Optically Triggering Spatiotemporally Confined GPCR Activity in a Cell And

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Optically triggering spatiotemporally conﬁned GPCR PNAS PLUS activity in a cell and programming neurite initiation and extension

W. K. Ajith Karunarathnea, Lopamudra Giria, Vani Kalyanaramana, and N. Gautama,b,1

Departments of aAnesthesiology and bGenetics, Washington University School of Medicine, St. Louis, MO 63110

Edited* by Peter N. Devreotes, Johns Hopkins University School of Medicine, Baltimore, MD, and approved February 12, 2013 (received for review November 29, 2012) G-protein–coupled receptor (GPCR) activity gradients evoke impor- due to the known high light sensitivity, slow deactivation (4), and tant cell behavior but there is a dearth of methods to induce such rapid bleaching (9) of rhodopsin. asymmetric signaling in a cell. Here we achieved reversible, rapidly Opsins are optimally activated at wavelengths that span the switchable patterns of spatiotemporally restricted GPCR activity in entire visual spectrum (4). We developed a strategy to identify a single cell. We recruited properties of nonrhodopsin opsins—rapid opsins with sufficient spectral selectivity, such that the wave- deactivation, distinct spectral tuning, and resistance to bleaching— length used to globally image cellular and molecular responses to activate native Gi, Gq, or Gs signaling in selected regions of a cell. using fluorescent protein reporters did not interfere with the Optical inputs were designed to spatiotemporally control levels of localized optical activation of an opsin. Using this strategy, we second messengers, IP3, phosphatidylinositol (3,4,5)-triphosphate, developed a set of optical triggers based on nonrhodopsin opsins and cAMP in a cell. Spectrally selective imaging was accomplished that are capable of selectively modulating the activity of all three to simultaneously monitor optically evoked molecular and cellular major heterotrimeric G proteins—Gi/o, Gq, and Gs—in selected response dynamics. We show that localized optical activation of an opsin-based trigger can induce neurite initiation, phosphatidy- regions of a cell. linositol (3,4,5)-triphosphate increase, and actin remodeling. Serial We then examined whether these optical triggers could be CELL BIOLOGY optical inputs to neurite tips can refashion early neuron differen- used to achieve optical control over polarized cell behavior that tiation. Methods here can be widely applied to program GPCR- is regulated by GPCR activity. Because global activation of Gi/ mediated cell behaviors. o-coupled GPCRs induces neurite outgrowth (10), we examined whether neurite growth can be spatially controlled by optically lo- optogenetics | cell polarity calizing GPCR signaling in a cell. We found that optical activation of the opsin-based signaling triggers can achieve coordinated con- -protein–coupled receptors (GPCRs) initiate most of the trol over early neuron differentiation events. Gsignaling in metazoans and regulate a wide variety of cel- Results lular responses that include differentiation, migration, secretion, Screening Opsins for Spectral Selectivity. The three human color and contraction. Asymmetric activation of GPCR signaling ac- fi tivity in a cell is thought to play a critical role in varied processes opsins, blue, green, and red have been identi ed and charac- such as cell polarization (1) and modulation of neuron function terized biochemically (11, 12). These opsins absorb maximally at ∼ ∼ (2). There is still limited information about activation of sig- 414 nm (blue), 540 nm (green), and 560 nm (red) (11, 12) naling that is restricted in space and time across a single cell. An (Fig. 1A). However, their ability to function heterologously in an impediment is the lack of methods to continuously vary signal intact cell has not been examined. Color opsins are coupled to α input to a single cell with high time resolution and precision to the G-protein subunit, G tc in the cone photoreceptor cells of α quantitate second messenger and cellular output from the same the mammalian retina (13). Because G tc is homologous to and cell. A method that provides reversible, temporal control over falls in the Gi subfamily (14), we examined whether the human GPCR activity in restricted regions of a single cell may help govern cell behavior and probe the cellular and molecular basis Significance of single-cell responses. Here we used a set of light-triggered GPCRs, human color G-protein–coupled receptors control a variety of important cell opsins, and related nonrhodopsin opsins, to achieve such confined behaviors. However, tools are not available to activate these GPCR activation in a single cell. In contrast to molecular gra- receptors in selected areas of a cell and exert control over cell dients, an optical signal provides higher spatiotemporal control behavior. Here we recruit unique properties of nonrhodopsin and it can be switched on or off or relocated almost instan- opsins to activate all the major types of G-protein signaling in taneously. We recruited the properties of color opsins to develop spatially confined regions of single cells. We show that this optical triggers that spatiotemporally confine signaling. These approach can be used to optically induce polarized cell be- opsins deactivate rapidly and demonstrate relatively low sensi- havior and refashion early neuron differentiation. This optical tivity to light (3, 4). The deactivation characteristics curtail the approach can be applied to control other cell behaviors such as diffusion of activated receptors across a cell and help localize immune cell migration and cardiomyocyte contraction. receptor activation to selected regions of a cell. Low susceptibility Author contributions: W.K.A.K. and N.G. designed research; W.K.A.K. performed re- to bleaching allows continuous reproducible activation. In addi- search; W.K.A.K. and V.K. contributed new reagents/analytic tools; W.K.A.K. and L.G. tion, nonrhodopsin opsins selectively activate different G-protein analyzed data; and W.K.A.K., L.G., and N.G. wrote the paper. types, suggesting that they can be individually used to regulate The authors declare no conflict of interest. distinct second messengers (4, 5). Rhodopsin or its chimeric *This Direct Submission article had a prearranged editor. – forms have been valuable for globally activating G proteins (6 8). 1To whom correspondence should be addressed. E-mail: [email protected]. However, we found that these receptors do not allow spatial This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. control over G-protein activation in a single cell. This is likely 1073/pnas.1220697110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1220697110 PNAS Early Edition | 1of10 Downloaded by guest on September 29, 2021 A B Red Green Blue Jellyfish Melanopsin Select an opsin ~560nm ~540 ~414 ~500 ~480 445 nm 488 nm Choose a wavelength 515 nm 595 nm αi/ο β γ αi/ο β γ α i /ο β γ αs β γ αq β γ Set condions for imaging Intensity (I), exposure (E), Change repeats (N) wavelength C Before After Retinal YFP Image FP-γ9 Change imaging + Green Opsin +YFP γ9 translocaon in cells condions (I, E, N)

opsin 515 1.2 Red opsin bOpsin+mChγ9 expressing the opsin +CFP γ9 (without retinal) NO CFP 1.0 bOpsin+mChγ9 + (with retinal) Is γ9 0.8 translocaon YES Lowest YES 445 detectable ? I, E, and N? mCh 0.6

