Single-component optogenetic tools for inducible RhoA GTPase signaling

Abstract

We created optogenetic tools to control RhoA GTPase, a central regulator of actin organization and actomyosin contractility. RhoA GTPase, or its upstream activating GEF effectors, were fused to BcLOV4, a photoreceptor that can be dynamically recruited to the plasma membrane by a light-regulated protein-lipid electrostatic interaction with the inner leaflet. Direct membrane recruitment of these effectors induced potent contractile signaling sufficient to separate adherens junctions in response to as little as one pulse of blue light. Cytoskeletal morphology changes were dependent on the alignment of the spatially patterned stimulation with the underlying cell polarization. RhoA-mediated cytoskeletal activation induced YAP nuclear localization within minutes and subsequent mechanotransduction, verified by YAP-TEAD transcriptional activity. These single-component tools, which do not require protein binding partners, offer spatiotemporally precise control over RhoA signaling that will advance the study of its diverse regulatory roles in cell migration, morphogenesis, and cell cycle maintenance.

Temperature-responsive optogenetic probes of cell signaling

“…Single-component membrane translocation was recently described using the BcLOV4 photoreceptor, which translocates from the cytoplasm to membrane phospholipids under blue light in mammalian cells 15 ( Figure 1A ). BcLOV4 has already served as a modular technology for light-induced activation of the Rho GTPases Rac1 28 , RhoA 29 , Cdc42 30 , suggesting that BcLOV4 may be adapted to regulate many additional pathways.…”

Optogenetic control of apical constriction induces synthetic morphogenesis in mammalian tissues

“…Similar tools have been developed to spatiotemporally control contractility in mammalian cells, mainly through recruiting RhoGEF or RhoA factors to the plasma membrane. The approach has been effectively used to study mechanotransduction 19 , cell junction remodeling 20 , and cytoskeletal dynamics 21 .…”

“…The approach has been effectively used to study mechanotransduction 19 , cell junction remodeling 20 , cytoskeletal dynamics 21 and cytokinesis 22 . However, in mammalian tissues this approach has been mainly applied to study cell-level events, and its application to induce morphogenesis in complex tissue shapes would require very precise patterns of illumination.…”

“…Similar tools have been developed to spatiotemporally increase or reduce contractility in mammalian cells, mainly through recruiting RhoGEF, RhoA or myosin regulators to the plasma membrane. The approach has been effectively used to study mechanotransduction 19 , cell junction remodeling 20 , cytoskeletal dynamics 21 and cytokinesis 22 . However, in mammalian tissues this approach has been mainly applied to study cell-level events, and its application to induce morphogenesis in complex tissue shapes would require very precise patterns of illumination.…”

“…The copyright holder for this preprint (which this version posted April 21, 2021. ; https://doi.org/10.1101/2021.04.20.440475 doi: bioRxiv preprint OptoShroom3 joins the expanding set of optogenetic tools capable of inducing actomyosin constriction. While pre-existing tools have mainly used plasma membrane recruitment of RhoGEF or RhoA factors for the activation of actomyosin constriction [18][19][20][21] , the recruitment of OptoShroom3 is specific to the apical junctions of epithelial cells. Therefore, OptoShroom3 does not require subcellular precision of stimulation with multi-photon microscopy to induce apical constriction.…”