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An optimized optogenetic clustering tool for probing protein interaction and

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Received 22 Jul 2014 | Accepted 6 Aug 2014 | Published 18 Sep 2014 DOI: 10.1038/ncomms5925

An optimized optogenetic clustering tool for

probing protein interaction and function
Amir Taslimi1, Justin D. Vrana1, Daniel Chen1, Sofya Borinskaya2, Bruce J. Mayer2, Matthew J. Kennedy1
& Chandra L. Tucker1

The Arabidopsis photoreceptor cryptochrome 2 (CRY2) was previously used as an

optogenetic module, allowing spatiotemporal control of cellular processes with light. Here we
report the development of a new CRY2-derived optogenetic module, ‘CRY2olig’, which
induces rapid, robust, and reversible protein oligomerization in response to light. Using this
module, we developed a novel protein interaction assay, Light-Induced Co-clustering, that can
be used to interrogate protein interaction dynamics in live cells. In addition to use probing
protein interactions, CRY2olig can also be used to induce and reversibly control diverse
cellular processes with spatial and temporal resolution. Here we demonstrate disrupting
clathrin-mediated endocytosis and promoting Arp2/3-mediated actin polymerization with
light. These new CRY2-based approaches expand the growing arsenal of optogenetic
strategies to probe cellular function.

1 Department of Pharmacology, University of Colorado School of Medicine, Aurora, Colorado 80045, USA. 2 Raymond and Beverly Sackler Laboratory of

Genetics and Molecular Medicine, Department of Genetics and Molecular Biology, University of Connecticut Health Center, Farmington, Connecticut 06030,
USA. Correspondence and requests for materials should be addressed to C.L.T. (email: chandra.tucker@ucdenver.edu).

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5925

P
rotein–protein interactions underlie nearly all molecular Results
and cellular processes. As such, tools and technologies While screening for CRY2 variants with longer signalling states in
allowing scientists to probe and control protein interactions a yeast two-hybrid assay (Supplementary Fig. 1), we identified
have fundamentally advanced our understanding of molecular CRY2olig, containing an E490G mutation that greatly enhances
and cellular mechanisms. Methods for detecting protein–protein light-induced clustering of CRY2. When fused to the
interactions such as yeast two-hybrid and co-immunoprecipita- reporter mCherry (mCh) and expressed in mammalian cells,
tion1–3 are commonly part of every experimental biologist’s CRY2olig–mCh shows diffuse expression in the dark, but
toolkit and have led to numerous new discoveries. In addition to upon blue light exposure it undergoes massive clustering,
tools for detecting protein interactions, technologies for redistributing a majority of protein in the cytosol and nucleus
controlling protein function have been instrumental in (70±15% of cytosolic protein) into large puncta within tens of
advancing scientific discovery. Chemical genetic tools allowing seconds following a pulse of light (Fig. 1a–c and Supplementary
inducible control of processes have been critical for dissecting Movie 1). Rate of clustering is dependent on protein
complex biochemical and signalling events within cells4,5; concentration, with half-maximal clustering time varying from
however, these tools can be expensive, difficult to deliver to and 75 to 15 s (Fig. 1c). Clusters appear to coalesce more rapidly and
remove from cells, and have limited temporal and spatial remain fixed in the nucleus, compared with the cytoplasm (Fig. 1a
resolution. and Supplementary Movie 1), perhaps due to interaction
Emerging optical tools allow much finer control and observa- with nuclear components15. Clustering is light dose-dependent
tion of cellular processes using light, an actuator that can be and maximally induced with only a 6 ms 488 nm light pulse at
delivered immediately and with subcellular spatial resolution. 5% laser power, conditions much lower than those typically
Optogenetic actuators allow light control of specific processes used to image green fluorescent protein (GFP) (Fig. 1d). After
such as neuronal firing6,7, but more general tools have also been clusters form with light, they reversibly dissociate in the dark but
developed to manipulate protein transcription, protein–protein can be restimulated by a second light application (Fig. 1e). The
interactions and a variety of other cellular biochemical events8. In half-life of dissociation from clusters (t½ ¼ 23.1 min) is longer
previous work, we developed one such tool, a system using the than that reported for wild-type CRY2 (t½B6 min)17,18,
Arabidopsis flavoprotein cryptochrome 2 (CRY2) and its consistent with our identification of CRY2olig as a mutant with
interacting partner CIB1 to inducibly control protein a longer signalling state.
interactions with light9. While these proteins do not interact in To directly compare clustering of CRY2olig with wild-type
the dark, blue light absorption triggers a conformational change protein (CRY2PHR, containing the photosensory domain), we
in CRY2, allowing binding to CIB1 that is reversible after several tested clustering under identical conditions in HEK293 cells
minutes (t½B5.5 min). When attached to diverse target proteins, (Supplementary Fig. 2 and Supplementary Movie 2). In the dark,
the CRY2 and CIB1 modules allow tight light regulation of target the proteins showed similar low levels of clustering (o1% of total
protein activity9–13. protein clustered), with the majority of clusters forming in cells
Wild-type CRY2 clusters in nuclear bodies in the nucleus of where the proteins were greatly overexpressed or concentrated
plant and animal cells with light14–16, and light-dependent (data not shown). In light, while only a small fraction (6±3%) of
clustering of CRY2 has also been adapted for light control, cytosolic protein in relatively few (12±7%) cells expressing
where it has been used to stimulate17 or inhibit18 protein CRY2PHR–mCh clustered (o1% of total cytosolic protein),
function. While clustering appears to be a broadly useful B40–90% of cytosolic CRY2olig–mCh redistributed into clusters
approach to inducibly modulate cell function, wild-type CRY2 in 100% of cells illuminated (B70% of total cytosolic protein;
clusters poorly on its own, requiring high local protein Supplementary Fig. 2a,b). Thus, the E490G mutation enhances
concentrations or association with a multivalent protein clustering in the cell population by nearly two orders of
partner18 to achieve robust clustering. Here we describe a new magnitude. The enhanced clustering capability of CRY2olig was
CRY2-based optogenetic module, ‘CRY2olig’, that can be used not due to enhanced expression: all cells expressing CRY2olig,
on its own to probe and detect protein interactions in live cells, even at very low levels, showed clustering (Supplementary
as well as to perturb protein function with light. When Fig. 2c). While it is possible that CRY2PHR forms smaller
stimulated by a pulse of light, CRY2olig undergoes rapid, oligomers with light that cannot be resolved, these results indicate
reversible, and robust clustering within seconds, redistributing a that the E490G mutation is a much more potent clustering
majority of cytosolic protein into clusters in every cell module with enhanced dynamic range that opens up new
illuminated. While wild-type CRY2 also clusters under certain possibilities for optogenetic applications.
conditions17, clustering of CRY2olig is dramatically enhanced, As our initial characterization of the E490G mutant (Fig. 1)
enabling an entirely new set of experimental approaches for was in the truncated CRY2PHR domain, we also tested full-length
probing cellular biochemistry that cannot be achieved with CRY2 with the E490G mutation (CRY2oligFL). CRY2oligFL
existing tools. In the manuscript, we describe a new optical clusters with similar kinetics, but forms smaller puncta with less
approach, Light-Induced Co-clustering (‘LINC’), using total protein redistributed (Supplementary Fig. 3a,b and
CRY2olig to query protein–protein interactions. We Supplementary Movie 3), suggesting that the C-terminal exten-
demonstrate the use of this approach to study protein sion may restrict self-association. A CRY2olig–GFP fusion also
interaction dynamics in live cells, as well as to query clustered, as did untagged CRY2olig coexpressed with GFP–
interactions in situ within small (sub-micrometer) intracellular CIBN, a light-dependent interaction partner of CRY2 (refs 9,19)
domains. In addition to use querying protein interactions, we (Supplementary Fig. 3c), consistent with prior results showing
also demonstrate that CRY2olig provides a powerful tool to CIB interacts with wild-type CRY2 clusters17,18. While we did not
transiently and reversibly control protein function with light. As observe robust clustering of wild-type CRY2PHR–mCh on its
a proof-of-principle demonstration of use, we apply this tool to own, CRY2PHR–mCh did strongly cluster when coexpressed
optically disrupt clathrin-mediated endocytosis and stimulate with CRY2olig–GFP (Supplementary Fig. 3d), indicating it could
Arp2/3-mediated actin polymerization in live cells. These be incorporated into CRY2olig clusters. Taken together, these
general optogenetic approaches are modular and can be used results indicate that CRY2olig is sufficient to induce clustering by
with a wide variety of target proteins, providing a powerful itself and that interacting proteins can be recruited into clusters.
means to probe protein function within living cells. As with the CRY2/CIB interaction9, CRY2olig clustering could be