- 0.4 5 μW OI NO 595 445nm The opsin is spectrally selecve 0.2 under these opcal condions Normalized IM fluorescence

bOpsin Red opsin Green + 0.0 mCh 595 0 3 6 9 12 20 40 60 80 100 Time (s) E Optical Imaging D Single point laser beam Designing an opcal input activation response (Unit opcal input) λ1 λ2 optical input (OI) cell Intensity 2 10

Duraon = t Time OI area (mm) X 0.87 (ms/mm)

0 1 0 5 t = + 0 1 2 mm 0 5 10 mm imaging exposure time (ms) FWHM=0.8 mm F Before After 1.0 G

0.8 βγ 9

0.6 γ 9 fluorescence 0.4 βγ 9 fluorescence

0.2 Internal membrane change in internal membranes Fractional GFP- 0.0 Time taken to reach max fluorescence intensity (s) 0246 445 nm Beam energy ( μW) Time (s)

Fig. 1. Screening opsins for spectral selectivity and optimizing conditions for OIs and imaging. (A) Opsins used, their λ-max, and Gα-subtype specificity are shown. Individual opsins are described in the text. (B) Schematic for screening opsins for spectral selectivity. (C) Representative images of HeLa cells expressing green opsin and YFP-γ9 (green), red opsin and CFP-γ9 (blue), and bOpsin and mCh-γ9 (red). Cells were incubated with (+) or without (−) 11-cis retinal as indicated. For green opsin and red opsin, FP-γ9 distribution in the first (before) and last (after) images during image capture is shown. For bOpsin, images show mCh-γ9 distribution before and after optical activation (at 20 s after initiating image acquisition) with 445-nm, 5-μW optical inputs. Translocation of FP- βγ to intracellular membranes (IM) is plotted on the right (n = 8). bOpsin-expressing cells with retinal were activated with a single 5-μW pulse whereas cells without retinal did not show translocation even after optical activation with 30 pulses (n = 8). Here and in all optical activation experiments below, n values represent number of cells. (D) Designing an optical input (OI) for opsin activation. (Left) Single-point laser beam energy density profile of 445 nm, 5 μWatthe image plane. Experimentally a cell can be exposed to this optical input by selecting the crosshair tool (┼) as the ROI. (Right) Energy density profile of square- shaped OI area (example: 3 × 3 μm) of laser raster scan. The galvo mirrors scan the ROI at 0.87 ms/μm2 and the area of the OI determines the duration of a single pulse. (E) Optical activation at one specific wavelength and intensity (purple) is followed by imaging at a different wavelength. (F) A single HeLa cell coexpressing bOpsin-mCh and YFP-γ9 was optically activated by varying laser intensities (445 nm). Individual cells were optically activated using a single-pulse OI that covered the entire cell (energy of the OI in microwatts is indicated on the image). After 20 s the cell was imaged to capture YFP-γ9 distribution. The cell was allowed a 1-min recovery and tested at the next intensity. The plot shows fractional YFP-γ9 intensity changes in internal membranes. The red arrow shows the selected intensity (5 μW) for optical activation of bOpsin in experiments below (n = 7). (G) Magnitude and duration of γ9 translocation can be controlled by varying the number of pulses in HeLa cells expressing bOpsin and mCh-γ9. The cell was initially imaged for 10 s (baseline reference) and then activated with 1, 5, and 10 (1 pulse every 5 s) OI pulses (5 μW). mCh-γ9 distribution was continually imaged. Plot shows internal membrane fluorescence. Bar chart shows averaged mCh-γ9 fluorescence rise time in response to number of pulses (n =6).

color opsins, blue, green, and red, activate endogenous Gi sig- assay detects opsin-induced G-protein activation in real time in naling activity in HeLa cells. a living cell. Cellular and molecular responses to localized op- We developed an assay to identify opsins that are spectrally tical activation in a single cell could be imaged without activating selective. We used G-protein βγ complex translocation away the opsin globally. As shown in the results, βγ translocation from the plasma membrane to internal membranes on receptor served as a reporter of the location, onset, extent, and termi- activation and reversal on receptor deactivation (15–17). This nation of activation of an opsin. By varying wavelengths, fre-

