For more details, see Supplementary Materials and Methods.

For more details, see Supplementary Materials and Methods.

Packaging plasmids: pMDLg/pRRE (Addgene #12251), pMD2. G (Addgene #12259), and pRSV-Rev (Addgene #12253) (Kato et al., 2014; Dull et al., 1998)

HEK293T cell line (obtained from ATCC).

Lipofectamine 3000 Transfection Reagent (Invitrogen #L3000008).

Opti-MEM reduced serum medium (Gibco #31985-062).

10 cm cell culture dish.

6-well plate (Greiner Bio-One #657160).

Polybrene (Sigma #TR1003).

48-well cell culture plate (Sarstedt #83.3923).

DMEM (Gibco #10569-010) supplemented with 10% FBS and 1% penicillin-streptomycin.

Neon™ transfection kit (Invitrogen #MPK10025B).

Neon™ electroporation system (Invitrogen #MPK5000).

Gene Pulser Electroporation Cuvettes, 0.1 cm gap (BIO-RAD #1652089).

Gene Pulser Xcell Electroporation System (BIO-RAD #1652660).

0.1 cm-gap cuvette (BIO-RAD, 1652089).

Lipofectamine 3000 Transfection Reagent.

10 cm cell culture dish.

48-well cell culture plate.

Opti-MEM reduced serum medium.

Amaxa cell line kit V (Lonza; #VACA-1003).

Amaxa Nucleofector II device (Amaxa Biosystems).

Zeiss LSM800 GaAsP single-point, laser scanning confocal microscope with a wide-field camera.

Zeiss LSM780-FCS single-point, laser scanning confocal microscope (Zeiss Axio Observer with 780-Quasar confocal module).

8-well chamber (Lab-Tek, #155409 PK).

200 μg/mL fibronectin (Sigma #F4759).

MCF-10CA1h imaging medium: phenol red-free DMEM/F-12 (Gibco #21041-025).

RAW 264.7 imaging medium: phenol red-free HBSS (Gibco # 14025092) supplemented with 1 g/L glucose.

BD FACSAria IIu cell sorter (Beckton Dickinson).

SH800S cell sorter (Sony).

40 µm nylon cell strainer (Corning #431750).

5 mL polystyrene round-bottom tube (Corning #352235).

100 μm cell sorting chip (Sony #LE-C3210).

Sorting buffer (1x PBS, Ca²⁺/Mg²⁺ free; 0.9% FBS; 2% penicillin-streptomycin).

Collection medium (RPMI medium 1640 supplemented with 20% FBS and 2% penicillin-streptomycin).

For more details, see Supplementary Materials and Methods.

For lentiviral constructs, CIBN-CAAX or LifeAct-miRFP703 ORF was cloned in NheI/EcoRI sites of pLJM1-eGFP plasmid, in place of the eGFP gene (Sancak et al., 2008; Idevall-Hagren et al., 2012; Shcherbakova et al., 2016; Pal et al., 2023). For transposon system, CRY2PHR-mCherry gene was first sub-cloned into the pPB-bsr2 transposon plasmid in XhoI/NotI sites. Next, at the C-terminal of CRY2PHR-mCherry gene in the transposon plasmid, we introduced full-length RasGRP4 gene at BspEI/SalI sites (Yusa et al., 2009; Kennedy et al., 2010; Bell et al., 2021; Pal et al., 2023).

For Dictyostelium constructs, N150-Venus-iLID gene was subcloned in pDM358 plasmid using AgeI/BamHI restriction digestion. The tgRFPt-SspB R73Q gene was subcloned in pCV5 or pDM335 plasmid using AgeI/BamHI or BglII restriction digestion (Guntas et al., 2015; Pal et al., 2023). Next, at the C-terminal of tgRFPt-SspB R73Q gene in pCV5, RacGEF (catalytic domain of RacGEF1) or KRas4B G12V ΔCAAX was PCR amplified and introduced via NheI/NotI digestion (Park et al., 2004; Miao et al., 2019). cAR1-CIBN was cloned into BglII/SpeI sites of pDM358 plasmid whereas CRY2PHR-mCherry gene was sub-cloned into XbaI/NheI in pCV5 plasmid (Banerjee et al., 2022).

