Optogenetic control of small GTPases reveals RhoA-mediated intracellular calcium signaling

Rho/Ras family small GTPases are known to regulate numerous cellular processes, including cytoskeletal reorganization, cell proliferation, and cell differentiation. These processes are also controlled by Ca2+, and consequently, crosstalk between these signals is considered likely. However, systematic quantitative evaluation is not yet reported. Thus, we developed optogenetic tools to control the activity of small GTPases (RhoA, Rac1, Cdc42, Ras, Rap, and Ral) using an improved light-inducible dimer system (iLID). Using these optogenetic tools, we investigated calcium mobilization immediately after small GTPase activation. Unexpectedly, we found that only RhoA activation induced a transient intracellular calcium elevation in RPE1 and HeLa cells. Transients were also observed in MDCK and HEK293T cells by RhoA activation, but interestingly, molecular mechanisms were identified to be different among cell types. In RPE1 and HeLa cells, RhoA directly activated phospholipase C (PLC)ε at the plasma membrane, which in turn induced Ca2+ release from the endoplasmic reticulum (ER). The RhoA-PLCε axis induced calcium-dependent NFAT nuclear translocation, suggesting it does activate intracellular calcium signaling.


Introduction 1
Small GTPases of the Ras superfamily have been identified as molecular switches 2 because they exist in two states, a GTP-binding state ("ON") and a GDP-binding state 3 ("OFF") (Mitin et al., 2005;Wennerberg et al., 2005). These states are known to be 4 regulated by activatorsguanine nucleotide exchange factors (GEFs)and inactivators, 5 that is, GTPase-activating proteins (GAPs). Rho and Ras subfamily small GTPases 6 localize at the plasma membrane (PM), respond to extracellular stimuli, and are 7 responsible for a variety of biological processes, including cytoskeletal reorganization, 8 cell proliferation, and cell differentiation (Mitin et al., 2005;Wennerberg et al., 2005). 9 Several of these biological processes are also regulated by a universal second messenger, 10 that is, calcium ion (Ca 2+ ) (Berridge et al., 2000;Clapham, 2007;Cullen and Lockyer, 11 2002). Thus, functional links must exist between these signaling pathways regulated by 12 was only induced by optogenetic RhoA activation. These RhoA-mediated calcium 1 transients were observed in all cell types examined, but the molecular mechanisms were 2 different among the cell types. Furthermore, we found that RhoA directly activated PLCe 3 in RPE1 and HeLa cells, which induced intracellular calcium signaling. (1) it is based on the AsLOV2 domain that can work without exogenously adding a 6 chromophore to mammalian cells; (2) iLID-SspB heterodimerization can be controlled 7 by blue-light with rapid on/off kinetics (seconds), which is suitable in controlling small 8 GTPase activity at high spatiotemporal resolution; and (3) the molecular weight of 9 proteins is small, allowing high-level expression in cells and relative ease of establishing 10 a lentivirus vector for stable cell lines.
3 4 Screening using opto-GTPases for induction of changes in intracellular calcium 5 concentrations 6 We monitored changes of intracellular calcium concentrations with a genetically encoded 7 red fluorescent calcium indicator R-GECO1 that is known to detect physiological calcium 8 changes as found during neural activity and muscle contraction (Zhao et al., 2011).  GTPases and the calcium reporter R-GECO1 were transfected into human non- RhoA was somewhat leaky, that is, opto-RhoA exerts background activity in the dark.

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The number of blebs was dramatically increased after the blue-light irradiation, indicating 12 that the blue-light irradiation did increase RhoA activity.

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Time-course analysis showed that initiation of an increase in intracellular 14 calcium concentration was observed within 10 seconds in RPE1, HeLa, and HEK293T 15 cells; meanwhile, maximum concentration was observed about 20 seconds after light 16 irradiation ( Figure 2D). In contrast, initiation of this increase was observed within 20-17 30 seconds, and the maximum increase was reached about 2 minutes after light irradiation in MDCK cells ( Figure 2D). Cellular responses differed among cell types. 1 2 Mechanisms of RhoA-induced calcium transients are different among cell types 3 We investigated molecular mechanisms of RhoA-induced intracellular calcium transients 4 using small molecule inhibitors (see Figure 7 for summary). Several calcium channels 5 are activated by RhoA and its downstream factors (Li and Brayden, 2017;Mehta et al., 6 2003;Wing et al., 2003). We initially examined the RhoA-ROCK-myosin II axis, the 7 major pathway of the RhoA signaling pathway. Both ROCK inhibitor, Y-276322, and 8 myosin II inhibitor, Blebbistatin, were observed to efficiently inhibit RhoA-induced 9 calcium transients in MDCK and HEK293T cells, but surprisingly not in RPE1 and HeLa 10 cells (Figure 3). Actomyosin-mediated cellular contraction activates mechanosensitive 11 (MS) calcium channels such as Piezo1 and transient receptor potential (TRP) family 12 channels in MDCK and HEK293T cells.