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1220697110 Karunarathne et al. Downloaded by guest on September 29, 2021 quency of image capture, laser intensities, and exposure times, To achieve better control over spatially restricted signaling PNAS PLUS we examined opsins to identify those that are not activated by activity, we experimentally determined the extent of confinement the wavelength used for imaging the cells (Fig. 1B). Even under of GPCR activity that can be achieved using optical activation of minimal exposure and intensity conditions both green and red an opsin in a single cell. To do so, we exposed a confined region opsins were activated by 445-, 488-, 515-, and 594-nm lasers (Fig. of the plasma membrane of a bOpsin-expressing cell to an OI of 1C and Table S1). In contrast, it was possible to identify con- 3-μm width (Fig. S1B, purple box). We measured GFP-γ9 loss in ditions where imaging fluorescent proteins at 488 and 594 nm the plasma membrane 5 s after activation and found that this did not interfere with localized blue opsin (bOpsin) activation at follows a Gaussian distribution with a full width at half maximum 445 nm (Table S1). Thus, GFP (488 nm excitation) and mCherry (FWHM) = 6.3 (Fig. 2E). This indicates that an OI can induce (mCh) (595 nm excitation)-based sensors could be used to image confined activation with a steep gradient of decreasing activity at molecular responses to bOpsin activation (Fig. 1C). In the ab- the boundary. sence of 11-cis retinal Gβγ translocation was not detected (Fig. To examine whether the bOpsin was coupled to Gαi/o in HeLa 1C, Left and Right). Cells without opsin also did not provide cells we treated the cells with pertussis toxin. bOpsin activation aGβγ translocation response on optical activation but sub- in these cells failed to evoke Gβγ translocation after treatment sequent activation of CXCR4 receptors with SDF-1α in the same with pertussis toxin, indicating that bOpsin activates endogenous cells evokes βγ translocation (Fig. S1A). Gi/o in HeLa cells as anticipated (Fig. S2). The optical input (OI) to optimally activate bOpsin (Fig. 1C) We examined whether we can use these properties of the OI to was designed by varying the size of the OI area, the intensity of elicit a gradient of signaling activity by placing it outside a single fi the beam, pixel dwelling time, and pulse frequency (Fig. 1 D and cell. We rst located the OI at the periphery of a HeLa cell and βγ E). Fig. 1E shows that the time taken for a cycle of optical ac- found that it elicited localized FP- translocation (Fig. S3). Next tivation and image capture depends on the size of the OI area. we located the OI near the edge of RAW 264.7 cells expressing We chose a repeating-pulse OI over a continuous one to extend bOpsin and a phosphatidylinositol (3,4,5)-triphosphate (PIP3) the lifespan of an activated opsin. We titrated the intensity of sensor, Akt-PH-GFP (18) and assayed the appearance of PIP3. 445-nm OI on a single cell expressing bOpsin-mCh and YFP-γ9 PIP3 is known to be activated by Gi-coupled GPCRs in immune to determine the optimum intensity for optical activation. The cells and accumulates at the cell periphery, which senses the more results show that increasing the beam intensity of OIs in a single intense signal (19). The results showed localized PIP3 increase cell increases the magnitude of YFP-γ9 translocation that rea- on the plasma membrane adjacent to the OI (Fig. 2F). The plot fl CELL BIOLOGY ches saturation at ∼5 μW (Fig. 1F). We then conducted a similar shows normalized PIP3 sensor uorescence changes at the single-cell experiment at 5 μW and used a different number of proximal plasma membrane. These results show that an optical pulses (Fig. 1G). The results demonstrate that increasing the input located externally to a bOpsin-expressing cell can evoke an number of pulses increases both magnitude and duration of asymmetric signaling response. translocation. Thus, both light intensity and number of pulses Optical Activation of Localized Gq Signaling Within a Single Cell. To can be used to modulate GPCR activity in a single cell. create a comprehensive set of optical triggers to induce spatial and temporal confinement of all of the major G-protein signaling Optical Activation of Localized Gi Signaling Within a Single Cell. Next in a single cell, we next searched for Gq- and Gs-coupled opsins we examined whether the spectral selectivity of bOpsin can be with similar properties. We used the methods described above to used to confine G-protein activity to a selected region of a single detect spectral selectivity as well as localized signaling (Figs. 1 and cell and globally image the response (Fig. 2A and Movie S1). We 2). We focused on melanopsin, a Gq-coupled opsin expressed in tested this approach in a HeLa cell expressing bOpsin and mCh- a subset of mammalian retinal ganglion cells, which has a λ-max γ9. A localized single-pulse OI (445 nm, 5 μW) (Fig. 2B, white of ∼480 nm (Fig. 1A) (20). Using the strategy developed in Fig. box) induced mCh-γ9 translocation only from the exposed area 1B, we determined the optimized imaging and optical activation (Fig. 2B, yellow arrow) compared with the unactivated area (Fig. < μ 2 conditions under which melanopsin could be used as a spectrally 2B, green arrow) as seen by imaging at 595 nm, 40 W/cm selective optical trigger. Optical activation of melanopsin (488 (Fig. 2B and Movie S1). These results showed that the properties nm, 27 μW) in a HeLa cell induced Gβγ translocation (Fig. 3A) of bOpsin allow localized G-protein activity evoked by this opsin and it was possible to activate melanopsin repeatedly (Fig. 3B). to be imaged at different wavelengths without global activation The rapid reversal of translocation after the activation showed of the opsin. ∼ γ that it could deactivate rapidly (t1/2 5 s) (Fig. 3B). Restricted We monitored the mCh- 9 intensity over time in internal optical activation of one of the cells (Fig. 3C, yellow box) coex- membranes adjacent to the activated plasma membrane (Fig. 2B, pressing melanopsin and the IP3 sensor, PLCδ−PH-mCh (21) white arrow) to examine whether bOpsin could be used to ach- showed a strong translocation of the PH-mCh sensor to the cy- ieve tight temporal control of G-protein activation. Results show tosol whereas a neighboring cell did not show a response (Fig. 3C γ ∼ that mCh- 9 starts returning to the plasma membrane 5 s after and Movie S2). This result shows that melanopsin can be used activation, suggesting that bOpsin deactivates rapidly (Fig. 2C). to evoke Gq signaling in a single cell or multiple cells in a tissue. In combination with slow transmembrane diffusion of GPCRs, In a separate experiment, when the neighboring cell was in- fi this facilitates con nement of G-protein activation to the opti- dependently subjected to localized optical activation (Fig. 3D, γ cally stimulated region of the cell. The decrease in mCh- 9 white box), it showed a rapid increase in IP3 in the optically ac- (yellow arrow) in Fig. 2B is due to translocation and not pho- tivated proximal region compared with a slower increase in the fl tobleaching of the uorescent protein because there is a corre- distal region (Δt1/2 ∼ 4s)(Movie S3). The difference in IP3 sponding increase of mCh-γ9 in intracellular membranes within concentration dissipated over time due to the rapid diffusion of the OI area (Fig. 2 B and C). IP3 and the bound sensor. These results clearly show that mela- In the absence of introduced 11-cis retinal, there is no de- nopsin induces localized IP3 activity and can be used to exercise crease detected in the intensity of the FP-γ9 signal from the reversible, repeatable localized control over Gq signaling within activated region, showing that βγ does not translocate in the a single cell. absence of functional opsin. Furthermore, it was possible to activate bOpsin repeatedly without bleaching or desensitization Reengineering Opsins to Obtain Specific Combinations of Spectral (Fig. 2D), thus facilitating control of signaling in a single cell over Tuning and G-Protein Coupling. The box jellyfish, Carybdea rastonii, extended periods of time. expresses an opsin that is Gs coupled (λ-max ∼ 500 nm) (Fig. 1A)

Karunarathne et al. PNAS Early Edition | 3of10 Downloaded by guest on September 29, 2021 A Global Restricted Global B Imaging Optical activation Imaging

α Active Inactive opsins opsins Asymmetric Cell signaling output

C D E t1/2=1.7s 1.0 5 OA1 OA2 OA3 OA4 3 μΜ 1.0 OI

0.8 4 9 loss γ 0.8 0.6 3 0.6 Confined

optical 9 fluorescence

γ 0.4 0.4 input 2 0.2 0.2 1

in internal membranes Average in internal membranes 0.0 Gaussian fit

mCh-γ9 mean fluorescence mCh-γ9 0.0 0 0 20406080 Mean YFP- 020 40 120 220 310 -10 -5 0 5 10 from the plasma membrane

Time (s) Time (s) β Normalized G protein Distance - μΜ F

Fig. 2. Spatiotemporally restricted Gi activation using bOpsin. (A) Schematic showing how spatially confined GPCR activity can be achieved using an appropriate opsin with desired spectral selectivity. Cell is globally imaged in the basal state with a light beam (orange) of wavelength per second and intensity characteristics that do not activate the opsin (gray). Opsin is activated (yellow) in a spatially confined area of the cell, using a wavelength close to its λ-max (blue). The response to local activation (green) is imaged globally. (B) Localized bOpsin activation with a confined single-pulse OI (white box, 445 nm, 5 μW) induced spatially restricted mCh-γ9 translocation (yellow arrow, activated proximal plasma membrane; green arrow, unactivated distal) in HeLa cells (n = 10). (C) Plot shows averaged mCh-γ9 intensity changes in internal membranes adjacent to the activated plasma membrane (white arrow in B)(n = 10). (D) Re- peated optical activation (OA) of bOpsin can be used to achieve repeated signaling in a single cell. After bOpsin activation for a period, recovery was allowed for 1 min before repeating activation (n = 20). (E) Determination of spatial confinement of optically induced GPCR activity using FP-γ9 translocation. Shown is extent of GFP-γ9 translocation from the plasma membrane of HeLa cells expressing bOpsin and GFP-γ9 before and 5 s after application of a confined 3-μm- wide OI (purple line) (Fig. S1B). Fractional GFP-γ9 loss was calculated. A fitted Gaussian distribution curve (red line) to the averaged experimental data points (dotted line) resulted in FWHM of 6.3 (n = 6). (F) OI (yellow box) (445 nm, 5-s interval pulses) applied asymmetrically to a RAW 264.7 cell expressing bOpsin- mCh and PIP3 sensor. Akt-PH-GFP evokes localized PIP3 production at the proximal region of the cell (Right)(n =5).

