Biology subjects

Pernier, J.

Publications and source records attributed to Pernier, J..

7 recordsLinked to original sources

bioRxiv2022

The adherens junction proteins α-catenin, vinculin and VASP cooperate to promote actin assembly

The cohesion of tissues requires that cells establish cell-cell junctions. Cells contact each other by forming Arp2/3-dependent lamellipodia before they initiate the formation of cadherin-based adherens junctions (AJs). Maturing AJs then assemble actin under force though the formation of a mechanosensitive complex comprising the actin-binding proteins -catenin, vinculin and VASP, which individually act on the nucleation, elongation and organisation of actin filaments in different ways. However, the activity of the ternary complex that these actin-regulatory proteins form has not been investigated due to the difficulty of assembling this complex in vitro in the absence of force. Here, we first designed mutants of these proteins that interact independently of force. We then studied their activity by combining actin polymerization kinetics in fluorescence spectroscopy with observation of single actin filaments in TIRF microscopy. Our results reveal how -catenin, vinculin and VASP combine their activities in a complex to inhibit Arp2/3-mediated branching, stimulate the nucleation and elongation of linear actin filaments from profilin-actin and crosslink these filaments into bundles. These findings shed light on the molecular mechanisms by which actin regulators synergistically control the transition of actin architecture and dynamics that accompanies the formation and maturation of AJs.

biochemistry↗

bioRxiv2022

Talin and vinculin combine their activities to trigger actin assembly

Focal adhesions (FAs) strengthen their link with the actin cytoskeleton to resist force. Talin-vinculin association could reinforce actin anchoring to FAs by controlling actin polymerization. However, the actin polymerization activity of the talin-vinculin complex is not known because it requires the reconstitution of the mechanical and biochemical activation steps that control the association of talin and vinculin and the exposure of their actin-binding domains. By combining kinetic and binding assays with single actin filament observations in TIRF microscopy, we show that the association of talin and vinculin mutants, mimicking different degrees of activation, results in a variety of activities. In particular, mechanically stretched talin and activated vinculin combine to stimulate actin assembly synergistically through a sequential mechanism in which filaments are nucleated, capped and released to elongate. Our findings suggest a versatile mechanism for the regulation of actin assembly in FAs subjected to various combinations of biochemical and mechanical cues.

biochemistry↗

bioRxiv2022

Talin and kindlin cooperate to control the density of integrin clusters.

Focal adhesions are important mechanosensitive structures, composed of transmembrane integrins, linking the extracellular matrix to the actomyosin cytoskeleton, via cytoplasmic proteins. Cellular adhesion to the extracellular matrix depends on the activation of integrins by intracellular mechanisms. Talin and kindlin are major activators of integrins that are recruited to the inner membrane and bind to {beta}-integrin cytoplasmic tails. Many studies showed the importance of integrin activation and clustering and how the organization of extracellular ligands guides the nanoscale organization of adhesion complexes. However, the roles of talin and kindlin in this process are poorly understood. To determine the contribution of talin, kindlin, lipids and actomyosin in integrin clustering, we performed experiments using a biomimetic in vitro system, made of Giant Unilamellar Vesicles, containing transmembrane integrins, on which purified talin, kindlin, and actomyosin assemble. Here we first show that talin and kindlin individually have the ability to cluster integrins. When added together, talin and kindlin synergize to induce the formation of larger integrin clusters containing the three proteins. Comparison of protein density in the talin-integrin, kindlin-integrin, and talin-kindlin-integrin clusters reveals that kindlin increases talin and integrin density, whereas talin does not affect kindlin and integrin density. Finally, kindlin significantly enhances the segregation of talin-integrin clusters induced by actomyosin contractility, suggesting that it increases the coupling of these clusters to the actin cytoskeleton. Our study unambiguously demonstrates how kindlin and talin cooperate to induce integrin clustering, which is a major parameter for cell adhesion.

biochemistry↗

bioRxiv2022

BIN1/Amphiphysin 2 and ezrin drive filopodia-like structures in myoblasts

Amphiphysin 2 (BIN1) is a membrane and actin remodeling protein mutated in congenital and adult centronuclear myopathies. Here, we report an unexpected function of this N-BAR domain protein BIN1 in filopodia formation. We demonstrated that BIN1 expression is necessary and sufficient to induce filopodia formation. BIN1 is present at the base of forming filopodia and all along filopodia, where it colocalizes with F-actin. We identify that BIN1-mediated filopodia formation requires IRSp53, which allows its localization at negatively-curved membrane topologies. Our results show that BIN1 bundles actin in vitro. Finally, we identify that BIN1 regulates the membrane-to-cortex architecture and functions as a molecular platform to recruit actin-binding proteins, dynamin and ezrin, to promote filopodia formation.

cell biology↗

bioRxiv2022

Activated I-BAR IRSp53 clustering controls the formation of VASP-actin-based membrane protrusions

Filopodia are actin-rich membrane protrusions essential for cell morphogenesis, motility, and cancer invasion. How cells control filopodia initiation on the plasma membrane remains elusive. We performed experiments in cellulo, in vitro and in silico to unravel the mechanism of filopodia initiation driven by the membrane curvature sensor IRSp53. We showed that full-length IRSp53 self-assembles into clusters on membranes depending on PIP2. Using well-controlled in vitro reconstitution systems, we demonstrated that IRSp53 clusters recruit the actin polymerase VASP to assemble actin filaments locally on membranes, leading to the generation of actin-filled membrane protrusions reminiscent of filopodia. By pulling membrane nanotubes from live cells, we observed that IRSp53 can only be enriched and trigger actin assembly in nanotubes at highly dynamic membrane regions. Our work supports a regulation mechanism of IRSp53 in its attributes of curvature sensation and partner recruitment to ensure a precise spatial-temporal control of filopodia initiation.

biophysics↗

bioRxiv2020

Myosin 1b Flattens and Prunes Branched Actin Filaments.

Motile and morphological cellular processes require a spatially and temporally coordinated branched actin network that is controlled by the activity of various regulatory proteins including the Arp2/3 complex, profilin, cofilin and tropomyosin. We have previously reported that myosin 1b regulates the density of the actin network in the growth cone. Using in vitro F-actin gliding assays and total internal reflection fluorescence (TIRF) microscopy we show in this report that this molecular motor flattens the Arp2/3-dependent actin branches up to breaking them and reduces the probability to form new branches. This experiment reveals that myosin 1b can produce force sufficient enough to break up the Arp2/3-mediated actin junction. Together with the former in vivo studies, this work emphasizes the essential role played by myosins in the architecture and in the dynamics of actin networks in different cellular regions. Short summaryUsing in vitro F-actin gliding assays and total internal reflection fluorescence (TIRF) microscopy we show that myosin flattens the Arp2/3-dependent actin branches up to breaking them and reduces the probability to form new branches

biophysics↗

bioRxiv2018

A new actin depolymerase: a catch bond Myosin 1 motor

The regulation of actin dynamics is essential for various cellular processes. Former evidence suggests a correlation between the function of non-conventional myosin motors and actin dynamics. We investigate the contribution of myosin1b to actin dynamics using sliding motility assays. We observe that sliding on myosin1b immobilized or bound to a fluid bilayer enhances actin depolymerization at the barbed end, while sliding on myosin II, although 5 times faster, has no effect. This work reveals a non-conventional myosin motor as a new type of depolymerase and points to its singular interactions with the actin barbed end.

biophysics↗