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.

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