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5925 ARTICLE

a CRY2olig–mCh (post 488 nm pulse)

Pre light 10 s 20 s 30 s 40 s 50 s

20 Before
50 s post
16

Fluorescence
intensity (a.u.)
12
8
4

0 4 8 12 16
Distance (µm)

b c d e
100 100

% Max clustering
60

clustering time (s)

80 80
Half-maximal
80
% Protein
clustered

% Protein
clustered
60 60 40
60
40 40 40 20
20 20 20 0
0 3 6 9 0 3 6 9 0 10 20 30 40 0 40 80 120 160
Fluorescence Fluorescence Illumination time Time (min)
intensity (a.u.) intensity (a.u.) (ms)

Figure 1 | CRY2olig undergoes rapid clustering with light. (a) HEK293 cells expressing CRY2olig–mCh pre and post blue light (25 ms pulse, 488 nm, 5%
laser power). Graph at right shows the relative fluorescence intensity under the line in light versus dark. Scale bar, 7.5 mm. (b) Percentage of cytosolic
CRY2olig–mCh depleted into clusters after light (25 ms pulse, 488 nm) correlated with mCh expression level (a.u.). (c) Graph showing dependency of
half-maximal clustering time of CRY2olig–mCh on cellular expression level. (d) Light dose dependence. HEK293 cells expressing CRY2olig–mCh were
exposed to 2.5–40 ms pulses of blue light (5% laser power), allowed to cluster, then illuminated with a saturating light pulse to induce maximal clustering.
(e) Clustering recovery quantification. HEK293 cells expressing CRY2olig–mCh were exposed to light at time t ¼ 0 (first arrow), followed by a dark
incubation for indicated times in the presence of 35 mg ml  1 cycloheximide. A second pulse of light applied after recovery at 160 min (second arrow)
induced re-clustering.

