Force tuning through regulation of clathrin-dependent integrin endocytosis.

Integrin endocytosis is essential for many fundamental cellular processes. Whether and how the internalization impacts cellular mechanics remains elusive. Whereas previous studies reported the contribution of the integrin activator, talin, in force development, the involvement of inhibitors is less documented. We identified ICAP-1 as an integrin inhibitor involved in mechanotransduction by co-working with NME2 to control clathrin-mediated endocytosis of integrins at the edge of focal adhesions (FA). Loss of ICAP-1 enables ß3-integrin-mediated force generation independently of ß1 integrin. ß3-integrin-mediated forces were associated with a decrease in ß3 integrin dynamics stemming from their reduced diffusion within adhesion sites and slow turnover of FA. The decrease in ß3 integrin dynamics correlated with a defect in integrin endocytosis. ICAP-1 acts as an adaptor for clathrin-dependent endocytosis of integrins. ICAP-1 controls integrin endocytosis by interacting with NME2, a key regulator of dynamin-dependent clathrin-coated pits fission. Control of clathrin-mediated integrin endocytosis by an inhibitor is an unprecedented mechanism to tune forces at FA.

CCM2-deficient endothelial cells undergo a ROCK-dependent reprogramming into senescence-associated secretory phenotype.

Cerebral cavernous malformation (CCM) is a cerebrovascular disease in which stacks of dilated haemorrhagic capillaries form focally in the brain. Whether and how defective mechanotransduction, cellular mosaicism and inflammation interplay to sustain the progression of CCM disease is unknown. Here, we reveal that CCM1- and CCM2-silenced endothelial cells expanded in vitro enter into senescence-associated secretory phenotype (SASP) that they use to invade the extracellular matrix and attract surrounding wild-type endothelial and immune cells. Further, we demonstrate that this SASP is driven by the cytoskeletal, molecular and transcriptomic disorders provoked by ROCK dysfunctions. By this, we propose that CCM2 and ROCK could be parts of a scaffold controlling senescence, bringing new insights into the emerging field of the control of ageing by cellular mechanics. These in vitro findings reconcile the known dysregulated traits of CCM2-deficient endothelial cells into a unique endothelial fate. Based on these in vitro results, we propose that a SASP could link the increased ROCK-dependent cell contractility in CCM2-deficient endothelial cells with microenvironment remodelling and long-range chemo-attraction of endothelial and immune cells.

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

The 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.

Immunofluorescence of Cell-Cell and Cell-Extracellular Matrix Adhesive Defects in In Vitro Endothelial CCM Model: Juxtacrine Role of Mutant Extracellular Matrix on Wild-Type Endothelial Cells.

Endothelial cells lining cerebral cavernous malformations (CCM) present strong adhesive and mechanical defects. Increased cell contractility is a driver to the onset and the expansion of the CCM lesions. 2D in vitro endothelial models have been developed from either endothelial cells isolated from ccm1-3 knock-out mice or CCM1-3-silenced primary endothelial cells. These in vitro models faithfully recapitulate the adhesive and contractile defects of the CCM-deficient endothelial cells such as increased cell-extracellular matrix (ECM) adhesion through ß1 integrin-anchored actin stress fibers, abnormal remodeling of the ECM, and destabilized VE-cadherin-dependent cell-cell junctions. Using such 2D in vitro CCM models, we have shown that the ECM remodeled by CCM-depleted endothelial cells can propagate CCM-like adhesive defects to wild-type endothelial cells, a process potentially pertinent to CCM lesion expansion. Here, we detail methods for studying the morphology of focal adhesions, actomyosin cytoskeleton, and VE-cadherin-dependent Adherens junctions by immunofluorescence and morphometric analyses. Moreover, we detail the protocols to produce and purify remodeled ECM and to test its effect on endothelial cell adhesion.

VEGF165b, a splice variant of VEGF-A, promotes lung tumor progression and escape from anti-angiogenic therapies through a ß1 integrin/VEGFR autocrine loop.