(9, 22). While following the same strategy described in Fig. 1B, wavelengths that excite mCh, YFP, or GFP. Localized optical we found that jellyfish opsin was activated under all imaging activation of CrBlue resulted in translocation of mCh-γ9from conditions used. As soon as imaging was initiated, FP-γ9 trans- proximal activated [Fig. 4E, red region of interest (ROI), yellow location was observed (Fig. 4A and Table S1), indicating lack of arrow] regions but not distal unactivated (blue ROI, white ar- sufficient spectral selectivity to achieve control over localized row) regions, indicating its suitability as an optical trigger of Gs signaling. spatially confined Gs signaling (Movie S4). We then used HeLa We therefore examined whether jellyfish opsin could be ge- cells coexpressing CrBlue and GFP-Δepac-mCh cAMP sensor netically redesigned to introduce spectral selectivity while retain- (24) to examine whether CrBlue regulated cAMP levels. For- ing Gs coupling. The conservation in the structure of GPCRs skolin was able to induce a FRET change in this sensor, con- has facilitated the design of chimeric receptors that alter specific- firming its ability to detect cAMP increase under similar ity for the extracellular signal and G-protein subtype (7, 8, 23). We experimental conditions (Fig. 4F). Results demonstrate that only synthesized a chimeric opsin, CrBlue, containing the chromo- optically activated cells show an increase in cAMP levels (Fig. phore-binding region of bOpsin and the Gs-coupling intracellular 4F). Together these results showed that it is possible to reengi- region of jellyfish opsin (Fig. 4 B and C). In contrast to jellyfish neer opsins with specific combinations of spectral sensitivity opsin, CrBlue was activated by 445 nm light (Fig. 4D) and not by and G-protein specificity.

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1220697110 Karunarathne et al. Downloaded by guest on September 29, 2021 A B lamellipodia and migration in immune cells (26). This suggested PNAS PLUS st nd that localized increase in PIP3 might regulate neurite initiation. Single pulse 1 OA 2 OA Before OA 12 25 OA t We coexpressed Gi/o-coupled bOpsin and Akt-PH-GFP in post- 10 1/2 20 t1/2 natal 1- to 2-d-old hippocampal neurons. The plasma membrane 8 15 of the stage I neuron showed patches of PIP3 accumulation (Fig.

6 9 fluorescence γ 9 fluorescence 5A). This is reminiscent of the similar distribution of PIP3 on the

γ 10 After OA 4 plasma membrane of basal-state migratory cells (26). It is also 2 5 consistent with the ability of these neurons to initiate neurites

Internal membranes in internal membranes 0 Plasma membrane 0 Mean mCh- 0 50 100 250 300 spontaneously (27). Mean mCh- 020406080100 Time (s) Initial activation resulted (Fig. 5A, upper part of cell) in little CrBlue Time(s) change in PIP3 level proximally but signiﬁcant decrease distally C 1.4 (Fig. 5A, yellow arrows). Switching the location of the optical t ~3.5 s signal (Fig. 5A, lower part of cell) resulted in signiﬁcant increase OA 1.3 1/2 Single proximally (Fig. 5A, yellow arrow) and decrease distally (Fig. 5A, 1.2 pulse OA Right). These results suggested that activation of Gi/o locally in

1.1 a neuron could evoke localized regulation of PIP3 levels and potentially neurite growth. 1.0 We therefore examined whether a conﬁned optical signal to Before OA 4 s 0.9 bOpsin could regulate neurite growth. Selected regions at the Plasma membrane periphery of bOpsin-expressing stage I hippocampal neurons 0.8 Cytosol with no neurites were optically activated (445 nm, 5 μW). The Normalized mean mCh fluorescence Normalized 01020304050 results show that continuous pulses (every 5 s) of the optical Time (s) input (Fig. 5B, yellow box) resulted in individual neurons responding with a protrusion followed by the formation of ex- 25 s Proximal tensive lamellipodia (yellow arrows). The changes in mean pixel D OA Distal intensity of bOpsin-mCherry that marks the plasma membrane Neighboring 30 were measured in a region of interest as shown. The neuronal

ﬁ CELL BIOLOGY 20 response to optical activation was quanti ed as described in SI Δdt Methods and Fig. S4. Plots in Fig. 5C show that neurite initiation 10 1/2 was not observed before optical activation. After optical activation was terminated, the lamellipodia consolidated into a neurite 0 0 20 40 60 80 100 (Fig. 5B, white arrows, and Fig. S4B). In an experiment where

