Optogenetic control of small GTPases reveals RhoA mediates intracellular calcium signaling

Hironori Inaba,Qianqian Miao,Takao Nakata

doi:10.1016/j.jbc.2021.100290

Hironori Inaba, Qianqian Miao + Show 1 more

Open Access

https://doi.org/10.1016/j.jbc.2021.100290

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Abstract

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, cross talk between these signals is considered likely. However, systematic quantitative evaluation has not yet been reported. To fill this gap, we constructed optogenetic tools to control the activity of small GTPases (RhoA, Rac1, Cdc42, Ras, Rap, and Ral) using an improved light-inducible dimer system (iLID). We characterized these optogenetic tools with genetically encoded red fluorescence intensity-based small GTPase biosensors and confirmed these optogenetic tools’ specificities. Using these optogenetic tools, we investigated calcium mobilization immediately after small GTPase activation. Unexpectedly, we found that a transient intracellular calcium elevation was specifically induced by RhoA activation in RPE1 and HeLa cells. RhoA activation also induced transient intracellular calcium elevation in MDCK and HEK293T cells, suggesting that generally RhoA induces calcium signaling. Interestingly, the molecular mechanisms linking RhoA activation to calcium increases were shown to be different among the different cell types: In RPE1 and HeLa cells, RhoA activated phospholipase C epsilon (PLCε) at the plasma membrane, which in turn induced Ca2+ release from the endoplasmic reticulum (ER). The RhoA–PLCε axis induced calcium-dependent nuclear factor of activated T cells nuclear translocation, suggesting that it does activate intracellular calcium signaling. Conversely, in MDCK and HEK293T cells, RhoA–ROCK–myosin II axis induced the calcium transients. These data suggest universal coordination of RhoA and calcium signaling in cellular processes, such as cellular contraction and gene expression.

Highlights

Extracellular stimuli, and regulate a variety of biological processes, including cytoskeletal reorganization, cell proliferation, and cell differentiation [1, 2]
We found that RhoA activated PLCε in RPE1 and HeLa cells, which induced intracellular calcium signaling
Among the several light-inducible heterodimerization systems, we chose the improved light-inducible dimer system (iLID) system because of the reasons that follow: (i) it is based on the AsLOV2 domain that can work without exogenously adding a chromophore to mammalian cells; (ii) iLID-SspB heterodimerization can be controlled by blue light with rapid on/off kinetics, which is suitable in controlling small GTPase activity at high spatiotemporal resolution; and (iii) the molecular weight of proteins is small, allowing high-level expression in cells and relative ease of establishing a lentivirus vector for stable cell lines

Summary

Results

Specific control of Rho/Ras family small GTPase activity at high spatiotemporal resolution was achieved using optogenetic tools. Blue light irradiation using a 458-nm laser induced iLID–SspB heterodimerization, which in turn induced the localization of LARG-DH to the plasma membrane, at which it could activate RhoA (Fig. 1B, 458-nm light ON). We characterized this light-induced translocation using the mCherry version of opto-RhoA, in which mVenus was replaced with mCherry (Fig. S1). LARG-DH of opto-RhoA was replaced with the GEF domains of specific GEFs for each small GTPase (see details in the Experimental procedure section) Most of these constructs were previously applied in optogenetic analyses [19, 26, 27]. P2A ddRFPB RBD ddRFPA small G opto-control opto-RhoA opto-Rac opto-Cdc opto-Ras opto-Rap

A B “active” small

Discussion

Experimental procedures