Vascular endothelial growth factor-A (VEGF-A) is highly subjected to alternative pre-mRNA splicing that generates several splice variants. The VEGFxxx and VEGFxxxb families encode splice variants of VEGF-A that differ only at the level of six amino acids in their C-terminal part. The expression level of VEGFxxx splice variants and their function as pro-angiogenic factors during tumor neo-angiogenesis have been well-described. The role of VEGFxxxb isoforms is less well known, but they have been shown to inhibit VEGFxxx-mediated angiogenesis, while being partial or weak activators of VEGFR receptors in endothelial cells. On the opposite, their role on tumor cells expressing VEGFRs at their surface remains largely unknown. In this study, we find elevated levels of VEGF165b, the main VEGFxxxb isoform, in 36% of non-small cell lung carcinoma (NSCLC), mainly lung adenocarcinoma (46%), and show that a high VEGF165b/VEGF165 ratio correlates with the presence of lymph node metastases. At the molecular level, we demonstrate that VEGF165b stimulates proliferation and invasiveness of two lung tumor cell lines through a VEGFR/ß1 integrin loop. We further provide evidence that the isoform-specific knockdown of VEGF165b reduces tumor growth, demonstrating a tumor-promoting autocrine role for VEGF165b in lung cancer cells. Importantly, we show that bevacizumab, an anti-angiogenic compound used for the treatment of lung adenocarcinoma patients, increases the expression of VEGF165b and activates the invasive VEGFR/ß1 integrin loop. Overall, these data highlight an unexpected role of the VEGF165b splice variant in the progression of lung tumors and their response to anti-angiogenic therapies.

The CCM1-CCM2 complex controls complementary functions of ROCK1 and ROCK2 that are required for endothelial integrity.

Endothelial integrity relies on a mechanical crosstalk between intercellular and cell-matrix interactions. This crosstalk is compromised in hemorrhagic vascular lesions of patients carrying loss-of-function mutations in cerebral cavernous malformation (CCM) genes. RhoA/ROCK-dependent cytoskeletal remodeling is central to the disease, as it causes unbalanced cell adhesion towards increased cell-extracellular matrix adhesions and destabilized cell-cell junctions. This study reveals that CCM proteins directly orchestrate ROCK1 and ROCK2 complementary roles on the mechanics of the endothelium. CCM proteins act as a scaffold, promoting ROCK2 interactions with VE-cadherin and limiting ROCK1 kinase activity. Loss of CCM1 (also known as KRIT1) produces excessive ROCK1-dependent actin stress fibers and destabilizes intercellular junctions. Silencing of ROCK1 but not ROCK2 restores the adhesive and mechanical homeostasis of CCM1 and CCM2-depleted endothelial monolayers, and rescues the cardiovascular defects of ccm1 mutant zebrafish embryos. Conversely, knocking down Rock2 but not Rock1 in wild-type zebrafish embryos generates defects reminiscent of the ccm1 mutant phenotypes. Our study uncovers the role of the CCM1-CCM2 complex in controlling ROCK1 and ROCK2 to preserve endothelial integrity and drive heart morphogenesis. Moreover, it solely identifies the ROCK1 isoform as a potential therapeutic target for the CCM disease.

CCM proteins control endothelial ß1 integrin dependent response to shear stress.

Hemodynamic shear stress from blood flow on the endothelium critically regulates vascular function in many physiological and pathological situations. Endothelial cells adapt to shear stress by remodeling their cytoskeletal components and subsequently by changing their shape and orientation. We demonstrate that ß1 integrin activation is critically controlled during the mechanoresponse of endothelial cells to shear stress. Indeed, we show that overexpression of the CCM complex, an inhibitor of ß1 integrin activation, blocks endothelial actin rearrangement and cell reorientation in response to shear stress similarly to ß1 integrin silencing. Conversely, depletion of CCM2 protein leads to an elongated "shear-stress-like" phenotype even in the absence of flow. Taken together, our findings reveal the existence of a balance between positive extracellular and negative intracellular signals, i.e. shear stress and CCM complex, for the control of ß1 integrin activation and subsequent adaptation of vascular endothelial cells to mechanostimulation by fluid shear stress.

Unraveling ICAP-1 function: toward a new direction?

Cell adhesion to either the extracellular matrix (ECM) or to neighboring cells is of critical importance during both physiological and pathological situations. Integrins are a large family of cell adhesion receptors composed of two non-covalently linked alpha and beta subunits. They have a well-identified dual function of mediating both firm adhesion and signaling. The short cytoplasmic domain of integrin can interact with cytoplasmic proteins that are either shared by several different integrins or specific for one type of integrin. Integrin cytoplasmic domain-associated protein-1 (ICAP-1) is a small cytoplasmic protein that specifically interacts with the beta1 integrin subunit. In this review we will discuss recent findings on ICAP-1, not only at the structural and functional level, but also its possible interconnection in other signaling pathways such as those that control cell proliferation.