Before OA After OA Mean PH-mCh fluorescence Time (s) the neurite was observed 2 h after consolidation, a growth cone Fig. 3. Localized activation of Gq signaling by melanopsin. (A) Single-pulse was observed (Fig. 5B, green arrow). optical activation (OA) of melanopsin (488 nm, 27 μW) induced mCh-γ9 In Akt-PH-GFP–expressing neurons, optical activation resul- translocation. (Right) Plot shows representative increase in mCh-γ9inin- ted in only limited lamellipodia formation (Fig. 5A) but not sig- tracellular membranes. Decrease in the plasma membrane is shown by yel- nificant neurite formation. This result is consistent with previous low arrows (Left)(n = 6). (B) Repeated activation (2 min apart) of melanopsin evidence that the Akt-PH-GFP competes with PIP effectors and γ ∼ 3 induces repeated translocation of mCh- 9(t1/2 = 6s,n = 6). (C) Optical inhibits neuron differentiation (28). Although it has been repor- activation of melanopsin induces PH domain translocation in HeLa cells. A HeLa cell expressing melanopsin and PH-mCh was optically activated (entire ted that lowering the level of the PIP3 sensor expression allowed cell, yellow box) with a single pulse of light. PH-mCh translocated to the PIP3 to be detected at growing neurite tips (25), we were unable cytosol (image: 4 s). There was complete reversal of PH-mCh to the plasma to evoke robust neurite initiation even in the presence of rela- membrane over time (image: 25 s). (Right) Plot shows mCh intensity changes tively low levels of the PIP3 sensor. in the plasma membrane and the cytosol (n = 7). (D) Localized melanopsin Neurons coexpressing bOpsin and a dominant-negative RacT17N activation (white box) induced localized PH-mCh translocation, indicating (29) failed to respond to optical activation compared with neu- IP3 production. (Right) Plot shows intensity changes in ROIs in the image rons expressing the wild-type Rac, confirming that optical acti- Δ ∼ ( t1/2 4s,n =7). vation-induced neurite initiation is associated with a Rac-mediated pathway. Optically induced neurite formation in GFP-β actin- expressing neurons showed extensive remodeling of the actin cy- The spectral selectivity properties of different opsins under toskeleton (Fig. 5D and Movie S5). Thus, the initial lamellipodia the experimental conditions used are shown in Table S1.The fi formation, Rac dependence, and actin cytoskeleton remodeling optimized condition for con ned optical activation of opsins as recapitulate the typical native properties seen during spontane- well as imaging responses using a set of FP tags are shown ous neurite growth (30). These results show that the optical ap- (Tables S2 and S3). proach developed here recruits the endogenous signaling network in the cell and executes behavioral changes that mimic native cell Optical Control of Neurite Initiation and Extension. The ability to behavior (Fig. 5 A, B, and D and Movie S5). optically confine G-protein signaling to a selected area of a cell fi Because Gi/o-coupled CXCR4 receptors are enriched at the for speci c durations of time using the optical triggers above sug- leading edge of neurites and are known to promote neurite gested that this approach could be used to induce symmetry- growth (31), we then examined whether bOpsin activation of Gi/o breaking cellular events. Exposure of neurons to neurotransmitters could induce neurite extension. In postnatal day 1–2 hippocampal that stimulate Gi/o encourages neurite outgrowth (10). We exam- neurons, optical activation of the tip of an existing neurite in ined whether spatially selective Gi/o-coupled receptor activity can a neuron expressing bOpsin and dendritic marker DenMark regulate the second messenger and actin dynamics to induce neu- shows an optically induced formation of an ∼75-μm-long neurite rite growth. We first tested whether localized optical activation of (Fig. 5E). In these stage II neurons, the DenMark-mCh dendritic bOpsin can regulate PIP3 formation in a hippocampal neuron. marker is globally distributed on the plasma membrane as ex- Selective accumulation of PIP3 has been shown at the tip of pected (32). Newly initiated growth had an inhibitory effect on growing neurites of a hippocampal neuron (25). PIP3 increase previous spontaneous extension of lamellipodia (Fig. 5E, yellow at the leading edge plays an important role in the formation of arrows). Optically induced neurite extension was examined in

Karunarathne et al. PNAS Early Edition | 5of10 Downloaded by guest on September 29, 2021 A Imaging at 595nm B 5 Jellyfish opsin CrBlue 4 ~414nm Before After Blue 3 Opsin 9 fluorescence γ 2 J.fish opsin αs 1 β γ

in internal membranes 0

Mean mCh- Mean 0481216 Time (s) C D Blue MRKMSEEEFYLFKNISSVGPWDGPQYHIAPVWAFYLQAAFMGTVFLIGFPLNAMVLVATL CrBlue V Jellyfish ------K MGANITEIL-----SGFLACVVFLSISLNMIVLITFY CrBlue MRKMSEEEFYLF NISSVGPWDGPQYHIAP WAFYLQAAFMGTVFLIGFPLNAMVLVAFY Before After --

Blue RYKKLRQPLNYILVNVSFGGFLLCIFSVFPV-FVASCNGYFVFGRHVCALEGFLGTVAGL Jellyfish RLRHKLAFKDALMASMAFSDVVQAIVG-YPLEVFTVVDGKWTFGMELCQVAGFFITALGQ CrBlue RLRHKLAFLNYILVNVSFGGFLLCIFSVFPV-FVASCNGYFVFGRHVCALEGFLGTVAGL ---IL-1-

Blue VTGWSLAFLAFERYIVICKPF--GNFRFSSKHALTVVLATWTIGIGVSIPPFFGWSRFIP Jellyfish VSIAHLTALALDRYFTVCRPFVATAIHGSMRNAGMVIFVCWFYASFWAVLPLVGWSNYDV CrBlue VTGWSLAFLAFERYIVICRPFVATAIHGSMRNALTVVLATWTIGIGVSIPPFFGWSRFIP 7 OA ------IL-2----

Blue EGLQCSCGPDWYTVGTKYRSESYTWFLFIFCFIVPLSLICFSYTQLLRALKAV------6 Jellyfish EGDGMRCSINW--ADDSPKSYSYRVCLFVFIYLIPVLLMVATYVLVQGEMKNMRGRAAQL CrBlue EGLQCSCGPDWYTVGTKYRSESYTWFLFIFCFIVPLSLICFSYTLVQGEMKNMRGRAAQL 5 ------IL-3--- 4 Internal Blue -AAQQQESATTQKAEREVSRMVVVMVGSFCVCYVPYAAFAMYM--VNNRNHGLDLRLVTI fluorescence membrane Jellyfish FGSESEAALKNIKAEKRHTRLVFVMILSFIVAWTPYTFVAMWVSFFTKQLGPIPLYVDTL 3 CrBlue FGSESEAALKNIKAEKRHTRMVVVMVGSFCVCYVPYAAFAMYM--VNNRNHGLDLRLVTI γ9 plasma ------IL-3------2 membrane Blue PSFFSKSASIYNPIIYCFMNKQFQACIMKMVCGKAMTDESDTCSSQKTEVSTVSSTQVGP 1 Jellyfish AAMLAKSSAMFNPIIYCFLHKQFRRAVLRGVCGRIVGGNA------IAPSSTAVEP CrBlue PSFFSKSASIYNPIIYCFMNKQFQRAVLRGVCGRIVGGNA------IAPSSTAVEP ------C-TERMINUS------0

Mean mCh- 0 40 80 120 Blue NETSQVAPA-- Time (s) Jellyfish GQTLASGTAES CrBlue NETSQVAPA— -C-TERMINUS- E F IBMX+Forskolin 110 OA 100 5 OA 90 80 4 70 3 Distal 60 9 fluorescence Proximal Normalized FRET γ 2 50 0 1 0 100 200 300 400 Time (s) 0 CrBlue+No OA

