title: A novel high-content analysis tool reveals Rab8-driven cytoskeletal reorganization through Rho GTPases, calpain and MT1-MMP.
abstract: Rab8 is a small Ras-related GTPase that regulates polarized membrane transport to the plasma membrane. Here, we developed a high-content analysis (HCA) tool to dissect Rab8-mediated actin and focal adhesion reorganization that revealed that Rab8 activation significantly induced Rac1 and Tiam1 to mediate cortical actin polymerization and RhoA-dependent stress fibre disassembly. Rab8 activation increased Rac1 activity, whereas its depletion activated RhoA, which led to reorganization of the actin cytoskeleton. Rab8 was also associated with focal adhesions, promoting their disassembly in a microtubule-dependent manner. This Rab8 effect involved calpain, MT1-MMP (also known as MMP14) and Rho GTPases. Moreover, we demonstrate the role of Rab8 in the cell migration process. Indeed, Rab8 is required for EGF-induced cell polarization and chemotaxis, as well as for the directional persistency of intrinsic cell motility. These data reveal that Rab8 drives cell motility by mechanisms both dependent and independent of Rho GTPases, thereby regulating the establishment of cell polarity, turnover of focal adhesions and actin cytoskeleton rearrangements, thus determining the directionality of cell migration.
authors.affiliation: Confocal Microscopy Unit, Biotechnology Programme, Spanish National Cancer Research Centre (CNIO), C/Melchor Fernéndez Almagro 3, Madrid E28029, Spain.
authors.forename: José J
authors.initials: JJ
authors.lastname: Bravo-Cordero
authors.affiliation: Cellomics Unit, Cell Biology & Physiology Program, Cell & Developmental Biology Area, Centro Nacional de Investigaciones Cardiovasculares CNIC, C/ Melchor Fernandez Almagro 3, Madrid E28029, Spain.
authors.forename: Marco
authors.initials: M
authors.lastname: Cordani
authors.affiliation: Integrin Signaling Laboratory, Cell Biology & Physiology Program, Cell & Developmental Biology Area, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Melchor Fernández Almagro 3, Madrid E28029, Spain.
authors.forename: Silvia F
authors.initials: SF
authors.lastname: Soriano
authors.affiliation: Cellomics Unit, Cell Biology & Physiology Program, Cell & Developmental Biology Area, Centro Nacional de Investigaciones Cardiovasculares CNIC, C/ Melchor Fernandez Almagro 3, Madrid E28029, Spain.
authors.forename: Begoña
authors.initials: B
authors.lastname: Díez
authors.affiliation: Cellomics Unit, Cell Biology & Physiology Program, Cell & Developmental Biology Area, Centro Nacional de Investigaciones Cardiovasculares CNIC, C/ Melchor Fernandez Almagro 3, Madrid E28029, Spain.
authors.forename: Carmen
authors.initials: C
authors.lastname: Muñoz-Agudo
authors.affiliation: Cellomics Unit, Cell Biology & Physiology Program, Cell & Developmental Biology Area, Centro Nacional de Investigaciones Cardiovasculares CNIC, C/ Melchor Fernandez Almagro 3, Madrid E28029, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Casanova-Acebes
authors.affiliation: Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), C/Melchor Fernández Almagro 3, Madrid E28029, Spain.
authors.forename: César
authors.initials: C
authors.lastname: Boullosa
authors.affiliation: Integrin Signaling Laboratory, Cell Biology & Physiology Program, Cell & Developmental Biology Area, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Melchor Fernández Almagro 3, Madrid E28029, Spain.
authors.forename: Marta C
authors.identifier: http://orcid.org/0000-0001-8464-3094
authors.initials: MC
authors.lastname: Guadamillas
authors.affiliation: Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), C/Melchor Fernández Almagro 3, Madrid E28029, Spain.
authors.forename: Iakes
authors.initials: I
authors.lastname: Ezkurdia
authors.affiliation: Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), C/Melchor Fernández Almagro 3, Madrid E28029, Spain.
authors.forename: David
authors.initials: D
authors.lastname: González-Pisano
authors.affiliation: Integrin Signaling Laboratory, Cell Biology & Physiology Program, Cell & Developmental Biology Area, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Melchor Fernández Almagro 3, Madrid E28029, Spain.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Del Pozo
authors.affiliation: Cellomics Unit, Cell Biology & Physiology Program, Cell & Developmental Biology Area, Centro Nacional de Investigaciones Cardiovasculares CNIC, C/ Melchor Fernandez Almagro 3, Madrid E28029, Spain mmontoya@cnic.es.
authors.forename: María C
authors.identifier: http://orcid.org/0000-0001-8878-7028
authors.initials: MC
authors.lastname: Montoya
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D002154:Calpain / Q000378:metabolism*; D002465:Cell Movement; D016764:Cell Polarity; D022001:Focal Adhesions / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D053511:Matrix Metalloproteinase 14 / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D022502:Stress Fibers / Q000378:metabolism; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D020662:Guanine Nucleotide Exchange Factors; C107953:RAC1 protein, human; D034741:RNA, Small Interfering; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; C087334:TIAM1 protein, human; C066737:RHOA protein, human; D002154:Calpain; C507086:MMP14 protein, human; D053511:Matrix Metalloproteinase 14; C082377:RAB8A protein, human; D020691:rab GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein
keywords: Actin; Cytoskeleton; Focal adhesion; Migration; Proteases; Rab8; Rho GTPases