4 mutant embryos fail to assemble the central spindle. Positional cloning and localization studies revealed that the cyk- 4 gene encodes a novel GTPase activating protein (GAP) for the Rho family of GTPases that localizes to the central spindle. The missense mutation in the cyk- 4 (tl 689ts) allele is found in a domain dispensable for GAP activity suggesting that Cyk-4 may have another function in addition to activating GTP hydrolysis by Rho family proteins. Accordingly, it was found that Cyk-4 and the kinesin-like protein Zen-4/CeMKlpl are interdependent for their proper localization. Based on these data, a model is proposed by which Cyk-4 acts in concert with Zen-4/CeMKlpl to assemble the central spindle. The concentration of Cyk-4 to the central spindle would then serve to target the GAP domain to the fully ingressed contractile ring where it could promote GTP hydrolysis by RhoA, thereby facilitating the completion of cytokinesis.
In further experiments, the biochemical state of CYK-4 and ZEN-4 has been examined and it was found that they do indeed associate in vivo. These proteins also efficiently associate in vitro and an in vitro binding assay has been used to molecularly dissect the regions of both proteins that are necessary and sufficient for this interaction. It is shown that the protein encoded by the ts allele cyk-4 (tl689) is defective in binding to ZEN-4. The identification of mutations in zen-4 that partially rescue cyk-4 (tl689) and map to the CYK-4 binding region provides strong evidence for the importance of the interaction between CYK-4 and ZEN-4 for progression of cytokinesis. It has also been shown that the human orthologs of CYK-4 and ZEN-4, HsCYK-4/MgcRacGAP and MKLPl, are in a complex which has been purified. The complex contains approximately stoichiometric amounts of HsCYK-4 and MKLPl . The human complex localizes to the central spindle and. to division remnants, as do the nematode proteins. Thus, in the present invention, an evolutionarily conserved complex that is essential for central spindle assembly has been identified.
The division of a cell into two daughters requires dynamic interactions between the microtubule-based mitotic spindle and the actin-based contractile ring.
In animal cells the position of the cleavage furrow, an actomyosin-based structure, is determined by the mitotic spindle in a manner that is poorly understood.
*
In recent years is has become clear that the central spindle, an array of microtubule bundles, that forms during anaphase, also plays an important role in cytokinesis. To gain insight into this fundamental cellular process, the cytokinesis-defective mutant, cyk-4, was analyzed. Cytological analysis reveals that cyk-4 mutant embryos fail to assemble the central spindle. Though lacking a central spindle, cyk-4 mutant embryos furrow extensively, but they fail to complete cytokinesis . The mutation responsible for the cyk-4 phenotype was found to be a missense mutation in a gene encoding a Rho family GAP that, in vitro, stimulates GTP hydrolysis by Rho, Rac, and Cdc42. CYK-4 localizes to the central spindle and to cell division remnants. CYK-4 colocalizes with the ZEN-4/CeMKLPl kinesin-like protein. Moreover, CYK-4 and ZEN-4/CeMKLPl are interdependent for their localization. It is concluded that the CYK-4 GAP and the ZEN-4/CeMKLPl kinesin-like protein cooperate to assemble the central spindle. Furthermore, it -is proposed that a concentrated source of CYK-4 GAP on the central spindle could downregulate the RhoA GTPase and thereby promote the late stages of cytokinesis. The findings of the present invention provide a model for CYK-4 dependent assembly of the central spindle:
In cyk-4 mutant embryos the robust microtubule bundles that constitute the central spindle do not form.
Instead, the spindle develops into two mitotic asters separated by a few overlapping, disorganized, microtubules. A similar phenotype is observed in zen-4 mutant embryos (Powers et'al., 1998; Raich et al., 1998) . Thus both the ZEN-4/CeMKLPl kinesin-like protein and the CYK-4 GAP are essential for this microtubule bundling. The Drosophila orthologue of ZEN-4/CeMKLPl is also required for cytokinesis, though it seems to be required for all aspects of furrow ingression (Adams et al., 1998). Members of the MKLPl subfamily of kinesin- like proteins have microtubule bundling activity in vitro (Kuriyama et al., 1994; Nislow et al., 1992). However, in vivo, ZEN-4 mediated microtubule bundling requires CYK-4.
In answering the question how CYK-4 and ZEN-4 could cooperate to assemble the central spindle, it is proposed that a complex containing multiple motor proteins could specifically localize to overlapping, antiparallel microtubules (figure 10) . If such a motor complex transits along a microtubule it might continue to an end and dissociate. However, if such a motor complex transited along a microtubule in the vicinity of an antiparallel microtubule, the complex might bind simultaneously to both microtubules and attempt to move alternately in opposite directions, the net result being that the complex would concentrate in the region of microtubule overlap. Since CYK-4 does not have a microtubule motor domain, yet it is essential for the formation of the central spindle, it is proposed that CYK-4 forms a complex with multiple ZEN-4 homodimers that localizes to and stabilizes overlapping antiparallel microtubules (figure 10) .
It was of interest to find out if the RhoGAP activity of CYK-4 necessary to promote microtubule bundling by ZEN-4. I,t is proposed that central spindle assembly is unlikely to require CYK-4 GAP activity. This is suggested by two lines of evidence. First, central spindle assembly is defective in the cyk-4 (tl 689ts) allele that carries a missense mutation at amino acid 15. This substitution is distant from the C-terminal GAP domain, and in vitro, the amino terminus of CYK-4 is dispensable for GAP activity. Thus this allele would be predicted to retain catalytic activity in vivo and therefore GAP activity is not sufficient for central spindle assembly. Moreover, Rho RNAi experiments reveal that central spindle assembly is Rho independent, suggesting that Rho GAP activity is not required for this process. Thus CYK-4 may act to promote central spindle assembly, independent of its GAP activity.
In the present invention, the function of the CYK-4 gap domain was analysed:
If CYK-4 function in central spindle assembly is independent of the Rho GTPase, it was of interest to determine the function of the CYK-4 GAP domain. CYK-4 • is likely bifunctional, one function being to promote the assembly of the central spindle, the second function being to promote GTP hydrolysis by Rho family members. These two functions might be related in that the first function would serve to concentrate CYK-4 at a site where GAP activity is required. It may be assumed that CYK-4 GAP activity is required late in cytokinesis, to promote GTP hydrolysis by a Rho family GTPase whose downregulation causes disassembly of the contractile ring and cell separation (fig. 10) .
It was determined which GTPase might CYK-4 act on to promote cytokinesis. The CYK-4 GAP domain has all the hallmarks of a Rho family GAP and it may therefore be expected that it will act on this subfamily of the GTPase superfamily. Like many other RhoGAPs, the GAP domain of CYK-4 is promiscuous in its ability to promote GTP hydrolysis on Rho, Rac, and Cdc42 (Lamarche and Hall, 1994) . The strongest piece of evidence that CYK-4 acts on Rho is based on the observation that of the GTPases tested, Rho is the only one that is clearly essential for cytokinesis. The requirement for Rho in cell division is well documented in a variety of experimental systems. To date there is no evidence that Rac is required for cytokinesis and the data using RNAi to deplete Rac also failed to detect a role for this GTPase in this process. Moreover, it has been recently reported that ced-10 mutants, which are defective in corpse engulfment subsequent to apoptosis and distal tip cell migration, contain mutations in the rac gene (Reddien and Horvitz, 2000) . ced-10 mutants do not have any gross phenotypes indicative of a role in cytokinesis. Moreover, Racl deficient mice are gastrulation defective, but the embryos do not contain multinucleate cells indicative of a cell division defect (Sugihara et al., 1998). The sum of these data argue that Rac is not an essential target of CYK-4 during cytokinesis. With regard to Cdc42, previous studies have implicated this GTPase in cytokinesis (Drechsel et al., 1997; Dutartre et al., 1996). Superficially, the weakly penetrant cytokinesis phenotype observed in Cdc42 (RNAi) embryos is consistent with these earlier data. However the Cdc42 (RNAi) embryos that are cytokinesis defective are also osmotically swollen and therefore cytokinesis defect may be indirect. Thus at this juncture it appears most likely that RhoA is the key substrate for the CYK-4 GAP activity.
Further support for the hypothesis that completion of cytokinesis requires downregulation of RhoA by CYK-4 would be supported by experiments in which the cyk-4 phenotype is phenocopied by RhoA mutants that are hydrolysis defective. However the genetic tools necessary to express such dominant mutants in the early C. elegans embryo are currently unavailable. It is surprising that the CYK-4 GAP domain is less active towards RhoA as compared to Rac or Cdc42, if indeed RhoA is the relevant target of its GAP activity. One possible explanation is that full length CYK-4 has a different activity profile as compared to the isolated GAP domain. A more interesting possibility is that CYK-4 localization is important for CYK-4 GAP activity. The phenotype of cyk-4 mutant embryos suggests that CYK-4 needs to act when the contractile ring is in close proximity to the central spindle. Since CYK-4 is concentrated on the central spindle at this time, its high local concentration might overcome its lower activity towards RhoA.
In the present invention, the central spindle was shown to be at the center of cytokinesis:
There appear to be at least two microtubule-dependent steps in cytokinesis, contractile ring positioning and completion of cytokinesis. In some cells both processes are dependent on the central spindle. An important open question is whether these two reactions1
/ are mechanistically similar. While assembly of the, contractile ring requires activation of RhoA, it was previously shown that the position of the contractile ring is specified in a RhoA-independent manner in Xenopus embryos (Drechsel et al., 1997). It has been shown here that a Rho GAP is required for the late stages of cytokinesis, suggesting that the second process, completion of cytokinesis, does involve RhoA. It is therefore assumed that the two microtubule- dependent steps in cytokinesis are distinct.
CYK-4 and ZEN-4 are not the only components of the central spindle, a number of other components, some of which are required for cytokinesis, are also present at this site. Polo kinase is known to associate with MKLPl and to concentrate in the central spindle (Adams et al., 1998; Lee et al., 1995), and this kinase is essential for cytokinesis. Rho associated kinase also localizes to this site (Kosako et al., 1999). The AIR-2 aurora-like kinase localizes to the central spindle (Schumacher et al., 1998). This kinase seems to be required primarily for chromosome segregation (Woollard and Hodgkin, 1999) , its direct involvement in cytokinesis requires further analysis. INCENP and the TD-60 antigen also localize to the central spindle and there is evidence that they may play a role in cytokinesis (Eckley et al., 1997; Mackay et al., 1998; Martineau-Thuillier et al., 1998; Savoian et al., 1999) . Interestingly, a Rho GEF that is required for cytokinesis, ECT2, also accumulates on the central spindle (Tatsumoto et al., 1999), however the Drosophila ortholog, Pebble, does not localize in this manner (Prokopenko et al., 1999).
Further analysis of the specific 'functions of all of these cytokinesis regulators is the basis to determine which of these proteins are functionally interdependent as has been shown is the case for Cyk-4 and Zen-4/CeMKlpl. Furthermore, it was shown that the central spindle is a structure that is essential for completion of cytokinesis. Two proteins required for central spindle assembly, CYK-4 and ZEN-4, were shown to co-localize to the central spindle and to somehow act in concert with one another. In experiments of the present invention, it was demonstrated that in vivo, CYK-4 and ZEN-4 are present in an evolutionarily conserved protein complex and the nature of this complex in C. elegans embryos and in human cells was characterized in detail. These data suggest that the CYK-4/ZEN-4 complex, which was named centralspindlin, consists of a two molecules each of CYK-4 and ZEN-4. CYK-4 binds to the neck region of the ZEN-4 kinesin/ raising the possibility that the motor activity of the ZEN-4 is directly regulated by the CYK-4 RhoGAP.
Analytical biochemistry of the native centralspindlin complex isolated from C. elegans embryos demonstrates the existence of a complex containing the ZEN-4 kinesin and the CYK-4 RhoGAP. In vitro binding experiments were used to define the critical determinants for this interaction and to demonstrate that the two subunits are able to self associate. Genetic and biochemical suppression of the CYK-4 S15L mutation by a second site mutation in ZEN-4' strongly argues that the interaction between CYK-4 a'nd ZEN-4 is critical for CYK-4 function. Indeed, in vivo, the majority of ZEN-4 is in a complex with CYK-4. Moreover, three lines of evidence indicate that HsCYK-4 and MKLPl are in a tetrameric complex. First, immunopurification of HsCYK-4 and MKLPl reveals equal amounts of the two proteins. Second, these two proteins co-migrate on sucrose density gradients with a similar S value as observed for the C. ' elegans proteins. Third, the two proteins precisely co-migrate on a gel filtration column and their fractionation behavior suggests a native molecular mass for the complex of -300 kDa. Previous determinations of the native molecular mass of MKLPl have been reported and the values are similar to those obtained in the present invention here (Chui et al., 2000; Kuriyama et al., 1994) . Previous studies had not taken the presence of CYK-4 into consideration and therefore they interpreted their data to indicate that MKLPl exists as a homotetramer. However, the data obtained in the experiments of the invention are not compatible with this interpretion, but they rather indicate that the centralspindlin complex is a tetramer containing two molecules of the kinesin ZEN-4/MKLP1 and two molecules of the RhoGAP CYK-4.
CYK-4 binds to ZEN-4 in a particularly interesting region of this kinesin family member. Rice et al., 1999 have established that a critical element of the kinesin molecule lies just C-terminal to the catalytic core; the neck linker region. ATP binding to one catalytic core induces a large scale conformational change in the neck linker region that causes the other catalytic core to extend towards the adjacent tubulin dimer situated on the plus side of the initial microtubule contact (Rice et al., 1999). In the present invention, it was found that CYK-4 binds to a region of ZEN-4 that includes the neck linker region. In conventional kinesin, the neck linker region corresponds to a region 15 amino acids long that connects the catalytic core of kinesin to the coiled coil stalk domain. Among the family of KIN-N motors, the MKLPl subfamily has a distinctly divergent neck linker region, it lacks several nearly invariant residues and the linker between the catalytic core and the coiled coil region is about 5 times longer than in other members on the
KIN-N family. The divergence of this critical region of the kinesin suggests that MKLPl-mediated microtubule motility may differ from that of other kinesins. Moreover, since CYK-4 binds to the neck linker region of ZEN-4 it is possible that CYK-4 binding may in fact regulate ZEN-4 motor activity.
Previous work had suggested that MKLPl may exist as a tetramer containing 4 motor domains (Chui et al., 2000; Kuriyama et al., 1994). If the two dimers were arranged in an antiparallel orientation, this arrangement could easily explain how this complex could crosslink antiparallel microtubules. However, the data obtained in the present invention are not consistent with this proposed structure. The complex that has been characterized appears to contain two kinesin motors and two RhoGAP molecules . Since most kinesin motors are dimers in which both catalytic cores interact with a single microtubule protofilament, the architecture that is described here can not easily explain how microtubule bundling is achieved. If the two kinesin subunits of centralspindlin bind to the same microtubule protofilament, then how might microtubule crosslinking occur? At this point, at least three possible mechanisms can be envisaged (fig. 18) .
The first possibility is that there is an additional microtubule binding site elsewhere in the CYK-4/ZEN-4 complex. No additional binding site has been identified yet in CYK-4 nor has MKLPl, ZEN-4, or Pav been found to have an additional microtubule binding site. However, it has been shown, and was confirmed by the inventors, that MKLPl interacts differently with microtubules than most kinesin-like proteins. Specifically, ATP is usually sufficient to elute most kinesins from microtubules, but in the case of MKLPl, both ATP and high salt are required (Kuriyama et al., 1994; Nislow et al . , 1992). Thus it is possible that
MKLPl interacts with two microtubules, one by the motor domain and another via a different interaction surface. Consistent with this possibility is the finding that Rab6KIFL kinesin, which is quite similar to MKLPl in primary structure as well as in its localization and proposed function (Fontijn et al., 2001; Hill et al., 2000) , has been reported to contain a second microtubule binding activity in the C-terminal half of the molecule (Echard et al., 1998). However this possibility does not explain why CYK-4 is required for central spindle assembly.