Mean mCh- 0 50 100 150 200 250 CrBlue+OA Time (s) +IBMX+Forskolin

Fig. 4. Reengineering of a spectrally selective opsin for localized Gs signaling. (A) Gs-coupled jellyﬁsh opsin activation induced mCh-βγ9 translocation in HeLa cells during imaging of mCh (n = 10). (Right) Plot shows increase in mCh-βγ9 in intracellular membranes without a baseline. (B) Designing Gs-coupled opsin, CrBlue using extracellular and retinal-binding transmembrane regions of blue opsin (blue) responsible for spectral tuning, and jellyﬁsh opsin (green) intracellular loops that couple to Gs. (C) Primary structures of opsins aligned. IL, intracellular loops. (D) Global CrBlue optical activation (OA) induced mCh-γ9 translocation in HeLa cells (n = 8). (Lower) Plot shows the ability to acquire images in the basal state. (E) Localized CrBlue activation (white box) induced spatially restricted mCh-γ9 translocation (red ROI and yellow arrow, activated proximal region; blue ROI and white arrow, unactivated distal area) in HeLa cells (n = 7). (Right) Plot shows βγ translocation away from the plasma membrane. (F) Optical activation of CrBlue (every 5 s) in HeLa cells induced FRET changes in

GFP-Δ-epac-mRFP cAMP sensor [GR(488/565)/GG(488/515] (red) (n = 6). Control cells (black) were similarly imaged without optical activation. To check the sensor functionality, FRET changes in the GFP-Δ-epac-mRFP cAMP sensor were examined in HeLa cells (green plot) by adding 25 μM Forskolin and 100 μM phos- phodiesterase inhibitor, IBMX (ﬁnal concentrations) at 100 s.

multiple neurons. As mentioned above, growth was measured Denmark-mCh, provided results consistent with those obtained using bOpsin-mCh or DenMark-mCh as a marker. The methods with bOpsin-mCh (Fig. S4). used to quantitate neuronal responses are described in detail in SI In neurons coexpressing mGFP-actin with bOpsin, extension Methods and Fig. S4. Plots show that neurite extension could be of the neurite was supported by actin-enriched ﬁlopodia mim- achieved with optical activation independently in several neurons icking spontaneous neurite growth (Fig. 5G and Movie S6). Only (Fig. 5F). After a period of extension, even in the presence of a neurite that was optically activated responded (Fig. 5 G and H) optical activation, growth ceases and consolidation of lamellipo- and not a neighboring neurite that was not activated, showing dia into a neurite begins (Fig. 5 C and F). These results also that extension was dependent on the optical stimulus. Further- demonstrated that an independent plasma membrane marker, more, basal imaging of a neurite did not show growth until op-

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1220697110 Karunarathne et al. Downloaded by guest on September 29, 2021 PNAS PLUS Before OA A 0:01:36 0:00:00 0:04:50 0:04:55 0:07:15 0:12:40 4 min 55 s

PIP3 sensor sensor PIP3 12 min 40 s fluorescence (line) (line) fluorescence Before OA First upper Last upper First lower 0204060 Distance ( μm) C OA B 1.0 0.8 0.6 0.4

(initiation) 0.2 0.0

D Normalized proximal growth 0 3 6 9 12 15 18 Time (min)

F OA 1.00

0.75

E 0.50 (extension) 0.25 Normalized proximal growth 0.00 CELL BIOLOGY 0369 Time (min) H 5 OA

4 G 00:01 01:25 03:00 04:00 min:sec 3

2 Activated Neighboring 1

of the proximal region 0 Before After first OA GFP-actin fluorescence 02 3 4 5 Time (min)

Fig. 5. Optical control of neurite initiation and extension in rat hippocampal neurons. (A) Localized PIP3 formation in response to confined optical activation (OA). Yellow box represents OI. (Right) Line plots show PIP3 changes over time. Line used is shown in basal image (Left). Time here and below is shown as h:min:s on the images. (B)(Left) Stage 1 neuron expressing bOpsin-mCh before optical activation. Cell was optically activated every 5 s (yellow box) and imaged. Selected area of the light-activated region (yellow lines) is shown. Lamellipodia (yellow arrows) form in the direction of optical input. After termination of optical activation at 3 min 45 s, lamellipodia consolidate into a neurite (white arrows) with a growth cone (green arrow) (n = 6). (C) Growth dynamics of the lamellipodia formation during neurite initiation on onset of optical activation in multiple neurons. Responses were normalized to whole-cell mean opsin-mCh or DenMark-mCh fluorescence and further normalized from 0 to 1 in the y axis (methods for quantitation are described in SI Methods and Fig. S4). (D) Optically induced neurite initiation and associated actin cytoskeleton remodeling in neuron expressing bOpsin-mCh and mGFP-actin. During optical activation, only GFP images were captured. Both mCh and GFP images were captured after terminating optical activation and overlaid (Right). Actin- rich lamellipodia (yellow arrow) later consolidated into a neurite (white arrow) (n = 7). (E) Optical activation induced neurite extension in a neuron expressing bOpsin and DenMark-mCh (dendritic marker). During optical activation (yellow box) of a selected region of a neuron, spontaneously growing lamellipodia at the opposite end of the neuron (yellow arrow) retracted. (F) Growth dynamics during optically induced neurite extension in multiple neurons. Neurons expressing bOpsin-mCh or DenMark-mCh were optically activated as described in E, using 5 μW, 445 nm OI. Responses were normalized to whole-cell mean opsin-mCh or DenMark-mCh fluorescence and further normalized from 0 to 1 in the y axis as above (SI Methods and Fig. S4). (G) Actin remodeling during optical activation stimulated neurite extension in a neuron expressing bOpsin-mCh and mGFP-actin. Shown is formation of actin-rich filopodia (white arrow) (n = 5). (H) Plot shows GFP fluorescence change in the activated neurite compared with an unactivated neighbor (red line).

tical activation. These results here and with neurite initiation feedback mechanisms maintains symmetric growth until one above suggest that the responses observed are not spontane- neurite grows into an axon (33). Experiments with laminin-coated ous growth. Overall, the results demonstrate that bOpsin can beads have provided support for such a negative feedback be used to localize GPCR activity to induce neurite growth mechanism that leads to the inhibition of other neurites when and investigate the causal relationship between molecular and a neurite elongates (25). The optical methods here overcome behavioral dynamics. spatiotemporal limitations of coated beads and allow the relationship between the extension and retraction cycles of growing Optically Reprogramming Extension–Retraction Cycles of Growth to areas to be examined directly. We designed a sequential pattern Refashion Differentiation of a Single Neuron. As a neuron differ- of discrete switchable OIs directed independently at individual entiates, neurites undergo sequential extensions and retractions neurite tips or growth areas of a neuron and quantitatively and it has been suggested that a balance of positive and negative monitored the response dynamics. The results show that localized

