WO2011130540A1 - Light stimulated protein interaction molecules and methods of use - Google Patents

Abstract

Light stimulated protein interaction polypeptides are provided herein. In particular, methods and systems of controlling an interaction between a first protein construct comprising a Cryptochrome domain and a second protein construct comprising a cryptochrome interacting polypeptide by regulating the expositre of the protein constructs to light are provided. In addition, polynucleotides encoding carboxy-temiinal truncated versions of CRY2 and CIB1, cells comprising these polynucleotides and transgenic organisms comprising the cells are provided. Finally, kits for performing the methods and systems described herein are disclosed.

Description

LIGHT STIMULATED PROTEIN INTERACTION MOLECULES AND

METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims the benefit of priority of United States Provisional Patent Application Nos. 61/323,943, filed April 14, 2010 and 61/348,033, filed May 25, 2010, which are incorporated herein by reference in their entireties.

SEQUENCE LISTING

A Sequence Listing accompanies this application and is incorporated herein by reference in its entirety. The Sequence Listing was filed with the application as a text file on April 14, 201 1.

INTRODUCTION

Systems for the spatial and temporal controlling of protein interactions have a variety of experimental and commercial applications. The majority of such systems that have been developed are based on the administration of chemical effectors which require diffusion onto the cell to provide appropriate, non-toxic concentrations of chemical at the dimer-interface site. In an alternative strategy, attempts have been made to use light to regulate protein interactions (see, e.g., Leung et al. (2008) Proc. Natl. Acad. Set, USA, 15: 12797-12802; Shimizu-Sato et al., (2002) Nature Biotech. 20: 1041-1044; Tyszkiewicz et al., (2008) Nature Methods 5:303-305).

One such system, derived from photoreceptor proteins from Arabidopsis thaliana, utilizes photochrome photoreceptor protein (PhyB) and its interacting domain, PIF6 (or a truncated domain of PIF6) to control protein interactions (Levskaya et al, 2009). In this particular system, the protein interaction is promoted by red light (~660nm) and reversed with far-red light (~730nm). While such systems offer rapid stimulation and reversibility, it also has a major limitation when used in non-plant organisms or derived cells. Specifically, in these organisms and/or cells, the interaction required an exogenous photolabile billin cofactor, phycocyanobilin (PCB). This cofactor is difficult to prepare and also difficult to deliver to cells in "in vivo" experiments in whole organisms. Another system currently available utilizes two interacting components Arabidopsis FKF1 , containing a blue-light responsive LOV domain, and GIGANTIA (see, e.g., Yazawa, M. et al. (2009) Nat. Biotech. 27:941- 945). In this system, the protein interaction is promoted by blue light (~450nm). However, this system has much slower kinetics than the PhyB-PIF6 interaction (e.g., when used to force a cytoplasmic protein to localize to the plasma membrane, maximum localization was observed after 30 minutes, with half-maximal localization after approximately 10 minutes). Further, the interaction is not immediately reversible and requires constant blue light over 5 minutes in order to drive the interaction. Lastly, the proteins used in the FKF1 /GIGANTIA system are large (the FKF1 protein is 613 amino acids and GIGANTIA is 1173 amino acids), and may present problems when trying to introduce them into and/or express them in a cell.

Therefore, it would be advantageous to develop a system for the temporal and spatial interaction of proteins that was fast, reversible, and did not require the addition of exogenous proteins. The present disclosure describes such a system which solves these problems.

SUMMARY

Light stimulated protein interaction polypeptides and methods of using these polypeptides are provided herein. In one aspect, methods of controlling an interaction between a first protein construct and a second protein construct are provided. In these methods the exposure of the protein constructs to light is regulated. When the light is increased the interaction between the protein constructs is increased and when the light is decreased, the interaction between the protein constructs is decreased. In one aspect, the first and second protein constructs are in a cell. The the first protein construct may include a first polypeptide and a Cryptochrome domain (CD) and the second protein construct may include a second polypeptide and a Cryptochrome interacting polypeptide (CIP).

In one embodiment the CD is CRY2 of SEQ ID NO: 2, a fragment of SEQ ED NO: 2 consisting of, or consisting essentially of, amino acid residues 1-498 of CRY2, a fragment of SEQ ID NO: 2, or a variant of one of these that maintains the ability to interact with its CEP counterpart, such as CIB1. In one embodiment, the CIP is CIB1 of SEQ ID NO: 4, a fragment consisting of, or consisting essentially of, amino acid residues 1-170 of SEQ ID NO: 4, a fragment of SEQ ID NO:4 or a variant of one of these that maintains the ability to interact with its CD counterpart, CRY2. In one embodiment, the CD and CIP are heterologous. A variant refers to molecules with some differences in their amino acid sequences as compared to a reference (e.g. native sequence) polypeptide. The amino acid alterations may be substitutions, insertions, deletions or any desired combinations of such changes in a native amino acid sequence.

The cells may be non-plant cells, non-yeast cells, non-bacteria cells, or may be yeast, insect, avian, fish, worm, xenopus, bacteria, algae or mammalian cells. At least one of the first polypeptide or the second polypeptide comprises at least a portion of a transcription factor, an enzyme, a recombinase or a cell-signaling protein. The first and second polypeptides may be portions of a split protein. In another aspect, the first and second ploypeptides are not a fluorescent protein.

In another aspect, isolated polynucleotides encoding a carboxy-terminal truncated CRY2 or CIB1 or a fusion protein comprising a CD or CIP linked to a polypeptide of interest are provided. The isolated polynucleotides may encode a polypeptide comprising or consisting essentially of amino acids 1-498 of SEQ ID NO: 2, amino acid residues 1-170 of SEQ ID NO: 4, a polypeptide with 90% identity to amino acids 1 -498 of SEQ ID NO: 2, or a polypeptide with 90% identity to amino acid residues 1-170 of SEQ ID NO: 4 and a polypeptide of interest.

In yet another aspect, cells comprising a first protein construct comprising a first polypeptide and a Cryptochrome domain (CD) and a second protein construct comprising a second polypeptide and a Cryptochrome interacting polypeptide (CIP) are provided. The cells may be any prokaryotic or eukaryotic cell. The cells may be non-plant cells, non-yeast cells, non-bacteria cells, or may be yeast, insect, avian, fish, worm, xenopus, bacteria, algae or mammalian cells. At least one of the first polypeptide or the second polypeptide comprises at least a portion of a transcription factor, an enzyme, a recombinase or a cell-signaling protein. The first and second polypeptides may be portions of a split protein.

In a further aspect, transgenic organisms comprising cells with a first protein construct comprising a first polypeptide and a Cryptochrome domain (CD) and a second protein construct comprising a second polypeptide and a Cryptochrome interacting polypeptide (CIP) are provided. The transgenic organisms may comprise the cells disclosed herein.

In a still further aspect, kits comprising a first polynucleotide encoding a Cryptochrome domain (CD) derived from Arabidopsis thaliana Cryptochrome 2 (CRY2) (SEQ ID NO: 2) and a second polynucleotide encoding a Cryptochrome interacting polypeptide (CIP) derived from the Cryptochrome-binding domain of CIB1 (SEQ ID NO: 4) are provided. Suitably the CD and CIP are carboxy-terminal truncated polypeptides of SEQ ID NO: 2 or 4.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic showing the concept of light-activated protein interaction modules in accordance with one embodiment of the present disclosure.

Figure 2a is a schematic showing mapping of the interacting domains of CRY2 and CIB1 with numbers indicating amino acid positions.

Figure 2b is a graph showing β-galactosidase activity of CRY2 and CIB1 constructs tested for interaction in the dark or in blue light (461 ran, 1.9 mW, 4 h). The Gal4 binding domain (Gal D-X) and Gal4 activation domain (Gal AD- Y) fusions used are indicated and the empty vector control was pGB T7rec containing no insert. Error bars, s.d. (n = 3 samples). Inset, immunoblot analysis of Gal4BD fusion proteins in yeast.

Figure 3 is a schematic representation showing the mutation strategy used to remove putative nuclear localization sequences. The numbers indicate the amino acid residue positions.

Figure 4a is a schematic showing the fusion proteins and in particular showing CIBN- pmEGFP contains a CaaX box prenylation motif for targeting to the plasma membrane.

Figure 4b is a set of fluorescence images of CIBN-pmEGFP and CRY2-mCh coexpressed in HEK293T cells. CRY2-mCh was imaged before light excitation and 20 s after a 100-ms pulse of blue light (488 nm, 25 μW). Scale bar, 5 um.

Figure 4c shows a set of photographs showing a time course of CRY2-mCh recruitment to the plasma membrane after a single 100-ms pulse of 488-nm light (25 μW). CIBN-pmEGFP localization is shown on the left. Scale bar, 2 μm.

Figure 4d shows the CRY2-mCh translocation kinetics after a 100-ms pulse of 488-nm light (arrow). The distribution of CIBN-pmEGFP and the line used to generate the CRY2-mCh kymograph is shown in the upper left image. Scale bar, 1 um. The graph on the bottom shows quantification of CRY2-mCh in the cytoplasm and at the plasma membrane, using the regions shown in c by the dotted and solid lines, respectively. Each fraction was normalized between 0 and 1.

Figure 4e shows a set of fluorescence images of cells expressing the indicated constructs before and after delivery of two 100-ms pulses of blue light (25 μW) spaced 12.5 min apart (top). Quantification of cytoplasmic CRY2PHR-mCh, with light pulses (arrows) delivered at 0 and 12.5 min is shown in the graph at the bottom.

Figure 5a is a graph showing that CRY2-CIBN and CRY2PHR-CIBN interactions show nearly identical activation (inset) and reversal kinetics. A blue light pulse (100 ms, 488 nm, 25 μW) was delivered at t = 0 (arrow). Cytoplasmic mCh signal was quantified at several 20 x 20 pixel regions drawn over cytosolic portions of the cell being careful to exclude the plasma membrane. Measurements from these regions were averaged for each cell. Measurements were repeated for at least 3 different cells and averaged to generate the traces shown. The data represents the mCh signal at time (t) divided by the average mCh signal prior to blue light illumination.

Figure 5b is a graph showing that translocation of CRY2PHR-mCh to the plasma membrane can be repeatedly induced with blue light stimulation. Cytoplasmic CRY2-mCh intensity was measured in response to a train of blue light pulses (100 ms, 488 nm, 25 μW) delivered every 200 s (arrows).