stimulated by two-photon excitation at 850 nm (Supplementary even CRY2PHR resulted in substantial pre-clustering in the dark
Fig. 3e), making the technology amenable to in vivo or tissue slice (data not shown), likely because CaMKII forms dodecamers that
preparations. Finally, CRY2olig was not toxic to COS-7 cells or increase the propensity for CRY2 self-association. As an alternate
within neurons (Supplementary Fig. 4). means to cluster CaMKII, we tethered CaMKII to CIBN, which
can be recruited into CRY2olig clusters. When coexpressed with
CRY2olig and CaM–yellow fluorescent protein (CaM–YFP) and
Visualization of protein interaction dynamics. As photo- illuminated with blue light, CIBN–mCh–CaMKIIa clustered, but
activation clusters both CRY2olig and any interacting protein, we very little CaM–YFP colocalized (Fig. 2d, post 488 nm).Addition
considered use of CRY2olig to probe protein–protein interac- of the Ca2 þ ionophore ionomycin prior to blue light clustering
tions. Such a strategy, in which an interaction is assessed by resulted in robust colocalization of CaM–YFP with CaMKII
inducing redistribution of one protein, and querying whether a clusters. In addition to use with overexpressed FP-tagged
second protein also redistributes, was used previously20,21. proteins, we also show that LINC can be combined with
However, while previous approaches require chemicals (or immunofluorescent staining to detect endogenous CaM protein
chemicals plus light) to induce redistribution, our ‘LINC’ assay co-clustering with CaMKII (Supplementary Fig. 6).
requires only light. As shown in Fig. 2a, a CRY2olig-tagged ‘bait’
protein and a fluorescent-tagged (FP-tagged) ‘prey’ are
coexpressed. The prey is imaged prior to and post blue light LINC–FRAP assay to query interactions at compact sites.
application, which clusters the CRY2olig-bait. The prey co- While many assays can probe interactions in heterologous cells,
clusters with the bait only if the proteins interact. Figure 2b shows fewer allow study of protein interaction dynamics in situ. To
a proof-of-concept LINC experiment, demonstrating co- interrogate protein interactions in compact macromolecular
clustering of mCh and GFP-tagged versions of homer1c, a structures (such as synapses, adherens junctions and kine-
synaptic protein that homodimerizes22. In contrast, homer1c tochores) where resident proteins already appear punctate, we
does not co-cluster with postsynaptic density protein 95 (PSD95), extended LINC for use with Fluorescence Recovery After Pho-
another synaptic protein that does not interact (Fig. 2c). LINC tobleaching (FRAP)25. In ‘LINC–FRAP’, we also coexpress a
also works with membrane proteins, which are difficult to analyze CRY2olig-tagged bait and FP-tagged prey, but rather than
by traditional methods, as demonstrated by co-clustering of assessing a change in localization of the FP–prey with light, we
stargazin and PSD95, two interacting membrane-associated use FRAP to monitor the exchange rate and mobile fraction of the
proteins23 (Supplementary Fig. 5 and Supplementary Movie 4). FP-tagged protein. The premise is that blue light will crosslink the
One advantage of LINC is that light may be applied at any CRY2olig-tagged bait, restricting its exchange, but also restricting
time, allowing visualization of dynamic changes in interactions in the exchange of any interacting protein, which is assessed before
response to a stimulus. To examine this, we visualized the and after exposure to blue light (Fig. 3a).
interaction between Ca2 þ /calmodulin-dependent protein kinase We used LINC–FRAP to examine interactions within the PSD
II (CaMKII) and calmodulin (CaM), which is dependent on of neuronal synapses, small subcellular regions with an average
Ca2 þ (ref. 24; Fig. 2d). Initial fusions of CaMKIIa to CRY2olig or diameter near the diffraction limit of light (B0.1–0.5 mm)

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a b c
Bait (mCh–CRY2olig-tag)
Pre light 60 s Post Pre light 60 s post
Prey (GFP-tag)

PSD95–GFP
Homer-GFP
No interaction

light Only red clusters

CRY2olig
CRY2olig
Homer–
Interaction

Homer-
–mCh

-mCh
Red and green clusters form

d CIBN–mCh
–CaMKII CaM–YFP Merge

CIBN–CaMKII Pre
488 nm

t
Ligh
CRY2olig Post
Lig
ht 488 nm
Ca2+
CaM
Post+
Ionomycin

Figure 2 | LINC co-clustering assay to detect protein interactions. (a) Schematic describing LINC. (b) Positive control. COS-7 cells expressing
homer1c–GFP co-cluster with homer1c–CRY2olig–mCh with blue light (50 ms pulse, 488 nm). Scale bar, 7.5 mm. (c) Negative control. PSD95–GFP does not
co-cluster with homer1c–CRY2olig–mCh. Scale bar, 7.5 mm. (d) Assessment of dynamic interactions between CaMKII and CaM in response to Ca2 þ .
COS-7 cells expressing CIBN–mCh–CaMKII, CRY2olig and CaM–YFP were imaged pre and post blue light exposure (500 ms pulse, 488 nm). At resting
Ca2 þ , only a small amount of CaM–YFP colocalizes with CaMKII clusters. Addition of ionomycin elevates intracellular Ca2 þ , resulting in increased
CaM–YFP localized to clusters. Scale bar, 5 mm.

