Actin Dynamics Associated with Focal Adhesions

Cell-matrix adhesion plays a major role during cell migration. Proteins from adhesion structures connect the extracellular matrix to the actin cytoskeleton, allowing the growing actin network to push the plasma membrane and the contractile cables (stress fibers) to pull the cell body. Force transmission to the extracellular matrix depends on several parameters including the regulation of actin dynamics in adhesion structures, the contractility of stress fibers, and the mechanosensitive response of adhesion structures. Here we highlight recent findings on the molecular mechanisms by which actin assembly is regulated in adhesion structures and the molecular basis of the mechanosensitivity of focal adhesions

The microtubule-binding protein CLIP-170 coordinates mDia1 and actin reorganization during CR3-mediated phagocytosis

Microtubule dynamics are modulated by regulatory proteins that bind to their plus ends (+TIPs [plus end tracking proteins]), such as cytoplasmic linker protein 170 (CLIP-170) or end-binding protein 1 (EB1). We investigated the role of +TIPs during phagocytosis in macrophages. Using RNA interference and dominant-negative approaches, we show that CLIP-170 is specifically required for efficient phagocytosis triggered by αMβ2 integrin/complement receptor activation. This property is not observed for EB1 and EB3. Accordingly, whereas CLIP-170 is dynamically enriched at the site of phagocytosis, EB1 is not. Furthermore, we observe that CLIP-170 controls the recruitment of the formin mDia1, an actin-nucleating protein, at the onset of phagocytosis and thereby controls actin polymerization events that are essential for phagocytosis. CLIP-170 directly interacts with the formin homology 2 domain of mDia1. The interaction between CLIP-170 and mDia1 is negatively regulated during αMβ2-mediated phagocytosis. Our results unravel a new microtubule/actin cooperation that involves CLIP-170 and mDia1 and that functions downstream of αMβ2 integrins

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

Funding Information: Acknowledgments: The computations were supported by the University of Chicago Research Funding Information: The computations were supported by the University of Chicago Research Computing Center (RCC). We thank E. Coudrier and C. Simon for insightful discussions. We also thank F. Di Federico for handling plasmids, F. Tabarin-Cayrac for cell sorting, and A.-S. Mace for ImageJ programming assistance. F.-C.T., C.L.C., and P.B. are members of the CNRS consortium AQV. F.-C.T. and P.B. are members of the Labex Cell(n)Scale (ANR-11-LABX0038) and Paris Sciences et Lettres (ANR-10-IDEX-0001-02). We acknowledge the Cell and Tissue Imaging Core facility (PICT IBiSA), Institut Curie, member of the French National Research Infrastructure France-BioImaging (ANR10-INBS-04). This work was supported by Human Frontier Science Program (HFSP) grant RGP0005/2016 (to F.-C.T., J.M.H., G.A.V., P.L., and P.B.), Institut Curie and the Centre National de la Recherche Scientifique (CNRS) (to F.-C.T., J.M.H., and P.B.), Marie Curie actions H2020-MSCA-IF-2014 (to F.-C.T.), EMBO Long-Term fellowship ALTF 1527-2014 (to F.-C.T.), Pasteur Foundation Fellowship (to J.M.H.), Agence Nationale pour la Recherche ANR-20-CE13-0032 (to J.M.H. and P.B.) and ANR-20-CE11-0010-01 (to F.-C.T), Université Paris Sciences et Lettres-QLife Institute ANR-17-CONV-0005 Q-LIFE (to P.B.), FY 2015 Researcher Exchange Program between the Japan Society for the Promotion of Science and Academy of Finland (to Y.S.), the Takeda Science Foundation (to Y.S.), the Wesco Scientific Promotion Foundation (to Y.S.), Agence Nationale pour la Recherche ANR-18-CE13-0026-01 and ANR-21-CE13-0010-03 (to C.L.C.), Cancer Society Finland 4705949 (to P.L.), and U.S. National Institutes of Health (NIH) Institute of General Medical Sciences (NIGMS) grant R01-GM063796 (to G.A.V. and Z.J.) Publisher Copyright: Copyright © 2022 The Authors, some rights reserved.Filopodia are actin-rich membrane protrusions essential for cell morphogenesis, motility, and cancer invasion. How cells control filopodium initiation on the plasma membrane remains elusive. We performed experiments in cellulo, in vitro, and in silico to unravel the mechanism of filopodium initiation driven by the membrane curvature sensor IRSp53 (insulin receptor substrate protein of 53 kDa). 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 (vasodilator-stimulated phosphoprotein) 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 filopodium initiation.Peer reviewe

Liability in Software Engineering: Overview of the LISE Approach and Illustration on a Case Study

© ACM – 2010. This is the authors' pre-version of the work. It is posted here by permission of ACM for your personal use. Not for redistribution. The definitive version was published in the Proceedings of the 32nd ACM/IEEE international Conference on Software Engineering (ICSE'10) - Volume 1 – 978-1-60558-719-6/10/05 – (May 2-8 – 2010) http://doi.acm.org/10.1145/1806799.1806823LISE is a multidisciplinary project involving lawyers and computer scientists with the aim to put forward a set of methods and tools to (1) define software liability in a precise and unambiguous way and (2) establish such liability in case of incident. This report provides an overview of the overall approach taken in the project based on a case study. The case study illustrates a situation where, in order to reduce legal uncertainties, the parties to a contract wish to include in the agreement specific clauses to define as precisely as possible the share of liabilities between them for the main types of failures of the system

Molecular Basis for the Dual Function of Eps8 on Actin Dynamics: Bundling and Capping