Figure 6a is a set of photographs showing that HE 293T cells expressing CRY2PHR- mCh and CIBN-pmGFP were excited with two-photon illumination at 860 nm to induce translocation of CRY2PHR-mCh to the plasma membrane (second panel). Following spontaneous dissociation of CRY2PHR-mCh from the plasma membrane, the same cells were excited with 488 nm light for comparison (third panel). The distribution of CIBN-pmGFPis shown in the far right panel. Scale bar 5 μm.

Figure 6b is a set of photographs showing the plasma membrane localization of CRY2PHR-mCh following two-photon excitation at 860 nm every 25 s (arrows). Scale bar 1 μm.

Figure 6c is a graph showing quantification ofCRY2PHR-mCh redistribution from the cytosol to the plasma membrane in response to two-photon excitation at 860 nm (arrows).

Figure 7a is a schematic of split Gal4 modules expressed in yeast cells containing a gene encoding a hemagglutinin (HA)-tagged reporter protein under control of a galactose-inducible promoter. UAS, upstream activating sequence.

Figure 7b(top) is an immunoblot analysis of the HA-tagged reporter (top) in response to blue-light pulses (10 s pulses, 1.7 mW, 8 min apart). The control was lysates from cells expressing only the reporter. The graph at the bottom shows quantification of western blot bands.

Figure 7c is a schematic showing the two split Cre recombinase constructs (CIBN-CreC and CRY2-CreN) and the reporter construct. IRES, internal ribosome entry site.

Figure 7d is a graph showing Cre reporter recombination measured 48 h after transfection of HEK293T cells with the Cre reporter and indicated constructs. Cells were exposed to blue- light pulses (450 nm and 4.5 mW) for the indicated durations or kept in the dark (-). Error bars, s.d. (n - 3) from three independent experiments.

Figure 7e is a set of photographs showing EGFP fluorescence images from samples containing both CRY2-CreN and CIBN-CreC that were exposed to 24 h of blue light or maintained in the dark. Scale bar, 20 μm.

Figure 8a is a set of photographs showing that CIBN fused to the YFP variant citrine (CIBN-pmCitrine) imaged with increasing intensities of 514 nm light (indicated above top panel). The bottom panel shows the CRY2PHR-mCh distribution following 20 frames of CIBN- pmCitrine imaging (514 nm, 100 ms integration time /frame). Note that at lower intensities, YFP can be visualized without triggering the CRY2-CIBN interaction. Higher intensities of 514 nm illumination triggered translocation of CRY2PHR-mCh to the plasma membrane.

Figure 8b is a graph showing quantification of CRY2PHR-mCh translocation following 20 frames of 514 nm illumination at the indicated powers. The dashed line represents the level of cytoplasmic CRY2PHR-mCh following a saturating pulse of 488 nm light.

DETAILED DESCRIPTION

The present disclosure includes a genetically-encoded, light-switchable assay system for modulating protein-protein interactions. The system is based upon a properly-titrated, high affinity but reversible binding between a Cryptochrome domain (CD) with a Cryptochrome- interacting polypeptide (CIP). The system allows spatio-temporal control of interaction at a fine resolution. Binding between the CD and the CIP is strong enough to result in a significant and detectable interaction, yet is reversible and shows fast association and dissociation rates. As described herein, the system can control protein interactions within a spatial resolution in the range of a micrometer and within a timescale in the range of a second. The system can be applied to control processes in living cells, tissues, or organisms, such as a process that is dependent on a recruitment event, but may also be used in vitro. Further, as shown in the examples, there is a direct relationship between the recruited fluorescent fraction and signaling activity which will allow measureable 'dosage' of signaling flux for quantitative perturbations. The examples herein demonstrate that the system works robustly in yeast and mammalian cells. Those of skill in the art will appreciate that the system could be applied in many other cell types including, but not limited to, insect, avian, fish, worm, amphibian, fungal, xenopus, bacteria, or algae. In addition, the system can be adapted to work in tissues, such as organs, engineered tissues, biopsy tissue and in whole organisms.

Among other things, the present disclosure provides systems, methods and materials for regulating the association between proteins using light. In an aspect, the present disclosure takes advantage of the ability of Cryptochromes to change conformation upon exposure to appropriate light conditions, and to bind in a conformation-dependent manner to cognate proteins referred to as Cryptochrome-interacting polypeptides (CIP).

In one aspect, the present disclosure comprises a method of regulating interaction between a first protein construct and a second protein construct by light. In some embodiments the methods are used within a cell, tissue or organism. Such a method may include (1) providing in the cell, tissue or organism a first protein construct which comprises the first polypeptide and a Cryptochrome domain (CD), and (2) providing in the cell, tissue or organism a second protein construct which comprises the second polypeptide and a Cryptochrome-interacting polypeptide (CIP) that can bind selectively to the Cryptochrome domain. In one embodiment, the first and/or second protein construct is a fusion protein. In another aspect, the binding of the CD to the CIP is reversible over time. In certain embodiments, the binding is reversible 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 minutes after light activation.

In another aspect, the first and/or second polypeptides are not endogenously present in the cell, tissue or organism, or are present at low levels that do not interfere with the purpose of the assay.

In other aspects, the first and second polypeptides do not normally associate or interact with each other. In cases where the first and second polypeptide can interact with each other in their naturally-occurring forms, either or both can be modified if desired in such a manner that they do not associate or interact with each other in the absence of association between the CD and the CEP. Those of skill in the art will appreciate that a variety of modifications could be made. In particular modifications made in experiments using chemical dimerizers could be used in the methods disclosed herein.

In one embodiment, and as shown in FIG 1, a CD (e.g., CRY2 1-613, i.e. a CD comprising CRY2 (SEQ ID NO: 2) sequence from amino acid residue 1 to 613) is fused to a first polypeptide in a first protein construct and a CIP (e.g., the Cryptochrome binding domain of SEQ ID NO: 4 (i.e., CIB1, 1-336)) is fused to a second polypeptide to form a second protein construct. A polypeptide need not correspond to a full-length protein found in nature, but can be derived from any portion thereof and can contain variations that do not eliminate binding activity. In other embodiments, and also shown in FIG 2, a CD or CIP molecule may comprise only a fragment of the full length protein. For example, only the N -terminal region of CRY2 comprising amino acids 1 to 498 of SEQ ID NO:2 may be used in place of the full length sequence. Similarly, the N-terminal region of CIB1 comprising amino acids 1 to 170 of SEQ ID NO:4 may be used. These carboxy-terminal truncations are shown to maintain their binding activity in the examples. It is also within the scope of the present disclosure that different combinations of the CD and CIP proteins may be used, for example, the full length protein for CD may be paired with the N-terminal region of the CIP protein, and so forth.

In other embodiments, the present disclosure includes the use of polypeptides with at least about 60%, 70%, 80%, 85%, 90%, 95% or at least 99% identity (at the amino acid level) with a known polypeptide (such as the Cryptochromes described herein). As used herein, the term "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the sequence (either polynucleotide or amino acid) in the comparison window may comprise additions or deletion (i.e., gaps) as compared to the reference sequence (which does not comprise any additions or deletions) for the optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

Optionally, the first and second polypeptides do not interact with each other. For example, the first and second polypeptides can be derived from non-interacting portions of one or more proteins. In other embodiments, the first and second protein constructs only interact with each other through the CD and CIP domains. Alternatively, the first and second polypeptides are capable of interacting with each other independently of any interaction between the CD and CIP. Association between the CD and CIP can serve to enhance or modify or impair the interaction between the first and second polypeptides. In certain embodiments, association of the CD and CIP serves to localize interaction between the first and second polypeptides to a specific sub-compartment within the cell, tissue or organism. Such localization may affect the activity of one or more of the polypeptides.

Association of the CD and CIP, and the resulting association between the first and/or second polypeptides can result in a biologically significant effect upon the cell, tissue or organism. Such an effect can be detected using assays such as those known to those of skill in the art. In one embodiment, the first and second polypeptides interact when associated via the CD or CIP, and the interaction produces an effect on a cell structure or process, or structure or function of a tissue or organism. For instance, the first polypeptide can cause the second polypeptide to be modified when both are brought into proximity by the association between the CD and CIP, or vice versa. In one such situation, the first polypeptide is a kinase and the second polypeptide is a substrate for the kinase, or vice versa. In another embodiment, the first and/or second polypeptide can associate or interact with a third protein only when the first and second polypeptides are brought together through an association between the CD and CIP. In yet another embodiment, the first polypeptide can dissociate from a third protein (e.g., an inhibitory protein) only when brought together with the second polypeptide through an association between the CD and the CIP, or vice versa.

In another aspect, dissociation (rather than association) between the first and second polypeptides results in a biologically significant effect. In one such example, association between the CD and CIP portions acts to prevent a different association or interaction between the first and second polypeptides. In another example, the first and/or second polypeptide can only associate with or interact with a third protein when separated from each other due to dissociation between the CD and CIP. In another example, the first and/or second polypeptide can dissociate from a third protein (e.g., an inhibitory protein) only when separated from each other due to dissociation between the CD and the CIP. The association between the polypeptides can modulate or have an effect on any biologically significant cellular process, which may have an effect at the cellular, tissue or organism level. In one aspect, the association (or dissociation) between the polypeptides or protein constructs can have an effect on a cellular signaling process (e.g., the first and/or second polypeptides are signaling proteins). In another aspect, the polypeptides may have an effect on transcription in a cell (e.g., the polypeptides may be transcription factors, such as the Gal4 polypeptides used in the Examples). In still another aspect, the polypeptides may affect an enzymatic process within the cell and the polypeptides may be an enzyme, an activator or an inhibitor. In the Examples, a split Cre recombinase was shown to exhibit recombination activity after light mediated regulation of the CD-CIP interaction. In a still further aspect, the first and second polypeptides may be a split protein such as the Gal4 and Cre recombinase used in the Examples. Those of skill in the art will appreciate that other split proteins are available or may be designed in the future and may be used in the methods and systems described herein. For example, transcription may be regulated using TetR and LexA-VP16 in the methods and systems described herein.

