Nils C. Gauthier

Clustering of α5β1 integrins determines adhesion strength whereas αvβ3 and talin enable mechanotransduction

A key molecular link between cells and the extracellular matrix is the binding between fibronectin and integrins α5β1 and αvβ3. However, the roles of these different integrins in establishing adhesion remain unclear. We tested the adhesion strength of fibronectin-integrin-cytoskeleton linkages by applying physiological nanonewton forces to fibronectin-coated magnetic beads bound to cells. We report that the clustering of fibronectin domains within 40 nm led to integrin α5β1 recruitment, and increased the ability to sustain force by over six-fold. This force was supported by α5β1 integrin clusters. Importantly, we did not detect a role of either integrin αvβ3 or talin 1 or 2 in maintaining adhesion strength. Instead, these molecules enabled the connection to the cytoskeleton and reinforcement in response to an applied force. Thus, high matrix forces are primarily supported by clustered α5β1 integrins, while less stable links to αvβ3 integrins initiate mechanotransduction, resulting in reinforcement of integrin-cytoskeleton linkages through talin-dependent bonds.

Temporary increase in plasma membrane tension coordinates the activation of exocytosis and contraction during cell spreading

Cell migration and spreading involve the coordination of membrane trafficking, actomyosin contraction, and modifications to plasma membrane tension and area. The biochemical or biophysical basis for this coordination is however unknown. In this study, we show that during cell spreading, lamellipodia protrusion flattens plasma membrane folds and blebs and, once the plasma membrane area is depleted, there is a temporary increase in membrane tension by over twofold that is followed by activation of exocytosis and myosin contraction. Further, an artificial increase in plasma membrane tension stopped lamellipodia protrusion and activated an exocytotic burst. Subsequent decrease in tension restored spreading with activation of contraction. Conversely, blebbistatin inhibition of actomyosin contraction resulted in an even greater increase in plasma membrane tension and exocytosis activation. This spatiotemporal synchronization indicates that membrane tension is the signal that coordinates membrane trafficking, actomyosin contraction, and plasma membrane area change. We suggest that cells use plasma membrane tension as a global physical parameter to control cell motility.

Integrin-dependent force transmission to the extracellular matrix by α-actinin triggers adhesion maturation

Significance Mechanical forces transmitted between a cell and its surrounding extracellular matrix determine functions like proliferation or differentiation, and drive processes in development, tumorigenesis, and wound healing. However, the molecules involved in this force transmission remain unclear. Here we show that forces exerted by cells are transmitted to the extracellular matrix through α-actinin molecules via the transmembrane protein integrins. Furthermore, this transmission enables the growth and maturation of adhesion sites to the matrix, and takes place in competition with another molecule submitted to force, talin. This force regulation mechanism may help us understand the role of force in different biological scenarios. Focal adhesions are mechanosensitive elements that enable mechanical communication between cells and the extracellular matrix. Here, we demonstrate a major mechanosensitive pathway in which α-actinin triggers adhesion maturation by linking integrins to actin in nascent adhesions. We show that depletion of the focal adhesion protein α-actinin enhances force generation in initial adhesions on fibronectin, but impairs mechanotransduction in a subsequent step, preventing adhesion maturation. Expression of an α-actinin fragment containing the integrin binding domain, however, dramatically reduces force generation in depleted cells. This behavior can be explained by a competition between talin (which mediates initial adhesion and force generation) and α-actinin for integrin binding. Indeed, we show in an in vitro assay that talin and α-actinin compete for binding to β3 integrins, but cooperate in binding to β1 integrins. Consistently, we find opposite effects of α-actinin depletion and expression of mutants on substrates that bind β3 integrins (fibronectin and vitronectin) versus substrates that only bind β1 integrins (collagen). We thus suggest that nascent adhesions composed of β3 integrins are initially linked to the actin cytoskeleton by talin, and then α-actinin competes with talin to bind β3 integrins. Force transmitted through α-actinin then triggers adhesion maturation. Once adhesions have matured, α-actinin recruitment correlates with force generation, suggesting that α-actinin is the main link transmitting force between integrins and the cytoskeleton in mature adhesions. Such a multistep process enables cells to adjust forces on matrices, unveiling a role of α-actinin that is different from its well-studied function as an actin cross-linker.

Cytoskeletal coherence requires myosin-IIA contractility

Maintaining a physical connection across cytoplasm is crucial for many biological processes such as matrix force generation, cell motility, cell shape and tissue development. However, in the absence of stress fibers, the coherent structure that transmits force across the cytoplasm is not understood. We find that nonmuscle myosin-II (NMII) contraction of cytoplasmic actin filaments establishes a coherent cytoskeletal network irrespective of the nature of adhesive contacts. When NMII activity is inhibited during cell spreading by Rho kinase inhibition, blebbistatin, caldesmon overexpression or NMIIA RNAi, the symmetric traction forces are lost and cell spreading persists, causing cytoplasm fragmentation by membrane tension that results in ‘C’ or dendritic shapes. Moreover, local inactivation of NMII by chromophore-assisted laser inactivation causes local loss of coherence. Actin filament polymerization is also required for cytoplasmic coherence, but microtubules and intermediate filaments are dispensable. Loss of cytoplasmic coherence is accompanied by loss of circumferential actin bundles. We suggest that NMIIA creates a coherent actin network through the formation of circumferential actin bundles that mechanically link elements of the peripheral actin cytoskeleton where much of the force is generated during spreading.