The second alternative is that MKLPl forms higher order structures and that the tetramer that has been characterized in the present invention is merely a building block. This possibility gains some support from localization studies. In both C. elegans and in mammalian cells, ring-like structures are found that are termed division remnants that persist in the cell cortex after division. These remnants appear to be large aggregates of centralspindlin which are not in direct association with microtubules. Higher order oligomers could potentially form in early anaphase and promote microtubule bundling.
The third possibility is that, unlike most N-terminal kinesins, the two catalytic cores of the kinesin subunits in the centralspindlin complex could bind to different microtubules. This would not be without precedent in that the KIN-N KIF1A moves processively along a microtubule using a single head (Okada and Hirokawa, 2000) . The association of the two catalytic cores of MKLPl with different microtubules is made feasible by the fact that the linker region between the catalytic core and the coiled coil domain is much longer than that present in most N-terminal kinesins. Perhaps CYK-4 ensures that the two motor domains are oriented in such a way to bind to antiparallel microtubules (fig 18) . Structural analysis and biochemical reconstitution of centralspindlin from purified components will allow further insight into mechanism of antiparallel microtubule bundling.
One critical question, which has not be rigorously explored to date is the function of the central spindle during the process of cytokinesis. The obtained data do not shed extensive new light on this question. However, it may be noted that the RhoGAP domain of CYK-4 is highly conserved at the primary sequence level. Interestingly CYK-4 is significantly less active at inducing GTP hydrolysis by RhoA1 as compared to Rac and Cdc42 (Jantsch-Plunger et al., 2000), and this feature of CYK-4 is conserved in HsCYK-4 (Toure et al., 1998). It has been proposed by the inventors that one function of the central spindle is to concentrate CYK-4 to a position in the cell where it could promote inactivation of RhoA at a late stage in cytokinesis. Consistent with this model, inactivation of RhoA by RNAi does not prevent CYK-4 localization nor does it impair central spindle assembly. In addition, the RhoGAP domain seems to be required at some stage of cytokinesis, since overexpression of a HsCYK- 4/MgcRacGAP bearing a mutation in the RhoGAP domain causes cytokinesis defects (Hirose et al., 2001). Further structure-function analysis of CYK-4 is necessary to test this model further. However such analyses must take into consideration that CYK-4 is able to dimerize or oligomerize and therefore such analysis is best done in the absence of endogenous CYK-4.
Although centralspindlin appears to be a simple tetrameric complex, there are several lines of evidence that suggest that it may be subject to regulation by at least two different kinases. Genetic analysis of Polo kinase in Drosophila indicates Pav localization is Polo dependent (Carmena et al., 1998) and moreover a biochemical interaction between Polo and MKLPl orthologs have been shown in extracts from both mammalian cell and Drosophila embryos (Adams et al., 1998; Lee et al., 1995) .• .In addition, the
Aurora-B/Incenp complex /is also required for the stable localization of centralspindlin in C. elegans embryos (Kaitna et al., 2000; Schumacher et al., 1998; Severson et al., 2000) and an in vitro biochemical interaction has been detected between Aurora-B (AIR-2) and ZEN-4 (Severson et al., 2000). A stable association between Polo kinase or Aurora-B kinase with centralspindlin has not been observed. Reconstitution of central spindle assembly in vitro will allow the role of these and other regulators of centralspindlin to be dissected.
In summary, the experiments of the present invention comprise an initial phenotypic, molecular, biochemical, and cell biological analysis of the cyk-4 gene. Furthermore, they comprise an analysis of the CYK-4/ZEN-4 complex (centralspindlin) that consists of two molecules of each CYK-4 and ZEN-4.
The experiments of the present invention (Examples 1-5) indicate that Cyk-4 is an active GTPase activating protein that is required for cytokinesis, likely by its ability to regulate the RhoA GTPase. Quite surprisingly, one additional function of this protein is to promote assembly of the central spindle. Cyk-4 is a key molecule required for cytokinesis that regulates both the structure of the late mitotic spindle and the function of the contractile ring.
Thus, in a first aspect, the present invention generally relates to Cyk-4 polypeptides, which have been shown to be essential for this second microtubule dependent' step and thus define a functional link between the central spindle and the contractile ring.
In a preferred embodiment, the invention relates to a mammalian Cyk-4 polypeptide, in particular murine and human Cyk-4. In a first aspect, the invention relates to the urine Cyk-4 polypeptide with the amino acid sequence as set forth in SEQ ID NO: 4 or with the amino acid sequence encoded by a polynucleotide which hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO: 3.
In a preferred aspect, the invention relates to the human Cyk-4 polypeptide with the amino acid sequence as set forth in SEQ ID NO: 2 or with the amino acid sequence encoded by a polynucleotide which hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO:l.
In a further aspect, the present invention relates to ah isolated DNA molecule comprising a polynucleotide with the nucleotide sequence as set forth in SEQ ID NO:l encoding human Cyk-4 polypeptide, or an isolated DNA molecule encoding human Cyk-4 polypeptide comprising a polynucleotide which hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO:l.
In a further aspect, the present invention relates to an isolated DNA molecule comprising a polynucleotide with the nucleotide sequence as set forth in SEQ ID NO: 3 encoding murine Cyk-4 polypeptide, or an isolated DNA molecule encoding murine Cyk-4 polypeptide comprising a polynucleotide which hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO: 3.
By "stringent hybridization conditions" as used herein is meant overnight incubation at 42 °C in a solution comprising: 50% formamide, 5x SSC (IX SSC = 150 mM NaCl, 15mM trisodium citrate) , 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65°C, or equivalent conditions.
In the following, if not otherwise stated, the term "Cyk-4" refers to both the murine and the human Cyk-4.
Homologues of the subject Cyk-4 polypeptides also include versions of the polypeptide which are resistant to post-translation modification or which alter the activity of the protein. Cyk-4 polypeptides may comprise a full length protein, such as represented in SEQ ID NO: 2 or 4, or they may comprise fragments or variants therof.
Beside DNA molecules, the present invention relates to nucleic acid molecules in the form of RNA, such as mRNA. The DNA molecules include cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA may be double-stranded or single-stranded.
Single-stranded DNA or RNA may be the coding strand, also known as the sense (or plus) strand, or it may be the non-coding strand, also referred to as the antisense (or minus) strand. The present invention also relates to preparations of double stranded Cyk-4 RNA or derivatives thereof that can be used to interfere with gene expression by ds-RNA mediated gene interference as described by Fire et al. 1998 and reviewed by Fire, 1999; Bosher and Labouesse, 2000; Sharp, 1999. By "isolated" nucleic acid molecule (s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. Recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells, and those DNA molecules purified (partially or substantially) from a solution whether produced by recombinant DNA or synthetic chemistry techniques.
Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. However, it is intended that "isolated" as used herein does not include the Cyk-4 cDNA present in a cDNA library or in a preparation of purified or isolated genomic DNA containing the Cyk-4 gene or a portion thereof in admixture with one or more other cDNA molecules or DNA fragments.
The nucleic acid molecules of the present invention further include genetic constructs comprising one or more Cyk-4 DNA sequences operably linked to regulatory DNA sequences (which may be heterologous regulatory sequences) , such as promoters or enhancers, wherein upon expression of these DNA sequences in host cells, preferably in bacterial, fungal (including yeast), -. * plant or animal (including insect or mammalian) cells, one or more Cyk-4 polypeptides are produced. In such constructs, the design of which is described in laboratory manuals (see e.g. Sambrook et al., 2000) and is routine to the skilled artisan, the regulatory sequences may be operably linked to a Cyk-4 polynucleotide encoding mature Cyk-4 polypeptide or any of its variants, precursors, fragments or derivatives described herein, which may include one or more polynucleotides having a nucleic acid sequence that is complementary to substantially all or a portion of a nucleic acid molecule having a nucleic acid sequence as shown in SEQ ID NO:l or 3. As used herein, the terms "a portion" or "a fragment" of a nucleic acid molecule or a polypeptide means a segment of a polynucleotide or a polypeptide comprising at least 15, and more preferably at least 20, contiguous nucleotides or amino acids of a reference polynucleotide or polypeptide (for example, the polynucleotide and polypeptide shown in SEQ ID NOs: 1 or 3, respectively, unless otherwise specifically defined below.)
Besides the DNA molecules having a nucleotide sequence corresponding to that depicted SEQ ID NO:l or 3, the invention also relates to DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the Cyk-4 mouse or human polypeptides. Since the genetic code is well known in the art, it is routine for one of ordinary skill in the art to produce the degenerate variants described above without undue experimentation.
". \ In addition, the invention relates /to Cyk-4 polypeptides which have deviations' from the sequence shown in SEQ ID NO: 2 or 4, caused by the conservative exchange of amino acids, if they are Cyk-4 derivatives or fragments or peptides with the properties which are desirable for their use in therapy or in screening assays, or isolated DNA molecules encoding such derivatitives or fragments with a polynucleotide sequence varying in their sequence from SEQ ID NO:l or 3.
Nucleic acid molecules of the present invention which encode a Cyk-4 polypeptide or a derivative or fragment thereof may include, but are not limited to, those encoding the amino acid sequence of the polypeptide by itself, together with additional, non-coding sequences, including for example introns and non-coding 5 ' and 3 ' sequences, such as the transcribed, untranslated regions (UTRs) or other 5' flanking sequences that may play a role in transcription (e.g., via providing ribosome- or transcription factor-binding sites) , mRNA processing ( e . g. splicing and polyadenylation signals) and stability of mRNA; the coding sequence for the
Cyk-4 polypeptide operably linked to a regulatory DNA sequence, particularly a heterologous regulatory DNA sequence such as a promoter or enhancer; and the coding sequence for the Cyk-4 polypeptide linked to one or more coding sequences which code for amino acids that provide additional functionalities. Thus, the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide. In certain embodiments' of this aspect of the invention, the marker amino /acid sequence may be a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described for instance in Gentz et al . , 1989, hexa-histidine provides for convenient purification of the fusion protein. The "HA" tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al . , 2984. Yet another useful marker peptide for facilitation of purification of Cyk-4 is glutathione S-transferase (GST) encoded by the pGEX fusion vector (see, e . g. , Winnacker, From Genes to Clones, New York: VCH Publishers, pp. 451-481 (1987)). As discussed below, other such fusion proteins include the Cyk-4 fused to immunoglobulin Fc at the N- or C-terminus.
A still further aspect of the present invention relates to antibodies and antibody preparations specifically reactive with an epitope of the Cyk-4 polypeptide.
Polyclonal "antibodies are conventionally obtained by immunising animals, particularly rabbits, by injecting the antigen or fragments thereof and subsequently purifying the immunoglobulin.
Monoclonal anti-Cyk-4-antibodies may be obtained by standard procedures following the principle described by Kohler and Milstein, 1975, by immunising animals, particularly mice, then immortalising antibody- producing cells from the immunised animals, e.g. by fusion with myeloma cells, and screening the ' supernatant of the hybridomas obtained by immunological '' standard assays for monoclonal anti- Cyk-4 -antibodies. I For therapeutic or diagnostic use in humans, these animal antibodies may optionally be chimerised in the conventional way (Neuberger et al., 1984, Boulianne et al., 1984) or humanised (Riechmann et al., 1988, Graziano et al., 1995). Cyk-4 specific antibodies can be used for both diagnostic and screening applications.
Cyk-4 specific antibodies are useful in clinical situations, where determining overexpression or underexpression of CYK-4 has predictive value. Anti- CYK-4 antibodies can be used on tissue of cell specimens to evaluate the level of expression or changes in the subcellular localization in various disease states. In addition, anti-CYK-4 antibodies can be used to detect the presence of CYK-4 in solid-phase binding assays as described in the subsequent subsection related to finding compounds that affect the interaction of CYK-4 with other proteins.
In a further aspect, the present invention provides screening methods for identifying compounds capable of modulating, in particular inhibiting-, cytokinesis by modulating, in particular inhibiting, the function of CYK-4.
In a first embodiment, the screening method of the invention is used to identify compounds that modulate the function CYK-4 to promote GTP hydrolysis by Rho family GTPases. (Due to the evolutionary conservation of the GTPase domain and the requirement for Rho in cytokinesis, it can be concluded from the findings of the invention that the ability of CYK-4 to promote GTP hydrolysis by Rho is crucial for CYK-4 function.)
In this embodiment of the invention, a compound's ability to promote GTP hydrolysis by a Rho family GTPase is determined by incubating a substrate selected from the members of the Rho family GTPases with GTP for a period of time sufficient to allow saturation of the substrate's GTP binding sites, adding Cyk-4 and allowing it to react in the presence or absence of the test compound, and determining the amount of hydrolyzed GTP.
This type of assay may be conducted on the basis of the so-called "GTP hydrolysis assay", which is a biochemical assay carried out according to standard protocols, as described, inter alia, by Self et al., 1995, or by Settleman and Foster, 1995. By way of example, this assay may be carried out as follows: In a first step, the substrate is incubated with GTP that carries a radioactive label (e.g. like the commercially available γ-32P-GTP, or an otherwise measurable, e.g. a fluorescent label, as described, inter alia , by Hazlett et al., 1993, under conditions and for a period of time sufficient to allow saturation of GTP binding sites. Subsequently, CYK-4 is added, optionally at various concentrations of from 0 to eg 1 uM; in the presence of absence of test compounds. After the appropriate time (e.g. 5 min.) the amount of GTP that has been hydrolysed is determined.
After the time required for CYK-4 stimulated GTP hydrolysis by Rho in the control reaction (absence of test compound) , the reaction solution may be filtered through a protein binding matrix, e.g. nitrocellulose, that is arranged in the same geometrical pattern as the original microtiter plate where the reaction took place, and the radioactivity retained in the filters, or the amount of liberated free phosphate (or other label as appropriate) is quantified. To simplify the screening assay, the Rhό protein may be immobilized on a solid matrix, either via a tag that allows for binding to a suitably modified solid support, e.g. by using a biotinylated Rho protein and a streptavidin-coated microtiter plate, the solid matrix may also be in the form of beads. In the above- described assays that measure GTP hydrolysis, the Rho protein is preferably human.
By way of example, the assay may be conducted as follows: A member of the Rho family GTPases is immobilized on a solid support, e.g. a SPA (proximity scintillation assay bead) using either an epitope tag such as His6 or other similar tags. The Rho protein is loaded with radioactive 32P-gamma-GTP in the presence of EDTA. Magnesium and Cyk-4 are added in the absence or presence of test compounds, incubated for various times at which point the amount of radioactivity or other suitable label remaining associated with the beads is measured. Other embodiments of the assay include using alternative labels such as fluorescent GTP or other radioisotopes and using alternative measures of GTP hydrolysis such as measuring the amount of free 32P released, as described above.
Alternatively to using the full-length Rho protein, a truncated version may be used, as long the GTPase
/ activity is maintained, e.g. a fragment that lacks the critical cysteine residue in the CaaX box at the C-terminus that is required for membrane targetting.