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Further, we tested the non-selective calcium channel blockers SKF96365 and 2-14 APB (Figure 3). SKF96365 was observed to inhibit the RhoA-induced calcium transients 15 in MDCK and HEK293T cells (Figures 3C, D). Conversely, 2-APB blocked transients 16 in RPE1 and HeLa cells (Figures 3A, B). Mechanisms of RhoA-induced calcium mechanisms of RhoA-induced calcium transients in RPE1 and HeLa cells. in this condition when incubated in Ringer's solution. As hypothesized, RhoA activates 12 PLC, which in turn induces calcium transients. 13 We also tested RhoA-induced calcium transients in calcium-free buffers 14 (Figures 3A, B). The percentage of cells exhibiting calcium transients significantly 15 decreased in both cell types. However, about 10 % of cells still showed transients, and 16 intracellular calcium stores have been identified as a source of these calcium transients.
We also knocked down PLCe in RPE1 and HeLa cells with small interference 1 RNA (siRNA) and examined calcium transients (Figures 4A, B). Blue-light irradiation   Figure 4C) of PLCe is 2 required for its activation by RhoA (Wing et al., 2003). In contrast, Ras and Rap1 are seen 3 to bind directly to the second Ras association domain (RA2 domain in Figure 4C  RhoA activation at the PM appears essential for calcium transients. 7 We next explored PI(4,5)P2 dynamics on the PM using the PI(4,5)P2 marker, a 8 PH domain of PLCd (PLCd-PH) (Stauffer et al., 1998) and an optogenetic tool for 9 controlling the inositol 5-phosphatase (opto-5-ptase) with the inositol 5-phosphatase  Finally, we examined RhoA-PLCe-mediated intracellular calcium transients for 8 activation of intercellular calcium signaling with an NFAT nuclear translocation assay.

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NFAT is identified as a transcription factor, and, after dephosphorylation by calmodulin- In this study, we have developed optogenetic tools to control Rho and Ras family GTPase 2 activity; we then performed cell-based systematic functional screening (Figure 2). We 3 focused on intracellular calcium signaling induced by Rho and Ras signaling. This 4 approach for cell-based screening is applicable for other signaling pathways as well if 5 both optogenetic tools and biosensors are available. Before the introduction of 6 optogenetic tools, single cell-based enzymology was difficult due to the lack of sufficient 7 spatiotemporal resolution and specificity. Optogenetics has resolved this issue by exerting 8 control over specific signaling molecules using a light stimulus with high spatiotemporal 9 resolution. This tool provides a powerful platform for cell-based enzymology.
10 Surprisingly, only RhoA activation was seen to induce calcium transients in effects. However, there were still cell populations that appeared healthy, did not highly express exogenous proteins, but still did not exhibit calcium transients.  Unexpectedly, molecular mechanisms underlying calcium transients were found 8 to be notably different among cell types (Figures 3, 7). In MDCK and HEK293 cells, the In contrast, the RhoA-PLCe axis was determined to be functional in RPE1 and 1 HeLa cells (Figures 3, 4 and 7, upper scheme). Calcium transients were totally blocked 2 by 2-APB and partially observed in Ca 2+ -free buffer, and transients were apparently 3 induced by the IP3-IP3R pathway that promotes Ca 2+ release from the ER. In the Ca 2+ -  PLCe has been identified to be activated by other small GTPases, including Ras, 11 Rap, and Ral (Jin et al., 2001;Kelley et al., 2004Kelley et al., , 2001, which is inconsistent with the 12 present results (Figure 2). This discrepancy is explained by the fact that there is no 13 evidence that small GTPases directly activate the PLCe. In previous studies,    Opto-5-ptase was constructed as previously reported for mCherry-PHR-iSH-2A-pEGFP-N1, and mCherry and iSH were replaced with ECFP and the inositol 5-phosphate 1 domain of OCRL (OCRLcat, aa 234-539), respectively.

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All cloned fragments were verified by sequencing.    The authors are grateful to Dr. Naofumi Yui (TMDU) for the kind gift of MDCK cells 1 and Toshiyuki Kakumoto for plasmid construction. We also thank Drs. Tomohiro Ishii 2 and Toshifumi Asano for the helpful discussion and Satoko Nakamura for secretarial 3 expertise.   15 The authors declare that they have no conflict of interests.   GTPases were activated by a multi-argon 458 nm laser every 10 sec for 2 minutes.

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Percentages of cells exhibiting intracellular calcium transients (F/F0 > 2) in response to 6 opto-GTPases activation are shown. Data are presented as means ± SEM from more than 7 three independent experiments. In total, >150 cells were analyzed for each photoswitch.  are shown as means ± SEM from more than three independent experiments. In total, >200 6 cells were analyzed for each condition. ***, p < 0.001; **, p < 0.01; and n.s., not 7 significant; two-tailed unpaired Student's t-test.  Figure 2). Data are presented as means ± SEM from three independent experiments. In 6 total, >150 cells were analyzed for each condition. ***, p < 0.001; and n.s., not 7 significant; two-tailed unpaired Student's t-test. Golgi, and R-GECO1 (see Figure 2). Data are presented as means ± SEM from three 17 independent experiments (in total >150 cells were analyzed for each photoswitch). ***, p < 0.001; **, p < 0.01; and n.s., not significant; one-way ANOVA and Tukey's test. (D) 1 Schematic of opto-5-ptase. OCRLcat, inositol 5-phosphatase catalytic domain of OCRL.   to cytosol ratio (C) and changes of this ratio (D) of NFAT-mCherry before (dark) and after transiently expressed opto-RhoA and NFAT-mCherry. Data from three independent 1 experiments is shown in box-and-whisker plots. Boxes represent the first to third quartiles 2 (interquartile range: IQR). The horizontal line inside the box represents the median-3 vertical lines above and below the box span 1.5×IQR. Dots represent outliers that are 4 above or below 1.5×IQR. In total, 61, 58, and 72 cells treated with si control, si PLCe 5 seq1, and seq3 were analyzed, respectively. ***, p < 0.001; **, p < 0.01; and n.s., not 6 significant; two-tailed paired Student's t-test (C) and one-way ANOVA followed by 7 Wilcoxon signed-rank test (D).