Cryptochrome Domains (CD)

In one aspect, the Cryptochrome domain (CD) of the present disclosure comprises a protein sequence derived from a Cryptochrome protein. In one aspect, a Cryptochrome domain (CD) of the present disclosure is capable of light-regulated, reversible interaction with a Cryptochrome-interaction polypeptide (CIP). In certain embodiments, the CD preferably binds reversibly to the CIP, although CDs that undergo irreversible binding can also be used in some situations, hence, the CD can comprise minimal portions of a Cryptochrome protein component to undergo reversible conversion. For example, residues 1 -613 of a Cryptochrome protein, e.g., CRY2 (SEQ ID NO:2), can bind reversibly to CIB1 (SEQ ID NO:4). CRY2 is homologous to CRY1 and both CRY1 and CRY2 have homologs in other plants. These CDs are highly homologous and have more than 85% identity, in fact many CRY2 CDs have over 90% identity. These homologs and variants thereof may also be used in the methods, systems and kits described herein. In certain embodiments, a strong affinity interaction between the CD and CIP may be desired, e.g., a strong interaction that results in a significant and visually detectable interaction inside a mammalian cell, tissue or organism. The Cryptochrome domain (CD) of the present disclosure may comprise any portion of a Cryptochrome protein, or any variant, or derivative of such a portion, that retains the ability to bind to a CIP. The CD may comprise a sequence derived from a CRY2 of SEQ ID NO:2. A sequence "derived from" another sequence includes the full-length sequence, fragments or portions of the full-length sequence, variants of the full-length or portion of the sequence (such as a variant comprising mutations to codon optimize the sequence, remove subcellular localization signals, increase or decrease expression in a cell, increase or decrease dark reversion rate or other mutations) that retain the ability to bind CIP. An exemplary Cryptochrome sequence (A. thaliana CRY2) is shown in FIG 2 and SEQ ID NO:2. In one embodiment, the CD can comprise the entire protein of CRY2 (e.g., residues 1-613; SEQ ID NO:2) or a portion thereof. In other embodiments, the CD comprises the N-terminal domain from or is a C-terminal truncation of a Cryptochrome protein, where the domain retains the ability to associate with a CIP. For example, the CD may comprise the N-terminal domain of CRY2 (e.g., residues 1-498 of SEQ ID NO:2).

In other embodiments, useful CRY2 fragments can comprise A. thaliana CRY2 sequence spanning from a first residue to a second residue. The first residue is for example residue 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or 20 of CRY2 (SEQ ID NO:2). The second residue is optionally any residue between 450 and 613, including residues 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610 and 613 of CRY2 torn A. thaliana (SEQ ID NO:2).

Useful CDs of the present disclosure can include (1) CRY2 full length sequence, residues 1-613 (SEQ ID NO: 2) and (2) N-terminal region of CRY2, residues 1-498 of SEQ ID NO:2.

Cryptochrome-interacting Polypeptide

A Cryptochrome-interacting polypeptide (CIP) of the present disclosure can comprise any protein sequence that can bind selective to a CD molecule. The CIP can comprise any Cryptochrome binding domains from Cryptochrome-interacting factors, or any portion, variant or derivative thereof. The CIP comprises a sequence derived from a CIB1 of SEQ ID NO:4. A sequence "derived from" another sequence includes the full-length sequence, fragments or portions of the full-length sequence, variants of the full-length or portion of the sequence (such as a variant comprising mutations to codon optimize the sequence, remove subcellular localization signals, increase expression in a cell or other mutations) that retain the ability to bind CD.

An exemplary Cryptochrome-interacting peptide, CIB1, is shown in FIG 2 and SEQ ID NO:4. In one embodiment, the CIP can comprise the entire protein of CIB1 (e.g., residues 1- 336; SEQ ID NO:4) or a portion thereof. In other embodiments, the CIP comprises the N- terminal domain from or a C-terminal truncation of a Cryptochrome-interacting polypeptide, where the domain retains the ability to associate with a CD. For example, the CIP may comprise the N-terminal domain of CIB1 (residues 1-170 of SEQ ID NO:4).

In another example, the CIP can comprise an antigen-binding site of an antibody that binds selectively to the CD.

CD-CIP Pairs

The present disclosure uses CD and CIP pairs that can associate together, which association can be regulated and/or monitored using the methods taught in the present disclosure.

Any reference to a CD or CIP protein is intended to include not only the full length protein, but also function fragments thereof. The fragment is optionally capable of binding robustly and reversibly to its corresponding bonding partner, for example, under intracellular conditions, such as within a mammalian cell, tissue or organism.

Suitable CDs or CIPs are not limited to full-length proteins or fragments encoded by naturally occurring genes. For example, techniques of directed evolution can be used to produce new or hybrid gene products. In addition, catalytically active fragments and variants of naturally occurring CDs or CIPs can be used. Partially or wholly synthetic CDs or CIPs, such as enzymes designed in silico or produced by using art-known techniques for directed evolution including gene shuffling, family shuffling, staggered extension process (StEP), random chimeragenesis on transient templates (RACHITT), iterative truncation for the creation of hybrid enzymes (ITCHY), recombined extension on truncated templates (RETT), and the like (see, e.g., Crameri et al. (1998) Nature 391:288-291; Rubin-Pitel et al. (2006) Comb. Chem. High Throughput Screen 9:247-257; Johannes and Zhao (2006) Curr. Opin. Microbiol. 9:261-267; Bomschuerer and Pohl (2001) Curr. Opin. Chem. Biol. 5: 137-143).

CD-CIP pairs can be identified using methods taught herein as well as those techniques known to those skilled in the art. For example, a first protein construct comprising a test CD, and second protein construct comprising a test CIP, can be introduced into a cell and exposed to blue light to promote binding. The test CDs and CEPs can be fragments or variants of known proteins, e.g., those described herein. The observation of association between the test CD and the test CIP indicates that binding has occurred. In addition, the CD-CIP pairs may be mutated for other reasons such as to remove subcellular localization signals, to improve expression in particular cell types, such as by codon optimization, or to improve interaction capabilities.

Association can be visualized using methods described herein as well as those techniques known to those skilled in the art, e.g., by adding appropriate labels or proteins to the first and/or second construct. For example, one protein construct can contain a membrane localization sequence, while the other protein construct can contain a detectable tag, e.g., Red or Green Fluorescent Protein (RFP and GFP, respectively), wherein binding can be detected by localization of the RFP or GFP to the membrane. In another example, established techniques can be used to determine association (and its reversibility) between a CD and a CIP, including bimolecular fluorescence complementation (BiFC), fluorescence resonance energy transfer (FRET), chemical cross- linking, dual polarization interferometry (DPI), static light scattering (SLS), or a yeast two-hybrid assay, affinity electrophoresis, label transfer, Immunoelectrophoresis, in vivo cross-linking of protein complexes using photo-reactive analogs, and others. For example, the K_D and binding affinity can be measured by known techniques such as fluorescence correlation spectrometry.

In one embodiment, the CD is for example CRY2 of A. thaliana. The CIP is for example a Cryptochrome binding protein such as CIB1. One such CRY2/CIB1 pair comprises at least the first 498 residues of CRY2 in conjunction with a peptide comprising at least the first 170 residues of CIB1. Other pairs include the full length CRY2 (e.g., residues 1 - 613 of SEQ ID NO:2) and the N-terminal region of CIB1 (e.g., residues 1-170 of SEQ ID NO:4); N-terminal fragment of CRY2 (e.g., residues 1-498 of SEQ ID NO:2) and the full length region of CIB1 (e.g., residues 1-336 of SEQ ID NO:4) and other combinations thereof.

Subcellular Localization Signals

In one aspect, the invention can include a first protein construct that comprises (1) a polypeptide, (2) a CD and (3) a subcellular localization signal (SLS). The SLS can localize the first protein construct to any subcellular compartment of interest. In such an embodiment, a second protein can optionally be attached to a corresponding CIP (to form a second protein construct). The localization of the second protein construct can then be regulated by exposure to light. In an alternative strategy, the second protein construct comprises (1) a polypeptide, (2) a CIP and (3) a subcellular localization signal (SLS), while the first protein construct comprises a polypeptide of interest and a CD.

Various SLSs are known that can direct proteins to subcellular compartments such as the extracellular space, cytoplasm, nucleus, mitochondria, Golgi apparatus, endoplasmic reticulum (ER), peroxisome, vacuoles, plastids, cytoskeleton, nucleoplasm, nucleolus, nuclear matrix, actin and tubulin filaments, endosomes or ribosomes. The SLS can be attached in the appropriate orientation, N or C terminal to the protein construct. In one aspect, the first and/or second protein construct does not comprise a nuclear localization signal. For example, the first and/or second protein construct can comprise an SLS that localizes to a non-nuclear subcompartment of the cell.

Specific examples of SLSs include: 1) Hras [Entrez Gene ID: 3265] palmitoylation CaaX sequence (GCMSC CVLS; SEQ ID NO: 27), ggc tgc atg age tgc aag tgt gtg etc; SEQ ID NO: 28, targets to the plasma membrane, endoplasmic reticulum membrane, and golgi membrane; 2) Kras4B [Entrez Gene ID: 3845] polybasic CaaX terminus (KKKKKK.SKTKC TM ; SEQ ID NO: 6) [ggt aaa aag aag aaa aag aag tea aag aca aag tgt gta art atg; SEQ ID NO:5], targets to the plasma membrane; 3) Lyn kinase [Entrez Gene ID: 4067] NT 13 plasma membrane targeting sequence MGCIKSKGKDSAGA; SEQ ED NO: 29 [atg gga tgt ata aaa tea aaa ggg aaa gac age gcg gga gca; SEQ ID NO: 30]i.4) a-actinin [Entrez Gene ID: 87] F-actin binding domain (ABD) (residues 33-245) was cloned from a vector bearing a partial region of the cDNA. This targets the actin cytoskeleton; 5) human β, 4- galactosyltransferase: the n-terminal 81 amino acids targets a protein to the trans-medial region of the Golgi apparatus; 6) mitochondrial targeting sequence from the precursor of subunit VIII of human cytochrome C oxidase. (Rizzuto, Brini et al. 1995); 7) endoplasmic reticulum targeting sequence of calreticulin (Fliegel, Burns et al. 1989). Other known SLSs can be found in public databases such as eSLDB (eukaryotic cells, http://gpcr.biocomp.unibo.it/esldb ) and pSORTb (bacterial signals, available at http://www.psort.org). Detectable Labels

In one aspect, one or more proteins (or protein constructs) of the invention is attached to a detectable label. A wide variety of detectable labels are known in the art. Such labels include molecules that can be attached to or form part of a protein or protein construct of the invention and are capable of being detected (or are capable of reacting to form a chemical or physical entity (e.g. , a reaction product) that is detectable) in an assay according to the instant disclosure. Representative examples of detectable labels or reaction products include precipitates, fluorescent signals, compounds having a color, and the like. Representative labels include, e.g., fluorophores (e.g., below), bioluminescent and/or chemiluminescent compounds, radioisotopes (e.g., ¹³¹L ¹²⁵1, ¹⁴C, ³H, ³⁵S, ³²P and the like), enzymes (e.g., below), binding proteins (e.g., biotin, avidin, streptavidin and the like), magnetic particles, chemically reactive compounds (e.g., colored stains), antibodies, labeled-oligonucleotides; molecular probes (e.g., CY3, Research Organics, Inc.), and the like.

