TY - JOUR
T1 - Opto-RhoGEFs, an optimized optogenetic toolbox to reversibly control Rho GTPase activity on a global to subcellular scale, enabling precise control over vascular endothelial barrier strength
AU - Mahlandt, Eike K.
AU - Palacios Martínez, Sebastián
AU - Arts, Janine J. G.
AU - Tol, Simon
AU - van Buul, Jaap D.
AU - Goedhart, Joachim
N1 - Funding Information: We want to thank Ronald Breedijk for the support at the van Leeuwenhoek Centre for Advanced Microscopy, Section Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam. We also want to thank Anne-Marieke van Stalborch, Jos van Rijssel, Max Grönloh, and Werner van der Meer for their support and advice on the work with endothelial cells, the permeability assay, and ECIS experiment. Funding: EM was supported by an Nederlandse Organisatie voor Weten-schappelijk Onderzoek ALW-OPEN grant (ALWOP.306). Funding Information: We want to thank Ronald Breedijk for the support at the van Leeuwenhoek Centre for Advanced Microscopy, Section Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam. We also want to thank Anne-Marieke van Stalborch, Jos van Rijssel, Max Grönloh, and Werner van der Meer for their support and advice on the work with endothelial cells, the permeability assay, and ECIS experiment. Funding: EM was supported by an Nederlandse Organisatie voor Weten-schappelijk Onderzoek ALW-OPEN grant (ALWOP.306).Nederlandse Organisatie voor Wetenschappelijk Onderzoek ALWOP.306 Jaap D van Buul Joachim GoedhartZonMw VICI grant #91819632 Jaap D van BuulThe funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Publisher Copyright: © 2023, eLife Sciences Publications Ltd. All rights reserved.
PY - 2023/7/14
Y1 - 2023/7/14
N2 - The inner layer of blood vessels consists of endothelial cells, which form the physical barrier between blood and tissue. This vascular barrier is tightly regulated and is defined by cell-cell contacts through adherens and tight junctions. To investigate the signaling that regulates vascular barrier strength, we focused on Rho GTPases, regulators of the actin cytoskeleton and known to control junction integrity. To manipulate Rho GTPase signaling in a temporal and spatial manner we applied optogenetics. Guanine-nucleotide exchange factor (GEF) domains from ITSN1, TIAM1, and p63RhoGEF, activating Cdc42, Rac, and Rho, respectively, were integrated into the optogenetic recruitment tool improved light-induced dimer (iLID). This tool allows for Rho GTPase activation at the subcellular level in a reversible and non-invasive manner by recruiting a GEF to a specific area at the plasma membrane, The membrane tag of iLID was optimized and a HaloTag was applied to gain more flexibility for multiplex imaging. The resulting optogenetically recruitable RhoGEFs (Opto-RhoGEFs) were tested in an endothelial cell monolayer and demonstrated precise temporal control of vascular barrier strength by a cell-cell overlap-dependent, VE-cadherin-independent, mechanism. Furthermore, Opto-RhoGEFs enabled precise optogenetic control in endothelial cells over morphological features such as cell size, cell roundness, local extension, and cell contraction. In conclusion, we have optimized and applied the optogenetic iLID GEF recruitment tool, that is Opto-RhoGEFs, to study the role of Rho GTPases in the vascular barrier of the endothelium and found that membrane protrusions at the junction region can rapidly increase barrier integrity independent of VE-cadherin.
AB - The inner layer of blood vessels consists of endothelial cells, which form the physical barrier between blood and tissue. This vascular barrier is tightly regulated and is defined by cell-cell contacts through adherens and tight junctions. To investigate the signaling that regulates vascular barrier strength, we focused on Rho GTPases, regulators of the actin cytoskeleton and known to control junction integrity. To manipulate Rho GTPase signaling in a temporal and spatial manner we applied optogenetics. Guanine-nucleotide exchange factor (GEF) domains from ITSN1, TIAM1, and p63RhoGEF, activating Cdc42, Rac, and Rho, respectively, were integrated into the optogenetic recruitment tool improved light-induced dimer (iLID). This tool allows for Rho GTPase activation at the subcellular level in a reversible and non-invasive manner by recruiting a GEF to a specific area at the plasma membrane, The membrane tag of iLID was optimized and a HaloTag was applied to gain more flexibility for multiplex imaging. The resulting optogenetically recruitable RhoGEFs (Opto-RhoGEFs) were tested in an endothelial cell monolayer and demonstrated precise temporal control of vascular barrier strength by a cell-cell overlap-dependent, VE-cadherin-independent, mechanism. Furthermore, Opto-RhoGEFs enabled precise optogenetic control in endothelial cells over morphological features such as cell size, cell roundness, local extension, and cell contraction. In conclusion, we have optimized and applied the optogenetic iLID GEF recruitment tool, that is Opto-RhoGEFs, to study the role of Rho GTPases in the vascular barrier of the endothelium and found that membrane protrusions at the junction region can rapidly increase barrier integrity independent of VE-cadherin.
KW - Rho GTPase
KW - RhoGEF
KW - VE-cadherin
KW - cell biology
KW - endothelium
KW - human
KW - optogenetics
KW - vascular barrier
UR - http://www.scopus.com/inward/record.url?scp=85164757757&partnerID=8YFLogxK
U2 - https://doi.org/10.7554/eLife.84364
DO - https://doi.org/10.7554/eLife.84364
M3 - Article
C2 - 37449837
SN - 2050-084X
VL - 12
JO - eLife
JF - eLife
M1 - RP84364
ER -