(Fig. 3). We first established that CRY2olig–mCh–homer1c clathrin function27. We reasoned that tethering CLC to CRY2olig
rapidly exchanges at synapses before blue light exposure, but would allow conditional disruption of endocytosis with light
exchange is restricted after blue light (Fig. 3b). When CRY2olig– (Fig. 4a). To assay endocytosis, we measured uptake of
homer1c is coexpressed with DsRed–homer1c, DsRed–homer1c transferrin, which is internalized via clathrin-dependent
also exchanges rapidly prior to blue light. Rate of exchange at endocytosis28. In COS-7 cells, CRY2olig–mCh–CLC clustered
synapses is similar to that previously observed with an enhanced with light and showed a B60% reduction in transferrin uptake in
GFP (EGFP)-tagged homer1c26. After blue light exposure, light compared with dark or untransfected cells (Fig. 4b–d).
exchange of DsRed–homer1c is severely limited, indicating Disruption of endocytosis was reversible, as cells exposed to blue
interaction (Fig. 3c). In contrast, clustering of CRY2olig– light followed by a 3 h dark incubation showed only a slight
homer1c does not affect the mobile fraction of PSD95–mCh, reduction in uptake (Fig. 4d). Using total internal reflection
and clustering of PSD95–CRY2olig does not affect the mobile fluorescence (TIRF) microscopy, we visualized clathrin pit
fraction of DsRed–homer1c (Fig. 3c), indicating that clustering dynamics at the plasma membrane. Prior to blue light
one PSD resident protein does not generally restrict exchange of treatment, CRY2olig–CLC puncta colocalized with endocytic
all PSD proteins. As interactions can be queried at different times, markers (Supplementary Fig. 7) and underwent similar levels of
this method also enables study of interaction changes after a dynamic events (appearing or disappearing from the surface) as a
stimulus. These results demonstrate that LINC–FRAP will be a DsRed–CLC control (Fig. 4e). With light, we saw a reduction in
powerful tool to study the protein interaction landscape within dynamic events, similar to what was previously observed with
compact subcellular domains in living cells. chemical crosslinking27. The overall fluorescence intensity of
clathrin pits also increased dramatically after light exposure
(Fig. 4f,g), suggesting that cytosolic CRY2olig–CLC subunits were
Transient perturbation of protein function using CRY2olig. recruited to CRY2olig–CLC within clathrin pits, disrupting the
Given the dramatic redistribution of CRY2olig with light, we precise architecture of these structures.
hypothesized that CRY2olig could be used not only to detect We next examined whether CRY2olig could also be used to
protein interactions, but also to acutely perturb them. While we induce processes that are naturally stimulated by clustering. In
were preparing this manuscript, another group independently one such example, antibody-mediated clustering of Nck SH3
demonstrated the use of CRY2 clustering to disrupt activity18, but domains resulted in localized induction of actin polymerization29.
the authors were unable to directly cluster targets fused to wild- Using a similar approach, we fused the three SH3 domains of Nck
type CRY2: clustering was induced by recruiting wild-type CRY2 to CRY2olig to generate CRY2olig–mCh–Nck, which showed
to a second CIB-fused multivalent protein. In our case, we diffuse localization in the dark but clustered with blue light
directly fuse CRY2olig to a target protein to allow inducible loss (Fig. 5a,b). Actin clusters that colocalized with Nck clusters
of function. As a proof-of-principle, we tested clathrin light chain formed within seconds after light exposure (Fig. 5b and
(CLC), involved in endocytosis. Previously, crosslinking CLC Supplementary Movie 5), as did clusters of Neuronal Wiskott–
using chemical dimerizers resulted in a B70% inhibition of Aldrich Syndrome protein (N-WASP) and N-WASP interacting

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a x
CRY2olig–fused

x
bait
PSD
FP–fused prey

Blue light

b Pre-bleach Post-bleach 15 min post Pre

Pre-Blue
Post
CRY2olig–mCh–homer1c

CRY2olig–mCh–homer1c
1.0
Pre Post
0.6
Post-Blue

0.2

0 5 10 15
Time (min)

c CRY2olig–homer1c CRY2olig–homer1c PSD95–GFP–CRY2olig

+dsred–homer1c +PSD95–mCh +dsred-homer1c

1.0 Pre Post 1.0

1.0
F/F0

F/F0

0.6 0.6

F/F0
0.6

0.2 0.2 0.2

0 10 20 0 10 20 30 0 4 8 12
Time (min) Time (min) Time (min)

Figure 3 | LINC–FRAP assay. (a) Schematic describing use of LINC–FRAP in synapses. Mobility of a CRY2olig-fused protein (blue) is restricted after
light-induced clustering, which restricts the mobility of an interacting FP-tagged protein (red). (b) LINC–FRAP control experiments in primary neuronal
cultures showing exchange of CRY2olig–mCh–homer1c is delayed at the indicated photobleached synapse (arrowhead) after blue light clustering. (c) LINC–
FRAP quantification before and after blue light, testing homer/PSD95 and homer/homer interactions at synapses.