Representative fluorophores include fluorescein isothiocyanate, succinyl fluorescein, rhodamine B, lissamine, 9,10-diphenlyanthracene, perylene, rubrene, pyrene and fluorescent derivatives thereof such as isocyanate, isothiocyanate, acid chloride or sulfonyl chloride, umbelliferone, rare earth chelates of lanthanides such as Europium (Eu) and the like. Representative labels that can be conjugated to include the enzymes in: IUB Class 1, especially 1.1.1 and 1.6 (e.g., alcohol dehydrogenase, glycerol dehydrogenase, lactate dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, glyceraldehyde- 3-phosphate dehydrogenase and the like); IUB Class 1.1 1.1 (e.g., catalase, peroxidase, amino acid oxidase, galactose oxidase, glucose oxidase, ascorbate oxidase, diaphorase, urease and the like); IUB Class 2, especially 2.7 and 2.7.1 (e.g., hexokinase and the like); IUB Class 3, especially 3.2.1 and 3.1.3 (e.g., alpha amylase, cellulase, β-galacturonidase, amyloglucosidase, β-glucuronidase, alkaline phosphatase, acid phosphatase and the like); IUB Class 4 (e.g., lyases); IUB Class 5 especially 5.3 and 5.4 (e.g., phosphoglucose isomerase, trios phosphatase isomerase, phosphoglucose mutase and the like.).

Useful labels also include labels whose products are detectable by fluorescent and chemiluminescent wavelengths, e.g., fluorescence emitting metals such as ¹⁵²Eu, or others of the lanthanide series; compounds such as luminol, isoluminol, acridinium salts, and the like. Fluorescent or bioluminescent proteins can be especially useful, such as luciferase, luciferin; fluorescent proteins; and the like. Fluorescent proteins include, but are not limited to the following: namely, (i) green fluorescent protein (GFP), i.e., including, but not limited to, a "humanized" versions of GFP wherein codons of the naturally-occurring nucleotide sequence are exchanged to more closely match human codon bias; (ii) GFP derived from Aequoria victoria and derivatives thereof, e.g., a "humanized" derivatives such as Enhanced GFP, which are available commercially, e.g., from Clontech, Inc.; (iii) GFP from other species such as Renilla reniformis, Renilla mulleri, or Ptilosarcus guernyi, as described in, e.g., WO 99/49019 and Peelle et al. (2001) J. Protein Chem. 20:507-519; (iv) "humanized" recombinant GFP (hrGFP) (Stratagene); and, (v) other fluorescent and colored proteins from Anthozoan species, such as those described in Matz et al. (1999) Nature Biotechnol. 17:969- 973; and the like. Also included are fluorescent proteins of other colors, e.g., mCherry, mStrawberry, mTangerine, mTomato, mOrange, mBanana and mHoneydew, EGFP, ECFP and EBFP. Yellow fluoresecent protein include the Citrine and Venus versions. Other examples of fluorescent proteins are listed in Shaner et al., Nature Methods, 2(12): 905-917 (2005), incorporated by reference in its entirety. Notably, the excitation spectra for some of the fluorescent labels and proteins may overlap with that capable of regulating the interaction of the CD-CIP interaction. Those skilled in the art will be able to choose appropriate fluorescent labels to design methods. As shown in Figure 8 and described in the Examples, excitation of YFP may induce CD-CIP interactions, but does not do so at low levels.

Where desired, the label reflects or emits a detectable light signal under appropriate conditions that allows the labeled protein to be detected. The label can for example be visually detected by itself instead of having its presence inferred through detection of another labeled product. Examples of appropriate conditions include exposure to light of excitatory wavelength in the case of a fluorescent label or exposure to visible light in the case of a colored label. The visually detectable label can for example emit light within the optically visible range of the spectrum. Optionally, the label emits light that can be detected by the human eye, with or without the aid of other instrumentation such as the microscope.

The labels are optionally detectable using a non-invasive method. One method is visual examination by eye, optionally with the aid of devices such as a microscope. Other methods include methods amenable to automation such as a spectrophotometric method, a fluorescence method, a chemiluminescent method, an electrical nanometric method involving e.g., a change in conductance, impedance, resistance and the like and a magnetic field method.

If desired, the labels can be attached to the protein or protein construct by any known method. The label can be attached for example by using a chemical linking method as discussed herein or if proteinaceous in nature, by generation of a fusion protein. Attaching certain labels to proteins can also be accomplished through metal chelating groups such as EDTA, linkers, etc.

Strength of Association

The interaction between the CD and the CIP optionally allows fine spatiotemporal control in vivo. For example, binding between the CD and CIP should be strong enough to result in a detectable and significant recruitment under intracellular conditions.

For example, the affinity of binding between the CD and CIP can be expressed in terms of a dissociation constants K_D. Optionally the K_D is at least about 500nM, for example at least about 250nM. Where fine spatiotemporal control (discussed below) is desired, especially useful binding partners can have an affinity of binding in the range of 10-250 nM.

The CD and CIP can specifically bind to each other. For example, the CD and CIP can preferentially bind to each other when present within a mixture of different proteins (for example the entire repertoire of proteins present within a cell). In some methods, the CD and CEP can specifically bind to each other instead of other proteins with a greater than about 10- to about 100-fold; sometimes greater than about 1000- to about 10, 000-fold increased affinity. In other cases, the CD and CIP show detectable levels of binding to each other in the presence of a repertoire of proteins present within a living cell, tissue or organism wherein neither protein shows detectable levels of binding to other proteins.

In some embodiments interaction between the CD? and CD can result in a visually detectable change in spatial and/or temporal distribution of one or more protein constructs within a cell, tissue or organism. For example, a first protein construct comprising a CD can comprise a subcellular localization signal that recruits it to a specific subcellular compartment, while a second protein construct containing a CIP can optionally be engineered to emit a visually detectable signal, or vice versa. Exposure to blue light stimulates CIP -CD interaction which can further result in the translocation of the detectable signal to the specific subcellular compartment. Optionally, the binding between the CD and the CIP is robust enough (i.e., of high enough affinity) to result in sufficient translocation within mammalian cells, tissues or organisms to produce a change in distribution that is optically visible to the human eye, (e.g., detectable photographically or microscopically, for example with the aid of confocal microscopy). For example, the detectable signal that is emitted from a subcellular compartment after recruitment can be at least about 1.5X, 2X, 3X, 5X or 10X higher than before recruitment. See, e.g., Fig. 4.

The interaction between the CIP and the CD optionally exhibits very quick rates of association or dissociation. The rate of association upon exposure to blue light can be for example within the range of 0.3-60s. For example, above 50% of fully-associated CD and CIP can bind together within 1, 5, 10, 30 or 60 seconds of exposure to blue light (for example a 100- millisecond pulse of blue light of about 10,000 micromoles of photons per square meter). Optionally, at least 90% of associated CD and CIP can bind together within about 10 seconds after such a pulse.

The rate of dissociation post-exposure to blue light is optionally 1-15 minutes. For example, above 50% of fully-dissociated CD and CIP can dissociate within 5,7, or 10 minutes after exposure to blue light (for example a 100-millisecond pulse of infrared light of about 10,000 micromoles of photons per square meter). Optionally, at least 90% of associated CD and CIP can dissociate within 12 minutes after such a pulse.

Where CEP -CD binding results in subcellular localization (e.g., membrane recruitment), it can yield time constants of 1 -5 seconds for recruitment to the subcellular compartment and time constants of 5-12 minutes for release from the subcellular compartment, demonstrating reversibility of the reaction.

In other embodiments, the CIP-CD binding may be "toggled," whereby repeated exposure to light results in the association of the proteins, followed by dissociation of proteins, repeatedly without loss of robustness (see, e.g., FIG 5).

Regulation of CD-CIP Association

According to the present disclosure, the overall extent of association between proteins or protein constructs can be precisely controlled in various ways, as defined below. Where desired, the extent of association can be precisely controlled by determining the exact parameters of wavelength, exposure time, and/or intensity of light used to control the interaction of CD and CIP. In one aspect, a substantial fraction, or a majority, or substantially all of the CD is associated with the CIP. For example, about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or above 99% of the CD is associated with the CIP, as desired.

Illumination with blue light promotes the association of the CD and CIP molecules. As used herein, the term "blue light" includes those wavelengths between 380 nm and 514 nm. In certain embodiments, frequency is between 445 nm and 490 nm. In preferred embodiments, the frequency is within 10 nm of 450 nm.

The methods discussed herein can allow extremely quick detection and/or control of protein interactions. In one aspect, binding between a first and a second protein construct can be quickly effected or impaired. In one embodiment, the first protein construct comprises a CD and the second protein construct a CIP, or vice versa. The quick association and dissociation of CDs and CIPs (and proteins comprising them) in response to light can allow control of interaction and/or localization within 1 minutes, or sometimes within 5-15 seconds, and sometimes even within 1 second.

In another aspect, the cellular localization of a first protein construct can be modified by allowing or disallowing binding to a second localized protein construct (e.g., a protein comprising a subcellular localization tag). The first protein construct may comprise a CD and the second protein construct a CEP, or vice versa. The quick association and dissociation of CDs and CEPs (and proteins comprising them) in response to light can allow control of protein's subcellular localization within 1 minute, or sometimes within 10-15 seconds, and sometimes within 1 second.

In another aspect, the interaction between two proteins can be selectively regulated within a localized portion of a cell, tissue or organism, by exposing only that portion of the cell, tissue or organism to blue light. For example, a portion of a cell can be exposed to blue light that induced protein interaction while another portion of the cell can be not illuminated by blue light.

In other examples, the interaction between two proteins can result in a detectable change within the cell, tissue or organism, such as change in cellular or tissue morphology or behavior. Regulation of such interactions can be used for example to control cell or tissue morphology and/or movements. In other examples, the interaction between the two proteins can result in changes in transcription of genes, cell signaling cascades or enzymatic activity which can be detected using methods within the skill of those skilled in the art. By exposing a cell, tissue or organism to focused blue light, it is further possible to localize the association or recruiting (localization) region to a small, diffraction- limit-sized membrane region or a specific subcellular organelle in the cell. This can be on the order of centimeters or millimeters (e.g., when exposing parts of a tissue or organism), down to micrometer or even sub-micrometer scales (e.g., when studying intracellular interactions or localization within subcellular compartments within a cell or membrane subdomains such as lipid rafts with slow diffusion rates).