All constructs were sequenced and verified at the JHMI Synthesis and Sequencing Facility.

For more details, see Supplementary Materials and Methods.

For more details, see Supplementary Materials and Methods.

For stable protein expression in HL-60 cells, a combination of lentiviral and transposon approaches was used sequentially (Bell et al., 2021; Hadjitheodorou et al., 2021; Li et al., 2021; Pal et al., 2023). The procedure involved three steps. In step 1, virus was made using HEK293T cells at ∼80% confluency. For each transfection, 2 µg pMDLg/pRRE, 4.64 µg pRSV-Rev, 3.32 µg pMD2. G, and 10 µg CIBN-CAAX/pLJM1 constructs were mixed with Lipofectamine 3000 transfection reagent as per manufacturer’s instructions (Dull et al., 1998). After 96 h, virus-rich culture medium was collected at 3000 rpm for 20 min at 4°C. Next, viral supernatant was added to 4 × 10⁶ HL-60 cells, seeded at a density of 0.25 × 10⁶ cells/mL, in a 6-well plate. 10 μg/mL polybrene was added to this cell-virus mixture. Post 24 hour-incubation, virus was aspirated, and infected cells were added to a mixture of conditioned and fresh (mixed) medium. To select CIBN-CAAX-expressors, 1 μg/mL puromycin was added to infected cells after a day of recovery. Infected cells were kept in presence of puromycin for 5 days till only resistant cells were alive. Next, cells were harvested and transferred to a 48-well cell culture plate without puromycin for ∼3 weeks till resistant cells grew to confluency. These CIBN-CAAX expressing cells were next infected with LifeAct-miRFP703-expressing virus as explained above. To select LifeAct-miRFP703 expressors, cells were sorted on the fifth day post-infection, and grown to confluency. Finally, cells expressing CIBN-CAAX and LifeAct-miRFP703 were maintained in puromycin.

Next, recruitable RasGRP4 was introduced in CIBN-CAAX and LifeAct-miRFP703 dual expressing HL-60 cells using PiggyBac transposon system. 5 μg CRY2PHR-mCherry-RasGRP4/pPB plasmid was co-electroporated with an equal amount of transposase expression plasmid (pCMV-hyPBase) into 2 × 10⁶ cells using Neon transfection kit. DNA-cell mixture was resuspended in buffer “R” before single-pulse electroporation was carried out in a 100 µL pipette at 1350 V for 35 m sec using Neon electroporation system. Cells were resuspended in mixed culture medium in a 6-well plate and selected in presence of 10 μg/mL blasticidine S, as described for puromycin. Finally, these triple expressors were cultured throughout in puromycin and blasticidine S.

Dictyostelium stable cell lines were generated by electroporation (Li et al., 2021; Banerjee et al., 2022; Banerjee et al., 2023; Pal et al., 2023). Briefly, 1 × 10⁷ cells were harvested, washed twice, and resuspended in 100 μL ice-cold H-50 buffer. Next, 2 μg iLID (tgRFPt-SspB R73Q/pDM335, tgRFPt-SspB R73Q-RacGEF/pCV5 or tgRFPt-SSPB R73Q-KRas4B G12V/pCV5 and N150-Venus-iLID/pDM358) or cryptochrome (CRY2PHR-mCherry/pCV5 and cAR1-CIBN/pDM358) constructs were mixed with the cell suspension, and moved to an ice-cold 0.1 cm-gap cuvette. Electroporation was done with two pulses at 0.85 kV/25 μF at 5 s interval. Cuvettes were then incubated on ice for 10 min. Next, cells were transferred to a 10 cm culture dish containing 10 mL HL5 culture medium supplemented with heat-killed bacteria. On the next day, 10–20 μg/mL G418 sulphate and 30–40 μg/mL hygromycin B were added to cells and selected over 3–4 weeks. To induce protein expression, doxycycline (50 μg/mL) was added to the selected cells, 8–12 h prior to imaging. Cells were placed in an 8-well cover slip chamber and allowed to adhere for 20 min. HL5 medium was aspirated and 300 μL DB was added to cells. After a further 30 min incubation in the dark, cells were taken for imaging.

