Single‐Component Optogenetic Tools for Inducible RhoA GTPase Signaling

Abstract

to the extracellular matrix (ECM) via focal adhesions. [3] Thus, new tools for inducible control over RhoA activity may greatly enhance understanding of cytoskeletal dynamics and mechanotransduction. [4,5] Optogenetics is highly attractive for this purpose owing to its high spatiotemporal precision versus pharmacological and genetic techniques that can be encumbered by slow uptake/washout kinetics and pleotropic effects. 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. [16][17][18] Previously, we reported the direct optogenetic control over RhoA GTPase itself by another mechanism of inducible clustering of RhoA fused to an oligomerizing form of plant cryptochrome, which presumably increases the binding avidity of the GTPase to membrane GEFs. However, this system has limited spatial resolution due to extensive cytosolic diffusion beyond the optical stimulation field prior to stable membrane localization post-oligomerization. [19] Recently, we reported that BcLOV4, a light-oxygen-voltage (LOV) flavoprotein from Botrytis cinerea, rapidly translocates to the plasma membrane in mammalian cells via a blue lightregulated electrostatic protein-lipid interaction (PLI) with the inner leaflet. [20,21] This direct interaction with the membrane itself is powerful for creating "single-component" tools for dynamic membrane recruitment of fused peripheral membrane proteins, without the obligate heterodimerization-or Optogenetic tools are created to control RhoA GTPase, a central regulator of actin organization and actomyosin contractility. RhoA GTPase, or its upstream activator ARHGEF11, is 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 proteins induces potent contractile signaling sufficient to separate adherens junctions with as little as one pulse of blue light. Induced cytoskeletal morphology changes are dependent o...

Designing Single-Component Optogenetic Membrane Recruitment Systems: The Rho-Family GTPase Signaling Toolbox

“…Initially reported using an engineered chimera of the AsLOV2 photosensory domain , fused to a phospholipid binding domain (of RIT GTPase), “single-component” recruitment by direct association with the inner leaflet, without a heterodimerization partner, offers experimental simplicity in that it requires only one transgene (to be delivered, expressed, and visualized). The endogenous binding partner is in natural excess at the membrane to avoid binding-site availability limitations that reduce signaling fold-change and compromise spatiotemporal resolution by increasing diffusional length/time before stable association. − …”

“…It is a natural single-component system whose rapid membrane association/dissociation kinetics (τ on ∼ seconds and τ off ∼ 1 min) and slow lateral diffusion ( D mem < 0.028 μm 2 /s) are favorable to high performance control. It is a versatile technology with which we have engineered numerous chimeric Rho-family GTPases and GEF proteins created by a common workflow that involved six engineered genetic constructs. − …”

Mechanotransductive feedback control of endothelial cell motility and vascular morphogenesis

“…Thus, cytoskeletal activation is likely non-rate-limiting in cytoskeletal feedback, in contrast to single cell tensional homeostasis (Webster et al, 2014). Likewise, acute optogenetic RhoA activation produces significant YAP/TAZ nuclear localization within minutes and induces subsequent co-transcriptional activity (Berlew et al, 2021; Valon et al, 2017). Thus, we posit that the dynamics of YAP/TAZ-dependent transcription and the translation of those target genes are rate-limiting for feedback execution, consistent with the dynamics of YAP/TAZ target gene induction by adhesion to variable stiffness matrices.…”

Optical regulation of endogenous RhoA reveals selection of cellular responses by signal amplitude

“…Alternatively, RhoA can be sequestered to mitochondria via a protein-protein interaction that occurs in the dark but not in blue light so that it can be released throughout the cell upon illumination (Wang et al, 2016). In another example, RhoA can be fused to BcLOV4 (Berlew et al, 2021), a light-oxygen-voltage (LOV) domain that interacts with phospholipid membranes after illumination. Light allows the RhoA-BcLOV4 protein to accumulate at the plasma membrane, leading to RhoA activation (Glantz et al, 2018(Glantz et al, , 2019.…”