Lasers

Any light source capable of emitting a wavelength of about 380 nm to 514 nm may be used, such as blue light emitting laser diodes. Such devices are well known to those skilled in the art. In one embodiment, the CD-CIP interactions are excited by two photon excitation using conventional pulsed laser. The use of blue light and two photon excitation allows the methods of the present disclosure to be used in vivo, since blue light is able to penetrate deeper into tissue. Hence, the present disclosure is suitable for both in vitro and in vivo model systems.

Two photon excitation with wavelengths between about 820 nm to about 980 nm was found to increase the interaction of CRY2 and CIB1 in the Examples. Suitably excitation uses a wavelength of about 860 nm.

The intensity of light to which the cell is exposed can be used to control the extent of association, e.g., the proportion of molecules in an associated and unassociated state. For example, low-intensity blue light will achieve only partial, titrated association. Total illumination doses less than 1,000 micromoles of photons per square meter can be regarded as low intensity blue light. Total illumination doses greater than 10,000 micromoles of photons per square meter can be regarded as high-intensity light that is sufficient for 100% conversion. The intensity of blue light required to convert a significant fraction or majority or substantially all CDs can be empirically determined using the methods taught herein and described in the Examples.

The time of exposure to light can be varied according to effect needed and light intensity chosen, e.g., for about 1 , 10 or 100 milliseconds, or about 1, 5 or 10 seconds, or about 1, 2, 3, 5, 10, 20 or 30 minutes, or about 1 , 2, 3 or 5 hours, or about 1 , 2, 3, or 5 days, or 1 , 2 or 3 weeks. In one embodiment, the cell is exposed for a short time. For example, the cell can be exposed to blue light for less than a minute, e.g., about 1, 5, 10, 20 or 40 seconds. The light can be delivered to a cell by known devices such as a laser or LEDs, in one or more pulses or individual portions.

Nucleic Acids and Vectors

The present disclosure also includes nucleic acids encoding the polypeptides and protein constructs of the disclosure. In one aspect, the proteins and/or proteins constructs of this disclosure are synthesized using recombinant expression systems. For example, this involves creating a DNA sequence that encodes the desired protein(s), placing the DNA in an expression cassette or vector under the control of a particular promoter, expressing the protein in a.host, and, if desired isolating the expressed protein. Using the information provided herein, the nucleic acids can be prepared using standard methods known to those of skill in the art. For example, the nucleic acid(s) may be cloned, or amplified by in vitro methods, such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (SSR), etc. A wide variety of cloning and in vitro amplification methodologies are well-known to persons of skill.

The term "protein construct" refers to an artificially made or recombinant molecule that comprises two or more protein sequences that are not naturally found within the same protein. The protein construct may be a fusion protein encoded by a single polynucleotide and may be made recombinantly. Alternatively, the protein construct may be made by chemically or otherwise linking the polypeptide to the CD or CIP. In either case, the polypeptide and CD or CIP may be linked via a protein or chemical linker molecule. In some embodiments, a protein construct can have non-proteinaceous elements as well as proteinaceous elements.

DNA encoding desired proteins described herein can be prepared by any suitable method as described above, including, for example, cloning and restriction of appropriate sequences or direct chemical synthesis.

Also provided herein are polynucleotides encoding a fusion protein comprising a CD or CIP linked to a polypeptide of interest. These polynucleotides may be used in any of the systems, methods and kits described herein. The polypeptides of interest may include at least a portion of a transcription factor, an enzyme, a recombinase or a cell-signaling protein. In one embodiment the polypeptide is not a fluorescent protein. The CD may include a sequence derived from Arabidopsis thaliana Cryptochrome 2 (CRY2) (SEQ ID NO: 2), the CRY2 from another plant or CRY1. In one embodiment the CD is 90% identical to amino acids 1-498 of SEQ ID NO: 2. The CIP may include a sequence derived from the Cryptochrome- binding domain of CIB1 (SEQ ID NO: 4) or a homolog or variant thereof. In one embodiment, the CIP is 90% identical to amino acids 1-170 of SEQ ID NO: 4.

Host Cells and Organisms

A variety of cells, tissues or organisms can be used in conjunction with the present disclosure. Useful cells can be eukaryotic, including yeast, algae, fungal, fish, insect, avian, worm, xenopus, plant, and mammalian cells. Prokaryotic cells include bacteria. In one embodiment, the host cell is not a plant cell, a bacterial cell or a yeast cell.

One or more proteins or protein constructs of the disclosure can be introduced into a host cell in a variety of ways. For example, a recombinant cell can be engineered that expresses one or more proteins or protein constructs. Alternatively, the proteins or protein constructs can be introduced by any known method, such as microinjection, transfection and/or transduction of nucleic acid and/or protein. Optionally, the host cell is cultured. The cells may be part of a tissue.

Protein constructs that comprise more than one protein can be made by any known method. The protein construct can for example be a fusion protein, or can be synthesized by solid phase synthesis methods, or made by conjugation or linkage of existing proteins, e.g., by chemical linkage. If desired, the individual proteins can be attached to each other by linker peptide sequences. Examples of linker sequences include standard polyglycine-serine flexible linkers, which can be made by, e.g., oligo annealing. Linkers may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

Living or dead, e.g., freshly killed, organisms can also be used, either in whole or in part, such as a tissue. The organism can comprise recombinant host cells that contain one or more nucleic acid or protein constructs of the invention. In one aspect, the organism can be a transgenic organism. Some organisms that are widely used in research include mice, rats, hamsters, monkeys, dogs, cats, and hydra. Animals that are naturally transparent at any stage of development can be especially useful in the invention, including zebrafish, jellyfish, and various embryos. Uses

The methods, materials and systems of the present disclosure can be used in a variety of ways. In an aspect, the present disclosure can be used as a research tool to study the biological role of a protein of interest, or the role of an interaction between a first and second protein of interest. Protein of interest and polypeptide of interest are used interchangeably herein and refer to options for the polypeptides for use in the methods, cell, organisms and kits described herein.

Other examples of uses that are suitable for study using the methods, materials and systems of the present disclosure include (1) the controlling of oligomerization of two proteins to allow protein activation in the cases of proteins such as caspases, EGF receptor, or other tyrosine kinase receptors; (2) the localization of proteins to different locations in the cell, e.g., forcing a constitutively active form of rac to the plasma membrane to cause the formation of lammellopodia; (3) forced localization to sequester proteins from a particular compartment, e.g., forcing TBP out of the nucleus (such sequestration/removal of protein allows for the creation of a knockout phenotype "at will"); (4) forced control of protein interactions to reconstitute activity of split protein (e.g., Gal4 in use of yeast two hybrid systems); forced control of split versions of enzymes, such as Cre recombinase or beta-galactosidase, and the like. The methods may be used in vitro, ex vivo or in vivo.

The systems, methods and materials of the present disclosure also have the benefit of allowing spatial resolution in a cell (e.g., a single cell), tissue or region of tissue or organism or region or the organism to be stimulated for a particular activity (or to eliminate a particular activity). As light-activation of proteins is typically extremely rapid, the methods described herein have the potential to allow control of proteins with millisecond time resolution. With the added temporal and spatial benefits of the light-regulation according to the present disclosure, these modules will be useful for delineating roles of proteins and cells in research areas such as neurobiology or cell polarity where it is important to delineate the roles of proteins at precise subcellular locations, or in developmental biology, where specific cells play precise roles in tissues and developing organelles. These methods are also useful in model organisms and transgenic mice, allowing control of transcription factors, signal transduction pathways, and enzymatic activities in a spatial and temporally restricted manner. For example, the systems and methods described herein can be used to turn on an enzyme such as Cre recombinase in a single cell (thus deleting or activating a gene), or used to allow expression of a constitutively active protein that would normally be lethal by restricting its activity to only a specific subcellular location.

The invention can be used in a variety of settings. For example, the invention can be used in vitro with cultured cells, or in vivo using organisms into which cells containing or expressing protein constructs of the invention have been introduced. Alternatively, the organism can be a transgenic organism that expresses one or more protein constructs of the invention.

Diagnostic uses include the introduction of the protein constructs into cells taken from a patient to detect abnormal effects.

Proteins of Interest

The present disclosure can be used to study a wide variety of proteins that are capable of interacting with other proteins. In another embodiment, interactions such as dimerization or multimerization can be studied, wherein the first and second protein constructs comprise the same protein and form homodimers or different proteins and form heterodimers.

In one embodiment, the first and/or second protein is involved in cell signaling. Molecules involved in signaling include receptors (both at the cell surface and intercellular). Such receptors include G-protein coupled receptors, e.g., chemokine receptors; receptor tyrosine kinases, e.g., growth factor receptors, integrins and toll-like receptors. Signaling proteins downstream of receptors include intracellular proteins activated by a ligand/receptor interaction; these often possess an enzymatic activity. These include small G proteins such as the Ras, Rho, and Ral families, Guanine nucleotide exchange factors such as SOS, eIF-2B, Ras-GRFl, GOCRs and Kalinin, tyrosine kinases, heterotrimeric G proteins, small GTPases, various serine/threoine protein kinases, phosphatases, lipid kinases, and hydrolases. Some receptor-stimulated enzymes create specific second messengers including cyclic nucleotides, such as cyclic AMP (cAMP) and cyclic GMP (cGMP), Phosphatidyl inositol derivatives, such as Phosphatidylinositol- triphosphate (PIP3), Diacylglycerol (DAG) and Inositol-triphosphate (IP3), IP3, controlling the release of intracellular calcium stores into the cytoplasm (see second messengers section later in this article).

Adapter proteins are another type of protein involved in signaling. Adapter proteins include GRAP - GRB2-related adaptor protein; GRAP2 - GRB2-related adaptor protein 2; LDLRAP1 - low density lipoprotein receptor adaptor protein 1 ; NCK1 - NCK adaptor protein 1 ; NC 2 - NC adaptor protein 2; NOS1AP - nitric oxide synthase 1 (neuronal) adaptor protein; PD 3AP1 - phosphoinositide-3 -kinase adaptor protein 1 ; SH2B1 - SH2B adaptor protein 1 ; SH2B2 - SH2B adaptor protein 2; SH2B3 - SH2B adaptor protein 3; SHB - Src homology 2 domain containing adaptor protein B; SLC4A1 AP - solute carrier family 4 (anion exchanger), member 1, adaptor protein; and GAB2, GRB2-associated binding protein 2.