RAW 264.7 cells were transfected by nucleofection using Amaxa cell line kit V (Banerjee et al., 2022; Pal et al., 2023). Briefly, 3 × 10⁶ cells were harvested and resuspended in 100 µL Nucleofector Solution V containing 1.8 µg cryptochrome [0.7 µg pCIBN-CAAX and 1.1 µg pCRY2PHR (W349R)-mCherry or pCRY2 (R489E, A491D)-tdTomato] or iLID (0.9 µg pLL7.0-tgRFPt-SspB R73Q and pLL7.0-Venus-iLID-CAAX each) construct (Guntas et al., 2015; Taslimi et al., 2016; Duan et al., 2017). DNA and cell were mixed gently, transferred to a Lonza cuvette, and electroporated with Amaxa Nucleofector II device using the pre-set program “D-32”. Cell-DNA mix was transferred to 500 µL pre-warmed culture medium after a single pulse, and incubated for 10 min at 37°C and 5% CO₂. Next, 2 × 10⁵ cells were added to an 8-well chambered cover glass and incubated for 1 h 500 μL culture medium was next added to each sample, after which cells were incubated for 4 h before imaging.

3 × 10⁴ MCF-10CA1h cells were allowed to attach to 8-well chambered cover-glass overnight, prior to transfection (Zhan et al., 2020). Cells were at ∼30% confluency at the time of transfection. Transient transfection of the cells with 80 ng each of pStargazin-GFP-LOVpep, p2XPDZ-mCherry-LARG (DH), or pLifeact-7-iRFP670 was performed using Lipofectamine 3000 following manufacturer’s instructions (Wagner and Glotzer, 2016; Padilla-Rodriguez et al., 2018). After 4 h, transfection medium was aspirated and cells were cultured in 500 µL fresh growth medium for 48 h before imaging.

All optogenetic experiments were done in the absence of any chemoattractant. Photoactivation was done with argon laser (488 nm excitation). All recruitable effectors, tagged with mCherry, RFP, or tdTomato were visualized with solid-state laser (561 nm excitation and 579–632 nm emission). miRFP703 or iRFP670 was excited with 633 nm diode laser and emission was collected at 659–709 nm. DIC was acquired using the T-PMT associated with red channel. 40X/1.30 Plan-Neofluar oil objective, along with digital zoom, was used. For imaging mammalian cells, both microscopes contained a temperature-controlled chamber held at 37°C and 5% CO₂. Zeiss 800 or 780 was operated by ZEN Blue or Black software, respectively (Kuhn et al., 2021; Banerjee et al., 2022; Banerjee et al., 2023; Pal et al., 2023).

For photoactivation of differentiated HL-60 cells, we discovered that their pre-treatment with heat-killed Klebsiella aerogenes greatly improved efficiency of cryptochrome system. Briefly, 10⁷ differentiated neutrophils, grown on 10 cm culture dish, were incubated with 13 μg/mL heat-killed bacteria for 7 h. Next, cells were allowed to adhere to chambered coverglass, coated with fibronectin at a density of 35–40 μg/cm², for 40 min. Dead bacteria and unattached neutrophils were rinsed off from fibronectin-coated surface before imaging. We performed global recruitment experiments on Zeiss LSM780 microscope. The 488 nm excitation laser was turned on after imaging for at least 5 min. Image acquisition and subsequent photoactivation were carried out once every 7 s. Laser intensity during image capture was maintained at a low level (laser power of 1.7%–2% or 0.14–0.17 W/cm² at the objective) and exposure time was set at ∼2 s. This ensured RasGRP4 recruitment over the cell boundary without causing light damage. All local recruitment experiments were done using the Zeiss LSM800 microscope. A small region of interest was drawn near the cell (shown as dashed white boxes in the images), which was illuminated with the 488 nm laser (power of 5%–7% or 0.6–0.8 W/cm² at the objective) in multiple iteration. All imaging was completed within 5 h (Pal et al., 2023).