protein, involved in Nck-mediated actin polymerization30,31 researchers, and is relatively quick and easy to perform. The
(Supplementary Fig. 8). In some cases, actin polymers could be clustering phenotype is robust, thus interactions are scored
observed that spanned NCK clusters (inset, Fig. 5b). With without complex image analysis. As many labs already have
prolonged light (41 h), COS-7 cells expressing CRY2olig–Nck numerous FP-tagged proteins available for use with other studies,
began to round up and lose their membrane extensions. To these can be readily used as preys. Finally, protein interactions
determine if local actin reorganization due to Nck clustering can be queried at user-defined timepoints in individual cells,
induced this phenotype, possibly by disrupting focal adhesion– allowing determination of how protein interactions change over
actin connections, we locally photostimulated CRY2olig–Nck in time in response to a stimulus or inhibitory drug.
cell extensions (Fig. 5c and Supplementary Movie 6), which The LINC–FRAP approach, used for probing interactions at
triggered retraction. To test the generality of this approach, we compact intracellular sites, also provides advantages over
induced clustering of a completely different target, the Verprolin- previous methods. A similar FRAP approach was previously
homology, Central, Acidic (VCA) domain of N-WASP. used to query interactions33,34, however, that method used
Oligomerization of VCA was previously shown to greatly antibodies to immobilize the bait proteins, and thus could only be
stimulate the actin nucleating activity of Arp2/3 (ref. 32). Upon used with transmembrane proteins. LINC–FRAP simplifies this
light application, we observed rapid formation of CRY2olig– approach (as light is used both to induce clustering and monitor
mCh–VCA clusters that colocalized with GFP–actin (Fig. 5d,e mobility), and extends its use to intracellular proteins. The most
and Supplementary Movie 7) and could not be reproduced by versatile and commonly used technology for probing interactions
substituting CRY2PHR (Supplementary Fig. 9). in vivo at compact sites is Förster Resonance Energy Transfer
(FRET), a powerful approach, but one that can be difficult to
optimize for specific molecules and difficult to interpret since
Discussion fluorophore crosstalk and differences in stoichiometry between
Here we introduce CRY2olig, a modular genetically encoded labelled proteins can greatly influence outputs35. In contrast,
protein tag that can be used to inducibly trigger oligomerization LINC–FRAP requires little optimization. While LINC–FRAP
of fused protein targets with light. We envision two broad uses for does not provide a continuous readout of protein interaction
this tool in live cells: interrogating protein–protein interactions dynamics such as can be achieved with FRET, by applying light at
and perturbing protein function. As a tool for querying protein– different times after a stimulus or treatment, changes in these
protein interactions, the LINC technology has a number of interactions over time can be monitored.
advantages. LINC requires no major equipment beyond a While LINC and LINC–FRAP have a number of advantages, we
fluorescent microscope, an instrument accessible to most note that the technologies do not discriminate between direct and

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a b
Dark Light Dark Light

CRY2olig-mCh-CLC

c d Dark
CRY2olig– Transferrin– ** Light
mCh–CLC Alexa488 40

Relative transferrin
fluorescence (a.u.)
Light/3 h dark
30

20

10

Mock CRY2-CLC

e f g
80 ** CRY2olig–mCh–CLC
% Dynamic pits

Time pre/post 488 nm pulse (s)

1 min
60

488 nm
–36 –33 –30 –24 –18 –12
40
Pre
20
Post
0
Dark Light
DsRed-
3 6 12 18 24 30
CRY2olig–CLC CLC

Figure 4 | Light-mediated disruption of clathrin-dependent endocytosis. (a) Schematic indicating light-mediated disruption of CLC function. (b) COS-7
cells expressing CRY2olig–mCh–CLC before and after blue light. Scale bar, 7.5 mm. (c) COS-7 cell expressing CRY2olig–mCh–CLC (inside dashed line) after
exposure to blue light is defective for uptake of Alexa 488-transferrin (green), while surrounding untransfected cells show efficient uptake. Scale bar,
7.5 mm. (d) Quantification of transferrin uptake in untransfected (mock) versus CRY2olig–CLC-expressing cells exposed to dark, blue light or light/3 h dark.
Data represents average and s.e.m., n ¼ 15. **Po0.01. (e) Quantification of clathrin-coated pit dynamics within cells expressing CRY2olig–mCh–CLC in dark
or after blue light (200 ms pulse, 488 nm), compared with a DsRed–CLC control. Data represents average and s.e.m., n ¼ 3 cells (over 30 clusters per cell
counted). **Po0.01. (f) TIRF images of COS-7 cells expressing CRY2olig–mCh–CLC, showing clathrin pits at the cell surface before and after blue light
exposure (488 nm, 350 ms). Arrow indicates mobile clathrin pit undergoing endocytosis. (g) Kymograph of CRY2olig–mCh–CLC-labeled clathrin pit
showing increase in fluorescence after light exposure.