Cell crawling mediates collective cell migration to close undamaged epithelial gaps

Fundamental biological processes such as morphogenesis and wound healing involve the closure of epithelial gaps. Epithelial gap closure is commonly attributed either to the purse-string contraction of an intercellular actomyosin cable or to active cell migration, but the relative contribution of these two mechanisms remains unknown. Here we present a model experiment to systematically study epithelial closure in the absence of cell injury. We developed a pillar stencil approach to create well-defined gaps in terms of size and shape within an epithelial cell monolayer. Upon pillar removal, cells actively respond to the newly accessible free space by extending lamellipodia and migrating into the gap. The decrease of gap area over time is strikingly linear and shows two different regimes depending on the size of the gap. In large gaps, closure is dominated by lamellipodium-mediated cell migration. By contrast, closure of gaps smaller than 20 μm was affected by cell density and progressed independently of Rac, myosin light chain kinase, and Rho kinase, suggesting a passive physical mechanism. By changing the shape of the gap, we observed that low-curvature areas favored the appearance of lamellipodia, promoting faster closure. Altogether, our results reveal that the closure of epithelial gaps in the absence of cell injury is governed by the collective migration of cells through the activation of lamellipodium protrusion.

Force generated by actomyosin contraction builds bridges between adhesive contacts

Extracellular matrices in vivo are heterogeneous structures containing gaps that cells bridge with an actomyosin network. To understand the basis of bridging, we plated cells on surfaces patterned with fibronectin (FN)‐coated stripes separated by non‐adhesive regions. Bridges developed large tensions where concave cell edges were anchored to FN by adhesion sites. Actomyosin complexes assembled near those sites (both actin and myosin filaments) and moved towards the centre of the non‐adhesive regions in a treadmilling network. Inhibition of myosin‐II (MII) or Rho‐kinase collapsed bridges, whereas extension continued over adhesive areas. Inhibition of actin polymerization (latrunculin‐A, jasplakinolide) also collapsed the actomyosin network. We suggest that MII has distinct functions at different bridge regions: (1) at the concave edges of bridges, MIIA force stimulates actin filament assembly at adhesions and (2) in the body of bridges, myosin cross‐links actin filaments and stimulates actomyosin network healing when breaks occur. Both activities ensure turnover of actin networks needed to maintain stable bridges from one adhesive region to another.

Plasma Membrane Area Increases with Spread Area by Exocytosis of a GPI-anchored Protein Compartment

The role of plasma membrane (PM) area as a critical factor during cell motility is poorly understood, mainly due to an inability to precisely follow PM area dynamics. To address this fundamental question, we developed static and dynamic assays to follow exocytosis, endocytosis, and PM area changes during fibroblast spreading. Because the PM area cannot increase by stretch, spreading proceeds by the flattening of membrane folds and/or by the addition of new membrane. Using laser tweezers, we found that PM tension progressively decreases during spreading, suggesting the addition of new membrane. Next, we found that exocytosis increases the PM area by 40-60% during spreading. Reducing PM area reduced spread area, and, in a reciprocal manner, reducing spreadable area reduced PM area, indicating the interconnection between these two parameters. We observed that Golgi, lysosomes, and glycosylphosphatidylinositol-anchored protein vesicles are exocytosed during spreading, but endoplasmic reticulum and transferrin receptor-containing vesicles are not. Microtubule depolymerization blocks lysosome and Golgi exocytosis but not the exocytosis of glycosylphosphatidylinositol-anchored protein vesicles or PM area increase. Therefore, we suggest that fibroblasts are able to regulate about half of their original PM area by the addition of membrane via a glycosylphosphatidylinositol-anchored protein compartment.

Early endosomes associated with dynamic F-actin structures are required for late trafficking of H. pylori VacA toxin

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are endocytosed by a clathrin- independent pathway into vesicles named GPI-AP–enriched early endosomal compartments (GEECs). We recently showed that the vacuolating toxin VacA secreted by Helicobacter pylori is endocytosed into the GEECs (Gauthier, N.C., P. Monzo, V. Kaddai, A. Doye, V. Ricci, and P. Boquet. 2005. Mol. Biol. Cell. 16:4852–4866). Unlike GPI-APs that are mostly recycled back to the plasma membrane, VacA reaches early endosomes (EEs) and then late endosomes (LEs), where vacuolation occurs. In this study, we used VacA to study the trafficking pathway between GEECs and LEs. We found that VacA routing from GEECs to LEs required polymerized actin. During this trafficking, VacA was transferred from GEECs to EEs associated with polymerized actin structures. The CD2-associated protein (CD2AP), a docking protein implicated in intracellular trafficking, bridged the filamentous actin (F-actin) structures with EEs containing VacA. CD2AP regulated those F-actin structures and was required to transfer VacA from GEECs to LEs. These results demonstrate that sorting from GEECs to LEs requires dynamic F-actin structures on EEs.

Gastric cell apoptosis and H. pylori: has the main function of VacA finally been identified?

Abstract The vacuolating cytotoxin VacA is one of the most important virulence factors of Helicobacter pylori , a bacterium causing severe gastric diseases such as ulcers and cancer. VacA forms large cytoplasmic vacuoles in cultured cells, although its effects on host cells in vivo remain to be elucidated. Three independent groups have reported that VacA induces epithelial cell apoptosis. In particular, a recent study has demonstrated unambiguously the role of VacA in inducing epithelial gastric cell apoptosis.

Filamin depletion blocks endoplasmic spreading and destabilizes force-bearing adhesions

Cells severely depleted of filamins were observed to have numerous motility-related defects, including a defect in endoplasmic spreading; smaller, more dynamic focal adhesions; and an inability to sustain high levels of traction force. The endoplasm as a separate mechanical unit spread by pulling forces is also discussed.