A number of Rho proteins are available that are suitable for use in a GTP hydrolysis assay as a substrate, they have been cloned from various species. Preferably, the are employed as recombinant proteins.
Examples for Rho substrates that may be employed are human RhoA (Swiss Prot Primary Accession Number P06749) , human RhoB (Swiss Prot Primary Accession Number P01121) , human RhoC (Swiss Prot Primary Accession Number P08134), human RAC1 (Swiss Prot Primary Accession Number P15154), human RAC2 (Swiss Prot Primary Accession Number P15153) , human RAC3 (Swiss Prot Primary Accession Number 014658) and human GB25 (Swiss Prot Primary Accession Number P21181.
Based on the sequence information, the Rho substrates and the potentially present co-factor (s) can be produced recombinantly in suitable host, cells, e.g. in E. coli or by means of baculovirus in insect cells, according to standard methods.
Compounds that have the ability to prevent CYK-4 stimulated GTP hydrolysis by Rho family GTPases are then tested for specificity in a similar assay format using other proteins that have Rho family GAP domains, e.g. pl90 or RhoGAP (Lamarche and Hall, 1994).
In an alternative embodiment of the screening method, the compound' s ability to inhibit Cyk-4 function is determined by determining the compound' s ability to interfere with the biochemical interaction of CYK-4
I with a member of the MKLPl subfamily.
The function of CYK-4 in cytokinesis is intimately tied to its ability to interact with MKLPl, a kinesin like protein that is also required for central spindle assembly. Therefore compounds that inhibit this interaction are expected to prevent cytokinesis and to inhibit cell proliferation.
Examples for members of the MLKP1 family are CeM03D4.1b (C. elegans; GenBank ID U61955, Protein ID 1397342) and HsMKLPl (human; GenBank ID X67155; SwissProt Q02241) . Preferably, HsMKLPl is employed.
In C. elegans extracts, CYK-4 and CeMKLPl are present in a protein complex and can be co-immunoprecipitated and co-purified. The nature of this protein interaction can be further characterized, in particular by determining whether additional proteins also interact with this complex.
In an assay that employs an additional protein component involved in this interaction, a compound's ability to modulate Cyk-4 function is determined by determining its effect on the interaction of said protein with the Cyk-4/MKLPl complex.
In carrying out an assay based on the Cyk-4/MKLPl interaction, a minimal set of proteins necessary to form a stable complex, i.e. Cyk-4, MLKP1, and additional potential protein co-factors are produced in recombinant 'form.
/
The domains required for the interaction of Cyk-4 and MKLPl have been identified by the present inventors.
In an embodiment of the invention, the domain of Cyk-4 that interacts with MKLPl (i.e. the N terminal Cyk-4 region containing at least amino acids 1-120) , or the whole Cyk-4 protein, is immobilized on a solid support, as described below. The domain of MKLPl that interacts with CYK-4 (i.e. the central MKLPl domain containing at least amino acids 503-603) or the whole MLKPl protein that is modified with a suitable label to allow for rapid detection (i.e. radiolabelled, fluorescently labeled, hapten labelled etc., as described below) is incubated in the presence or absence of the test compounds .
After an incubation period that allows for interaction of the proteins, e.g. for about 20 minutes at 25°C, the amount of MKLPl bound to the immobilized CYK-4 is measured by use of the label outlined above or by the use of suitable antibodies in an ELISA type assay. The assay may also be setup in the reverse, e.g. with Cyk-4 being labeled or with MKLPl immobilized, or by performing the binding reaction in solution and then capturing one of the components on a solid support and measuring the amount of the other component that is co-immobilized.
According to another aspect, the invention relates to a screening method that takes advantage of the biochemical multimerization of CYK-4 on the one hand and the biochemical multimerization of the MKLPl subfamily of the kinesin-like protein superfamily on the other hand. This approach is based on the finding that the domain of CYK-4 that is required for interaction with MKLPl contains a coiled-coil domain that mediates self association. Likewise the domain of MKLPl that is required to interact with CYK-4 contains a coiled-coil domain that mediates self association. Since the ability of these proteins to 'self association is expected to be required for their function in vivo, compounds that inhibit the self assocation of either one or both of the interaction partners Cyk-4 and MKLPl are candidates for perturbing cytokinesis.
In an embodiment of the invention that is based on determining whether a test compound has the ability to interfere with the self association of Cyk-4, the N- terminal domain of CYK-4 (containing at least amino acids 1-120) , or the whole Cyk-4 protein, is immobilized on a solid support, either directly or through a tag. (Suitable tags are commercially available, e.g. the FLAG, HA, MYC, HIS tag, etc.). Examples for solid supports useful in the present invention are commercially available immunobeads or immunoplates, e.g. 96- well immunoplates, or microchips, which are coated with an antibody directed against one of the above-listed tags fused to the interacting protein.
A second CYK-4 fragment consisting of or containing the N terminal domain of CYK-4, i.e. amino acids 1-120, or the whole Cyk-4 protein is modified with a suitable label that allows for rapid detection. (The immobilized and the labeled Cyk-4 protein (fragments) may be identical or different.)
Examples for suitable labels are commercially available radioactive or fluorescence labels, hapten labels, or peptide labels, e.g. Europium or the Green Fluorescent Protein (GFP), an enzyme label, e.g. luciferase, alkaline phosphatase etc.) The labeled Cyk-4 (fragment) is incubated with the immobilized CYK-4 in the presence or absence of the test compounds .
After an incubation period that allows for interaction of the proteins, e.g. for ca. 20 min, the amount of CYK-4 bound to the immobilized CYK-4 is determined by measuring the signal of the label or by the use of suitable antibodies in an ELISA type assay.
In a further embodiment of the invention, the screening method to identify modulators of Cyk-4 function is based on determining whether a test compound has the ability to interfere with the self association of MKLPl. In this embodiment, the central domain of MKLPl (containing at least amino acids 503-603) or the whole MKLPl protein, is immobilized on a solid support.
A second MKLPl fragment, consisting of or containing the above-defined central domain of MKLPl, or the whole MKLPl protein is modified with a suitable label that allows for rapid detection. (The immobilized and the labeled MKLPl protein (fragments) may be identical or different) .
The assay for identifying inhibitors of MKLPl self assembly may be conducted in the the same way as described above for identifying inhibitors of Cyk-4 self assembly.
All embodiments of the screening methods of the invention may be performed in the high throughput format by automation of the reaction steps. In this case, a great number of compounds, e.g. from compound or natural product libraries, are applied to microtiter plates containing the components necessary for the reaction.
Due to their ability of inhibiting the function of Cyk-4 to promote GTP hydrolysis by Rho family GTPase, of interfering with the interaction between Cyk-4 and MKLPl, or by interfering with self assembly of Cyk-4 and/or MKLPl, respectively, compounds identified in the above screens have the potential to perturb cytokinesis. In tumor cells, this effect may result in a decrease or a stop of tumor growth. In addition, inhibition of cytokinesis may cause the activation of a cell cycle arrest check point that will trigger apoptosis of the tumor cells.
In light of the recent observation (Van de Putte et al 2001) that murine CYK-4 is highly expressed in Purkinje cells of the brain, in particular in cells that have abundant antiparallel bundles of microtubules (as is the case in the central spindle) , it may be assumed that compounds that inhibit CYK-4 function in cytokinesis may also impair CYK-4 function in neuronal cells. Inhibitors of CYK-4 function in these cells may have have utility in the treatment of neurological disorders, e.g. neuronal hyperactivity.,
Compounds that have been identified in the above- described assays to specifically affect CYK-4 function are drug candidates which may be further developed, e.g. in a first step, by structure-function analysis.
To further evaluate the potential of the compounds as drugs, the candidate compounds can be assayed for their effect on cytokinesis and other cellular processes in tissue culture of normal or transformed cells. To test the inhibition of tumor cell proliferation, primary human tumor cells, are incubated with the compound identified in the screen and the inhibition of tumor cell proliferation is tested by conventional methods, e.g. bromo-desoxy-uridine or 3H thymidine incorporation.
Compounds that exhibit an anti-proliferative effect in these assays may be further tested in tumor animal models and used for the therapy of tumors.
Thus, in a further aspect, the invention relates to compounds identified in the above screens for the therapy of tumors and any other situation in which cell overproliferation is observed.
Toxicity and therapeutic efficacy of the compounds identified as drug candidates by the screening methods of the invention can be determined by standard pharmaceutical procedures, which include conducting cell culture and animal experiments to determine the IC50, LD5O, the EDso- The data obtained are used for determining the human dose range, which will also depend on the dosage form (tablets, capsules, aerosol sprays, ampules, etc.) and the administration route (oral, buccal, nasal, paterental or rectal) . A pharmaceutical composition containing the compound as the active ingredient can be formulated in conventional manner using one or more physologically active carriers and excipients. Methods for making such formulations can be found in manuals, e.g. Remington Pharmaceutical Sciences, 1980; Remington: The Science and Practice of Pharmacy, 2000.
Brief description of the figures.
Figure 1 :
cyk-4 (tl 689ts) mutant embryos fail to complete cytokinesis .
Figure 2 :
cyk-4 mutant embryos produce deeply ingressing cleavage furrows, but do not form a prominent central spindle.
Figure 3:
Positional cloning of the cyk-4 locus.
Figure 4 :
cyk-4 (RNAi) causes disorganization of the proximal gonad and affects formation of the central spindle.
Figure 5:
CYK-4 enhances GTP hydrolysis by Rho, Rac, and Cdc42.
Figure 6:
rho (RNAi) causes cytokinesis defects and cdc42 (RNAi) causes defects in spindle positioning.
Figure 7:
CYK-4 localizes to the central spindle and division remnants. Figure 8 :
Time lapse analysis of CYK-4: GFP.
Figure 9:
CYK-4 and ZEN-4/CeMKLPl colocalize and are interdependent for their localization.
Figure 10:
Model for the function of CYK-4 in central spindle formation and cytokinesis.
Figure 11:
RhoA does not have a role in the formation of central spindle
Figure 12 :
CYK-4 and ZEN-4 associate in vivo and in vitro
Figure 13:
The N- terminus of CYK-4 is necessary and sufficient to bind to ZEN-4
Figure 14:
The central region of ZEN-4 is necessary and sufficient to bind to CYK-4
Figure 15:
A complex of CYK-4 and ZEN-4 is required for function in vivo
Fiure 16:
Self-association of CYK-4 and ZEN-4
Figure 17:
A complex of MKLP-1 and HsCYK-4 in HeLa cells
Figure 18:
Centralspindlin and its role in the formation of central spindle
In the Examples 1 - 5, if not otherwise stated, the following materials and methods were used:
a) Strains and alleles
The cyk-4 (tl 689ts) allele was identified in a search for maternal effect lethal mutations on chromosome III (see (Gδnczy et al., 1999b) for details). The strains DR104, BW1535, BW1369, and RW7000 were obtained from the CGC (C. elegans Genetics Center) . The strain EU699 containing the zen-4 (orl53ts) allele was described by Severson, et al., 2000. The end points of the deficiency tDflO (Heinke Schnabel, unpublished data) are not molecularly defined, but it uncovers cyk-4, lit-1 , and bli-5 and it does not uncover unc-64. b) Antisera
Cyk-4 specific antisera were produced in rabbits using a His6-Cyk-4 fusion protein as immunogen (containing amino acids 407-613 of cyk-4) . A GST-Cyk-4 fusion containing amino acids 407-681 of Cyk-4 was coupled to a Hi-Trap NHS resin (Amersham-Phamacia) and used to affinity purify anti-Cyk-4 antibodies which were used at a final concentration of 1:300. The antibodies used for the studies are specific for Cyk-4 since the staining can be blocked with antigen, a similar pattern is observed when anti-GFP antibodies are used to detect a Cyk-4: GFP fusion construct, and the staining pattern is disrupted in cyk-4 mutant embryos. The rat monoclonal YOL 1/34 anti-tubulin antibody was used at a dilution of 1:200-500. Anti-GFP antibodies (Roche) were used at a dilution of 1:500. Antisera specific for Zen-4/MKlpl was generously provided by Bill Saxton and Susan Strome (Univ. of Indiana) and used at a dilution of 1:4000. Antisera specific for Air-2 was generously provided by Andy Golden (NIH) and used at a dilution of 1:1000.
c) Genetic mapping of Cyk-4
The cyk-4 locus map's under the deficiency tDf6 which deletes a large fraction of the distal right arm of LGIII. Recombination mapping using unc-32 (el 89) cyk-4 (tl 689ts) /dpy-18 (e364) unc-25 (el56) placed cyk-4 distal to (or very close to) unc-25 (23/23 Dpy non Unc's carried the cyk-4 mutation). Recombination between dpy-18 cyk-4/RV11000 which carries several Tel elements including one on the cosmid F14F7 gave rise to 45 Dpy non-Ts animals, 3 of which lacked the TCI insertion on F14F7, indicating that the cyk-4 gene is distal to this cosmid. Crosses to strains carrying the deficiencies ctDf3, ctDf2, tDflO revealed that cyk-4 (tl 689ts) is not uncovered by ctDf3 and is uncovered by both ctDf2 and tDflO. Since tDflO does not uncover unc-64, cyk-4 (tl 68 ts) must be distal to unc-64.
d) Time Lapse recordings
Time lapse Nomarski imaging was performed as described previously (Jantsch-Plunger and Glotzer, 1999) . Time lapse imaging of Cyk-4: GFP was performed on a Zeiss Axiovert microscope using a 100X/1.3 neofluar objective. The illumination source, an Atto-arc HBO-103, was reduced to 50% intensity. An intensified cooled CCD camera (GenlV pentamax, Princeton Instruments) was used for image acquisition. The camera and other electronics were controlled with MetaMorph software (Universal Imaging) . Typical acquisition times were 40-80 msec. Each 10 seconds, 4-5 fluorescent images were acquired at different focal planes and a nomarski image was acquired. The fluorescent images1 were projected onto a single frame using the maximum intensity from the stack of images. Under these j conditions, embryos could be filmed for more than one hour without affecting the cell cycle timing or pattern of cell divisions. e) Rescue Experiments
To identify the cyk-4 gene in this region cosmid DNA (from stocks kindly provided by Alan Coulson, Sanger Center) was coinjected with the rol-6 (sulOOβ) dominant marker (Mello et al., 1991) into the gonad of unc-32 cyk-4/qCl worms. Heterozygous FI hermaphrodites that carried the rol-6 dominant marker were cloned to individual plates at 25°C and the presence of Unc progeny, indicating zygotic rescue of the cyk-4 mutation, was assessed. Individual unc-32, cyk-4 worms carrying the extrachromosomal arrays were cloned to individual plates to assess the extent of germline rescue. The cyk-4 genomic rescue construct MP17, contains a 4.9 kb genomic Xbal fragment excised from K08E3 and inserted into pBS-KS+.
f) RNA interference
Approximately 500 bp of DNA corresponding to predicted coding regions of Rho (Y51H4A.B), Rac-1 (C09G12.8B), Cdc42 (R07G3.1), F22E12.2, Y32F6B.3, K08D3.9, K08E3.2, K08E3.3, K08E3.4, K08E3.6, K08E3.7, K08E3.8 were amplified by PCR and cloned into pGEM-T (Promega) . Double stranded RNA was transcribed (Ambion) and annealed, and injected into the gonads of wildtype N2 hermaphrodites as described (Fire et al., 1998). g) Production of Cyk-4: GFP transgenes
The GFP cassette from vector pPD119.16 was excised with BspLUIII and inserted into the unique Ncol site of MP17 (see above) . This construct was linearized with Xbal, and complex arrays containing linearized genomic DNA and linearized rol-6 (sulOOδ) DNA were mixed in a ratio of 1:100:1 and injected into unc-32 (el89) cyk-4 (tl 689 ts) /qCl hermaphrodites. Rolling FI heterozygotes were singled out at 25°C and rolling Unc F2 animals were picked. A line MG110, was obtained that gave stable rescue of the cyk-4 (tl 689ts) mutation.
The structure of the cyk-4 gene was established by analysis of a large number of EST sequences available in the sequence databases at the Sanger Center and the National Institute of Genetics and by sequencing the clones yk63D6 and ykl04gl2 (kindly provided by Yuji Kohara) . The structure of the gene is identical to the structure predicted by the C. elegans genome consortium.
h) Immunolocalization
Immunolocalization studies were performed as previously
* described (Jantsch-Plunger and Glotzer, 1999) . Briefly, gravid hermaphrodites were placed on aminoproyl-silanej treated slides, a coverslip was added, and sufficient pressure to extrude the embryos was applied. The slide was placed into liquid nitrogen. The coverslip was removed while the sample was still frozen, the preparation fixed with -20°C methanol and then antibody staining was performed according to standard procedures.
i) Biochemical analysis of Cyk-4