Many proteins involved in signaling possess specialized protein domains that bind to specific secondary messenger molecules. For example, calcium ions bind specifically to the EF hand domains of calmodulin, allowing this molecule to bind and activate Calmodulin- dependent kinase. PIP3, PIP2 and other phosphoinositides may bind to the Pleckstrin homology domains of proteins such as the kinase protein AKT. Examples of specific signaling proteins of interest include a G protein, Rho Guanine nucleotide Exchange Factors (GEF), or any other signalling proteins of interest. Racl [Entrez Gene ID: 5879], Cdc42 [Entrez Gene ID: 998], RhoA [Entrez Gene ID: 387]. Examples of GEFs include Tiam [Entrez Gene ID: 7074], Intersectin [Entrez Gene ID: 6453], and Tim [Entrez Gene ID: 7984]. Other Signalling Factors include NckaplL (Heml) [Entrez Gene ID: 3071], G-gamma2 [Entrez Gene ID: 54331], and inter-SH domain (residues 420- 615) from p85alpha [Entrez Gene ID: 5295]. Other polypeptides that may be used include but are not limited to Src family tyrosine kinases, Raf, IKKs and caspases.

Biologically significant effects that result from signal transduction include activation of genes, alterations in metabolism, the continued proliferation and death of the cell, and the stimulation or suppression of locomotion.

As described above and in the examples, transcription factors may also be used as the polypeptides used herein. A split Gal4 was linked to CD and CIP in the examples and exposure to light was able to activate transcription in the cell. Those of skill in the art are aware of several other transcription factors that may be used in the methods and systems described herein. For example, the TetR or LexA-VP16 systems may be adapted for use in the methods. These transcription systems may be used in combination with the methods described herein to allow tunable expression of a gene of interest. Cell specific, location specific and in vivo transcription regulation will allow in depth study of the physiologic role of the gene and the protein it encodes. Kits

The present disclosure also includes kits containing any of the proteins, protein constructs, nucleic acids, cells, reagents or materials of the invention or any combination thereof. The kit optionally contains instructions that instruct a user to introduce proteins, protein constructs, nucleic acids, and/or reagents of the invention into cells and/or to regulate association of the proteins or protein constructs of the invention by regulating exposure to light (e.g., blue light).

EXAMPLES

Materials and Methods

Strains and Plasmids. For yeast two-hybrid studies, strains AH 109 (MATa, trp 1-901, leu2-3, 112, ura3-52, his3-200, gal4Δ gal80Δ LYS2::GAL1_UAS-GAL1_TATA-HIS3, GAL2_UAS- GAL2TATA-ADE2, URA3::MELl_USs-MELl_TATA-lacZ, MEL1) and Y187 (MAT , ura3-52, his3- 200, ade2-101, trpl-901, leu2-3, 112, g lrΔ, gal80Δ, met-, URA3::GALl_UAS-GALl_TATA-lacZ, MEL1) were used (Clontech). The light inducible transcription study used yeast strain PJ69-4a (MATa trpl-901 le 2-3,112, ura3-52, his3-200, gal40Δ, g l80Δ, LYS2::GAL1-HIS3, GAL2- ADE2, met2::GAL7-lacZ).

Plasmids used in this study are described in Table 1 and will be available from Addgene. Oligos are listed in Table 2. Gal4 binding domain fusions (Gal4BD-X) were in pDBTrp, a version of pDBLeu (Invitrogen) with a Trp+ selection marker. Gal4 activation domain fusion proteins (Gal4AD-Y) were in pGADT7rec (Clontech). To generate Gal4BD and Gal4AD constructs, Cry2 and Cibl were PCR amplified from Arabidopsis thaliana cDNA using gene specific primers. PCR products from this amplification were then used as template for a second round of PCR using oligos designed to allow expression (via homologous recombination in yeast) of full length CRY2 or CRY2_PHR (amino acids 1-498) at the C-terminus of Gal4BD, or full length CIB1 or CIBN (amino acids 1-170) at the C-terminus of Gal4AD.

For expression in mammalian cell lines, full length Cry2 and amino acids 1 -498 of Cry2 were PCR amplified and ligated into vector pmCherry-Nl (Clontech) at Xho I and Xma I sites. CibN was cloned in a similar manner using Nhe I and Age I sites into a version of eGFP-Cl that contained a CaaX polybasic sequence from KRas4B ( KKKKKSKTKCVIMM ; SEQ ID NO: 6) at the C-terminus. To mutate the NLS sequences, oligos CRY2dNLSf and CRY2dNLSr were used for CRY2 and CIBdNLSf and CIBdNLSr were used for CIB1. PCR amplification was carried out using mutagenic oligos and forward and reverse oligos from two-hybrid cloning to generate two overlapping fragments of DNA, which were joined via homologous recombination in yeast. Constructs were tested for interaction in yeast, then moved to mammalian vector systems as previously described.

The Cre recombination constructs were first assembled in yeast (in vector p414ADH) via homologous recombination of two overlapping fragments that had been generated by PCR. For the CRY2-CreN construct, the first fragment contained (in order) 33 bp of homology to p414ADH, a Sac I site, a Kozak sequence, full length (+NLS) CRY2, a flexible linker (GGGGSGGGGSGG; SEQ ID NO: 14)). The second PCR fragment contained the flexible linker, a Not I site, amino acids 19-104 of Cre recombinase, a stop codon, a Xma I site, followed by 33 base pairs of homology to the yeast vector. The CIBN-CreC construct was assembled identically, except CIBN was used in place of CRY2, and amino acids 106-343 of Cre were used in place of 19-104. After recombination in yeast, inserts containing fusion proteins were cut out of p414ADH using Sac I and Xma I, and cloned into the MCS (Sac I / Xma I sites) of pmCherryCl, downstream from an IRES2 element that was placed between mCherry and the MCS.

Yeast two-hybrid experiments. GaWBD plasmids containing vector only, CRY2, or CRY2PHR were expressed in strain AH 109 and patched on YPD plates. On top of patches, Y 187 yeast expressing Gal4AD fusions with CIB1, CIBN, or empty vector control were patched. Yeast were mated overnight at 30°C, then streaked on SD -Trp/-Leu plates to select for diploid cells that contained both Gal4AD and Gal4BD plasmids. Colonies that grew on SD -Trp/-Leu were then streaked on SD -Trp/-Leu/-His/ + 3 mM 3-aminotriazole (3-AT) plates, β- galactosidase assays were performed by growing yeast cells overnight in SD -Trp/-Leu medium, followed by dilution to 0.2 OD₆₀₀ in SD -Trp/-Leu medium the next morning. Following an initial 3 hour growth period in the dark, cultures were either kept in the dark or continuously exposed to an LED blue light source (461 nm, 1.9 mW) for 4 hours. At the conclusion of the 4 hour period, cultures were harvested and lysed with Y-PER reagent (Thermo Scientific). The assay for β-galactosidase activity was then carried out following a standard protocol for liquid cultures (Clontech Laboratories, protocol #PT3024-1) using ONPG (Sigma- Aldrich) as a substrate. Experiments were carried out at least three times with similar results to those shown. Samples incubated in dim room light (0.25 μW) gave results indistinguishable from samples incubated in total darkness. In constrast, bright room light (34 μW) activated reporters -30% as well as blue LED treated samples.

Live cell imaging. Live cell imaging was performed on a custom built spinning disc confocal microscope with a Yokogawa CSUIO scan head mounted on a Nikon TE300 inverted stand as previously described by Kennedy et al., Cell 141, 524-535 (2010). Images were acquired using a 60x Plan Apochromat 1.4 NA objective. A 1.5x tube lens between the filter wheel and camera focused light on the chip of a Hamamatsu C9100 EM -CCD camera giving a pixel size of 86 x 86 nm. The focal plane was controlled by a piezo-driven Z-stage (Applied Scientific Instruments). The EM-CCD Camera, filter wheel, stage, and AOTF laser line switching were controlled by Metamorph software (Molecular Devices). An environmental chamber (In Vivo Scientific) enclosing the microscope stand maintained the temperature at 37°C. HEK293T cells were grown on glass coverslips (Deckglaser #1, 18 mm) and maintained in DMEM containing 10% FBS. Cells were transfected with Lipofectamine 2000 (Invitrogen) when 50-80% confluent according to the manufacturers protocols and imaged 24 hours following transfection.

Excitation was provided by solid state 488 nm (Coherent) or 561 nm (Spectraphysics) lasers shuttered via an acousto-optical tunable filter (AOTF) (Neos Technologies), with emission directed through a filter wheel (Applied Scientific Instrumentation) holding either band pass or long pass filters (Chroma). Power used for stimulation of translocation was equivalent to that used for imaging GFP (25 μW measured 1 cm from the objective). Wavelengths tested for triggering translocation were consistent with the absorbance profile of cryptochrome, which responds to UV A/blue light with a peak at 450 nm, and weakly above 500 nm— i.e. 405 nm illumination triggered interaction, but illumination at 561 nm did not. Illumination at 514 nm (which would be used with YFP for two-color imaging) triggered translocation at high intensity illumination, but not at lower intensity (under 2 μW) (Fig. 8).

Reports in the plant literature suggest that light activation of cryptochromes may be reversed with green light, however we did not observe any effect of illumination with green light (514 or 540 nm) on the reversal time of the CRY2-CIBN interaction. We also tested the interaction of CIBN-mCh with CRY2-pmGFP. While these proteins expressed well, they did not show light-induced interaction. It is possible that membrane association of CRY2 sterically hinders the association with CIB in this conformation, and adjustment of the linker length may remedy this.

Dose-dependent activation of transcription. pDBTrp-CRY2 and pGBKT7rec-CIBl constructs, along with a plasmid from a galactose-inducible yeast overexpression library (Gelperin, D.M. et al, Genes Dev. 19, 2816-2826 (2005)) expressing the protein Snll from a galactose-inducible promoter, were co-transformed into strain PJ694-a and plated on SD -Trp/- Leu/-Ura plates. The triple transformed yeast were grown overnight at 30°C in media containing SD -Trp/-Leu/-Ura, then diluted to 0.1 OD₆oo in SD -Trp/-Leu/-Ura and placed in the dark. Following an initial 3 hour growth period in the dark, yeast cells were exposed to pulses of blue light from a fluorescent microscope beam (Leica MZFLIII) equipped with a GFP filter (10 s in duration, spaced 8 min apart, 1.7 mW). Cultures remained in the dark a total of four hours following the initial light exposure, at which point they were harvested for immunoblotting. Yeast were lysed in 2% SDS by glass bead disruption (425-600 μηι beads, Sigma), after which samples were boiled for 3 minutes, placed in 2x Laemmli Sample Buffer, boiled for 1 minute, and centrifuged at 14,000 rpm for 5 minutes. Equal amounts of total protein were run on a 12% SDS-PAGE gel and immunoblotted using standard procedures using a mouse anti-HA primary antibody (Covance) and an IRDye 700CW goat anti-mouse IgG secondary antibody (Li-COR). Proteins were visualized using an Odyssey infrared imaging system (Li-COR).

