Regulation of β1 Integrin-Klf2-mediated angiogenesis by CCM proteins

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Recent insights into cerebral cavernous malformations: a complex jigsaw puzzle under construction.: Emerging signaling pathways regulated by CCM proteins

International audienceCerebral cavernous malformations (CCM) are common vascular malformations with an unpredictable risk of hemorrhage, the consequences of which range from headache to stroke or death. Three genes, CCM1, CCM2 and CCM3, have been linked to the disease. The encoded CCM proteins interact with each other within a large protein complex. Within the past 2 years, a plethora of new data has emerged on the signaling pathways in which CCM proteins are involved. CCM proteins regulate diverse aspects of endothelial cell morphogenesis and blood vessel stability such as cell-cell junctions, cell shape and polarity, or cell adhesion to the extracellular matrix. Although fascinating, a global picture is hard to depict because little is known about how these pathways coordinate to orchestrate angiogenesis. Here we present what is known about the structural domain organization of CCM proteins, their association as a ternary complex and their subcellular localization. Numerous CCM partners have been identified using two-hybrid screens, genetic analyses or proteomic studies. We focus on the best-characterized partners and review data on the signaling pathways they regulate as a step towards a better understanding of the etiology of CCM disease

: Rap1 regulates Krit1 microtubule and lipid binding

International audienceThe small G protein Rap1 regulates diverse cellular processes such as integrin activation, cell adhesion, cell-cell junction formation and cell polarity. It is crucial to identify Rap1 effectors to better understand the signalling pathways controlling these processes. Krev interaction trapped 1 (Krit1), a protein with FERM (band four-point-one/ezrin/radixin/moesin) domain, was identified as a Rap1 partner in a yeast two-hybrid screen, but this interaction was not confirmed in subsequent studies. As the evidence suggests a role for Krit1 in Rap1-dependent pathways, we readdressed this question. In the present study, we demonstrate by biochemical assays that Krit1 interacts with Rap1A, preferentially its GTP-bound form. We show that, like other FERM proteins, Krit1 adopts two conformations: a closed conformation in which its N-terminal NPAY motif interacts with its C-terminus and an opened conformation bound to integrin cytoplasmic domain associated protein (ICAP)-1, a negative regulator of focal adhesion assembly. We show that a ternary complex can form in vitro between Krit1, Rap1 and ICAP-1 and that Rap1 binds the Krit1 FERM domain in both closed and opened conformations. Unlike ICAP-1, Rap1 does not open Krit1. Using sedimentation assays, we show that Krit1 binds in vitro to microtubules through its N- and C-termini and that Rap1 and ICAP-1 inhibit Krit1 binding to microtubules. Consistently, YFP-Krit1 localizes on cyan fluorescent protein-labelled microtubules in baby hamster kidney cells and is delocalized from microtubules upon coexpression with activated Rap1V12. Finally, we show that Krit1 binds to phosphatidylinositol 4,5-P(2)-containing liposomes and that Rap1 enhances this binding. Based on these results, we propose a model in which Krit1 would be delivered by microtubules to the plasma membrane where it would be captured by Rap1 and ICAP-1

Calcium signaling mediates a biphasic mechanoadaptive response of endothelial cells to cyclic mechanical stretch

International audienceThe vascular system is precisely regulated to adjust blood flow to organismal demand, thereby guaranteeing adequate perfusion under varying physiological conditions. Mechanical forces, such as cyclic circumferential stretch, are among the critical stimuli that dynamically adjust vessel distribution and diameter, but the precise mechanisms of adaptation to changing forces are unclear. We find that endothelial monolayers respond to cyclic stretch by transient remodeling of the vascular endothelial cadherin-based adherens junctions and the associated actomyosin cytoskeleton. Time-resolved proteomic profiling reveals that this remodeling is driven by calcium influx through the mechanosensitive Piezo1 channel, triggering Rho activation to increase actomyosin contraction. As the mechanical stimulus persists, calcium signaling is attenuated through transient down-regulation of Piezo1 protein. At the same time, filamins are phosphorylated to increase monolayer stiffness, allowing mechanoadaptation to restore junctional integrity despite continuing exposure to stretch. Collectively, this study identifies a biphasic response to cyclic stretch, consisting of an initial calcium-driven junctional mechanoresponse, followed by mechanoadaptation facilitated by monolayer stiffening

Novel and accesible 3D Traction Force Microscopy applied to an in vitro vascular disease model

Due to COVI19 the Conference did not take place in Milan. Rather, a webinar where the beest 4 selected student abstracts was held. This work won the ESB 2020 student Award.status: Published onlin