The coding regions of Rho, Rac, and Cdc42 were PCR amplified and cloned into pET28b with an C-terminal polyhistidine tag. The GTPases were expressed at 25°C and purified using Ni2+-NTA-agarose (QIAGEN) . Proteins were dialyzed into 50 mM Tris pH 7.5, 50 mM NaCl, 5 mM MgCl2 and quick frozen. The GAP domain of Cyk-4 (amino acids 407-681) was cloned into pGEX4T-l. Proteins were expressed at 25°C and purified using GSH-agarose (Sigma) . Proteins were dialyzed into 50 mM Tris pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mM DTT and quick frozen. To assess GAP activity, 15 pmol of the GTPases were loaded with 1 pmol 32P- -GTP in 20 mM Tris pH 7.6, 4 mM EDTA, 25 mM NaCl, 1 mM DTT, 1 mM ATP, 0.1 mg/ml BSA at room temperature. The sample was place on ice and MgCl2 was added to 17 mM. GST-Cyk-4-GAP was added at the indicated concentrations and, at intervals, samples were taken by dilution into 2% SDS, 20 mM EDTA. Aliquots were spotted onto TLC plates (PEI-cellulose, Machery-Nagel) and developed in 1M LiCl. The plates were dried and exposed using a Storm Phosphoimager: (Molecular Dynamics) and the data analysis was performed using the public domain NIH Image program (developed at the U.S. National Institutes of Health; http : //rsb . info . nih. gov/nih-image/) . Example 1
cyk-4 mutants initiate, but do not complete, cytokinesis
The cyk-4 (tl 689ts) allele was isolated in a screen for 5 maternal effect embryonic lethal mutations on chromosome III (Gδnczy et al., 1999b). The cyk-4 locus is defined by a single, temperature sensitive (ts) , allele. The phenotype of embryos derived from homozygous cyk-4 hermaphrodites at the restrictive
10 temperature (hereafter referred to as cyk-4 mutant embryos) during the first division is shown in figure IB. cyk-4 mutant embryos appear normal until cytokinesis, except that defects in polar body extrusion are frequently observed (data not shown) .
15 The first cleavage furrow forms at the correct time and place, it ingresses extensively, but, invariably, it regresses. Quantitation of the extent of furrow ingression in cyk-4 mutant embryos reveals that, on average, furrows ingress to 73+/-13% (n=14) of the egg
20 diameter. A multipolar spindle develops in the second cell cycle and the process of furrow ingression and regression occurs again. This pattern is repeated until the embryos become grossly disorganized. (Figure 1 shows that cyk-4 (tl 689 ts) mutant embryos fa.il^
25. to complete cytokinesis. Wildtype embryos (A) and embryos from cyk-4 mutant hermaphrodites (B) were dissected from young adults, mounted on agarose pads and observed by time-lapse nomarski microscopy. The ingressing cleavage furrow is indicated (arrows).10 μm
30 scale bars. ) Although the majority of cell divisions in worm development occur early in embryogenesis, cells of the germline and and many cells in the nervous system are produced during post embryonic development (Sulston and Horvitz, 1977) . A number of mutations in genes required for cell division cause worms to become sterile and uncoordinated (O'Connell et al., 1998; Woollard and Hodgkin, 1999) . To determine if cyk-4 is required post embryonically, temperature shift experiments with worms homozygous for the cyk-4 (tl 689ts) allele were performed (table 1) . Homozygous animals grown at 16°C are viable and fertile. Homozygous cyk-4 animals shifted to 25°C at the L4 stage produce embryos with the cytokinesis defects described above. Homozygous animals shifted to 25°C at earlier stages either fail to hatch or become sterile and uncoordinated, depending on the time of the temperature shift. Thus cyk-4 is required postembryonically, perhaps due to its role in cell division. Unexpectedly, animals shifted at the L2/L3 stages become highly uncoordinated adults, even though most motor neurons would be expected to have completed all their divisions at the time of the shift. This may suggest that CYK-4 has additional roles besides its role in cell division. However, not out the possibility cannot be ruled out that some of the cells in the ventral nerve cord divide later in cyk-4 mutants.
To investigate why cyk-4 mutant embryos fail to complete cytokinesis, actin and tubulin were localized in wild-type and cyk-4 mutant embryos. Both wild-type and mutant embryos contained deeply ingressing cleavage furrows that stain with an anti-actin antibody; the results are shown in figure 2. Figure 2A,B shows that cyk-4 mutant embryos produce deeply ingressing cleavage furrows, but do not form a prominent central spindle. Wild type (A, C) and cyk-4 mutant embryos (B, D) were fixed and stained for actin (green) and DNA (blue) (A,B); tubulin (green) and DNA (blue) (C,D). 10 μm scale bars.
The etaphase spindles of cyk-4 mutant embryos appeared normal. However, spindle morphology during early anaphase was significantly different in mutant embryos as compared to wild type. In wild-^type embryos (20/20) prominent microtubule bundles form between the separating masses of chromatin, forming the central spindle (figure 2C) . In cyk-4 mutant embryos (9/10) such bundles were largely reduced and disorganized
(figure 2D) . It is concluded that CYK-4 is required for the organization of the central spindle during anaphase. Since the central spindle is required for cytokinesis, it is possible that the cyk-4 mutant embryos fail to complete cytokinesis because they fail to assemble the central spindle.
Example 2
Cloning of the cyk-4'gene
To investigate the (molecular basis for the phenotypes described above, it was first sought to map the cyk-4 locus and clone the affected gene (see methods for details) . The cyk-4 gene maps distal to unc-64 on the extreme right arm of LG III. Figure 3 shows the positional cloning of the cyk-4 locus. (A) A schematic of LGIII showing the positions of various loci and the extent of the deficiencies, cyk-4 is uncovered by tDflO, tDfδ, and ctDf2, but not ctDf3. (B) An enlargement of the physical map from the unc-64 locus until the end of LGIII. The ability of various cosmid pools to rescue cyk-4 is indicated. (C) A map of the predicted genes on K08E3. All the predicted genes except for K08E3.1 and K08E3.5 were inactivated by RNAi and only RNAi of K08E3.6 produced multinucleate embryos. (D) A schematic representation of the domain structure of CYK-4 and its human ortholog. The position of the point mutation identified in cyk-4 (tl 689ts) is indicated.
The cyk-4 gene was then identified by functional rescue of the zygotic requirement for cyk-4 using pools of cosmids. A pool of three cosmids (ZK520, W06F12, and K08E3) allowed cyk-4 homozygotes to hatch and develop to adulthood at 25°C. These cosmids were injected individually and cosmid K08E3 contained rescuing activity. Further subcloning revealed that a 4.9 kb genomic fragment, predicted to contain the complete K08E3.6 gene and no other intact gene, could rescue the cyk-4 zygotic and germline phenotypes. Finally, the coding region was amplified from DNA derived from cyk-4 (tl 689) homozygotes and sequenced and a single point mutation was identified that differed from the, sequence provided by the genome project; this mutation was not observed in another line derived from the same parental strain. These data show that the defect in cyk-4 (tl 689ts) embryos is due to a point mutation in the K08E3.6 gene. The predicted protein product of the cyk-4 gene has a C-terminal domain that contains the consensus motifs of GTPase activating proteins for Rho family GTPases (figure 3) . Adjacent to the C-terminal GAP domain is a 5 Cl domain that is predicted to bind to diacylglycerol or phorbol esters. At the amino terminus of the protein is a 90 amino acid region predicted to form a coiled-coil domain. The S15L point mutation found in cyk-4 (tl 689ts) , is located just amino terminal to the
10 coiled-coil domain. Human and mouse proteins with structural similarities to cyk-4 have been described (Toure et al., 1998; Wooltorton et al., 1999). These genes are expressed in a variety of proliferating tissues. In addition, the Drosophila sequence database
1.5 contains an entry (ascension no. AC005977 (CLOT 94)) that, together with CYK-4 and the previously mentioned human gene, share a common structure consisting of approximately 650 amino acids, an NH2-terminal coiled- coil domain and a conserved C-terminus containing Cl
20 and GAP domains. The structural conservation of CYK-4 suggests that its function is conserved among metazoans.
In the present invention, the the human and mouse cDNA's were identified by searching the DNA databases 25 with the C. elegans CYK-4 protein sequence.
Since the cyk-4 (tl 689ts) mutation may contain residual activity RNA-mediated interference (RNAi) was used to deplete embryos of CYK-4 protein.
Figure 4 shows that cyk-4 (RNAi) causes disorganization 30 of the proximal gonad and affects formation of the central spindle. Young adults were injected with cyk-4 dsRNA and the injected animals were analyzed 30 hours after injection (B) . The gonad of an uninjected worm is shown for comparison (A) . Wild-type embryos (C) and embryos from cyk-4 (RNAi) injected animals (D) were fixed and stained for tubulin. 10 μm scale bars.
RNAi of the predicted open reading frame K08E3.6 generated multinucleate embryos which exhibited a similar phenotype to that of the cyk-4 mutant, including loss of the central spindle (figure 4 D) and incomplete cytokinesis (not shown) . Interestingly, the gonads of cyk-4 (RNAi) animals become disorganized 30 hours post-injection (figure 4 B) and irregularly sized embryos are produced, suggesting that CYK-4 acts not only during embryonic and zygotic development, but also in the female germline.
Example 3
Biochemical activity of the Cyk-4 GAP domain
The presence of a Rho-family GAP domain suggests that
CYK-4 may regulate one or more GTPases of Rho branch of the GTPase superfamilγ. To determine whether CYK-4 is active as a GAP and,whether its GAP activity is
/ restricted to particular members of the Rho subfamily, a recombinant fusion protein containing GST and the
CYK-4 GAP domain was prepared and GTP hydrolysis assays were performed with recombinant C. elegans Rho, Rac, and Cdc42. The GAP domain of CYK-4 promotes GTP hydrolysis by all three tested GTPases. Figure 5 shows that CYK-4 enhances GTP hydrolysis by Rho, Rac, and Cdc42. The GTPases were preloaded with 32P-α-GTP and then GST-CYK-4-GAP or GST was added at the indicated concentration. Samples were taken at two minute intervals, the labeled nucleotide was resolved by thin layer chromatography, the fraction of GTP and GDP was quantitated. This graph shows the fraction of GTP hydrolyzed at the 2 minute time point as a function of CYK-4 concentration. These data are representative of at least three independent experiments.
However kinetic differences were observed. At the conditions' used in the assays it was found that the CYK-4 GAP domain is more active towards Rac and Cdc42 than towards Rho. The human ortholog has a similar activity profile in vitro (Toure et al., 1998).
However, since CYK-4 has activity towards all three GTPases, these data are not sufficient to determine the in vivo target (s) of the CYK-4 GAP domain.
Next RNAi was used to determine which, if any, of the Rho family GTPases are required for cytokinesis in the C. elegans embryo. RNAi experiments were performed with RhoA, Racl, Cdc42 and three additional GTPases found in the genome that fall into the Rho subfamily. Approximately 90% of Rho (RNAi) embryos exhibit cytokinesis defects in the first and/or second cell cycle (table 2 and figure 6) .
Figure 6 shows that rho (RNAi) causes cytokinesis defects and cdc42 (RNAi) causes defects in spindle positioning. Young adults were injected with the indicated dsRNAs (see table 2) and the embryos produced by the injected worms were analyzed by time lapse nomarski microscopy. Images from a wild-type (A) , rho f- NΛi) (B) and a cdc42 (RNAi) (C) embryo are shown. 10 μm scale bars.
In most embryos, furrow ingression was inhibited.
Interestingly, central spindles assemble in RhoA (RNAi) embryos (data not shown) . In contrast, 88% of Cdc42 (RNAi) embryos complete cytokinesis normally. However, a distinct defect in the early embryo is observed in 54% of Cdc42 (RNAi) embryos; defects in spindle positioning are observed in P0 and/or PI. A minority (12%) of Cdc42 (RNAi) embryos fail to initiate cytokinesis; in most cases, these embryos appear osmotically swollen even when provided with osmotic support. Rac (RNAi) embryos hatch with high efficiency and did not exhibit a detectable phenotype in the early embryo. RNA interference experiments with the additional GTPases either alone or in combinations did not reveal any additional defects in the early embryo. Thus RhoA is the only member of the Rho family that is clearly required for cytokinesis and is therefore likely to be the critical target for the CYK-4 GAP domain.
Example 4
The subcellular localization of Cyk-4
Next the subcellular localization of CYK-4 protein was determined. CYK-4 localization is cell cycle dependent Figure 7 shows that CYK-4 localizes to the central spindle and division remnants. (A-F) Wild-type embryos were fixed and stained for CYK-4 (green) , tubulin (red) and DNA (blue) . The localization of CYK-4 to the central spindle (arrow) in a one cell embryo (D) and a two cell embryo (F) is shown. The localization of CYK-4 to the division remnant from the polar body (A) , and between the AB and Pi blastomeres is indicated with arrows. (E) . (G) An embryo from a line expressing CYK-4: GFP is stained with anti-GFP antibodies. The same structures are seen as with CYK-4 antibodies (arrow) . (H) The intrinsic fluorescence of the .gonad of a worm expressing CYK-4: GFP. CYK-4 is seen at the incomplete membranes of the syncytial gonad (arrow) and in oocyte nuclei. 10 μm scale bars.
In interphase cells, CYK-4 is present in the cytoplasm and slightly concentrated in the nucleus. CYK-4 is also highly concentrated in a spot at the anterior of the embryo, DNA labeling reveals that this localization corresponds to the site of polar body extrusion. As embryos enter mitosis, CYK-4 protein concentrates around the mitotic spindle. In early anaphase, CYK-4 concentrates to the central spindle. As the cleavage furrow ingresses, CYK-4 becomes highly concentrated on the central spindle 'into a structure that often appears ring shaped (not shown) . Upon completion of cytokinesis, CYK-4 staining persists at division remnants. CYK-4 organized in ring-like structures averaging 1.2 μm in diameter are occasionally observed in the cytoplasm (not shown) . To determine if CYK-4 localizes to the central spindle prior to the onset of cleavage furrow ingression , the dynamics of CYK-4 localization in live embryos was investigated. To accomplish this goal a transgenic line expressing a CYK-4: GFP fusion was generated and its localization followed by time lapse microscopy. The CYK-4: GFP fusion is partially functional since cyk-4 xsExl [cyk-4 : GFP] animals are viable and fertile at 25°C whereas the parental cyk-4 strain is inviable at 25°C. However, the fusion construct does not fully rescue the mutation, since about 40% of embryos produced by this line fail to hatch (table 1) .
Low light level fluorescence microscopy was used to visualize CYK-4: GFP in living embryos. Embryos were imaged using a multi-mode imaging system whereby a series of z-sections and a nomarski image were recorded every 10 seconds. The fluorescent images from each time point were projected to form a single image.
Figure 8 shows the time lapse analysis of CYK-4: GFP. An embryo from a line expressing CYK-4: GFP was imaged using low light level microscopy. The central spindle localization of CYK-4 is observed prior to furrow ingression. 10 μm scale bars.
These recordings reveal that CYK-4 accumulates on the central spindle prior to the initiation of furrowing (figure 8; 3:40). The CYK-4 that localizes to the central spindle becomes compressed into a bright spot which persists at the division remnant. The remnant persists for several cell cycles although instances were observed whereby the remnant (sometimes from the 5.6
polar body) detaches from the cortex arid is observed as a discrete spot in the cytoplasm. This detachment of CYK-4 from division remnants likely accounts for the CYK-4 rings seen in fixed specimens. It is concluded that CYK-4 localization on the central spindle precedes furrow ingression.
Example 5
Cyk-4 and zen-4/CeMklpl are functionally interdependent
There are remarkable similarities between CYK-4 and the kinesin-like protein ZEN-4/CeMKLPl (Powers et al., 1998; Raich et al., 1998). zen-4 mutant embryos also initiate, but fail to complete cytokinesis. They also fail to assemble a robust central spindle in early anaphase. Furthermore, ZEN-4 localizes to the central spindle and persists at division remnants after completion of cytokinesis. To test whether these proteins functionally interact, it was first assessed whether CYK-4 and ZEN-4/CeMKLPl co-localize."
Embryos expressing CYK-4: GFP were fixed and CYK-4 and ZEN-4 were localized simultaneously (using an anti-GFP antibody to detect CYK-4: GFP). The two proteins co- localize both on central spindle structures and on division remnants (Figure 9A-C) . It was next investigated if ZEN-4 localization requires functional CYK-4 protein. ZEN-4 staining of cyk-4 mutant embryos reveals that ZEN-4 localization to the central spindle is absent (figure 9E) , although staining of some microtubule bundles in the spindle midzone could be detected using an AIR-2 antibody (figure 9G) . Thus, recruitment of ZEN-4 to the central spindle is CYK-4 dependent .
Figure 9 shows an embryo expressing CYK-4: GFP stained for GFP (A) , ZEN-4/CeMKLPl (B) and the merged image (C) . CYK-4 and ZEN-4/CeMKLPl colocalize at division remnants (arrow) and a central spindle (arrowheads) structures. ZEN-4 localization to the central spindle is CYK-4 dependent (arrow) . Wild-type (D) and cyk-4 (tl 689ts) embryos (E) were fixed and stained for ZEN-4/CeMKLPl (green) , tubulin (red) and DNA (blue) . AIR-2 localization to the central spindle is CYK-4 independent (arrow). Wild-type (F) and cyk-4 (tl 689ts) embryos (G) were fixed and stained for AIR-2 (green) , tubulin (red) and DNA (blue) . CYK-4 maintenance to division remnants (arrowheads) is ZEN-4/CeMKLPl dependent. zen-4 (orl53ts) worms were maintained at 16°C and either fixed immediately (H) or shifted to 25°C for 18 minutes (I) . Embryos were fixed and stained for CYK-4 (green), tubulin (red) and DNA (blue). 10 μm scale bars.