Before imaging RAW 264.7 cells, culture medium was removed and 450 µL pre-warmed HBSS buffer was added to cells. For MCF-10CA1h cell imaging, culture medium was aspirated and replaced with 500 µL pre-warmed imaging medium. Image acquisition and photoactivation were carried out once every 7 s or 8 s for macrophage or MCF-10CA1h cells, respectively. Other parameters for recruitment studies were similar to HL-60 cells.

Vegetative and developed Dictyostelium cells were allowed to adhere on chambered coverglass for 30 min before imaging. For recruitment, 488 nm laser was switched on after imaging for at least 5–10 min. Photoactivation during single plane imaging was carried out once every 5–15 s since half-life of iLID-SspB was ∼30 s. Since Dictyostelium are light sensitive, very low laser intensity (0.017–0.030 W/cm² or 0.06–0.08 W/cm² at the objective on the Zeiss LSM780 or LSM800, respectively) was used to stably recruit SspB-RacGEF over the cell boundary without any light damage.

HL-60 cells, 5 days after infection, were harvested, washed twice and resuspended in sorting buffer at a density of 15 × 10⁶ cells/mL. Cells were declumped by passing suspension through 40 μm cell strainer once and collected in 5 mL round-bottom tubes. Wild type HL-60 cells (2 × 10⁵ cells resuspended in 300 µL sorting buffer) was used as unstained control. Sorting was done at the Ross Flow Cytometry Core and Centre for Cell Dynamics, JHU using 100 μm microfluidic sorting chip. 561 nm excitation laser was used to sort and collect RFP expressing cells, whereas 633 nm excitation was used to sort miRFP703 expressors. The detector has 735LP and 780/60BP. High expressors (top 1%–10%) were taken in 0.5–5 mL collection medium, spun down, and medium was discarded. Sorted cells were resuspended in fresh collection medium and grown to confluency.

For more details, see Supplementary Materials and Methods.

For Figure 1, Figure 2D, Figure 2I, Figure 3E, Figure 5D, and Supplementary Figure S6B, linescans were created on Fiji/ImageJ 1.52i software (https://imagej.nih.gov/ij/) (Schneider et al., 2012). On the red channel, a “straight line” segment (12-pixel width) was drawn across the cell using the ‘line tool’ option. Next, we obtained the average intensity value along that line using the “Plot Profile” option. Values were normalized and graphed in Microsoft Excel.

For Supplementary Figure S3C, turn-off rate (membrane-cytosol cycling) for the mutants was calculated by counting the number of frames from the last flash of the laser till the signal in the red channel was no longer present on the cell boundary. The frame with the last flash of the 488 nm laser was considered as “0 s” and frame rate was 7 s. For Supplementary Figure S3E, red signal intensity in the cytosol and nucleus was measured immediately before or after turning on the laser. This allowed us to calculate percentage decrease in signal intensity in the cytosol/nucleus with photoactivation. We used these values to obtain the percentage of total recruitable protein translocated to membrane for each cell. GraphPad Prism 8 was used to prepare box-and-whisker plots.

For Supplementary Figure S4B, acquired images from different experimental conditions were adjusted to the same contrast by ImageJ software. Cells were outlined by “polygon tool” in ImageJ and the mean intensity for each was recorded. GraphPad Prism 8 (https://www.graphpad.com/) was next used to prepare the scatter plot.

For Figure 4B, cell was first segmented against the background with help of a custom code written in MATLAB 2019b (https://www.mathworks.com/). Next, membrane kymograph was created from the segmented cell as described previously (Banerjee et al., 2022; Banerjee et al., 2023; Pal et al., 2023). A linear color map for normalized intensities was used; blue denoted the lowest intensity whereas yellow denoted the highest.