Karunarathne et al. PNAS Early Edition | 7of10 Downloaded by guest on September 29, 2021 stimulation of Gi/o signaling induces lamellipodia formation and mation with an increase in width but without significant change in neurite growth (Fig. 6 A and B). This growth is accompanied by length (Fig. 6C). When the same optical input was moved in the retraction of a distal growing area in the same neuron (Fig. 6B direction of the neurite axis, the lamellipodia extended toward the and Figs. S4 C and D and S5). Movie S7 shows one optically in- optical signal (Fig. 6D). During this process, the average rate of duced extension–retraction cycle. neurite elongation and the OI movement showed similar veloci- To estimate the correlation between the proximal growth and ties (∼0.05 μm/s). The result shows that appropriate signaling distal retraction of lamellipodia, we implement a bivariate mixed input characteristics can be designed to program complex events model, using the SAS Proc Mixed program. In this single-cell repeatedly in the same neuron (Fig. 6F). experiment, we found a strong correlation (correlation coeffi- cient = −0.822 ± 0.211) between the trends in four growth and Discussion < retraction pairs (P 0.0001) over time, suggesting synchroniza- The approach developed here can be used to control Gi/o, Gq, tion of these two events. Because the distance between an acti- and Gs signaling activity in a confined region of a single cell, an vated area and the induction of growth collapse is at times over μ entire cell, or a portion of a tissue. It further allows the entire 75 m (Fig. 6A), these results suggest that mechanisms may exist gamut of cellular events that constitute complex single-cell be- that are capable of communicating across relatively long dis- havior to be optically executed. tances within seconds. Using this optical approach, we quantified The bOpsin-based trigger allows asymmetric signaling to be dynamic parameters of Gi-mediated multiple extensions and rapidly switched on and off. Reversibility allows the responses to corresponding retractions in a single neuron (Table S4). Selective optical control over neurite extension allowed us to be measured after termination of the signal. Reproducibility further examine the patterning of the input signal that controls allows the same input to be provided to different parts of a cell neurite extension. Application of a stationary optical input pulsed or to different cells. The resistance to desensitization allows the every 5 s to a neurite tip resulted in fan-like lamellipodia for- opsin to be triggered repeatedly. Because single-cell responses

Fig. 6. Extension and retraction of growth in response to spatially discrete sequential optical activation. (A) Rat hippocampal neuron (1–2 d postnatal) expressing bOpsin-mCh. Images show optical activation (OA) induced lamellipodial growth (blue arrows) at the activated site and retraction (green arrows) at distal sites. Mean pixel intensities were determined in regions that cover fully grown lamellipodia (R1–R5). Images are representative of >10 experiments. In other experiments only one or two cycles of extension–retraction were examined. (B) Synchronization of multiple-lamellipodia extension and retractions in a single neuron during application of optical inputs. Image at 47 min 30 s shows optical activation induced three extended (yellow arrows) and one new (white arrow) neurite. (C) Determination of single-neurite growth dynamics to restricted optical input function varying in time and space. Stationary optical input induced lamellipodia growth, resulting in an increase in width of the lamellipodia without signiﬁcant elongation. (D) Elongation of lamellipodia and directed neurite extension were achieved by step-like movement of the optical input away from the growth region. The slope of lamellipodia elongationand the slope of optical input (∼0.05) varying along the neurite axis were found to be similar. (E) By varying the optical input in space and time, neurite initiation and extension can be reprogrammed.

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1220697110 Karunarathne et al. Downloaded by guest on September 29, 2021 can ensue over minutes or hours, this allows cells to be stimu- vated molecular pathways mediating complex single-cell events PNAS PLUS lated repeatedly over long periods. are essentially “hardwired” and the optical methods here can be There are several benefits to an optical trigger being a GPCR. used to control other heterotrimeric G-protein–regulated pro- As opsins activate endogenous G proteins, native pathway in- cesses such as cell migration, cardiomyocyte contraction, and tegrity is wholly maintained. The cell responds to an extracellular hormone secretion and probe their mechanistic bases. signal, and hence results from using this method are directly relevant to native cell behavior. Because the receptor rather than Methods a downstream protein is being modulated, the response of the Time-Lapse Imaging and Optical Activation. For imaging, cells were seeded in entire molecular network that governs cell behavior is measur- 29-mm glass-bottom culture dishes. Hanks’ balanced salt solution (HBSS) able. We are not aware of existing methods that possess the supplemented with 1 g/L glucose was used as the imaging buffer in all combination of properties mentioned above. experiments. All imaging was performed with a spinning-disk confocal im- The methods developed here have been validated by demon- aging system comprising a Leica DMI6000B inverted microscope, a Yokogawa CSU-X1 spinning-disk unit, an Andor fluorescence recovery after photo- strating that localized optical activation of a GPCR can initiate bleaching (FRAP) and photoactivation (PA) (FRAPPA) unit, a laser combiner neurites and can be used to achieve coordinated control over with 50 mW 445-, 488-, 515-, and 594-nm solid-state lasers, and an iXon+ early neuron differentiation events. Although receptors like EMCCD camera. This system is capable of high-speed 4D image acquisition, CXCR4 are thought to be involved in neuron differentiation, it is exposing a stationary or moving selected area to a light beam of desired difficult to spatiotemporally control the activation of these li- intensity, wavelength for defined durations of time, and live data acquisition. gand-binding receptors in a selected region of a single neuron. The environmental chamber on the microscope was at 37 °C and dishes were fi Although coated beads and micropatterning have been valuable masked with a transparent CO2 mask to maintain humidi ed 5% CO2 over in identifying mechanisms at the basis of neuron differentiation the cells. Adaptive corrective focus was used to prevent focus drift during – time-lapse imaging experiments. All imaging experiments were conducted (33 35), the optical method here allows continual relocation of × the neurite growth-inducing signal with precise temporal control. using a 63 , 1.4HCX apochromat objective. In experiments involving opsin activation, dishes were kept completely in the dark from the time of addition This capability allows models of neuron differentiation to be of 11-cis retinal. Depending on the opsin, wavelengths other than its λ-max tested directly. were used to visualize cells. A number of such models have been proposed to explain the Spectral selectivity of opsin was detected by determining opsin’s ability to characteristic patterns of neurite initiation, extension, and re- induce FP-γ9 translocation during time-lapse imaging of cells transfected traction leading to the establishment of neuronal polarity (27, 33, with the appropriate opsin and FP-γ9 at 1-s intervals (Fig. 1B). Scanning of

