Mechanotransduction pathways are activated in response to biophysical stimuli during the development or homeostasis of organs and tissues. In zebrafish, the blood-flow-sensitive transcription factor Klf2a promotes VEGF-dependent angiogenesis. However, the means by which the Klf2a mechanotransduction pathway is regulated to prevent continuous angiogenesis remain unknown. Here we report that the upregulation of klf2 mRNA causes enhanced egfl7 expression and angiogenesis signaling, which underlies cardiovascular defects associated with the loss of cerebral cavernous malformation (CCM) proteins in the zebrafish embryo. Using CCM-protein-depleted human umbilical vein endothelial cells, we show that the misexpression of KLF2 mRNA requires the extracellular matrix-binding receptor β1 integrin and occurs in the absence of blood flow. Downregulation of β1 integrin rescues ccm mutant cardiovascular malformations in zebrafish. Our work reveals a β1 integrin-Klf2-Egfl7-signaling pathway that is tightly regulated by CCM proteins. This regulation prevents angiogenic overgrowth and ensures the quiescence of endothelial cells.

Loss of CCM1/2 leads to destabilization of ICAP-1 and up-regulation of β1 integrin, resulting in the destabilization of intercellular junctions due to increased cell contractility and aberrant extracellular matrix remodeling.

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Matrix-bound BMP-2 is sufficient to induce β3 integrin–dependent, Cdc42/Src/FAK/ILK-mediated cell spreading by overriding the stiffness response through actin and adhesion site dynamics, showing BMP receptors and integrins work together to control signaling and tensional homeostasis, thereby coupling cell adhesion and fate commitment.

The small G protein Rap1 (Krev-1), a member of the Ras superfamily, has been brought to the forefront subsequent to the discovery that it regulates diverse cellular processes such as integrin activation and cell adhesion, cell spreading, cell polarity and cell-cell junction formation [1][2][3]. To gain more insight into these pathways, a variety of effector proteins that interact with the active Rap1GTP-bound form has been identified. Among them, RAPL, which is enriched in lymphoid tissues, activates the integrin aLb2, most likely by interacting with aL integrin [4] and RIAM, which binds to different actin regulators, and The 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.Abbreviations BHK, baby hamster kidney; CFP, cyan fluorescent protein; FERM, four-point-one protein ⁄ ezrin ⁄ radixin ⁄ moesin; ICAP, integrin cytoplasmic domain associated protein; Krit1, Krev interaction trapped gene; MT, microtubules; PTB, phosphotyrosine-binding domain.

Cerebral 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.

We have produced a recombinant transducin alpha subunit (rT alpha) in sf9 cells, using a baculovirus system. Deletion of the myristoylation site near the N‐terminal increased the solubility and allowed the purification of rT alpha. When reconstituted with excess T beta gamma on retinal membrane, rT alpha displayed functional characteristics of wild‐type T alpha vis à vis its coupled receptor, rhodopsin and its effector, cGMP phosphodiesterase (PDE). We further mutated a tryptophan, W207, which is conserved in all G proteins and is suspected to elicit the fluorescence change correlated to their activation upon GDP/GTP exchange or aluminofluoride (AlFx) binding. [W207F]T alpha mutant displayed high affinity receptor binding and underwent a conformational switch upon receptor‐catalysed GTP gamma S binding or upon AlFx binding, but this did not elicit any fluorescence change. Thus W207 is the only fluorescence sensor of the switch. Upon the switch the mutant remained unable to activate the PDE. To characterize better its effector‐activating interaction we measured the affinity of [W207F]T alpha GDP‐AlFx for PDE gamma, the effector subunit that binds most tightly to T alpha. [W207F]T alpha still bound in an activation‐dependent way to PDE gamma, but with a 100‐fold lower affinity than rT alpha. This suggests that W207 contributes to the G protein effector binding.