For Figures 4C,D, and Figures 6B–D, cells were segmented in Fiji/ImageJ 1.52i software (Pal et al., 2023). This was done in a stepwise manner. First, using the “Threshold” option, the image stack was thresholded. We made sure that the “Calculate threshold for each image” box was kept unchecked, and the range was not reset. Second, using the “Analyzed Particles” option, we created cell masks by size-based thresholding. Third, these binary masks were optimized by performing “Fill holes”, “Dilate”, and “Erode” multiple times. Fourth, “Area”, “Shape descriptors”, “Centroid”, “Min and max gray value” and “Mean gray value” boxes in the “Set Measurements” tab under “Analyze” were checked. This allowed us to obtain values for aspect ratio and centroid coordinates. Using GraphPad Prism 8, mean and SEM obtained from aspect ratio value replicates were plotted. We calculated velocity by quantifying displacement between two consecutive frames. Subsequently, cell speed was acquired by dividing displacement with time interval. Using GraphPad Prism 8, average cell speed values, obtained from time-averaging cell speeds over all the frames, were plotted as box-and-whisker plots.

For Figure 4F and Figure 6, local protrusion formation was analyzed in a stepwise manner (Banerjee et al., 2022; Pal et al., 2023). First, using the “segmented line” tool in Fiji/ImageJ software, GEF recruitment region in the red channel was marked. Second, the midpoint of the recruited region was determined with the sequential use of customized macros, “Fit Spline” and “Straighten”. Third, after having determined the centroid with assistance from another Fiji/ImageJ macro, the angle between the nascent protrusion and recruitment region midpoint was calculated using the “angle” tool, holding the centroid as vertex. Using “polarhistogram” in MATLAB, these values were plotted. Atleast 35 fresh protrusions were considered for each histogram, and the minimum number of bins for each plot was calculated by Sturges’ formula.

In Figures 5E,F, cell was binarized using ImageJ based on the LifeAct channel. One typical Life-act labelled actin patch, as indicated by pink arrows in Figure 5B, was quantified throughout the video to show the change of newly formed cortical F-actin. Graphs were plotted using Microsoft Excel.

All statistical analyses were done on GraphPad Prism 8 software. For Supplementary Figures S3D-E, unpaired 2-tailed non-parametric test (Mann-Whitney test) was done. For Figures 4C,D, Figures 6B–D, and Supplementary Figure S4B, paired 2-tailed non-parametric test (Wilcoxon matched-pairs signed rank test) was used. Results are expressed as mean ± SD from at least 3 independent experiments. ns denotes p > 0.05, ** denotes p ≤ 0.01, *** denotes p ≤ 0.001, **** denotes p ≤ 0.0001.

In this study, we developed and utilized blue light-inducible dimerization systems to acutely perturb Ras superfamily GEFs in migrating cells. As discussed in the methods section, we generated stable cell lines, co-expressing cryptochrome optogenetic system with F-actin biosensor, LifeAct-miRFP703, in differentiated HL-60 neutrophils. Interestingly, western blot and confocal imaging results showed that treating these triple expressors with heat-killed bacteria, before experimentation, improved expression levels of CRY2PHR recruitable component as well as LifeAct, in a time-dependent manner (Supplementary Figures S1A,B). We presume that this pre-treatment would have a similar effect on expression of the membrane anchor, CIBN-CAAX, but we could not test that in our study. When we turned on the 488 nm laser on the entire cell, it caused cytosolic CRY2PHR to recruit globally on the plasma membrane within seconds. LifeAct expression strongly defined the front of these migrating cells (Figures 2A,B).

Could we spatially confine cryptochrome recruitment to a certain region on the neutrophil membrane? To answer this, we selectively turned on the blue light at the back of the cell, as denoted by the dashed white box. Time lapse imaging and linescan analysis clearly showed that nearly half of the total cytosolic CRY2PHR protein translocated to the illuminated region of the membrane within 20 s (Figures 2C–E). This result led us to ask whether the distance of the illuminated region (dashed white box) from the cell boundary would have any effect on localized recruitment. When the laser was directed far from the cell boundary, we did not observe any appreciable recruitment (Supplementary Figure S2A). Once the blue light was applied near the boundary, CRY2PHR recruitment was strongly localized near the illumination region (Figure 2C; Supplementary Figure S2B). However, when we shined the laser within the cell boundary, it led to global recruitment of the CRY2PHR protein (Supplementary Figure S2C). These data suggest that blue light is travelling past the edges of the region of illumination, upto a certain distance, leading to unwanted recruitment. Hence, we performed all localized illumination experiments by applying blue light just outside the cell boundary.