35–39). In the case of neurite initiation, growth occurs only at each opsin was initially done under the following conditions: 594 nm, 19 μW, CELL BIOLOGY certain points on the surface of the cell and it has been suggested 40 ms; 515 nm, 125 μW, 50 ms; 488 nm, 3.5 μW, 30 ms; and 445 nm, 10 μW, that this is due to the presence of discretely distributed micro- 30 ms (Table S2). If the opsin got activated, either intensity or the exposure domains of signaling and other molecules (27). The extension and was reduced and the experiment was performed again. To selectively acti- fi retraction of growth seen during the establishment of neuronal vate spectrally selective opsins, we delivered user-de ned OIs, using a con- polarity have been suggested to occur due to either competition tinuous-wave laser by using a computer-steered dual head, fast-scanning galvo mirror. Laser power for optical activation was measured at the image for limited resources or feedback loops of local activation and plane, choosing the crosshair as the ROI. Laser power was measured using global inhibition (33, 36, 37). Neuronal polarity has also been a light meter (Ophir Nova II). We generated a variety of optical input thought to arise from specific patterns of second messenger gra- functions by varying the size of the input area intensity of the beam, pixel dients (35). dwelling time, and pulse frequency and achieved subsecond OI pulse rates Using the opsin-based method, molecular distribution at the (Fig. 1 E and F). Optical input area for spatially restricted activation varied cell periphery can be imaged while selectively initiating neurites from 9 to 100 μm2 (for a single cell) whereas restricted whole-cell activation to detect microdomains. The ability to optically direct specific input sizes were similar to cell size. The time required for the optical input to 2 extension–retraction cycles while imaging molecular changes will complete scanning a 1-μm (22 pixels) area was ∼0.87 ms. Multiband dichroic fi allow computational models of early neuron differentiation to be lters and 10-ms switching were used for OI and subsequent imaging. In- tensities of OI laser beams were titrated against FP-γ9 translocation and the tested. Optical control can be used to perturb signaling gradients fi optimum intensity was selected for further studies (Table S3). It was also globally or in de ned directions so that the role of spatiotem- ensured that, at these intensities, there was no photobleaching of tagged poral dynamics of signaling and cytoskeleton in governing neu- fluorescent proteins. Before and after neuronal imaging, Z-stack images were ronal polarity can be identified. Control over neurite growth can obtained by capturing images at 0.2-μm intervals by using a Prior Piezo stage. be of value in regenerating connections in the case of injury or disease and for generating de novo neuronal networks. Finally, Measuring Space- and Time-Variant GPCR Activity Using a Gβγ9 Translocation optical control allows the extracellular signal to be repeated re- Assay. The Gβγ9 translocation directly reflects the active status of GPCRs in producibly and switched spatiotemporally at high speed. This can living cells. We used this property to quantify GPCR activity in real time in facilitate high-throughput assays of neuron differentiation. a selected region of a cell or in a whole cell. FP-γ9 is present on the plasma A collection of fluorescent proteins of spectrally distinct ex- membrane when GPCRs are inactive. On GPCR activation βγ9 translocates to fl citability has been central to our capability to image the spatio- internal membranes, drastically decreasing the uorescence on the plasma membrane and increasing the fluorescence in the Golgi and the endoplasmic temporal dynamics of signaling activity in cells (40). In contrast, reticulum. In contrast to cytosolic secondary messengers, GPCRs and heter- tools to exert experimental control over the spatiotemporal dy- otrimeric G proteins possess slow plasma membrane diffusion rates. Here we namics of signaling activity in a single cell have been limited. The used these properties to develop Gβγ9 translocation as a fast transient assay opsin-based triggers described here help overcome this limitation. to detect global as well as localized GPCR activity in single cells. Quantifi- The large nonrhodopsin opsin family can be a spectrally diverse cation of GPCR activity included the following steps. First, a confocal image resource to probe the network-level control of a variety of GPCR- of the FP-γ9 distribution was captured. Second, areas for OI of GPCRs were initiated cell behaviors at the single-cell level. drawn on the initial image, using Andor IQ. Third, a time-lapse imaging It has been suggested that attaining optical control over sig- protocol was created that usually contained two segments: (i) capturing a series of basal-state images to monitor basal FP-γ9 distribution and (ii) naling may help control cell behaviors in useful ways (41). We γ demonstrate here that evolutionary conservation among GPCRs single- or multiple-pulse OI to the areas drawn followed by global FP- 9 imaging. Multiple segments were created if necessary with or without OI and G-protein subunits allows an optical trigger based on a color and imaging, by varying the pulse frequency and intervals between OI opsin from the human retina to recruit an entire native G-protein cycles. Finally, the protocol was executed and the time-lapse image series signaling network in hippocampal neurons. Localized activation was analyzed by calculating mean Gβγ9 fluorescence intensities in selected of an appropriate G protein is sufficient to orchestrate intricate OI areas while subtracting background. Due to cell-to-cell variation, Gβγ9 patterns of single-cell behavior. This suggests that GPCR-acti- fluorescence intensities were usually normalized to their basal level.

Karunarathne et al. PNAS Early Edition | 9of10 Downloaded by guest on September 29, 2021 FRET Imaging to Measure Optically Induced cAMP Production. HeLa cells on Data Analysis and Statistics. All intensity recordings were background sub- 23-mm glass-bottom dishes were transfected the cAMP FRET sensor, GFP- tracted. Image analysis was performed using Andor IQ v2.4.1 and task-specific ΔEpac-mCh using the protocol described above. Twenty-four hours after Python scripts. Data analyses, curve fitting, and statistical analysis associated transfection, dishes were transferred to an incubator in a dark room and 11- with the corresponding functions were performed using OriginPro 8.6 and cis retinal was added to the medium (3 ng/mL). After incubation with 11-cis Matlab (R2011b). Cell and optical input coordinates were determined using retinal for 30 min, the medium was replaced with HBSS warmed to 37 °C. the Tracker video analysis and modeling tool. To calculate the correlation cAMP binds to the GFP-ΔEpac-mCh sensor, resulting in FRET decrease. FRET between optically induced local neurite growth and distal lamellipodia re- was continually measured by exciting at 488 nm while measuring donor traction, we implemented a bivariate mixed model, using SAS Proc Mixed. emission using 515-nm (GG) filters and acceptor emission using 595-nm (GR) Error bars represent SEM. filters. Of several cells expressing the FRET sensor and CrBlue in the field of vision, a few cells were randomly chosen for OI. Separate areas were drawn ACKNOWLEDGMENTS. We thank P. Nanda and Andor Technology for around those cells for selective OI. After imaging basal FRET every second for customizing Andor IQ software and discussions; S. Mennerick for neurons; 100 s, selected cells were optically activated using a 445-nm beam (12.5 μW, D. Oprian, I. Provencio, A. Terakita, K. Jalink, K. Deisseroth, S. Karnik, and B. Hassan for cDNAs; the National Eye Institute, National Institutes of Health every 1 s) and FRET imaging was continued. FRET was calculated as GG/GR (NIH), and R. Crouch for 11-cis retinal; S. Karnik and P. O’Neill for discussions; fi ratio. Cells in the same eld that express the constructs but are not optically and H. Paulding for experimental assistance. We thank G. Zhou for assistance activated were considered as control cells. FRET sensor functionality was in statistical analysis. This work was supported by NIH Grants GM069027 and assessed by measuring FRET after acceptor photobleaching. GM080558 (to N.G.).

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