Next it was tested whether maintenance of ZEN-4 at division remnants requires functional CYK-4. cyk-4 mutant embryos grown at 16°C were shifted to 25°C for 15 minutes prior to fixation and staining with anti- ZEN-4 antibodies. The number of cells and number of division remnants labeled with the anti-ZEN-4 antibody were counted. Embryos maintained at the permissive temperature had a large number of ZEN-4 staining division remnants (table 3) whereas the embryos shifted to the non-permissive temperature lacked defined staining of division remnants. Thus both recruitment of ZEN-4 to the central spindle and its maintenance at division remnants is CYK-4 dependent. The reverse experiment was conducted with a temperature sensitive allele of zen-4 (orl53ts) . In this case CYK-4 staining at division remnants was observed in zen-4 mutant embryos at the permissive temperature but this staining disappeared upon a brief shift to the non-permissive temperature (figure 9H, I and table 3). Thus maintenance of CYK-4 at division remnants is ZEN-4 dependent. It is concluded that CYK-4 and ZEN-4/CeMKLPl colocalize and that the two proteins are interdependent for their localization.
Next, it was determined if embryos carrying mutations in both cyk-4 and zen-4 are distinguishable from the single mutants. Two strains were built, one strain was homozygous for zen-4 (orl53ts) and heterozygous for cyk-4 (tl 689 ts) and the second strain was homozygous for cyk-4 (tl 689ts) and heterozygous for zen-4 (orl53ts) . Both strains were viable at 16°C, but they failed to produce doubly homozygous larvae. It was found that worms of genotype unc-32 (el89) cyk-4 (tl 689 ts) I ' qCl III, zen-4 (orl53ts) laid a fraction of embryos that arrested during embryonic development, typically before the comma stage. Thus cyk-4 tl 689ts) and e zen 4 (orl53ts) are synthetically lethal.
/
In the subsequent Examples, the following Materials and Methods were used: i) Worm Strains and Alleles
The following alleles were used: N2 (Bristol), CB4856, cyk-4 (tl 689ts) , unc-64, bli-6 (scl 6) , unc-24 (el38) , unc-44 (el260) , lag-1 (q385) , and mlsll IV. Some strains were obtained from the C. elegans Genetics Center.
ii) Isolation and mapping of cyk-4 suppressor alleles
Suppressors of the cyk-4 (tl 689ts) mutation were obtained by mutagenizing cyk-4 (tl 689ts) animals with 30-40 mM EMS or 0.5 mM ENU. P0 animals were allowed to self fertilize for two generations at the permissive temperature. When F2 animals reached early adulthood, the population was shifted 20°C and fertile animals were selected. Approximately 110,000 FI genomes were screened and 18 suppressor mutations, all but 2 being unambiguously independent, were isolated. While all of the suppressor strains were viable and fertile at 20 °C, none were able to grow at 25°C, suggesting that none of them precisely reverted the original mutation. Several intragenic suppressor mutations were identified. The ZEN-4 coding region of the remaining mutants were sequenced and 7 strains were found to contain substitutions within the CYK-4 binding region of ZEN-4 and one contained a substitution in the catalytic domain. One suppressor strain, xs82, was characterized in detail. Suppressor activity was mapped using single nucleotide polymorphisms (SNPs) to the central region of chromosome IV (see methods) . The ZEN-4 coding region was sequenced in this strain and a point mutation was found that causes a substitution of glutamic acid for a lysine at position 502 (fig 15A) .
iii) Cell Culture
HeLa cells were routinely grown in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin.
iv) Antibodies
Rabbit CYK-4 specific and ZEN-4 specific antisera were reported previously (Jantsch-Plunger et al., 2000).
CYK-4 and ZEN-4 antibodies were affinity-purified with His6-CYK-4 (407-613) or His6-ZEN-4 (578-775) immobilized on NHS-Sepharose (Pharmacia) .
MKLP-1 specific antisera and HsCYK-4 specific antisera were produced in rabbits and mice (Gramsch
Laboratories, Schwabhausen, Germany) using C-terminal peptides (QLGPGYQHHAQPKRKKP and SKSKSATNLGRQGNFFASPMLK, respectively) conjugated to keyhole limpet hemocyanin as immunogens. Rabbit antibodies were affinity purified using peptides immobilized on Poros epoxide resin.
v) Plasmids
A cDNA clone for ZEN-4 in pBluescript (yk391b3) which lacks 13 nts at 5' of the coding region which were introduced by PCR. The pCBD-TEV vector was constructed by inserting the sequences encoding the chitin-binding domain (CBD) and a TEV protease site into pET28a (Novagen) . ZEN-4 fragments were amplified by PCR and cloned into pCBD-TEV.
vi) In Vitro Binding Assay
Full length and fragments of CYK-4 and ZEN-4 were expressed by in vitro transcription and translation system with reticulocyte lysates using the TNT® Coupled Reticulocyte Lysate Systems (Promega) or the PROTEINscript™ II (Ambion) kits, typically in 20 μl reactions. Full length proteins without tag were expressed from the T3 promoter of pBluescript SK(-).
Fragments of CYK-4 and ZEN-4 tagged with chitin-binding domain (CBD) at N-terminus were expressed from the T7 promoter of pET-CBD. In some experiments (Fig. 12C, 13C and 16) , CYK-4 and ZEN-4 were co-expressed in the same reactions. In other experiments, they were separately expressed and mixed. Following incubation at 20°C for 30 min in 100 μl buffer A (20 mM Hepes, 150 mM NaCl, 2 mM MgC12, 10 mM EDTA, 1 mM DTT, 1 mM PMSF, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml chymostatin) with 0.5% (w/v) Triton X-100, ZEN-4 or CYK-4 proteins were immunoprecipitated with specific antibodies, or affinity-purified by chitin beads. For immunoprecipitation, 0.5 μg antibody was added to the reactions and incubated on ice^ for 1 hr, followed by incubation with 5 μl protein---A-Sepharose beads at 4°C for Ih. For CBD-affinity purification, 5 μl of chitin beads (New England Biolabs)1 were added. In both cases, the beads were washed three times with buffer A containing 0.5% (w/v) Triton X-100. Proteins bound to the beads were analyzed by SDS-PAGE followed by autoradiography with Phospholmager . vii) Preparation of Cell Lysates
C. elegans embryos were prepared by bleaching a synchronous culture of adult worms grown on "egg plates" as described (Lewis and Fleming, 1995) . The embryos were washed with buffer A without Triton-X 100 and frozen with liquid nitrogen and kept at -80°C until use. Embryos were crushed by grinding in a mortar and pestle in liquid nitrogen.
HeLa cells were synchronised by treatment with 0.1 μg/ml nocodazole for 16 h. Cells were washed twice with cold PBS, frozen by liquid nitrogen and kept -80 °C until use. Cells were lysed and thawed by suspending into 10 volumes of buffer A with 0.5% (w/v) Triton X-100.
viii) Preparation of Microtubule-binding Fraction
Frozen pellet (0.5 ml) of nocodazole arrested M-phase cells was thawed and lysed in 5 ml ice-cold BRB80 (80 mM K-Pipes, 1 mM MgCl2, 1 mM EGTA, pH 6.8) with 0.1% (w/v) Triton X-100. The lysate was clarified by centrifuging at 10,000 x g for 15 min. The resulting supernatant was centrifuged at 25,000 x g for 20 min at 4°C in S100AT6 rotor (Hitachi) . Microtubules polymerized with glycerol and taxol was added to the supernatant. After incubating at 20°C for 20 min, microtubules were pelleted by centrifuging at
25,000 x g for 20 min at 20°C. The bound proteins were released from microtubules by incubation in 0.5 M NaCl, 5 mM ATP in BRB80 at 20°C for 20 min, followed by centrifugation at 25,000 x g for 20 min at 20°C. ix) Mass Spectrometry analysis
Hydrodynamics
Sedimentation coefficient was estimated by ultracentrifugation through 2 ml linear gradient of 5 to 20% (w/v) sucrose in buffer A without Triton X-100 using S55S rotor (Hitachi) . 150 μl cell lysate or microtubule binding fraction was applied. Chicken ovalbumin (3.4 S) , bovine gamma globulin (7.1 S) , bovine catalase (11 S) and bovine thyroglobulin (19 S) were used as standards. The diffusion coefficient was estimated by gel filtration using Superdex 200 column (30 ml) in FPLC system (Pharmacia) .
Immunoprecipitation and Western Blotting
Lysates (500 μl) were precleared with 50 μl protein A-Sepharose beads. For immunoprecipitation from worm embryos, 1 μg affinity-purified anitibody was added to the lysate. After incubation on ice for 1 hr, immunocomplex was recovered by incubation with 5 μl protein A-Sepharase (Pharmacia) at 4°C for 1 hr. For immunoprecipitation from HeLa cells, antibodies were covalently immobilized on protein A-beads (1 μg per 1 μl beads) with dimethyl pimelimidate. The precleared lysate was incubated antibody-beads at 4°C for 1 to 4 h. The beads were washed briefly with buffer A plus 0.5 % Triton X-100 three times.
For western blotting, samples were run on 7.5% SDS-PAGE gel and electrotransfered to a nitrocellulose membrane (HiBond ECL, Amersham) . x) Immunolocalization
Immunolocalization studies in worm embryos were performed as previously described ref. In brief, gravid hermaphrodites were placed on aminopropyl-silane treated slides, a coverslip was added, and sufficient pressure to extrude the embryos was applied (Jantsch- Plunger et al., 2000). The slide was placed into liquid nitrogen. The coverslip was removed while the sample was still frozen, the preparation was fixed with -20°C methanol, and antibody staining was performed according to standard procedures. Immunolocalization studies using HeLa cells were performed according to standard procedures following fixation in -20 °C methanol.
Example 6
Central spindle assembly is independent of RhoA
The process of central spindle assembly is dependent on the RhoGAP protein CYK-4. In the previous Examples, it was found that RhoA is essential for cytokinesis while Rac and Cdc42 are not (Jantsch-Plunger et al., 2000). Since RhoA is required for cytokinesis, it was tested if this GTPases is also required for central spindle assembly. To inactivate RhoA worms were grown on bacteria expressing dsRNA from the coding region of the gene (Timmons and (Fire, 1998) . Embryos were fixed and immunoflorescence-stained for CYK-4, microtubules and DNA with anti-CYK-4 antibody, anti-α-tubulin antibody and Hoechst 33342) . In both control embryos and rhoA (RNAi) embryos, central spindles formed and were prominently labeled with anti-CYK-4 antibodies.
Although RhoA dsRNAi causes a fully penetrant inhibition of cytokinesis, central spindle assembly occurs normally, and CYK-4 localizes to the central spindle (figure 11) . Thus the requirement for RhoA in cytokinesis can not be accounted for by a requirement for RhoA in central spindle assembly. These data indicate that the requirement for CYK-4 in central spindle assembly is independent of its ability to regulate RhoA.
Example 7
CYK-4 and ZEN-4 form a complex in vivo and in vitro
Since central spindle assembly and cytokinesis requires both CYK-4 and ZEN-4 and these two proteins are interdependent for their proper localization, it is possible that these factors exist in a stable biochemical complex. In order to test this possibility, immunoprecipitation experiments were performed using extracts prepared from early C. elegans embryos. When embryo extracts were immunoprecipitated using anti-CYK-4 antibodies, it was found that significant amount of ZEN-4 co-immunoprecipitated suggesting the existence, in vivo, of a stable complex containing CYK-4 and ZEN-4 (fig. 12A) .
To determine whether CYK-4 and ZEN-4 are competent to interact in the absence of other nematode proteins, the two proteins were produced by in vitro translation and subjected to immunoprecipitation. When CYK-4 and ZEN-4 (fused to the chitin binding domain (CBD) ) were translated in vitro and subsequently mixed, both proteins could be recovered in high yield by chitin beads (fig. 12B) . The co-precipitation of CYK-4 was dependent on ZEN-4; CYK-4 was not recovered on chitin beads when ZEN-4 was substituted by CBD alone. Moreover, when luciferase was substituted for CYK-4, it did not co-precipitate with ZEN-4. The association between CYK-4 and ZEN-4 could also be detected when anti-CYK-4 antibodies were used to retrieve the complex (fig. 12C) .
Figure 12 shows that CYK-4 and ZEN-4 bind to each other in vivo (A) and in vitro (B and C) . (A) CYK-4 and ZEN-4 were immunoprecipitated from worm embryo extracts with anti-CYK-4 antibody, anti-ZEN-4 antibody, or with non specific rabbit IgG as a control. The immunoprecipitates were resolved on SDS-PAGE gels followed by western blotting with an anti-ZEN-4 antibody. Mock immunoprecipitation without extract was included to control for cross reactivity with the antibodies. ZEN-4 specifically co-immunoprecipitated with CYK-4. (B) 35S-labeled CYK-4 (or luciferase, LUC) and chitin-binding domain (CBD) -tagged ZEN-4 (or CBD alone) were separately expressed by in vitro translation (left panel) . Translation reactions were mixed and incubated in the indicated combinations and CBD-ZEN-4 or CBD alone were recovered with chitin beads (right panel) . In this and all gels in this paper the input lanes contain the same amount of translation product as was added to the beads used for precipitation. The precipitates were resolved on SDS-PAGE gels and the labeled products detected using a phosphoimager. CYK-4 co-purified with CBD-ZEN-4 while the luciferase control did not. (C) CYK-4 (or luciferase) and CBD-ZEN-4 (or CBD alone) were co-expressed as 35S-labeled proteins in the indicated combinations (left panel) . CYK-4 was immunoprecipitated with an anti-CYK-4 antibody (right panel). ZEN-4 co-immunoprecipitated with CYK-4.
Example 8
Delineation of the region of CYK-4 necessary to bind to ZEN-4
Since the binding between CYK-4 and ZEN-4 could be easily reconstituted using in vitro translated proteins, this simple assay was used to dissect the regions of the two proteins that mediate this interaction. The experiment first concentrated on CYK- 4. The N-terminal 30 amino acids of CYK-4 are poorly conserved and are followed by a 90 amino acid region predicted to form a coiled-coil (see schematic fig.
13D) . The C-terminal 250 amino acids contains a RhoGAP domain that is active in vitro against RhoA, Rac, and Cdc42 (Jantsch-Plunger et al., 2000). The RhoGAP domain is preceded by a Cl domain, a cysteine-rich domain that mediates interactions with diacylglycerol (Hurley and Meyer, 2001) other ref. Truncations of the C-terminus of CYK-4 revealed that the ZEN-4 binding region is contained within the N-terminal 232 amino acids (fig. 13A) . Further truncations within this region indicated that residues 1-120 (from the N-terminus to the end of the coiled-coil region) are sufficient to bind efficiently to ZEN-4 (fig. 13B) . Deletion of the N-terminal 35 amino acids of CYK-4 prevented the interaction with ZEN-4. A near full length version of CYK-4 lacking only the N-terminal 35 amino acids does not bind ZEN-4 (fig 13C) . Thus the ability of CYK-4 to interact with ZEN-4 depends on the N-terminal 120 residues of CYK-4 (fig. 13D) .
Figure 13 shows the results of the in vitro binding assay that was used to define the ZEN-4 binding region of CYK-4.
(A) Full-length ZEN-4 and C-terminal deletion fragments of CYK-4 (1-232, 1-373 and 1-468) were separately expressed as 35S-labeled proteins by in vitro translation (left two panels) . The reaction products were mixed and after incubation ZEN-4 was immunoprecipitated with an anti-ZEN-4 antibody (right panel) . The three fragments of CYK-4 co- immunoprecipitated with ZEN-4. (B) Full length CYK-4 (1-681) and CYK-4 with N-terminal deletion (35-681) were expressed as 35S-labeled proteins and mixed with unlabeled CBD-ZEN-4 (left panel) . After incubation, CBD-ZEN-4 was affinity purified on chitin beads (right panel) . CYK-4 (35-681) did not copurify with CBD-ZEN- 4, but full length CYK-4 did. (C) CBD-CYK-4 fragments (1-232, 35-232, (121-232, 1-120 and 35-120) and full length ZEN-4 (o'r luciferase as a control) were co- expressed as 35S-labeled proteins by in vitro translation (left panel) . CBD-CYK-4 fragments were affinity purified (right panel) . ZEN-4 copurified with CYK-4 (1-232) and (1-120), but not with CYK-4 (35-232), (121-232) or (35-120) . (D) A schematic summary of the CYK-4 derivatives tested. The ZEN-4 binding region was defined as residues 1-120 of CYK-4. This region contains a coiled coil region and a N-terminal extension.
Example 9
Delineation of the ZEN-4 region required to bind CYK-4
Next, the in vitro assay was used to define the region of ZEN-4 that binds to the N-terminal binding of CYK-4. Initially the CYK-4 binding activity of full length ZEN-4 was compared to that of three C-terminal deletion fragments of ZEN-4: the N-terminal catalytic core alone (1-434), the catalytic core domain and the linker region (1-507), and a longer derivative that also includes the coiled coil domain (1-603) . The full length protein bound CYK-4 with high affinity, as did the next longer fragment (1-603), but the two smaller fragments did not interact with CYK-4 (fig. 14A) . Next it was examined whether the binding activity could be further localized within the central region of ZEN-4. A minimal binding domain consisting of 169 amino acids was defined (fig. 14B) . This region appears to consist of two elements, the linker region and the coiled'coil; neither of these individual elements had detectable binding activity on their own.
The interaction of CYK-4 with the neck linker/coiled coil region of ZEN-4 is particularly interesting in • light of recent studies that indicate that in conventional kinesin, the neck linker region is critical for transducing chemical energy into mechanical energy (Case et al., 2000; Rice et al., 1999) (see discussion) .
