Palacios Martinez, S. | Search bioRxiv

bioRxiv2022

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

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 to allow the passage of essential molecules like oxygen, carbon-dioxide, water, ions, and nutrients. The vascular endothelial barrier is defined by cell-cell contacts through adherens and tight junctions. To further investigate the signaling in the endothelium that regulates vascular barrier strength, we focused on Rho GTPases, regulators of the actin cytoskeleton and known to control junction integrity. Rho GTPase signaling is confined in space and time. To manipulate the signaling in a temporal and spatial manner we applied optogenetics. Guanine exchange factor (GEF) domains from ITSN1, TIAM1 and p63RhoGEF, activating Cdc42, Rac and Rho respectively, were integrated into the optogenetic recruitment tool iLID. This tool allows for activation at the subcellular level in a reversible and non-invasive manner and thereby to recruit a GEF to local areas at the plasma membrane, enabling the local activation of specific Rho GTPases. The membrane tag of iLID was optimized and a HaloTag was applied to gain more flexibility for multiplex imaging. The resulting 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, -roundness, local extension, and cell contraction. In conclusion, we have optimized and applied the optogenetic iLID GEF recruitment tool i.e. 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.

cell biology↗

bioRxiv2022

Endothelial transmigration hotspots limit vascular leakage through heterogeneous expression of ICAM1

Upon inflammation, leukocytes leave the circulation by crossing the endothelial monolayer at specific transmigration hotspot regions. Although these regions support leukocyte transmigration, their functionality is not clear. We found that endothelial hotspots function to limit vascular leakage during transmigration events. Using the photo-convertible probe mEos4b, we traced back and identified original endothelial transmigration hotspots. Using this method, we show that the heterogeneous distribution of ICAM-1 determines the location of the transmigration hotspot. Interestingly, loss of ICAM-1 heterogeneity either by CRISPR/Cas9-induced knockout of ICAM-1 or equalizing the distribution of ICAM-1 in all endothelial cells results in loss of TEM hotspots but not necessarily in reduced TEM events. Functionally, loss of endothelial hotspots results in increased vascular leakage during TEM. Mechanistically, we demonstrate that the 3 extracellular Ig-like domains of ICAM-1 are crucial for hotspot recognition. However, the intracellular tail of ICAM-1 and the 4th Ig-like dimerization domain are not involved, indicating that intracellular signalling or ICAM-1 dimerization is not required for hotspot recognition. Together, we discovered that hotspots function to limit vascular leakage during inflammation-induced extravasation.

immunology↗