indirect protein–protein interactions. Co-clustering or co-immo- In addition to disrupting protein function, we also show
bilization can occur either between direct binding partners, or CRY2olig efficiently induces oligomeric-dependent processes. We
through an intermediate partner. In this sense, the technology is demonstrate this by manipulating actin polymerization, a process
not different from co-immunoprecipitation or yeast two-hybrid, in known to be stimulated by clustering of Nck SH3 domains or the
which non-direct interactions are also a possibility. We also note VCA domain of N-WASP. As we show in Fig. 5c, we can focally
that LINC does not allow verification of subcellular localization of deliver light to induce clustering in a small region of the cell, thus
interactions, as localization can potentially be altered by clustering. enabling subcellular control of oligomeric-dependent processes.
Finally, while this method as it stands is amenable to high- CRY2olig has advantages over other clustering tools such as
throughput screening using automated microscopy systems, we antibody-mediated clustering29,36 or chemical inducers of
have not tested the use of this method with fluorescence-activated dimerization37, which require addition of chemicals or
cell sorting-based sorting or other selection schemes. antibodies, have no spatial control and limited temporal
While we expect LINC and LINC–FRAP to be useful control, and in the case of antibodies, can only be used with
technologies, perhaps an even more powerful application of cell surface proteins. We also find CRY2olig to be a much more
CRY2olig is the ability to control cellular function with light using potent inducer of clustering than wild-type CRY2, which has been
a single, genetically encoded tag. While a number of different utilized to confer light control over signalling pathways
optical dimerizer systems for controlling the interactions of two stimulated by oligomerization17. For example, while we could
proteins have emerged (see ref. 8 for a review), there remains an induce actin reorganization with CRY2olig–VCA, we were unable
unmet need for optical tools to rapidly disrupt protein function to accomplish the same result using CRY2PHR–VCA
that can be applied to any target. Because clustering triggered by (Supplementary Fig. 9). Adding to its versatility, clustered
CRY2olig is reversible, protein activity can be perturbed for CRY2olig retains interaction with CIB1, a light-dependent
precise user-defined periods of time. As light can be focally binding partner of wild-type CRY2, and thus can be used along
delivered to specific cells or even subcellular regions, CRY2olig with the CRY2/CIB system9 for more complex control.
can enable entirely new studies into spatial requirements of While further studies are needed to address the precise
protein activity. For example, the CRY2olig–CLC tool may be mechanism of CRY2olig clustering—as CRY2 can dimerize in
used to study the consequences of blocking endocytosis at specific the dark, we hypothesize that light may induce a multivalent state
times within specific subcellular regions. and trigger a sol-gel phase transition38—ultimately, the strategies

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5925 ARTICLE

a b CRY2olig–
CRY2olig Nck SH3 mCh–Nck GFP–actin Merge

mCh

Dark
Light

(60 min post)

Light
N-WASP/Arp2/3-
mediated
actin polymerization

c Post 488 nm (min) ***

70
Pre light 1 12 27

% Retraction
50
NS
30

10
CRY2olig CRY2– mCh
–Nck olig
d CRY2olig– e
mCh–VCA GFP–Actin Merge
Pre Post
Light Light
Dark
(6 min post)
Light

mCh–Actin

Figure 5 | Induction of actin cytoskeletal changes using CRY2olig. (a) Strategy for clustering Nck SH3 domains. (b) Cells expressing CRY2olig–mCh–Nck
and GFP–actin in dark, or 60 min post blue light (500 ms pulse, 488 nm, every 3 min). Scale bar, 20 mm. (c) Local photostimulation (within circle) of COS-7
cell expressing CRY2olig–mCh–Nck results in retraction of cell extension. Graph at right shows quantification of retraction (average and s.e.m., n ¼ 16) in
cells expressing CRY2olig–mCh–Nck, or controls CRY2olig–mCh or mCherryN1 45 min post initial light exposure. ***Po0.001. NS, not significant. (d) Cells
expressing CRY2olig–mCh–VCA, CRY2olig and GFP–actin in dark or 6 min post blue light (500 ms pulse, 488 nm, every 3 min). Inset images at right show
detail within white square. Scale bar, 20 mm. (e) Stress fibres within cells expressing CRY2olig–GFP–VCA, CRY2olig and mCherry–actin are disrupted with
light exposure.

outlined here can be applied to any number of diverse targets to ACC CCG TGC TGC TCC GAT CAT GAT CTG TGC TCC ACG GGT TCT T
disrupt or stimulate protein activities with light, and to probe a G-30 ). The construct expressing CRY2olig (without a fluorescent tag) was generated
by placing a stop codon at the end of the CRY2 domain of CRY2olig–mCh, before
variety of complex protein interactions. We anticipate that mCh. CRY2olig–GFP was generated by cloning CRY2olig (containing M354I) at
CRY2olig may be used in diverse ways to organize protein XhoI—XmaI sites9 into EGFP–N1 (Clontech). CIBN–GFP was generated by
complexes within cells, for example, to assemble synthetic replacing the GFP–CAAX sequence in CIBN–pmGFP9 with EGFP. CRY2oligFL–
scaffolds of metabolic enzymes, allowing efficient substrate mCh was generated by PCR-based mutagenesis of CRY2–mCh (ref. 9) to add
channelling through a metabolic pathway39,40. Given the E490G. CRY2olig–mCh–CLC was generated by cloning CLC into a version of
CRY2olig–mCh, containing residues CRY2 (with M354I) at BsrGI and NotI sites
growing recognition of the importance of scaffolding and (50 -CGG TGT GTA CAA GTC CGG TGG AAT GGC CGA GTT GGA TCC ATT
compartmentalization in biology41–43, tools such as CRY2olig C-30 ) (50 -GGA CCT GCG GCC GCT TAG TGC ACC AGA GGG GCC TG-30 ).
allowing rapid, reversible assembly or disruption of multi-protein CRY2olig–mCh–Nck and CRY2olig–mCh–VCA were generated by amplifying
complexes will enable a wealth of new studies. either the SH3 domains of Nck (residues 1–258) (50 -GACG AGC TGT ACA AGG
GAT CCA CCA TGG CAG AAG-30 ) (50 -GAGTC GCG GCC GCT CAT TAA GC
GTA ATC CGG AAC ATC GTA TGG GTA GGT ACC ACC TGA AGT TAA
Methods TGG ATT ATT CTG-30 ) or the VCA domain of N-WASP (residues 388–501)
Generation of constructs. CRY2olig consists of CRY2PHR (residues 1–498 of (50 -CGG CGG CAT GGA CGA GC TGT ACA AGG GAT CCC CTT CTG ATG
CRY2) containing a E490G mutation. Some constructs (noted below) also contain GTG ACC ATC-30 ) (50 -CTAGA GTC GCG GCC GCT CAT TAA GCG TAA TCC
a second M354I mutation originally found with E490G, which does not appear to GGA ACA TCG TAT GGG TAG GTA CCG TCT TCC CAC TCA TCA TCAT
affect clustering or photoresponse. CRY2olig–mCh was generated by generating a C-30 ) by PCR to add BsrGI and NotI restriction sites, then ligating into CRY2olig–
E490G mutation in CRY2PHR–mCh9, containing CRY2PHR in the pmCherryN1 mCh (containing M354I) cut with BsrGI and NotI. CRY2olig–mCh–CaMKIIa was
vector backbone (Clontech), using PCR with mutagenic oligos (50 -CTTGG CTC cloned by digesting CRY2olig–mCh with NheI and BsrGI and ligating into a
GAG GCC ACC ATG AAG ATG GAC AAA AAG ACT ATA GTT-30 ) (50 -TCG pEGFP (Clontech) backbone containing CaMKIIa. CIBN–mCh–CaMKII was