Figure 14 shows the results of the in vitro binding assay that was used to show that the central region of ZEN-4 is necessary and sufficient to bind to CYK-4.
Full length ZEN-4 (1-775) and C-terminal deletion fragments of ZEN-4 (1-603, 1-507 and 1-434) were 35S~labeled by in vitro translation and incubated with unlabeled CYK-4 (or luciferase as a control) (left panel) . CYK-4 was precipitated with anti-CYK-4 antibody (right panel) . Full length ZEN-4 and ZEN-4 (1-603) co-precipitated with CYK-4, while shorter fragments (1-507, 1-434) did not. (B) CBD-tagged fragments of ZEN-4 (1-603, 1-434, 435-603, 435-507 and 503-603) were expressed in an unlabeled form and incubated with 35S-labeled CYK-4 (1-232) (left panel) . ZEN-4 fragments were pulled down by affinity chromatography using chitin beads (right panel) . CYK-4 (1-232) copurified with ZEN-4 (1-603) and (435-603), but not with ZEN-4 (1-434), (435-507) or (503-603). (C) A schematic summary of the ZEN-4 derivatives tested. ZEN-4 fragments containing residues (435-603) associate with CYK-4, while the fragments lacking thj-β ^ region did not. This region contains a "neck" region C-terminal to the kinesin catalytic core and a coiled coil region. Example 10
a) The product of cyk-4 (tl689τs) does not interact with ZEN-4.
Although the amino terminal region of CYK-4 exhibits poor sequence conservation at the primary sequence level, earlier studies had indicated that this region is crucial for function since the mutant allele that was used to identify the cyk-4 gene had a Ser to Leu substitution at position 15. Since this point mutation maps into the ZEN-4 binding determinant and since, in vivo, this mutation seems to affect the ability of CYK- 4 and ZEN-4 to colocalize, it was tested whether this mutation also affects the ability of these two proteins to interact in vitro. The S15L mutation was introduced into the amino terminal fragment of CYK-4 and used for ZEN-4 binding assays. Whereas an interaction between the N-terminal domain of CYK-4 and the central region of ZEN-4 can be readily detected, the S15L mutant does not interact with ZEN-4 (fig. 15B) . This experiment indicates that the primary cause for the defect in cyk- 4 (tl 689ts) is a defect in the interaction with ZEN-4.
b) In vivo, ZEN-4 is in a stochiometric complex with Cyk-4
To gadn further insight into the molecular architecture
/ of the CYK-4/ZEN-4 complex and to determine whether a the 'majority of ZEN-4 is in a complex with CYK-4 in vivo, sucrose density gradient centrifugation experiments were performed with extracts prepared from C. elegans embryos. When wild-type extracts are prepared in the presence of 0.6 M NaCl, ZEN-4 migrates as a symmetrical 9 S peak (fig. 15C) . CYK-4 migrates at an identical S-value (data not shown) . To determine whether the migration of ZEN-4 on sucrose gradients was dependent on its association with CYK-4, advantage was taken of the finding that the cyk-4 (tl 689) allele is specifically defective in its ability to bind to ZEN-4. When extracts were prepared from cyk-4 mutant embryos and subjected to sucrose density centrifugation, ZEN-4 migrates as a symmetrical peak with a sedimentation value of 6 S, significantly less dense than ZEN-4 sediments in wild type extracts (fig. 15C) . These data indicate that, in vivo, the majority of ZEN-4 is in a complex with CYK-4.
c) In vivo evidence for the CYK-4/ZEN-4 interaction domains
To gain further insight into the mechanism of CYK-4 function in vivo, it was sought to identify extragenic suppressors of the cyk-4 (tl 689ts) mutation. Homozygous cyk-4 (tl 689 ts) animals were mutagenized and allowed to self fertilize for two generations at the permissive temperature. When the F2 animals reached early adulthood, the temperature was shifted to 20°C to impose a selection for fertile animals carrying presumptive suppressor mutations. By screening through approximately 100,000 FI genomes, 18 suppressor mutations were identified (see methods) . Intragenic suppressors were identified by sequencing of the cyk-4 locus PCR amplified from suppressor strains. Six suppressor strains contain additional point mutations in the N-terminus of CYK-4; all of the mutations code for amino acid substitutions within the biochemically defined ZEN-4 binding region.
In addition to the mutations found within the cyk-4 gene itself, extragenic suppressor mutations were also identified. One suppressor strain that does not contain any mutations in the cyk-4 gene was characterized in more detail. The suppressor activity was mapped using single nucleotide polymorphisms (SNPs) to the central region of chromosome IV (see methods) . Since the zen-4 gene maps to this region, the coding region of the zen 4 gene was sequenced. This strain, xs82, was found to contain a point mutation that causes a substitution of glutamic acid for a lysine at position 502 (fig. 15A) . Importantly, this substitution maps directly to the region of ZEN-4 that was found to mediate the interaction with CYK-4 in vitro. To confirm that the substitution in zen-4 (xs82) is responsible for the suppressing activity, the position of the suppressor was mapped to less than 1 cM surrounding the zen-4 locus. This mapping data combined with the fact that the sequence polymorphism maps to the CYK-4 binding region suggests strongly that these point mutations are responsible for the suppressing activity.
To determine whether the substitution-, *E502K, has any phenotype on its own, in the absence of the cyk-4 (tl 689) allele, cyk-4 (tl 689) Was crossed out and worms homozygous for zen-4 (xs82) were identified. These worms grew normally at 16°C, 20°C, and 25°C. d) ZEN-4 (xs82) partially restores binding to CYK-4 in vitro
As mentioned above, CYK-4 S15L is unable to interact with ZEN-4 in the in vitro binding assay. If the immediate consequence of the S15L substitution in the N-terminus of CYK-4 is defective ZEN-4 binding, then an extragenic suppressor mutation in zen-4 that rescues cyk-4 (tl 689) might be expected to restore binding to CYK-4 S15L. To test this possibility a N terminal fragment of CYK-4 carrying the S15L point mutation was translated and this product was mixed with a C terminal fragment of ZEN-4 carrying either the wild-type glutamic acid at position 502 or the lysine substitute allele. The wild-type CYK-4 fragment bound equally well to wild-type and the E502K derivative of ZEN-4. As discussed above the CYK-4 S15L is unable to bind to wild-type ZEN-4. However, CYK-4 S15L was able to bind to ZEN-4 E502K, though this interaction was weaker than that seen with the wild-type proteins (fig. 15A) . These data, together with the genetic suppression, prove that the interaction between the N-terminus of CYK-4 and the central region of ZEN-4 is crucial for its function in vivo.
Figure 15 shows that a complex of CYK-4 and ZEN-4 is required for function' in vivo
(A) A screen for suppressors of cyk-4 (tl 689ts) identified a point mutation of zen-4 (xs82) . This allele has a G to A point mutation, resulting in substitution of glutamate at residue 502 to lysine. (B) The E502K mutation partially restores binding to CYK-4 S15L. CYK-4 fragments (1-232, 1-232 with a point mutation S15L, 35-232) were expressed as 35S-labeled protein and incubated with unlabeled ZEN-4 fragments (435-775, 435-775 with the E502K mutation, 609-775) . ZEN-4 fragments were precipitated with anti-ZEN-4 antibody. CYK-4 (1-232) co-precipitated with ZEN-4 (435-775) but not with ZEN-4 (609-775) as shown in Figure 13. The S15L mutation in CYK-4 abolishes its ability to bind to ZEN-4 (CYK-4 1-232 S15L and ZEN-4 435-775 wt) . However, the suppressor mutation in ZEN-4 (E502K) partially recovered this interaction ( (CYK-4 1-232 S15L and ZEN-4 435-775 E502K) . The ZEN-4 E502K mutation does not affect the binding to wild-type CYK-4. (C) The majority of ZEN-4 is in a complex with CYK-4 in vivo. Extracts were made from wild type (N2) embryos or cyk-4 (tl 689ts) embryos. Sedimentation of ZEN-4 was analyzed by sucrose density gradient centrifugation followed by western blotting. The samples were spiked with the indicated standards and they ran identically in the two gradients. ZEN-4 from wild type embryos sediments at 8 S, whereas ZEN-4 from cyk-4 mutant embryos sediments at 7S.
Example 11
CYK-4 and ZEN-4 self-associate
thus far it has been established that central spindle assembly is dependent on an interaction between CYK-4 and ZEN-4 and have defined the regions of both proteins that are required for this interaction. Interestingly, both binding domains contain coiled coil regions; these regions are required for binding activity. Since coiled coils are often dimerization motifs, and since kinesin motors are often composed of two motor domains tethered together through a coiled coil, it was examined whether CYK-4 and ZEN-4 are individually able self-associate.
To assess if CYK-4 self associates, full length CYK-4 and the CYK-4 derivative 1-232 were co-expressed. As a control, the full length protein was substituted by a truncated version lacking the N-terminal 120 amino acids. The longer CYK-4 fragments were immunoprecipitated with an antibody directed against the C-terminus of CYK-4 and the co-immunoprecipitation of the short N-terminal fragment was assessed. The N-terminal fragment bound to full length CYK-4 but not to CYK-4 lacking the N-terminal domain (figure 16A) . These data demonstrate that CYK-4 self associates. This association is likely mediated by the coiled-coil domain.
The capacity of ZEN-4 to self-associate was examined using a similar strategy. Full length ZEN-4 was co- expressed with affinity-tagged deletion derivatives of ZEN-4. Neither the N-terminal region (1-507) nor the C-terminal region (604-775) of ZEN-4 bound appreciably to full length ZEN-4, but two internal fragments of
ZEN-4 (503-775; 435-603) did bind to full length ZEN-4 (figure 16B) . The region common to these two fragments consists of residues 503-603 which is the region predicted to form a coiled coil domain. The ability of CYK-4 and ZEN-4 to self associate in vitro raise the possibility that in vivo, the CYK-4/ZEN-4 complex may contain, at a minimum, two molecules each of CYK-4 and ZEN-4.
Figure 16 shows the self-association of both CYK-4 and ZEN-4
(A) CYK-4 (1-232), which does not contain the epitope recognized' by the anti-CYK-4 antibody, was co-expressed as a 35S-labeled protein with CYK-4 (1-681) or (121-681) , which do contain the epitope for this antibody (left panel) . CYK-4 (1-681) and (121-681) were immunoprecipitated. CYK-4 (1-232) co- immunoprecipitated with CYK-4 (1-681) , but not with CYK-4 (121-681) . (B) CBD-tagged fragments of ZEN-4 (1-507, 503-775, 435-603 or 604-775) were co-expressed with untagged ZEN-4 (1-775) (left panel) . CBD-tagged fragments were purified by affinity chromatography with chitin beads (right panel) . Untagged ZEN-4 (1-775) copurified with CBD-ZEN-4 (503-775) and (435-603), but not with ZEN-4 (1-507) or (604-775) . The low extent of labelling of 604-775 as compared to 435-603 is attributable to low cysteine and methionine content of the C-terminal fragment.
Example 12
The human orthologs of CYK-4 and ZEN-4, HsCYK-4 and MKLPl, interact in vivo and colocalize.
In C. elegans, CYK-4 and ZEN-4 associate in vivo and in vitro and this complex is important for central spindle assembly and cytokinesis. To extend these findings it was evaluated whether mammalian cells contain a stable complex containing the human orthologs of these proteins, HsCYK-4/MgcRacGAP (Hirose et al., 2001; Toure et al., 1998; Wooltorton et al., 1999) and MKLPl, respectively. Extracts were prepared from mitotic Hela cells and antibodies directed against HsCYK-4 or MKLPl were used for immunoprecipitation followed by western blotting. MKLPl could be co-immunoprecipitated with anti-HsCYK-4 antibodies and vice versa (fig. 17a) . The immunoblots reveal that small amounts of HsCYK-4 and MKLPl migrate with reduced mobility, likely due to phosphorylation.
To obtain evidence that these proteins play a similar role in human cells as was shown previously for the nematode proteins, the localization of HsCYK-4 and MKLPl was examined in cultured (HeLa) human cells. During all stages of the cell cycle the two proteins precisely co-localize (insets fig 17b) . In early anaphase, the two proteins localize to a discrete, central region of the central spindle. Three dimensional reconstruction of optically sectioned anaphase cells reveals that each bundle of microtubules in the central spindle is labeled at its distal end by MKLPl antibodies. During mid-to-late anaphase, the
MKLPl staining region is 0.93+0.25 μm in length (n=56) . This region largely corresponds to the region that appears deficient in tubulin staining, but this is a known artifact caused by epitope masking (Saxton and Mclntosh, 1987) . The extent of staining is somewhat narrower than previous ultrastructural determinations of the extent of microtubule overlap (> 2 μM) (Mastronarde et al., 1993). Later in cleavage, both proteins localize to a discrete, central portion of the midbody. During interphase, some cells contain HsCYK-4 and MKLPl in the nucleus, whereas others are do not. This is likely due to cell cycle regulated accumulation of these factors since, upon G2 arrest, HsCYK-4 and MKLPl accumulate in the nucleus of all cells (data not shown) . In addition, most cells contain brightly- staining, cortical ring-like structures. These structures likely correspond to division remnants; previous time-lapse microscopy with GFP-tagged CYK-4 and ZEN-4 in nematode embryos revealed that the central spindle matures into the midbody that later develops into a persistent spot or ring in 'the cell cortex. Thus there is a striking concordance between the biochemical properties and the subcellular localization of CYK-4/ZEN-4 in C. elegans embryos and HsCYK-4/MKLPl in human cells. Since the names of the individual proteins vary from species to species, it is proposed to call this complex "centralspindlin". It is reasonable to speculate that centralspindlin performs a similar function in these two systems.
To gain insight into the architecture of centralspindlin, the hydrodynamic properties of its constituents were investigated. , Eysates were prepared from mitotic Hela cells and run on sucrose density gradients. Western blotting of the gradient fractions indicates that HsCYK-4 and MKLPl precisely co-migrate on the gradients with an S-value of 8 S (fig. 17b) . This sedimentation behavior is similar to that of centralspindlin isolated from C. elegans embryos. The two proteins also co-migrated on a gel filtration column with an apparent molecular weight of 800 kD (data not shown) . Gel filtration chromatography can not accurately estimate native molecular mass of asymmetric particles, however, gel filtration data, combined with S-value measurements, allows a more accurate estimation of the native molecular mass. When the experimental values of centralspindlin are combined in this way, the complex is estimated to have a native molecular weight of -300 kDa.
To determine if equimolar amounts of HsCYK-4 and MKLPl are present in centralspindlin and whether or not other proteins are also present, the complex was immunopurified from a mitotic MAP (microtubule- associated protein) fraction that was eluted with ATP and 500 mM NaCl. Antibodies specific for HsCYK-4 and MKLPl were used in parallel for the immunoprecipitation. Coomassie blue staining of both immunoprecipitates revealed two major bands and one minor band (fig. 17C) . Mass spectrometry analysis revealed that the slower migrating major band and the minor band are MKLPl and the faster migrating major band is HsCYK-4. No additional bands reproducibly co precipitated with HsCYK-4 or MKLPl. Given that centralspindlin has a native molecular mass of 300 kD and that it dotes not appear to contain any proteins in addition to/HsCYK-4 (70 kD) and MKLPl (100 kD) and given that both components are capable of self- association, it is concluded that centralspindlin is a tetramer containing two molecules each of HsCYK-4 and MKLPl. Figure 17 shows that there exists a complex of MKLP-1 and HsCYK-4 in HeLa cells
HsCYK-4/MgcRacGAP and MKLP-1, the human orthologs of CYK-4 and ZEN-4, respectively, exist as a complex in mitotic HeLa cells. (A) HsCYK-4 and MKLP-1 were immunoprecipitated from HeLa cell lysate with specific rabbit antibodies (IP:HsCYK-4 or MKLP-1) and blotted with specific mouse antibodies (blot: HsCYK-4 or MKLP-1) . HsCYK-4 co-immunoprecipitated with MKLP-1 and vice versa. (B) HsCYK-4 and MKLPl co-localize to the central spindle and the midbody. Hela cells were fixed and immunostained with anti-CYK-4, anti-MKLPl, anti- tubulin antibodies and DNA was stained with Hoechst. Shown are cells in interphase (a) and various stages of cytokinesis (b-f) . In main panels, staining of CYK-4 (green) , microtubules (red) and DNA (blue) was shown. In inset, staining of CYK-4 (green) and MKLPl (red) was shown. (C) HeLa cell lysates were analyzed by sucrose density gradient centrifugation followed by blotting with anti-MKLP-1 antibody or anti-HsCYK-4 antibody.
HsCYK-4 and MKLP-1 co-sediment at 8S. The lowest band reacting with the CYK-4 antibody is caused by proteolysis of the N-terminus. (D) MKLP-1 and HsCYK-4 complex was immunoprecipitated from a microtubule- binding fraction (MTfr.) of mitotic HeLa cells with specific antibodies. Major 110 kDa and 75 kDa bands were identified by mass spectrometry as MKLP-1 and HsCYK-4, respectively. The minor band at 115 kDa also corresponds to MKLPl. Broad bands around 50 kDa and bands near gel bottom are heavy (IgGH) and light (IgGL) chains of immunoglobulin leaked from antibody beads. Table 1
genotype stage at percent phenotype of N time of viable surviving shift to progeny
25°C
N2 (wild type) embryos 98.8% wild-type 485 cyk-4 (not 70.2% fertile 198 shifted) cyk-4 embryos 1.5% sterile, highly 324 Unc cyk-4 LI larvae 100.0% sterile, highly 89 Unc cyk-4 L2/L3 100.0% sterile, highly 55 Unc cyk-4 L4 100.0% fertile, lay 52 dead embryos cyk-4 , embryos 59.4% fertile 350 xsExl [cyk-
