Optically inducible membrane recruitment and signaling systems

Single-component optogenetic tools for inducible RhoA GTPase signaling

“…Optogenetics is highly attractive for these purposes owing to its high spatiotemporal precision vs. pharmacological and genetic techniques, which can be encumbered by slow uptake/washout kinetics and frequent pleotropic effects. Because small GTPases and their activating GEFs (guanine nucleotide exchange factors) signal at the plasma membrane, optogenetic membrane localization techniques are effective for inducible control over their function, where cytosol-sequestered effector proteins are dynamically recruited to the cytosolfacing inner leaflet of the plasma membrane to upregulate effector signaling 6 . Based on earlier reported chemically inducible dimerization (CID)-based approaches for RhoA membrane recruitment 7,8 , heterodimerization between a photosensory protein and a protein binding partner (one of which is membrane localized) has been widely used to control upstream RhoA-activating GEFs 9–13 and phosphatases 14 .…”

“…This direct interaction with the membrane itself is powerful for creating “single-component” tools for dynamic membrane recruitment of peripheral membrane proteins, without the obligate heterodimerization- or self-oligomerization protein partners of the aforementioned (PPI) proteinprotein interaction-based systems. We previously leveraged the intrinsic membrane-binding capability of BcLOV4 fused to Rho-family Cdc42-GEF and Rac1 GTPase proteins to induce filopodial and lamellipodial protrusions 6,21 . Here, we report the engineering of single-component optogenetic RhoA GTPase and GEFs to potently drive actomyosin contractility, stress fiber formation, and rapid activation of transcriptional mechanotransduction ( Figure 1a, Supplementary Figure 1 ).…”

Single‐Component Optogenetic Tools for Inducible RhoA GTPase Signaling

“…Because small GTPases and their activating guanine nucleotide exchange factors (GEFs) signal at the plasma membrane, optogenetic membrane localization techniques are effective for inducible control over their function, where cytosol-sequestered proteins are dynamically recruited to the cytosol-facing inner leaflet of the plasma membrane to upregulate their signaling. [6] Based on earlier reported chemically inducible dimerization (CID)-based approaches for RhoA membrane recruitment, [7,8] optogenetic heterodimerization and photoactivatable chemically induced dimerization between a photo-responsive protein and a protein-binding partner (one of which is membrane localized) have been widely used to control upstream RhoA-activating GEFs [9][10][11][12][13][14] and phosphatases. [15] The heterodimerization strategy is sensitive to the stoichiometry of the two components, and thus may require expression leveltuning by clonal cell line selection and/or multiple fluorescent tags (i.e., separate for each component) at the expense of optical bandwidth otherwise useful for visualizing other fluorescent probes.…”

Lights up on organelles: Optogenetic tools to control subcellular structure and organization

“…Created with BioRender these reasons, a vast assortment of optogenetic tools has been developed to dynamically control protein localization to the plasma membrane. The purpose of this approach is often to optogenetically control signaling cascades originating at the plasma membrane (Hannanta-Anan, Glantz, & Chow, 2019;Mühlhäuser, Fischer, Weber, & Radziwill, 2017) or alter cell membrane dynamics (Ueda & Sato, 2018). As many of these strategies have been reviewed elsewhere (Hannanta-Anan et al, 2019;Mühlhäuser et al, 2017;Ueda & Sato, 2018), this section will provide a brief update on novel optogenetic tactics which affect the plasma membrane.…”