Light activation of split Cre recombinase. HE 293T cells were transfected with the Cre reporter and indicated constructs, and % Cre reporter recombination (# of GFP expressing cells / # of mCherry expressing cells) was measured 48 hours after transfection. For light treated samples, blue light pulses (2 s pulse delivered every 3 min, 450 nm, 4.5 mW) were administered by a custom LED array light source. For 24 hour experiments, light was administered from 24 to 48 hours following transfection. For pulse experiments, samples were exposed to pulsed light (15 min or 1 hr) at 24 hours post transfection, then incubated in the dark until 48 hours post transfection to allow reporter expression. Nontreated (-) samples were kept in the dark for the duration. Samples were fixed in 4% paraformaldehyde and the ratio of cells expressing GFP to cells expressing mCherry was calculated, based on the average counts from three wells. The results reported are the average and standard deviation from three independent experiments. To calculate the fold activation, the ratio of the percent of cells activated in the light to the percent activated in the dark was calculated after the background was subtracted. For the trypan blue cell counts described in Table 3 below, cells were harvested at 24 hours post-transfection and the harvested cell cultures were incubated with 0.4% trypan blue solution (Sigma) for 3 minutes, then the number of blue vs. white cells was determined using a hemocytometer. The results are reported as percentage of viable cells based on three independent counts.

Two-photon microscopy. Two-photon microscopy was performed using a Zeiss LSM 710 confocal scanhead mounted on an Axio-observer microscope a with Chameleon II ultra laser source using a 20x 1.0 NA Apochromat objective (Zeiss). IR laser power was normalized by measuring the power at the sample using a FiedMaxII power meter (Coherent) tuned to the corresponding wavelength. All two-photon experiments were performed using equivalent power, unless otherwise stated. HEK293T cells expressing CRY2_PHR-mCh and CIBN-pmGFP were imaged using 561 nm excitation with emission collected through a band-pass filter set for 570-610 nanometers. We collected 10 images of CIBN-mCh prior to exposure with 800-1000 nm excitation (-15% AOM at 930 nm for 30 iterations). Two-photon excitation was repeated every 25 images (acquired at 1 Hz) for the duration of the experiment. Translocation occurred within the range of 830-980 nm. After the time series completion, the localization of CIBN- pmGFP was determined by acquiring a single image with 488 nm exposure (-1.5 pixel dwell time for 1 s) with emission collected through a band pass filter set for 500-550 nm.

Organotypic slice culture was carried out as previously described except that rat pups were used instead of mouse pups (Gogolla,et al., Nat. Protoc. 1, 1 165-1171 (2006)). Briefly, hippocampi from postnatal day 5-6 rat pups were dissected and slices (350 μιη) were prepared using a Mcllwain tissue chopper and cultured on 0.4 μm millicell membrane inserts (Millipore). After 6 days in culture, slices were biolistically transfected with CIBN-pmGFP/CRY2_PHR-mCh constructs (Helios gene gun, Biorad). Following biolistic transfection, slices were either maintained in darkness or photostimulated 3-4 days following transfection. Following light stimulation, slices were immediately fixed in PBS containing 4% paraformaldehyde in PBS for 10-15 min on ice. The CRY2_PHR-mCh signal was enhanced with a rabbit polyclonal anti-RFP antibody (MBL) prior to mounting the slices for imaging.

Identification of Minimal Interaction Domains for Crv2 and CIB

Minimal interaction domains for the light-induced CRY2-CIB1 interaction were identified using the yeast two-hybrid assay (Fig. 2). Plant cryptochromes contain a conserved N- terminal photolyase homology region (PHR) that binds flavin and pterin chromophores and mediates light-responsiveness. Full length CRY2 and the PHR domain (CRY2PHR, aa 1-498) were tested for interaction with full length CIB1 or a truncated version (CIBN, aa 1-170) missing the conserved bHLH domain which mediates dimerization and DNA binding (Fig. 2a). Reporter levels of CRY2-CIB1 and CRY2-CIBN were indistinguishable from controls in the dark, but showed clear activation upon blue light stimulation (461 nm, 1.9 mW, 4 h) (Fig. 2b). CRY2PHR also interacted in a light-dependent manner with CIB1 and CIBN, indicating that this domain alone is sufficient to confer light-dependent specificity to the interaction. While CRY2 expression levels were very low, contributing to low levels of reporter activation, CRY2PHR expressed much better, resulting in higher levels of reporter activation in light-treated samples, but also higher basal activity with CIB1 and CIBN in dark-treated samples.

Localization of mCherry-CIBN to plasma membrane

To test the CRY2-CIB1 interaction in mammalian cells, we coexpressed full-length CRY2 or CRY2_PHR fused to the fluorescent protein mCherry (CRY2-mCh or CRY2_PHR-mCh), and CIBN fused to a prenylated version of EGFP that localizes to the plasma membrane (CEBN- pmGFP) (Fig. 4a). Initial experiments revealed nuclear localization of CRY2-mCh and CIBN- pmGFP, but cytosolic localization for CRY2p_HR-mCh. We mutated predicted NLS residues in CRY2-mCh and CIBN-pmGFP (Fig. 3; SEQ ID NO: 15-22), which resulted in robust cytoplasmic and plasma membrane expression, respectively, when cells were maintained in the dark (Fig. 4b). We also examined a NLS deleted version of full length CIB1 (CIBl-pmGFP), but did not use this construct further as it showed punctate perinuclear localization.

Light triggered translocation of CRY2 in mammalian cells

The ability of blue light to induce interaction between CRY2-mCh and membrane associated CIBN-pmGFP was also tested. Within 300 ms after blue light illumination (488 nm, 25 μW, 100 ms pulse), CRY2-mCh began accumulating at the plasma membrane, where it co- localized with CIBN-pmGFP (Fig. 4b, c). Greater than 95% of cells exhibited robust translocation of CRY2-mCh to the plasma membrane, while the distribution of CIBN-pmGFP did not change following illumination. Translocation was more than 90% complete within 10 s (Fig. 4d). Dim room light (5.2 μW) did not trigger translocation, indicating samples do not require excessive light shielding. CRY2-mCh and CRY2p_HR-mCh behaved similarly in translocation experiments, with similar activation and reversal kinetics (Fig. 5a). The complete time course of plasma membrane association and dissociation for the CRY2PHR-CIBN interaction is shown in Figure 4e. After an initial pulse of light, CRY2p_HR-mCh rapidly translocated to the plasma membrane, then slowly dissociated over -12 minutes. A subsequent light pulse triggered a second round of translocation nearly identical in magnitude to the first (Fig. 4e), and the interaction could be repeatedly induced at least six times with little or no loss in efficacy (Fig. 5b). While blue light has poor tissue penetration ability, multi-photon excitation can allow precise three-dimensional cell targeting in tissues with high cellular densities. The CRY-CIB modules can be activated by two-photon stimulation at 860 nm (range, 820-980 nm) in cell culture and organotypic cultured hippocampal slices (Fig. 6), suggesting the potential for precise spatial activation of protein dimerization in whole organisms.

One powerful application of artificial dimerizers is to allow inducible control of a 'split' protein, where two inactive fragments are brought together to reconstitute a functional protein activity. In principle, using this approach with optically controlled dimerizers would confer light-dependent activity to a diverse range of target proteins. The CRY-CIB modules were used to reconstitute split versions of two proteins, a Gal4 transcription factor and Cre recombinase, seeking light-dependent control of transcription and DNA recombination. For transcriptional control, our approach was similar to a prior light-regulated system using Arabidopsis PHYB and PIF3, but based on the transient nature of the CRY-CIB interaction (Fig. 4e), we speculated that light pulses would induce transcription in a dose-dependent fashion, allowing precise control of protein expression. We coexpressed in yeast the split Gal4 partners Gal4BD-CRY2 and Gal4AD-CIBl , as well as a reporter protein (Snll) under control of a galactose-inducible promoter (Fig 7a). Yeast were incubated in the dark or subjected to up to 20 blue light pulses (10 s pulses, 8 min apart) over four hours (Fig. 7b). Immunoblot analysis of Snll revealed a strong dose-dependence of protein expression in response to light, indicating that the CRY-CIB modules allow dose-dependent as well as spatiotemporal control of biological systems.

The ability of the CRY-CIB modules to induce dimerization of a split Cre recombinase was tested for its ability to allow light-dependent control of DNA recombination. Based on a previous split Cre recombinase activated by rapamycin, we fused CRY2 to amino acids 19-104 of Cre (CRY2-CreN), and CIBN to amino acids 106-343 of Cre (CIBN-CreC) (Fig. 7c). The Cre modules showed no toxicity in cells after transfection with the indicated plasmids and either kept in the dark or exposed to blue light pulses (lhr, 450 nm, 4.5 mW) at 20 hours post transfection (Table 3).

As a reporter of Cre recombinase activity, we used a plasmid containing a transcriptional stop sequence flanked by loxP sites preceding EGFP. When transfected into HE 293T cells, the reporter construct alone displayed low levels of recombination (0.6 ± 0.4 % of transfected cells, on average) (Fig. 7d), as did control cells expressing either CIBN-CreC or CRY2-CreN alone (data not shown). Cells containing both CRY2-CreN and CIBN-CreC incubated in the dark showed equivalent levels of recombination as control cells (0.7 ± 0.4 % of transfected cells), indicating minimal or no light-independent CRY2-CIBN interaction. Cells containing CRY2- CreN and CIBN-CreC exposed to continuously pulsed blue light for 24 hours (2 s pulses every 3 min) showed a 158-fold increase in the number of EGFP-positive cells (16.4 ± 1.6 % of transfected cells) compared to dark treated samples, indicating robust light-dependent activation that was comparable to the activity of the rapamycin dimerized split Cre under similar transient transfection conditions. Importantly, cells exposed to pulsed light for shorter times, mimicking activation of Cre in a tissue or in vivo, showed a substantial increase in activity over dark treated samples. Exposure to light pulses for only 15 mins resulted in a 25-fold increase in EGFP- positive cells over dark treated samples (3.5 ± 1.3 % of transfected cells), while exposure to pulses for one hour gave a ~50-fold increase over dark samples (5.1 ± 1.1 % of transfected cells). We expect that further optimization of these constructs such as linker modification and mutagenesis may allow even more robust light-dependent activation in future iterations. Furthermore, as CRY1 has been observed to dimerize in plants, optimization can also include modification of dimerization motifs.