With our success in neutrophils, we attempted to establish the CRY2-CIBN system in Dictyostelium amoeba. We were successfully able to express CRY2PHR (red) and CIBN-CAAX (untagged) in developed and vegetative Dictyostelium as demonstrated by our global and local recruitment experiments, respectively (Figures 2F,H). CRY2PHR recruitment was fairly uniform upon global illumination with blue light, whereas it could be spatially constricted to a specific part of the cell membrane by localized irradiation (Figures 2F–J). Moreover, light-sensitive Dictyostelium seemed to be unaffected with the blue laser as suggested by their polarized morphology and motility (Figures 2F,H).

To aid our investigation, we characterized several fast-cycling cryptochrome mutants. In RAW 264.7 macrophages, both CRY2PHR (W349R)-mCherry and CRY2PHR (R489E, A491D)-tdTomato mutants translocated back to the cytosol within 3 min and 4.5 min of switching off the blue laser, respectively. This is a significant improvement from the turn-off rate of ∼9.5 min for CRY2PHR-mCherry (Supplementary Figures S3A–D). Moreover, 40% of total CRY2PHR (R489E, A491D)-tdTomato mutant translocated to the membrane after irradiation, compared to 21% or 23% for CRY2PHR-mCherry or CRY2PHR (W349R)-mCherry, respectively (Supplementary Figure S3E). The primary reason for this is that a major portion of total CRY2PHR-mCherry or CRY2PHR (W349R)-mCherry localized within the nucleus upon expression, whereas CRY2PHR (R489E, A491D)-tdTomato expressed entirely in the cytosol and was excluded from the nucleus (Supplementary Figures S3A–C). Thus, we identified CRY2PHR (R489E, A491D)-tdTomato mutant to be the most recruitable cryptochrome with a relatively fast membrane-cytosol turnover, making it ideal for studying time-critical biological reactions.

In addition to the CRY2-CIBN system, we established an improved Light Induced Dimerization (iLID) optogenetic system in mammalian and amoeba cells to examine GEF function. As shown in Figures 3A,F, SspB component was optically recruited uniformly on the macrophage cell membrane, and soon after switching off the light source, translocated back to the cytosol. SspB could be subsequently recruited after switching on the laser again. With localized illumination, as shown with the dashed white box, we noticed a distinct crescent of recruited SspB on the membrane, which gradually diminished within a minute of taking away the light (Figures 3B,G). In Dictyostelium, we integrated SspB with a doxycycline-inducible expression system under the inducible promoter, TRE-P_min, which consists of seven repeats of the TetO operator fused to a small fragment of the act15 promoter (Veltman et al., 2009). This allowed us to tightly control expression of recruitable SspB in a time-dependent manner upto 14 h, making this a useful tool for preventing build-up of potentially toxic effector proteins for optical studies (Supplementary Figures S4A,B). Irradiating 488 nm laser on these doxycycline-treated cells elicited uniform membrane recruitment within 10 s (middle panel; Figure 3C). With prolonged exposure to light, we observed recruitment in more cells in the population (right panel; Figure 3C). When we restricted the laser at a certain location, it induced a gradient of recruited SspB near the illuminated membrane. Once this was turned off, SspB again translocated back to the cytosol (Figures 3D,G). Linescan analysis clearly underlines SspB recruitment and shows that ∼50% of total cytosolic SspB was recruited to the membrane (Figure 3E). Moreover, upon testing SspB recruitability with varying blue laser strengths (0.2%–5% of total laser intensity or 0.024–0.639 W/cm² laser power density), we identified a laser power density of ∼0.061 W/cm² (0.5%) to be ideal (Supplementary Figure S5C); a weaker laser strength was unable to efficiently recruit SspB (Supplementary Figures S5A, B) whereas higher intensities compelled Dictyostelium to move away from the light source (Supplementary Figures S5D,E). These results suggested easy recruitability and high membrane-cytosol turnover of the iLID system. Overall, both CRY2-CIBN and iLID-SspB systems provided us with tightly-regulated, spatiotemporal control necessary to investigate GEF function in different cell types.