The unusual dual functions of the actin-binding protein EPS8 as an actin capping and actin bundling factor are mapped to distinct structural features of the protein and to distinct physiological activities in vivo

Motilité liée à la polymérisation de l'actine (régulation des réseaux branchés d'actine par le complexe Arp2/3 en réponse à la signalisation)

Le complexe Arp2/3 (Actin-related protein 2/3 complex) joue un rôle majeur dans la réorganisation du cytosquelette d'actine en réponse à la signalisation dans la motilité cellulaire. Les protéines de la famille WASP (Wiskott-Aldrich Syndrom Proteins) relient le complexe Arp2/3 à une grande variété de signaux extracellulaires. Le complexe Arp2/3 activé par les protéines WASP multiplie les filaments d'actine par branchement. La formation d'un réseau polarisé dendritique de filaments d'actine et la force qui en résulte permet de déformer la membrane plasmique. Ce processus est utilisé par les cellules motiles pour étendre le lamellipode. Nos travaux sur les protéines WASP ont montré que la protéine N-WASP fixe simultanément des activateurs comme Grb2 et des inhibiteurs comme WIP (WASP Interacting Protein) pour réguler de manière fine l'activation du complexe Arp2/3. En plus de ces protéines régulatrices, la protéine WASP coopère avec la protéine VASP (Vasodilator-Stimulated Phosphoprotein) pour transformer la force de polymérisation de l'actine en force de protrusion pour déformer la membrane plasmique. Dans une deuxième partie, nous avons étudié les réactions associées à l'activation du complexe Arp2/3 par les protéines de la famille WASP. Le complexe Arp2/3 contient sept sous-unités dont deux protéines apparentées à l'actine, Arp2 et Arp3. Nous avons montré que dans le complexe Arp2/3, Arp2 et Arp3 fixent le nucléotide ATP et que la fixation de la protéine N-WASP à Arp2/3 augmente l'affinité de Arp2 pour l'ATP. La fixation de l'ATP sur Arp2 est nécessaire à la formation de filaments d'actine branchés par le complexe Arp2/3. Enfin, l'hydrolyse de l'ATP sur Arp2 provoque le débranchement des filaments d'actine. Nos travaux suggèrent que les cycles d'échange et d'hydrolyse de l'ATP sur le complexe Arp2/3 et sur l'actine gouvernent la structure et la stabilité du réseau d'actine dans le lamellipode et jouent un rôle majeur dans la motilité cellulaire.The Arp2/3 complex (Actin-related protein 2/3 complex) plays a key role in the spatially controlled actin polymerisation in response to extracellular signals leading to cell motility. The WASP (Wiskott-Aldrich Syndrom Proteins) family proteins connect the Arp2/3 complex to a variety of extracellular signals. Upon activation by WASP proteins, Arp2/3 complex multiplies actin filaments by branching. Growth of the resulting polarised dendritic array of branched filaments produces a mechanical force on the plasma membrane, allowing the formation of cell protrusions like lamellipodia and filopodia. In the first part of this work, we demonstrate that N-WASP can bind activators like the adaptor Grb2 and inhibitors like WIP (WASP Interacting Protein) simultaneously to regulate Arp2/3. In addition to these regulators, VASP (Vasodilator-Stimulated Phosphoprotein) cooperates with WASP to transform actin polymerisation into protrusive force. In a second part, we have studied the elementary reactions associated to Arp2/3 complex activation by WASP proteins. The Arp2/3 complex is composed of seven subunits including two actin related proteins, Arp2 and Arp3. We demonstrate that in Arp2/3 complex, Arp3 and Arp2, bind ATP. The binding of N-WASP to Arp2/3 enhanced the affinity ofArp2 for ATP. The binding of ATP to Arp2 is required for the branching activity of Arp2/3 complex. Finally, we show that ATP hydrolysis on Arp2 provokes debranching of actin filaments. In conclusion this work suggests that cycles of ATP exchange and hydrolysis on actin as well as on Arp2/3 complex govern the structure and the stability of the actin array in lamellipodia and thus play an important role in cell motility.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Integrin Facilitates the Internalization of TAT Peptide Conjugated to RGD Motif in Model Lipid Membranes

Abstract In recent years, targeted drug delivery has attracted a great interest for enhanced therapeutic efficiency, with diminished side effects, especially in cancer therapy. Cell penetrating peptides (CPPs) like HIV1−TAT peptides, appear to be the perfect vectors for translocating drugs or other cargoes across the plasma membrane, but their application is limited mostly due to insufficient specificity for intended targets. Although these molecules were successfully used, the mechanism by which the peptides enter the cell interior still needs to be clarified. The tripeptide motif RGD (arginine−glycine−aspartate), found in extracellular matrix proteins has high affinity for integrin receptors overexpressed in cancer and it is involved in different phases of disease progression, including proliferation, invasion and migration. Discovery of new peptides with high binding affinity for disease receptors and permeability of plasma membranes is desirable for both, development of targeted drug delivery systems and early detection and diagnosis. To complement the TAT peptide with specific targeting ability, we conjugated it with an integrin‐binding RGD motif. Although the idea of RGD−CPPs conjugates is not entirely new, [1] here we describe the permeability abilities and specificity of integrin receptors of RGD−TAT peptides in model membranes. Our findings reveal that this novel RGD sequence based on TAT peptide maintains its ability to permeate lipid membranes and exhibits specificity for integrin receptors embedded in giant unilamellar vesicles. This promising outcome suggests that the RGD−TAT peptide has significant potential for applications in the field of targeted drug delivery systems