The above demonstrations of the CRY-CIB modules indicate that they allow temporal, spatial, and dose-dependent optical control of protein dimerization, with time scales in the sub- second range and without the requirement for exogenous ligand seen with chemical dimerizers or the PHY-PIF system. As this platform is entirely genetically encoded and can be activated using commonly available light sources used for GFP imaging, these modules will be useful for controlling a broad range of biological phenomena. Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present examples along with the methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.

Claims

CLAIMS We claim:

1. A method of controlling an interaction between a first protein construct and a second protein construct comprising regulating the exposure the first protein construct and the second protein construct to light, wherein the first protein construct comprises a first polypeptide and a Cryptochrome domain (CD), wherein the second protein construct comprises a second polypeptide and a Cryptochrome interacting polypeptide (CIP), and wherein exposure to light controls the interaction between the first protein construct and the second protein construct.

2. The method according to claim 1 , wherein the CD comprises a sequence derived from Arabidopsis thaliana Cryptochrome 2 (CRY2) (SEQ ID NO: 2).

3. The method of claim 3, wherein the CD is 90% identical to amino acids 1-498 of SEQ ID NO: 2.

4. The method according to any one of the preceding claims, wherein the CIP comprises a sequence derived from the Cryptochrome-binding domain of CIB1 (SEQ ID NO: 4).

5. The method of claim 4, wherein the CIP is 90% identical to amino acids 1-170 of SEQ ID NO: 4.

6. The method of any one of the preceding claims, wherein the light comprises blue light.

7. The method according to any one of the preceding claims, wherein the light

comprises a wavelength of about 380 nm to about 514 nm.

8. The method according to any one of the preceding claims, wherein the light

comprises a wavelength of about 450 nm to about 488 nm.

9. The method according to any one of the preceding claims, wherein light is from

multi-photon excitation.

10. The method of claim 9, wherein the light from multi-photon excitation comprises a wavelength of about 820 nm to about 980 nm.

1 1. The method according to claim 9, wherein the light from multi-photon excitation comprises a wavelength of about 860 nm.

12. The method of any one of the preceding claims, wherein the interaction can be detected within one minute after exposure to light.

13. The method of any one of the preceding claims, further comprising detecting a

change in the interaction between the first protein construct and the second protein construct.

14. The method of any one of the preceding claims, wherein the first polypeptide and the second polypeptide are not fluorescent proteins.

15. The method of any one of the preceding claims, wherein the first polypeptide and the second polypeptide are portions of a split protein.

16. The method of any one of the preceding claims, wherein at least one of the first

polypeptide or the second polypeptide is at least a portion of a transcription factor, an enzyme, a recombinase or a cell-signaling protein.

17. The method of any one of the preceding claims, wherein the first protein construct and the second protein construct are within a cell.

18. The method of claim 18, wherein the cell is within a tissue or an organism.

The method of any one of claims 17-18, wherein only a portion of a cell, tissue or organism is exposed to light.

20. The method of claim 19, wherein the interaction is spatially localized within a few microns of the exposed portion.

21. The method of any one of the preceding claims, wherein the first protein construct or the second protein construct comprises a subcellular localization tag (SLT), whereby the protein construct may be localized to a subcellular compartment.

22. The method of claim 21 , wherein the subcellular localization tag is selected from a plasma membrane localization tag, a nuclear localization tag, a mitochondrial membrane tag, a nuclear export signal, and an endoplasmic reticulum localization tag.

23. The method of any one of claims 21 or 22, wherein one of the first protein construct and the second protein construct lacks a subcellular localization tag and comprises a detectable label, whereby the interaction can be detected by detecting the label in the subcellular compartment.

24. The method according to any one of the preceding claims, wherein the first protein construct or the second protein construct comprises a detectable label.

25. The method of claim 24, wherein the detectable label is optically detectable and wherein the change in the interaction is detected by optically detecting the spatial distribution of the label.

26. The method according to any one of claims 17-25, wherein the cell is selected from the group consisting of yeast, insect, avian, fish, worm, xenopus, bacteria, algae and mammalian cells.

27. The method according to any one of claims 17-26, wherein the cell is not a plant cell, not a bacterial cell or not a yeast cell.

28. The method according to any one of the preceding claims, wherein the first

polypeptide or the second polypeptide comprises a Gal4 binding domain (SEQ ID NO: 10) or the amino terminus of Cre recombinase (SEQ ID NO: 8, amino acids 19- 104).

29. The method according to any one of the preceding claims, wherein the first

polypeptide or the second polypeptide comprises a Gal4 activation domain (SEQ ID NO: 12) or the carboxy-terminus of Cre recombinase (SEQ ID NO: 8 amino acids 106-343).

30. A cell comprising a first protein construct and a second protein construct, wherein the first protein construct comprises a first polypeptide and a Cryptochrome domain (CD), wherein the second protein construct comprises a second polypeptide and a Cryptochrome interacting polypeptide (CIP).

31. The cell of claim 30, wherein the first polypeptide and the second polypeptide are portions of a split protein.

32. The cell of claim 30 or 31 , wherein at least one of the first polypeptide or the second polypeptide comprises at least a portion of a transcription factor, an enzyme, a recombinase or a cell-signaling protein.

33. The cell of any of claims 30-32, wherein the CD comprises a sequence derived from Arabidopsis thaliana Cryptochrome 2 (CRY2) (SEQ ID NO: 2).

34. The cell of claim 33, wherein the CD is 90% identical to amino acids 1-498 of SEQ ID NO: 2.

35. The cell of any one of claims 30-34, wherein the CIP comprises a sequence derived from the Cryptochrome- binding domain of CIB1 (SEQ ID NO: 4).

36. The cell of claim 35, wherein the CIP is 90% identical to amino acids 1-170 of SEQ ID NO: 4.

37. The cell of any one of claims 30-36, wherein the cell is selected from the group

consisting of yeast, insect, avian, fish, worm, amphibian, xenopus, bacteria, algae and mammalian cells.

38. The cell of any one of claims 30-37, wherein the cell is not a plant cell, not a bacterial cell or not a yeast cell.

39. The cell of any one of claims 30-38, wherein the first polypeptide or the second

polypeptide comprises a Gal4 binding domain (SEQ ID NO: 10) or the amino- terminus of Cre recombinase (SEQ ID NO: 8 amino acids 19-104).

40. The cell of any one of claims 30-39, wherein the first polypeptide or the second

polypeptide comprises a Gal4 activation domain (SEQ ID NO: 12) or the carboxy- terminus of Cre recombinase (SEQ ID NO: 8 amino acids 106-343).

41. The cell of any one of claims 30-40, wherein the cell is within a tissue or an

organism.

42. A non-human transgenic organism comprising the cell of any one of claims 30-41.

43. The transgenic organism of claim 42, wherein the organism is an insect, fish, bird, worm, amphibian, xenopus or non-human mammal.

44. A polynucleotide encoding a polypeptide comprising a carboxy-terminal truncated CRY2 or CIB1 polypeptide.

45. The polynucleotide of claim 44, wherein the polynucleotide further encodes a

polypeptide of interest.

46. A polynucleotide encoding a fusion protein comprising a CD or CIP linked to a

polypeptide of interest.

47. The polynucleotide of any one of claims 44-46, wherein the polypeptide of interest comprises at least a portion of a transcription factor, an enzyme, a recombinase or a cell-signaling protein.

48. The polynucleotide of any one of claims 44-47, wherein the polypeptide of interest is not a fluorescent protein.

49. The polynucleotide of any of claims 44-48, wherein the CD comprises a sequence derived from Arabidopsis thaliana Cryptochrome 2 (CRY2) (SEQ ID NO: 2).

50. The polynucleotide of claim 49, wherein the CD is 90% identical to amino acids 1- 498 of SEQ ID NO: 2.

51. The polynucleotide of any one of claims 44-50, wherein the CIP comprises a

sequence derived from the Cryptochrome- binding domain of CIB1 (SEQ ID NO: 4).

52. The polynucleotide of claim 51, wherein the CIP is 90% identical to amino acids 1- 170 of SEQ ID NO: 4.

53. A kit comprising a first polynucleotide encoding a Cryptochrome domain (CD)

derived from Arabidopsis thaliana Cryptochrome 2 (CRY2) (SEQ ID NO: 2) and a second polynucleotide encoding a Cryptochrome interacting polypeptide (CIP) derived from the Cryptochrome-binding domain of CIB1 (SEQ ID NO: 4).

54. The kit of claim 53, wherein the CD comprises a sequence derived from Arabidopsis thaliana Cryptochrome 2 (CRY2) (SEQ ID NO: 2).

55. The kit of claim 53, wherein the CD is 90% identical to amino acids 1-498 of SEQ ID NO: 2.

56. The kitof any one of claims 53-55, wherein the CIP comprises a sequence derived from the Cryptochrome- binding domain of CIB1 (SEQ ID NO: 4).

57. The kit of any one of claims 53-56, wherein the CIP is 90% identical to amino acids 1-170 of SEQ ID NO: 4.

58. The kit of any of claims 53-57, wherein a first expression vector comprises the first polynucleotide and a second expression vector comprises the second polynucleotide.

59. The kit of claim 58, wherein a third polynucleotide encoding a first polypeptide can be inserted into the first expression vector to generate a fusion protein of the first polypeptide and the Cryptochrome domain.

60. The kit of claim 58 or 59, wherein a fourth polynucleotide encoding a second

polypeptide can be inserted into the second expression vector to generate a fusion protein of the second polypeptide and the Cryptochrome-interacting polypeptide.

61. The kit of any of claims 58-60, wherein the third polynucleotide or the fourth

polynucleotide encode at least a portion of a transcription factor, a recombinase or a cell-signaling protein. 62 The kit of any one of claims 53-61 , wherein the first polypeptide or the second polypeptide comprises a Gal4 binding domain (SEQ ID NO: 10) or the amino- terminus of Cre recombinase (SEQ ID NO: 8 amino acids 19-104).

63 The kit of any one of claims 53-62, wherein the first polypeptide or the second polypeptide comprises a Gal4 activation domain (SEQ ID NO: 12) or the carboxy- terminus of Cre recombinase (SEQ ID NO: 8 amino acids 106-343).