Nuclear translocation of integrin cytoplasmic domain-associated protein 1 stimulates cellular proliferation.

Integrin cytoplasmic domain-associated protein 1 (ICAP-1) has been shown to interact specifically with the beta1 integrin cytoplasmic domain and to control cell spreading on fibronectin. Interestingly, ICAP-1 also is observed in the nucleus, by immunocytochemical staining, and after biochemical cell fractionation, suggesting that it has additional roles that have yet to be determined. We show that the nucleocytoplasmic shuttling capability of ICAP-1 is dependent on a functional nuclear localization signal. In addition, overexpression of beta1 integrin strongly reduced this nuclear localization, suggesting that integrin activity could modulate ICAP-1 shuttling by sequestering it in the cytoplasm. Indeed, the nuclear localization of ICAP-1 is dependent on the stage of cell spreading on fibronectin, and we also show that ICAP-1 expression stimulates cellular proliferation in a fibronectin-dependent manner. This function is dependent on its nuclear localization. Moreover, ICAP-1 is able to activate the c-myc promoter in vitro. Together, these results demonstrate that ICAP-1 shuttles between the nucleus and cytoplasm in a beta1 integrin-dependent manner. It could act as a messenger that relays information from sites of integrin-dependent cell adhesion to the nucleus for controlling gene expression and cell proliferation.

Disruption of focal adhesions by integrin cytoplasmic domain-associated protein-1 alpha.

Regulation of integrin affinity and clustering plays a key role in the control of cell adhesion and migration. The protein ICAP-1 alpha (integrin cytoplasmic domain-associated protein-1 alpha) binds to the cytoplasmic domain of the beta(1A) integrin and controls cell spreading on fibronectin. Here, we demonstrate that, despite its ability to interact with beta(1A) integrin, ICAP-1 alpha is not recruited in focal adhesions, whereas it is colocalized with the integrin at the ruffling edges of the cells. ICAP-1 alpha induced a rapid disruption of focal adhesions, which may result from the ability of ICAP-1 alpha to inhibit the association of beta(1A) integrin with talin, which is crucial for the assembly of these structures. ICAP-1 alpha-mediated dispersion of beta(1A) integrins is not observed with beta(1D) integrins that do not bind ICAP. This strongly suggests that ICAP-1 alpha action depends on a direct interaction between ICAP-1 alpha and the cytoplasmic domain of the beta(1) chains. Altogether, these results suggest that ICAP-1 alpha plays a key role in cell adhesion by acting as a negative regulator of beta(1) integrin avidity.

Integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha ) interacts directly with the metastasis suppressor nm23-H2, and both proteins are targeted to newly formed cell adhesion sites upon integrin engagement.

Cell adhesion-dependent signaling implicates cytoplasmic proteins interacting with the intracellular tails of integrins. Among those, the integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha) has been shown to interact specifically with the beta(1) integrin cytoplasmic domain. Although it is likely that this protein plays an important role in controlling cell adhesion and migration, little is known about its actual function. To search for potential ICAP-1alpha-binding proteins, we used a yeast two-hybrid screen and identified the human metastatic suppressor protein nm23-H2 as a new partner of ICAP-1alpha. This direct interaction was confirmed in vitro, using purified recombinant ICAP-1alpha and nm23-H2, and by co-immunoprecipitation from CHO cell lysates over-expressing ICAP-1alpha. The physiological relevance of this interaction is provided by confocal fluorescence microscopy, which shows that ICAP-1alpha and nm23-H2 are co-localized in lamellipodia during the early stages of cell spreading. These adhesion sites are enriched in occupied beta(1) integrins and precede the formation of focal adhesions devoid of ICAP-1alpha and nm23-H2, indicating the dynamic segregation of components of matrix adhesions. This peripheral staining of ICAP-1alpha and nm23-H2 is only observed in cells spreading on fibronectin and collagen and is absent in cells spreading on poly-l-lysine, vitronectin, or laminin. This is consistent with the fact that targeting of both ICAP-1alpha and nm23-H2 to the cell periphery is dependent on beta(1) integrin engagement rather than being a consequence of cell adhesion. This finding represents the first evidence that the tumor suppressor nm23-H2 could act on beta(1) integrin-mediated cell adhesion by interacting with one of the integrin partners, ICAP-1alpha.