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5925

generated by replacing CRY2olig in CRY2olig–mCh–CaMKII with CIBN at AgeI after transfection, neurons were either kept in the dark or exposed to light (1 s
and NheI sites. DsRed–homer1c was generated by cloning homer1c at BsrGI and pulse, every 1 min, 461 nm, 1.1 mW cm  2) for 3 min, then treated 24 h later with
NotI sites into a vector containing the pcDNA3.1 backbone with dsRed inserted at PI and quantified as described with COS-7 cells. All animal procedures were
the MCS. CRY2olig–mCh–homer1c was generated by cloning homer1c into performed in accordance with the University of Colorado School of Medicine
CRY2olig–mCh (containing M354I) using BsrGI and NotI sites. PSD95–mCh was guidelines.
generated by cloning PSD95 into HindIII and EcoRI sites of mCherryN1
(Clontech). PSD95–mCh–CRY2olig was generated by amplifying CRY2olig
(containing M354I) by PCR and cloning into PSD95–mCh at BsrGI and NotI sites LINC protein interaction studies. LINC studies were carried out in COS-7 cells
(50 -GGT TAA TGT ACA GTG CTG GTA GTG CTG GTA GTG CTG GTA TGA transiently transfected with indicated constructs. Cells were imaged before blue
AGA TGG ACA AAA AGACT-30 ) (50 -AAT TAA GCG GCCGC TCA TGC TGC light exposure (with the ‘Dark’ GFP image initiating CRY2olig photoactivation)
TCC GAT CAT GAT CTG-30 ). Dynamin–mCherryN1 was obtained from then every 10 s afterwards. COS-7 cells were transiently transfected with CIBN–
Christien Merrifield (Addgene, plasmid 27697). TfR–SEP was described mCh–CaMKII, unlabelled CRY2olig and CaM–YFP, and CaMKII clustering was
previously44. induced by recruitment of CIBN into CRY2olig clusters. For ionomycin experi-
ments, cells were incubated with 2 mM of ionomycin Ca2 þ salt for 1 min before
light-induced clustering. For LINC–FRAP analysis at synapses, primary hippo-
Yeast two-hybrid experiments. GalBD plasmids containing CRY2PHR, a CRY2 campal neurons were prepared as described in ‘Neuronal toxicity studies’. Pho-
mutagenic library (generated by error-prone PCR), or CRY2olig along with a tobleaching of individual synapses was carried out using galvometric steered laser
pGADT7rec-CIB1 plasmid9 were expressed in strain MaV203 (MATa leu2-3,112 excitation (FRAPPA, Andor) at a single spot for 1 ms. Photobleaching pulses were
trp-901 his3-200 ade2-1 gal4D gal80D SPAL10-URA3 GAL1-lacZ. LYS2::GAL1- calibrated so that they bleached no more than 50–75% of the original signal.
HIS3 can1R cyh2R). Yeast were grown at 30 °C for 72 h on SD–Trp/–Leu/–Ura Recovery was monitored following photobleaching every 15–60 s (depending on
under indicated light conditions (Dark; frequent light (1 s pulse every 3 min); the recovery kinetics of the molecule under investigation). FRAP recovery rates
Infrequent light (1 s pulse every 20 min). Illumination was applied using a 461 nm, were determined for different sets of synapses from the same neuron before and
1.1 mW cm  2 light-emitting diode array. after blue light exposure. Quantification was performed using ImageJ.

