RESUMO
The contractile actin cortex is a thin layer of filamentous actin, myosin motors, and regulatory proteins beneath the plasma membrane crucial to cytokinesis, morphogenesis, and cell migration. However, the factors regulating actin assembly in this compartment are not well understood. Using the Dictyostelium model system, we show that the three Diaphanous-related formins (DRFs) ForA, ForE, and ForH are regulated by the RhoA-like GTPase RacE and synergize in the assembly of filaments in the actin cortex. Single or double formin-null mutants displayed only moderate defects in cortex function whereas the concurrent elimination of all three formins or of RacE caused massive defects in cortical rigidity and architecture as assessed by aspiration assays and electron microscopy. Consistently, the triple formin and RacE mutants encompassed large peripheral patches devoid of cortical F-actin and exhibited severe defects in cytokinesis and multicellular development. Unexpectedly, many forA- /E-/H- and racE- mutants protruded efficiently, formed multiple exaggerated fronts, and migrated with morphologies reminiscent of rapidly moving fish keratocytes. In 2D-confinement, however, these mutants failed to properly polarize and recruit myosin II to the cell rear essential for migration. Cells arrested in these conditions displayed dramatically amplified flow of cortical actin filaments, as revealed by total internal reflection fluorescence (TIRF) imaging and iterative particle image velocimetry (PIV). Consistently, individual and combined, CRISPR/Cas9-mediated disruption of genes encoding mDia1 and -3 formins in B16-F1 mouse melanoma cells revealed enhanced frequency of cells displaying multiple fronts, again accompanied by defects in cell polarization and migration. These results suggest evolutionarily conserved functions for formin-mediated actin assembly in actin cortex mechanics.
Assuntos
Citoesqueleto de Actina/genética , Proteínas de Transporte/genética , Proteínas Contráteis/genética , Melanoma Experimental/genética , Citoesqueleto de Actina/química , Actinas/genética , Animais , Sistemas CRISPR-Cas , Movimento Celular/genética , Polaridade Celular/genética , Proteínas Contráteis/química , Dictyostelium/genética , Modelos Animais de Doenças , Forminas , Humanos , Melanoma Experimental/patologia , Camundongos , Microscopia Eletrônica , Contração Muscular/genética , Proteína rhoA de Ligação ao GTP/química , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Cell migration entails networks and bundles of actin filaments termed lamellipodia and microspikes or filopodia, respectively, as well as focal adhesions, all of which recruit Ena/VASP family members hitherto thought to antagonize efficient cell motility. However, we find these proteins to act as positive regulators of migration in different murine cell lines. CRISPR/Cas9-mediated loss of Ena/VASP proteins reduced lamellipodial actin assembly and perturbed lamellipodial architecture, as evidenced by changed network geometry as well as reduction of filament length and number that was accompanied by abnormal Arp2/3 complex and heterodimeric capping protein accumulation. Loss of Ena/VASP function also abolished the formation of microspikes normally embedded in lamellipodia, but not of filopodia capable of emanating without lamellipodia. Ena/VASP-deficiency also impaired integrin-mediated adhesion accompanied by reduced traction forces exerted through these structures. Our data thus uncover novel Ena/VASP functions of these actin polymerases that are fully consistent with their promotion of cell migration.
Assuntos
Actinas/metabolismo , Adesão Celular , Movimento Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Capeamento de Actina/metabolismo , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Fibroblastos , Adesões Focais , Técnicas de Inativação de Genes , Integrinas/metabolismo , Melanoma Experimental , Camundongos , Células NIH 3T3 , Polimerização , Pseudópodes/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Dictyostelium discoideum has proven to be an excellent model to study amoeboid cell migration. During their life cycle, Dictyostelium cells exhibit distinct modes of motility. Individual growth-phase cells explore new territories by random cell migration using the core cell motility machinery, but they can also hunt bacteria by detection and chemotaxis toward the by-product folate. After depletion of nutrients, the cells initiate a developmental program allowing streaming of the cells into aggregation centers by chemotaxis toward cAMP and by cell-to-cell adhesion. Subsequent development is associated with complex rotational movement of the compacted aggregates to drive cell type specific sorting, which in turn is necessary for terminal culmination and formation of fruiting bodies. Here we describe a protocol for the analyses of cell motility of vegetative Dictyostelium cells in unconfined and mechanically confined settings.