Optogenetic Rac1 engineered from membrane lipid-binding RGS-LOV for inducible lamellipodia formation

Berlew, Erin E.; Кузнецов, И. А.; Yamada, Keisuke; Bugaj, Lukasz J.; Chow, Brian Y.

doi:10.1039/c9pp00434c

Cited by 23 publications

(49 citation statements)

References 53 publications

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“…We fused wildtype RhoA over constitutively active mutants to limit the probability of RhoA signal transduction in the dark by diffusive membrane contact alone, which was evident in cells expressing a BcLOV4-fused constitutively active G14V RhoA mutant with elevated basal stress fiber levels ( Supplementary Figure 9 ). The dynamic range improvement by using wildtype GTPase was also observed with our previously reported opto-Rac1 21 .…”

Section: Discussionsupporting

confidence: 83%

“…Cells were imaged at room temperature in CO2-independent media (phenol-free HBSS supplemented with 1% l-glutamine, 1% penicillin-streptomycin, 2% essential amino acids, 1% nonessential amino acids, 2.5% HEPES pH 7.0, and 10% serum). The custom spatially patterned illuminator was (DMD) digital micromirror device-based and constructed from a digital light processor (DLP, Digital Light Innovations CEL5500), as previously described 21 .…”

Section: Materials and Methods Onlinementioning

confidence: 99%

“…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 ).…”

Section: Introductionmentioning

confidence: 99%

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Single-component optogenetic tools for inducible RhoA GTPase signaling

Berlew

Кузнецов

Yamada

et al. 2021

Preprint

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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.

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Section: Discussionsupporting

confidence: 83%

Section: Materials and Methods Onlinementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Single-component optogenetic tools for inducible RhoA GTPase signaling

Berlew

Кузнецов

Yamada

et al. 2021

Preprint

Self Cite

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“…Recently a LOV protein containing a RGS (regulator of G-protein signaling) domain has been identified in B. cinerea and other fungi (Table S2), indicated as BcLOV4 [37]. These fungal RGS-LOV proteins are associated with anionic plasma membrane phospholipids, when photoactivated with BL [132], and are now being used to engineer systems for optogenetic applications [133].…”

Section: Photoresponses and Photoreceptors In Plant-associated Fungimentioning

confidence: 99%

A light life together: photosensing in the plant microbiota

Losi

Gärtner

2021

Photochem Photobiol Sci

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Bacteria and fungi of the plant microbiota can be phytopathogens, parasites or symbionts that establish mutually advantageous relationships with plants. They are often rich in photoreceptors for UVA–Visible light, and in many cases, they exhibit light regulation of growth patterns, infectivity or virulence, reproductive traits, and production of pigments and of metabolites. In addition to the light-driven effects, often demonstrated via the generation of photoreceptor gene knock-outs, microbial photoreceptors can exert effects also in the dark. Interestingly, some fungi switch their attitude towards plants in dependence of illumination or dark conditions in as much as they may be symbiotic or pathogenic. This review summarizes the current knowledge about the roles of light and photoreceptors in plant-associated bacteria and fungi aiming at the identification of common traits and general working ideas. Still, reports on light-driven infection of plants are often restricted to the description of macroscopically observable phenomena, whereas detailed information on the molecular level, e.g., protein–protein interaction during signal transduction or induction mechanisms of infectivity/virulence initiation remains sparse. As it becomes apparent from still only few molecular studies, photoreceptors, often from the red- and the blue light sensitive groups interact and mutually modulate their individual effects. The topic is of great relevance, even in economic terms, referring to plant-pathogen or plant-symbionts interactions, considering the increasing usage of artificial illumination in greenhouses, the possible light-regulation of the synthesis of plant-growth stimulating substances or herbicides by certain symbionts, and the biocontrol of pests by selected fungi and bacteria in a sustainable agriculture.

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“…[12] Membrane translocation of BcLOV4-Rac1 leads to actin polymerization and lamellipodia formation in HEK cells. [13] Photoactivated adenylyl cyclases (PACs) have been used to manipulate cellular processes through light-dependent cAMP production. Flavin-based PACs have been successfully utilized in mammalian cells, but these PACs can only be turned off by spontaneous thermal decay.…”

Section: New Photoactivatable Proteinsmentioning

confidence: 99%

Steering Molecular Activity with Optogenetics: Recent Advances and Perspectives

Fan

Skeeters

et al. 2021

Advanced Biology

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In a transparent medium, lensbased optical microscopy could focus a coherent light beam (e.g., laser) into a tiny spot, whose dimension is comparable to the size of the wavelength of the light. The diameter of the smallest beam waist is about half the size of the wavelength, which is referred to as the diffraction limit. Thus, for visible light, the theoretical diffraction limit is between 200 and 400 nm, much smaller than the size of a single cell (Figure 1A). However, in biological tissues that significantly scatter and absorb visible light, the spatial resolution could be compromised. In multicellular organisms, light absorption limits the penetration depth. The scattering of the light by the opaque biological tissues would expand the volume of light stimulation and reduce the spatial resolution.

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Optogenetic Rac1 engineered from membrane lipid-binding RGS-LOV for inducible lamellipodia formation

Abstract: We report the construction of a single-component optogenetic Rac1 (opto-Rac1) to control actin polymerization by dynamic membrane recruitment.

Cited by 23 publications

References 53 publications

Single-component optogenetic tools for inducible RhoA GTPase signaling

Single-component optogenetic tools for inducible RhoA GTPase signaling

A light life together: photosensing in the plant microbiota

Steering Molecular Activity with Optogenetics: Recent Advances and Perspectives

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