Live cell imaging. HEK293 or COS-7 cells were cultured in Dulbecco’s Modified Immunocytochemistry. COS-7 cells grown on coverslips were transiently trans-
Eagle’s Medium (DMEM) with 10% foetal bovine serum, then seeded onto 18-mm fected with CIBN–mCh–CaMKIIa and CRY2olig, then incubated the next day with
coverslips or 35-mm glass bottom dishes (Mattek) in 12-well plates and transfected 2 mM ionomycin for 1 min. Samples were either maintained in the dark or treated
using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s with blue light for 5 min (1 s pulse every 1 min, 461 nm, 1.1 mW cm  2). Cells were
protocol. Cells were incubated in dark (wrapped with foil) and manipulations were fixed with 4% paraformaldehyde for 10 min, permeabilized and blocked (PBS, 5%
carried out under dim light or using a red safelight. Twelve to twenty-four hours normal goat serum, and 0.1% triton X-100) for 1 h, then incubated with an
after transfection, cells were moved to Hepes-buffered Saline (HBS) with 1 mM a-calmodulin monoclonal antibody (1:50, 2D1, Pierce) followed by an a-mouse
CaCl2 for imaging studies. Live cell imaging was performed at 33.5 °C on an IgG AlexaFluor-488 secondary antibody (1:1,000, Jackson ImmunoResearch).
Olympus IX71 microscope equipped with a spinning disc scan head (Yokogawa
Corporation of America) with a  60/NA 1.4 objective. Excitation illumination
was delivered from an AOTF-controlled laser launch (Andor Technology) and Endocytosis studies. COS-7 cells were seeded onto 18-mm coverslips, transfected
images were collected on a 1,024  1,024 pixel EM-CCD camera (iXon; Andor with CRY2olig–mCh–CLC using Lipofectamine 2000, and incubated in the dark.
Technology). For photoactivation studies (Fig. 5c), laser illumination (488 nm) was Twenty-four hours after transfection, samples were either maintained in the dark
delivered using galvometric laser-scanning mirrors (FRAPPA; Andor Technology) or treated with blue light for 10 min (1 s pulse, every 1 min, 461 nm,
at 1–2% of maximum power with a 100 ms dwell time. Two-photon excitation was 1.1 mW cm  2), followed by incubation with 50 mg ml  1 transferrin-AlexaFluor-
performed on a Zeiss LSM510 microscope containing a Chamelon Ultra II laser 488 (Life Technologies) for 10 min. Cells tested for dark recovery were exposed to
(Coherent) tuned to 850 nm and 60% power with a 50 ms pixel dwell time. For 10 min of blue light pulses, then incubated in the dark for 3 h before transferrin
light-sensitive experiments, cells were protected from ambient light sources by labelling. After transferrin labelling, coverslips were washed in PBS and cells were
focusing in the presence of filtered light (572/28 bandpass filter, Chroma). fixed in 4% paraformaldehyde. For TIRF studies, we used an Olympus IX81
inverted motorized microscope equipped with laser illumination of 10 mW 488 nm
argon, 543 nm HeNe coupled to a manual TIRF illuminator. A temperature of
Image analysis. Data acquisition and analysis were performed using MetaMorph 34 °C was maintained throughout experiments. Images were recorded at 2 Hz on a
(Molecular Devices), ImageJ or Fiji. Per cent of protein in puncta was calculated by CCD camera [Orca ER, Hamamatsu Photonics) using Slidebook software.
first determining the total fluorescence within puncta using manual ImageJ
thresholding (Otsu’s method) to delineate protein within puncta contained with the
cell cytosol. The total fluorescence within puncta was divided by the total fluores- Actin reorganization studies. For global activation and clustering of CRY2olig–
cence within each analyzed region. In some cases (Fig. 1e) to calculate protein in mCh–Nck and CRY2olig–mCh–VCA, CRY2olig was initally photoactivated by a
puncta, we also used ‘FindPeaks’ (http://www.sussex.ac.uk/gdsc/intranet/micro- 500 ms pulse of 488 nm laser illumination at 50% of maximum laser power, then
scopy/imagej/gdsc_plugins), an ImageJ plugin, with the following parameters: imaged every 3 min afterwards for mCh and GFP. For local photoactivation, a
background calculation, one s.d. above mean; autothreshold, Otsu method; mini- B15 mM-diameter region was photoactivated at the farthest tip of cell extensions
mum peak height relative above background, 0.10; minimum above saddle, 0.20. and the change in cell extension area was quantified after 45 min. With VCA
domain experiments, initial experiments using COS-7 cells expressing CRY2olig–
mCh–VCA showed no light-dependent clustering, possibly due to steric effects of
Comparison studies of CRY2PHR–mCh and CRY2olig–mCh. For comparison VCA fusions on CRY2 self-association. To circumvent this, we coexpressed
studies, we transiently transfected CRY2PHR–mCh or CRY2olig–mCh into CRY2olig–mCh–VCA along with unlabelled CRY2olig in a 1:1 ratio, so as to dilute
HEK293 cells, under identical conditions. After 24 h, we recorded time-lapse any steric issues caused by CRY2olig–mCh–VCA: CRY2olig–mCh–VCA associa-
movies at random-sampled locations on the coverslip, recording an initial mCh tion. This strategy allowed clustering and was used for all VCA experiments.
image, followed by a GFP image that stimulated CRY2, followed by a mCh image
series (every 30 s for 3 min). Fluorescence in puncta in dark and light were reported
for sampled areas. References
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