4 : GFP]
Except for the N2 control, the complete genotype of the strain was unc-32 (el89) cyk-4 (tl 689ts) . Gravid hermaphrodites were allowed to lay embryos for 2 hours at the permissive temperature. The adult was then removed and the number of embryos counted. After 24 hours at the indicated temperature the number of unhatched embryos was then counted. Table 2
injected locus percent number of number additional dsRNA cytokinesis time cytokinesi phenotypes defective (N) lapse s recording defective
RhoA Y51H4A. B 95% ( 175 ) 15 15 (100%)
Rac-1 C09G12 . 8 Not emb . B lethal
Cdc42 R07G3 . 1 12% ( 74 ) 22 3 (14%) symmetric 1st division (5/22); no rotation in PI (12/22) ; rotation of AB (2/22) others F22E12.2 Not emb. lethal Y32F6B.3 Not emb. lethal
K03D3.9 Not emb. lethal
Young adult hermaphrodites were injected with the indicated dsRNAs and broods of laid embryos were scored for embryonic lethality. dsRNAs that induced embryonic lethality were further characterized by dissecting embryos and scoring for multinucleate embryos and by performing time lapse recordings and evaluating cytokinesis, spindle orientation in the one-cell embryo (symmetric or asymmetric first cleavage) , and spindle positioning in the PI and AB blastomeres. Table 3
genot-ype temperature total total average nutribe. number of number of number of etribrj stained cells remnants/ remnants cell
N2 (wildtype) 25°C 110 137 0.80 16
zen-4 (orl98ts) 16°C 61 83 0.73 10
zen-4 (orl98ts) 25°C (18 min. ) 11 147 0.07 24
N2 (wildtype) 25°C 150 205 0.73- 22
cyk-4 (tl689ts) 16°C 61 98 0.62 10
cyk-4 (tl689ts) 25°C (15 min. ) 10 176 0.06 19
Wild-type and zen-4 (orl53ts) embryos grown at the indicated temperatures were fixed and stained for Cyk-4 . Similarly, Wild-type and cyk-4 (tl 689ts) grown at the indicated temperatures were fixed and stained forZen-4 /CeMKlpl . The number of cells in each embryo and the number of remnants staining with Cyk-4 or Zen-4 antibodies was counted. References
Adams, R. R., Tavares, A. A., Salzberg, A., Bellen, H. J., and Glover, D. M. (1998). pavarotti encodes a kinesin-like protein required to organize the central spindle and contractile ring for cytokinesis. Genes Dev 12, 1483-94. Aktories, K., and Hall, A. (1989). Botulinum ADP- ribosyltransferase C3: a new tool to study low molecular weight GTP-binding proteins. Trends
Pharmacol Sci 10, 415-8. Bosher, J.M. and Labouesse, M., (2000), Nat Cell Biol,
Feb; 2(2) :E31-6 Busson, S., Dujardin, D. , Moreau, A., Dompierre, J. , and De Mey, J. R. (1998) . Dynein and dynactin are localized to astral microtubules and at cortical sites in mitotic epithelial cells. Curr Biol 8,
541-4. Cao, L. G., and Wang, Y. L. (1996). Signals from the spindle midzone are required for the stimulation of cytokinesis in cultured epithelial cells. Mol
Biol Cell 7, 225-32.
Carmena, M., Riparbelli, M. G., Minestrini, G., Tavares, A. M. , Adams,1 R., Callaini, G. , and
/ Glover, D. M. (1998) . Drosophila polo kinase is required for cytokinesis. J Cell Biol 143, 659-71.
Carminati, J. L., and Stearns, T. (1997). Microtubules orient the mitotic spindle in yeast through dynein- dependent interactions with the cell cortex. J Cell Biol 138, 629-41. Case, R. B., Rice, S., Hart, C. L., Ly,' B., and Vale, R. D. (2000) . Role of the kinesin neck linker and catalytic core in microtubule-based motility. Curr Biol 10, 157-60. Castrillon, D. H., and Wasserman, S. A. (1994). Diaphanous is required for cytokinesis in Drosophila and shares domains of similarity with the products of the limb deformity gene. Development 120, 3367-77. Chang, F., Drubin, D., and Nurse, P. (1997). cdcl2p, a protein required for cytokinesis in fission yeast, •is a component of the cell division ring and interacts with profilin. J Cell Biol 137, 169-82. Chui, K. K. , Rogers, G. C, Kashina, A. M., Wedaman, K. P., Sharp, D.-J., Nguyen, D. T., Wilt, F. , and
Scholey, J. M. (2000) . Roles of two homotetrameric kinesins in sea urchin embryonic cell division. J Biol Chem 275, 38005-11. Drechsel, D. N., Hyman, A. A., Hall, A., and Glotzer, M. (1997) . A requirement for Rho and Cdc42 during cytokinesis in Xenopus embryos. Curr Biol 7, 12- 23. Dutartre, H., Davoust, J. , Gorvel, J. P., and Chavrier, P. (1996) . Cytokinesis arrest and redistribution of actin-cytoskeleton regulatory components in cells expressing the Rho GTPase CDC42HS. J Cell Sci 109, 367-77.
Echard, A., Jollivet, F., Martinez, 0., Lacapere, J.
J., Rousselet, A., Janoueix-Lerosey, I., and Goud, B. (1998) . Interaction of a Golgi-associated kinesin-like protein with Rab6. Science 279, 580- 5. Eckley, D. M., Ainsztein, A. M., Mackay, A. M.,
Goldberg, I. G., and Earnshaw, W. C. (1997). Chromosomal proteins and cytokinesis: patterns of cleavage furrow formation and inner centromere protein positioning in mitotic heterokaryons and mid-anaphase cells. J Cell Biol 136, 1169-83. Evangelista, M., Blundell, K. , Longtine, M. S., Chow, C. J. , Adames, N., Pringle, J. R., Peter, M. , and Boone, C. (1997) . Bnilp, a yeast formin linking cdc42p and the actin cytoskeleton during polarized morphogenesis. Science 276, 118-22. Field, C, Li, R. , and Oegema, K. (1999). Cytokinesis in eukaryotes: a mechanistic comparison. Curr Opin Cell Biol 11, 68-80. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A.,
Driver, S. E., and Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-11. Fire, A., (1999), Trends Genet, Sep; 15(9): 358-63 Fontijn, R. D., Goud, B., Echard, A., Jollivet, F., van Marie, J., Pannekoek, H., and Horrevoets, A. J. (2001) . The Human Kinesin-Like Protein RB6K Is under Tight Cell Cycle Control and Is Essential for Cytokinesis. Mol Cell Biol 21, 2944-55. Fujiwara, T., Tanaka, K., Inoue, E., Kikyo, M., and Takai, Y. (1999) . Bnilp Regulates Microtubule- Dependent Nuclear Migration through the Actin Cytoskeleton in Saccharomyces cerevisiae. Mol Cell Biol 19, 8016-8027. Giansanti, M. G., Bonaccorsi, S., Williams, B.,
Williams, E. V., Santolamazza, C, Goldberg, M. L., and Gatti, M. (1998). Cooperative interactions between the central spindle and the contractile ring during Drosophila cytokinesis. Genes Dev 12, 396-410. Glotzer, M. (1997) . The mechanism and control of cytokinesis. Curr Opin Cell Biol 9, 815-23.
Glotzer, M. (1997). Cytokinesis. Curr Biol 7, R274-6. Gδnczy, P., Pichler, S., Kirkham, M., and Hyman, A. A.
(1999a) . Cytoplasmic dynein is required for distinct aspects of MTOC positioning, including centrosome separation, in the one cell stage
Caenorhabditis elegans embryo. J Cell Biol 147,
135-50. Gδnczy, P., Schnabel, H., Kaletta, T., Mores, A. D.,
Hyman, T., and Schnabel, R. (1999b). Dissection of cell division processes in the one cell stage
Caenorhabditis elegans embryo by mutational analysis. J Cell Biol 144, 927-46.
Hill, E., Clarke, M., and Barr, F. A. (2000). The Rab6- binding kinesin, Rab6-KIFL, is required for cytokinesis. Embo J 19, 5711-9.
Hirose, K., Kawashima, T., Iwamoto, I., Nosaka, T., and Kitamura, T. (2001) . MgcRacGAP is involved in cytokinesis through associating with mitotic spindle and midbody. J Biol Chem 276, 5821-8. Hurley, J. H., and Meyer, T. (2001). Subcellular targeting by membrane lipids. Curr Opin Cell Biol 13, 146-52. Hyman, A. A., and White, J. G. (1987). Determination of cell division axes in the early embryogenesis of Caenorhabditis elegans. J Cell Biol 105, 2123-35. Hyman, A. A. (1989) . Centrosome movement in the early divisions of Caenorhabditis elegans: a cortical site determining centrosome position. J Cell Biol 109, 1185-93. Imamura, H., Tanaka, K., Hihara, T., Umikawa, M.,
Kamei, T., Takahashi, K., Sasaki, T., and Takai, Y. (1997) . Bnilp and Bnrlp: downstream targets of the Rho family small G-proteins which interact with profilin and regulate actin cytoskeleton in Saccharomyces cerevisiae. Embo J 16, 2745-55.
Jantsch-Plunger, V., and Glotzer, M. (1999). Depletion of syntaxins in the early Caenorhabditis elegans embryo reveals a role for membrane fusion events in cytokinesis. Curr Biol 9, 738-45.
Jantsch-Plunger, V., Gonczy, P., Romano, A., Schnabel, H. , Hamill, D. , Schnabel, R. , Hyman, A. A., and Glotzer, M. (2000). CYK-4. A rho family gtpase activating protein (gap) required for central spindle formation and cytokinesis. J Cell Biol 149, 1391-404.
Kaitna, S., Mendoza, M., Jantsch-Plunger, V., and Glotzer, M. (2000) . Incenp and an aurora-like kinase form a complex essential for chromosome segregation and efficient completion of cytokinesis [In Process Citation] . Curr Biol 10, 1172-81. Kishi, K., Sasaki, T., Kuroda, S., Itoh, T., and Takai, Y. (1993) . Regulation of cytoplasmic division of Xenopus embryo by rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI) . J Cell Biol 120, 1187-95. Kosako, H., Goto, H., Yanagida, M., Matsuzawa, K., Fujita, M., Tomono, Y., Okigaki, T., Odai, H., Kaibuchi, K. , and Inagaki, M. (1999). Specific accumulation of Rho-associated kinase at the cleavage furrow during cytokinesis: cleavage furrow-specific phosphorylation of intermediate filaments. Oncogene 18, 2783-8.
Kull, F. J., Sablin, E. P., Lau, R., Fletterick, R. J., and Vale, R. D. (1996) . Crystal structure of the kinesin motor domain reveals a structural similarity to myosin. Nature 380, 550-5.
Kuriyama, R., Dragas-Granoic, S., Maekawa, T.,
Vassilev, A., Khodjakov, A., and Kobayashi, H. (1994) . Heterogeneity and microtubule interaction of the CHOI antigen, a mitosis-specific kinesin- like protein. Analysis of subdomains expressed in insect sf9 cells. J Cell Sci 107, 3485-99.
Lamarche, N., and Hall, A. (1994). GAPs for rho-related GTPases. Trends Genet 10, 436-40. Larkin, K. , and Danilchik, M.V. (1999), Dev Biol. 214:215-226
Lee, K. S., Yuan, Y. L., Kuriyama, R., and Erikson, R. L. (1995) . Plk is an M-phase-specific protein kinase and interacts with a kinesin- like protein, CHOl/MKLP-1. Mol Cell Biol 15, 7143-51.
Lewis, J. A., and Fleming, J. T. (19,9*5). Basic culture methods. Methods Cell Biol 48, -29.
\ Mabuchi, I., Hamaguchi, Y., Fujimoto, H., Morii, N.,
Mishima, M., and Narumiya, S. (1993). A rho-like protein is involved in the organisation of the contractile ring in dividing sand dollar eggs.
Zygote 1, 325-31. Mackay, A. M. , Ainsztein, A. M., Eckley, D. M., and
Earnshaw, W. C. (1998) . A dominant mutant of inner centromere protein (INCENP) , a chromosomal protein, disrupts prometaphase congression and cytokinesis. J Cell Biol 140, 991-1002.
Martineau-Thuillier, S., Andreassen, P. R., and
Margolis, R. L. (1998) . Colocalization of TD-60 and INCENP throughout G2 and mitosis: evidence for their possible interaction in signalling cytokinesis. Chromosoma 107, 461-70.
Mastronarde, D. N., McDonald, K. L., Ding, R., and Mclntosh, J. R. (1993) . Interpolar spindle microtubules in PTK cells. J Cell Biol 123, 1475- 89. Mello, C. C, Kramer, J. M. , Stinchcomb, D., and
Ambros, V. (1991) . Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. E bo J 10, 3959-70. Miller, R. K., Matheos, D., and Rose, M. D. (1999). The cortical localization of the microtubule orientation protein, Kar9p, is dependent upon actin and proteins required for polarization. J Cell Biol 144, 963-75. Moorman, J. P., Boba?, D. A., and Hahn, C. S. (1996). Inactivation of the small GTP binding protein Rho induces multinucleate cell formation and apoptosis
I in murine T lymphoma EL4. J Immunol 156, 4146-53.
Nislow, C, Lombillo, V. A., Kuriyama, R., and Mclntosh, J. R. (1992) . A plus-end-directed motor enzyme that moves antiparallel microtubules in vitro localizes to the interzone of mitotic spindles. Nature 359, 543-7.
O'Connell, C. B., Wheatley, S. P., Ahmed, S., and Wang, Y. L. (1999) . The small GTP-binding protein rho regulates cortical activities in cultured cells during division. J Cell Biol 144, 305-13.
O'Connell, K. F. , Leys, C M., and White, J. G. (1998). A genetic screen for temperature-sensitive cell- division mutants of Caenorhabditis elegans. Genetics 149, 1303-21.
Okada, Y., and Hirokawa, N. (2000). Mechanism of the single-headed processivity: diffusional anchoring between the K-loop of kinesin and the C terminus of tubulin. Proc Natl Acad Sci U S A 57, 640-5. Powers, J. , Bossinger, 0., Rose, D., Strome, S., and
Saxton, W. (1998) . A nematode kinesin required for cleavage furrow advancement. Curr Biol 8, 1133-6. Prokopenko, S. N., Brumby, A., O'Keefe, L., Prior, L., He, Y., Saint, R., and Bellen, H. J. (1999). A putative exchange factor for Rhol GTPase is required for initiation of cytokinesis in Drosophila. Genes Dev 13, 2301-14. Raich, W. B., Moran, A. N., Rothman, J. H., and Hardin, J. (1998) . Cytokinesis and midzone microtubule organization in Caenorhabditis elegans require the kinesin-like protein ZEN-4. Mol Biol Cell 9, 2037-49. 'Rappaport, R. (1985) . Repeated furrow formation from a single mitotic apparatus in cylindrical sand dollar eggs. J Exp Zool 234, 167-71.
Reddien, P., and Horvitz, H. (2000). CED-2/CrkII and
CED-10/Rac control phagocytosis and cell migration in Caenorhabditis elegans. Nature Cell Biology 2, 131-136.
Remington's Pharmaceutical Sciences, 1980, Mack Publ.
Co. Easton, PA, Osol (ed. )
Remington: The Science and Practice of Pharmacy, 2000
(20th edition; Book News, Inc.)
Rice, S., Lin, A. W., Safer, D., Hart, C. L., Naber,
N., Carragher, B. 0., Cain, S. M., Pechatnikova, E., Wilson-Kubalek, E. M., Whittaker, M., Pate,
E., Cooke, R., Taylor, E. W., Milligan, R. A., and Vale, R. D. (1999) . A structural change in the kinesin motor protein that drives motility. Nature 402, 778-84. Rieder, C. L., Khodjakov, A., Paliulis, L. V., Fortier, T. M., Cole, R. W., and Sluder, G. (1997). Mitosis in vertebrate somatic cells with two spindles: implications for the metaphase/anaphase transition checkpoint and cleavage. Proc Natl Acad Sci U S A 94, 5107-12.
Sablin, E. P., Kull, F. J., Cooke, R., Vale, R. D., and Fletterick, R. J. (1996) . Crystal structure of the motor domain of the kinesin-related motor ncd. Nature 380, 555-9.
Sambrook et al., 2000, Molecular Cloning, Third Edition Savoian, M. S., Earnshaw, W. C, Khodjakov, A., and Rieder, C. L. (1999) . Cleavage furrows formed between centrosomes lacking an intervening spindle and chromosomes contain microtubule bundles, INCENP, and CHOI but not CENP-E. Mol Biol Cell 10, 297-311. Saxton, W. M. , and Mclntosh, J. R. (1987). Interzone microtubule behavior in late anaphase and telophase spindles. J Cell Biol 105, 875-86.
Schumacher, J. M., Golden, A., and Donovan, P. J. (1998) . AIR-2: An Aurora/Ipll-related protein kinase associated with chromosomes and midbody microtubules is required for polar body extrusion and cytokinesis in Caenorhabditis elegans embryos. J Cell Biol 143, 1635-46. Settleman, J. , and Foster, R. , (1995), Methods Enzymol; 256:105-13
Severson, A. F., Schumacher, J., Hamill, D. R., Carter,
J. C, and Bower an, B. (2000). The Aurora/Ipllp- Related Kinase AIR-2 Functions at Metaphase to Recruit theKinesin-like Protein ZEN-4 to the
Mitotic Spindle Midzone and is Required forCytokinesis. Current Biology, Oct 5;10(19): 1162-71.
Sharp, D. J. , Kuriyama, R. , and Baas, P. W. (1996). Expression of a kinesin-related motor protein induces Sf9 cells to form dendrite-like processes with nonuniform microtubule polarity orientation. J Neurosci 16, 4370-5. Sharp, P.A., (1999), Genes Dev, Jan 15; 13(2): 139-41 Skop, A. R., and White, J. G. (1998). The dynactin complex is required for cleavage plane specification in early Caenorhabditis elegans embryos. Curr Biol 8, 1110-6. Sugihara, K., Nakatsuji, N., Nakamura, K., Nakao, K., Hashimoto, R., Otani, H., Sakagami, H., Kondo, H., Nozawa, S., Aiba, A., and Katsuki, M. (1998). Racl is required for the formation of three germ layers during gastrulation. Oncogene 17, 3427-33. Sulston, J.E., and Horvitz, H.R., (1977), Dev Biol. 56:110-156 Swan, K. A., Severson, A. F., Carter, J. C, Martin, P. R. , Schnabel, H., Schnabel, R., and Bowerman, B. (1998). cyk-1: a C. elegans FH gene required for a late step in embryonic cytokinesis. J Cell Sci 111, 2017-27. Tatsumoto, T., Xie, X., Blumenthal, R., Okamoto, I., and Miki, T. (1999) . Human ECT2 is an exchange factor for rho GTPases, phosphorylated in G2/M phases, and involved in cytokinesis [In Process Citation]. J Cell Biol 147, 921-8.
Timmons, L., and Fire, A. (1998). Specific interference by ingested dsRNA. Nature 395, 854. Toure, A., Dorseuil, 0., Morin, L., Timmons, P., Jegou,
B., Reibel, L., and Gacon, G. (1998). MgcRacGAP, a new human GTPase-activating protein for Rac and Cdc42 similar to Drosophila rotundRacGAP gene product, is expressed in male germ cells. J Biol
Chem 273, 6019-23.
Vale, R. D., and Fletterick, R. J. (1997). The design plan of kinesin motors. Annu Rev Cell Dev Biol 13, 745-77.
Vale, R. D., and Milligan, R. A. (2000). The way things move: looking under the hood of molecular motor proteins. Science 288, 88-95. Van Aelst, L., and D'Souza-Schorey, C. (1997). Rho GTPases and signaling networks. Genes Dev 11,2295-322.
Van de Putte T, Zwijsen A, Lonnoy 0, Rybin V, Cozijnsen M, Francis A, Baekelandt V, Kozak CA, Zerial M, Huylebroeck D.Mice with a homozygous gene trap vector insertion in mgcRacGAP die during pre- implantation development .Mech Dev. 2001 Apr;102(l- 2):33-44
Waddle, J. A., Cooper, J. A., and Waterston, R. H.
(1994) . Transient localized accumulation of actin in Caenorhabditis elegans blastomeres with oriented asymmetric divisions. Development 120, 2317-28.
Watanabe, N., Madaule, P., Reid, T., Ishizaki, T.,
Watanabe, G., Kakizuka, A., Saito, Y., Nakao, K., Jockusch, B. M., and Narumiya, S. (1997). pl40mDia, a mammalian homolog of Drosophila diaphanous, is a target protein for Rho small
GTPase and is a ligand for profilin. Embo J 16, 3044-56. Wheatley, S. P., and Wang, Y. L. (1996). Midzone microtubules are continuously required for cytokinesis in cultured epithelial cells. J. Cell Biol. 135, 981-989. Woollard, A., and Hodgkin, J. (1999). Stu-7/air-2 is a C. elegans aurora homologue essential- for chromosome segregation during embryonic and post- embryonic development. Mech Dev 82, 95-108.