We first examined the effects of an opto-RasGEF in neutrophil morphology and migration (Pal et al., 2023). Blue light-driven global recruitment of full-length RasGRP4 resulted in increased spreading and F-actin patches in these cells (Figure 4A). A representative membrane kymograph showed increase in cell area with a concomitant increase in LifeAct patches after RasGRP4 recruitment (Figure 4B). Across the population, RasGRP4 recruitment induced a ∼90% or ∼20% increase in neutrophil cell area or aspect ratio (which serves as a proxy for cell polarity), respectively (Figures 4C,D). Next, selectively recruiting RasGRP4 to the back of the cell, as shown by dashed white box, caused new protrusions to arise locally with a concomitant disappearance of mature protrusions from the other end (Figure 4E). Angular histogram analysis showed that probability of fresh protrusion formation is highest at or near the RasGRP4 recruitment site (Figure 4F). Thus, opto-RasGRP4 increased cell area, F-actin polymerization, and reversed pre-exiting polarity. Since RasGEFs activate small Ras GTPase by exchanging GDP to GTP, we hypothesized that cytoskeletal effects induced by opto-RasGRP4 should be similar to an optically-recruited constitutively active Ras, KRas4B G12V. Upon locally recruiting CAAX-deleted KRas4B G12V to the Dictyostelium membrane, we noticed new protrusions appearing at the recruitment site (denoted by pink arrow in Supplementary Figure S6).

We next investigated how optically recruiting the catalytic Dbl homology (DH) domain of LARG would affect cell shape or size (O'Neill et al., 2018; Wagner and Glotzer, 2016; Rich et al., 2020; Inaba et al., 2021; Valon et al., 2017; Ridley, 2015). During imaging, we focused near the substrate-attached bottom surface of the epithelial cell as opposed to a middle section of the cell, as shown previously. Time-lapse imaging and linescan analysis demonstrated that the red fluorescence intensity of the LARG DH domain increased by ∼30% on the bottom surface of the MCF-10CA1h cell, after blue light was switched on, suggesting its recruitment from cytosol to the membrane (Figures 5A,D). This reduced the number of distinct F-actin patches at the cell cortex as denoted by pink arrows in Figure 5B. Overall, there was a >50% reduction in cortical F-actin intensity upon LARG (DH) recruitment (Figure 5E). This caused increased blebbing (shown with pink arrows in Figure 5C) and cell shrinkage (Figure 5F).

We next focused on opto-RacGEF mediated Rac GTPase activation and its role in regulating actin cytoskeleton and cell movement (Park et al., 2004; Pankov et al., 2005; Wu et al., 2009; Wang et al., 2010; Yoo et al., 2010; Kato et al., 2014). Upon global recruitment of the GEF domain of RacGEF1, the Dictyostelium cell triggered more protrusions around its periphery, as shown by pink arrows, and demonstrated appreciable spreading (Figure 6A). Across the population, RacGEF1 recruitment resulted in ∼20% increase in cell area (Figure 6B). There was a concomitant ∼10% or ∼40% reduction in cell polarity or average speed, respectively (Figures 6C,D). Next, we looked at the local effects of RacGEF1 recruitment on cell migration. Since RacGEFs trigger actin polymerization usually at the front of migrating cells, we anticipated that locally recruiting RacGEF1 catalytic domain would either enforce or oppose the pre-existing direction of migration. Indeed, when we locally recruited RacGEF1 to the back of the migrating cell, it first induced a protrusion at the recruitment site (denoted by pink arrows near the dashed white box), then changed the direction of migration by generating bigger membrane ruffles, and finally made the cell move towards the light source (Figure 6E). Angular histogram analysis confirmed that the probability of new protrusion generation was greatest at or near the site of GEF recruitment (Figure 6F).