Claims

Claims
1. Human poypeptide designated Cyk-4, which is a
GTPase activating protein (GAP) for Rho family of GTPases, with the amino acid sequence as set forth in SEQ ID NO: 2 or with the amino acid sequence encoded by a polynucleotide which hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO:l.
2. Murine Cyk-4 poypeptide designated Cyk-4, which is a GTPase activating protein (GAP) for Rho family of GTPases, with the amino acid sequence as set forth in SEQ ID NO: 4 or with the amino acid sequence encoded by a polynucleotide which hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO: 3.
3. An isolated DNA molecule comprising a polynucleotide with the nucleotide sequence as set forth in SEQ ID NO:l encoding human Cyk-4 polypeptide, or an isolated DNA molecule encoding human Cyk-4 polypeptide comprising a polynucleotide which hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence as set forth in SE§ ID NO:l.
4. An isolated DNA molecule comprising a polynucleotide with the nucleotide sequence as set forth in SEQ ID NO: 3 encoding murine Cyk-4 polypeptide, or an isolated DNA molecule encoding murine Cyk-4 polypeptide comprising a polynucleotide which hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO: 3.
5. An antibody which is specifically reactive with an epitope of the human Cyk-4 polypeptide of claim 1.
6. An antibody which is specifically reactive with an epitope of the murine Cyk-4 polypeptide of claim 2.
7. A method for identifying a compound capable of modulating cytokinesis, wherein the compound's ability to modulate the function of CYK-4 is determined.
8. The method of claim 7 wherein the compound's ability to promote GTP hydrolysis by a Rho family GTPase is determined by incubating a substrate selected from the members of the Rho family GTPases with GTP for a period of time sufficient to allow saturation of the substrate's GTP binding sites, adding Cyk-4 and allowing it to react in the presence or absence of the test compound, and determining the amount of hydrolized GTP.
9. The method of claim 7 wherein the compound's ability to inhibit Cyk-4 function is determined by determining the compound' s ability to interfere with the biochemical interaction of CYK-4 and a member of the MKLPl subfamily.
10. The method of claim 7 wherein the compound's ability to inhibit CYK-4 function is determined by determining the compound' s ability to interfere with the biochemical multimerization of CYK-4.
11. The method of claim 7 wherein the compound's ability to inhibit MKLPl function is determined by determining the compound' s ability to interfere with the biochemical multimerization of a member of the MKLPl subfamily.
12. A compound identified in the method of any one of claims 7 to 11 for use in cancer therapy.
EP01947374A 2000-06-19 2001-06-15 Cyk-4 polypeptides, dna molecules encoding them and their use in screening methods Withdrawn EP1297131A2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP01947374A EP1297131A2 (en) 2000-06-19 2001-06-15 Cyk-4 polypeptides, dna molecules encoding them and their use in screening methods

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
EP00112880 2000-06-19
EP00112880A EP1167525A1 (en) 2000-06-19 2000-06-19 Cyk-4 polypeptides, DNA molecules encoding them and their use in screening methods
EP01110554 2001-04-30
EP01110554A EP1254912A1 (en) 2001-04-30 2001-04-30 Cyk-4 polypeptides, DNA molecules encoding them and their use in screening methods
EP01947374A EP1297131A2 (en) 2000-06-19 2001-06-15 Cyk-4 polypeptides, dna molecules encoding them and their use in screening methods
PCT/EP2001/006797 WO2001097608A2 (en) 2000-06-19 2001-06-15 Cyk-4 polypeptides, dna molecules encoding them and their use in screening methods

Publications (1)

Publication Number Publication Date
EP1297131A2 true EP1297131A2 (en) 2003-04-02

Family

ID=26071059

Family Applications (1)

Application Number Title Priority Date Filing Date
EP01947374A Withdrawn EP1297131A2 (en) 2000-06-19 2001-06-15 Cyk-4 polypeptides, dna molecules encoding them and their use in screening methods

Country Status (4)

Country Link
EP (1) EP1297131A2 (en)
JP (1) JP2003535606A (en)
AU (1) AU2001269078A1 (en)
WO (1) WO2001097608A2 (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005017161A2 (en) * 2003-08-13 2005-02-24 Children's Hospital Medical Center Chimeric peptides for the regulation of gtpases
WO2005017160A2 (en) 2003-08-13 2005-02-24 Children's Hospital Medical Center Mobilization of hematopoietic cells
WO2011083830A1 (en) * 2010-01-06 2011-07-14 学校法人関西文理総合学園 Novel anticancer agent and method for screening same

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6238881B1 (en) * 1996-11-06 2001-05-29 Onyx Pharmaceuticals, Inc. Nucleic acids and polypeptides related to a guanine exchange factor of Rho GTPase
GB9807848D0 (en) * 1998-04-09 1998-06-10 Medical Res Council Assay method and means
EP1248798A2 (en) * 1999-08-18 2002-10-16 Fraunhofer-Gesellschaft Zur Förderung Der Angewandten Forschung E.V. Human dna sequences

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0197608A2 *

Also Published As

Publication number Publication date
JP2003535606A (en) 2003-12-02
AU2001269078A1 (en) 2002-01-02
WO2001097608A3 (en) 2002-12-05
WO2001097608A2 (en) 2001-12-27

Similar Documents

Publication Publication Date Title
Jantsch-Plunger et al. CYK-4: A Rho family gtpase activating protein (GAP) required for central spindle formation and cytokinesis
Geisbrecht et al. Drosophila ELMO/CED-12 interacts with Myoblast city to direct myoblast fusion and ommatidial organization
Gruneberg et al. The CeCDC-14 phosphatase is required for cytokinesis in the Caenorhabditis elegans embryo
US6703487B2 (en) Human pellino polypeptides
EP0832222B1 (en) Linc-53 from c. elegans and its uses in listing compounds involved in the control of cell behaviour and pharmaceutical compositions
US20030077699A1 (en) PAAD domain-containing polypeptides, encoding nucleic acids, and methods of use
JP4314386B2 (en) Bcl-2 regulator (BMF) sequences and their use in the regulation of apoptosis
US7183046B2 (en) Methods for identifying inhibitors of cytokinesis using CYK-4 proteins
US20030028904A1 (en) Genes involved in engulfment of dying cells and cell migration
EP1297131A2 (en) Cyk-4 polypeptides, dna molecules encoding them and their use in screening methods
Sumerel et al. Cyclin E and its associated cdk activity do not cycle during early embryogenesis of the sea urchin
US20020076794A1 (en) Mammalian pro-apoptotic Bok genes and their uses
WO1999055728A2 (en) Ese genes and proteins
EP1254912A1 (en) Cyk-4 polypeptides, DNA molecules encoding them and their use in screening methods
US7670778B2 (en) Tumor suppressor pathway in C. elegans
Ricard et al. Drosophila rab GDI mutants disrupt development but have normal Rab membrane extraction
US6579701B1 (en) Drosophila homologues of genes and proteins implicated in cancer and methods of use
WO2001085912A2 (en) Presenilin enhancers
AU2001262988A1 (en) Presenilin enhancers
EP1167525A1 (en) Cyk-4 polypeptides, DNA molecules encoding them and their use in screening methods
CA2448360A1 (en) Modulation of cell division by an early mitotic inhibitor protein
US7118886B1 (en) Ese genes and proteins
US7041783B2 (en) Survivin-binding proteins, encoding nucleic acids, and methods of use
WO2004024084A2 (en) Rb pahtway and chromatin remodeling genes that antagonize let-60 ras signaling
AU2002304971B2 (en) Bcl-2-modifying factor (Bmf) sequences and their use in modulating apoptosis

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Extension state: RO

17P Request for examination filed

Effective date: 20030605

17Q First examination report despatched

Effective date: 20040824

RTI1 Title (correction)

Free format text: USE OF CYK-4 POLYPEPTIDES IN SCREENING METHODS

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20090430