Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 5 de 5
Filtrar
1.
J Cell Sci ; 133(7)2020 04 09.
Artículo en Inglés | MEDLINE | ID: mdl-32094266

RESUMEN

Efficient migration on adhesive surfaces involves the protrusion of lamellipodial actin networks and their subsequent stabilization by nascent adhesions. The actin-binding protein lamellipodin (Lpd) is thought to play a critical role in lamellipodium protrusion, by delivering Ena/VASP proteins onto the growing plus ends of actin filaments and by interacting with the WAVE regulatory complex, an activator of the Arp2/3 complex, at the leading edge. Using B16-F1 melanoma cell lines, we demonstrate that genetic ablation of Lpd compromises protrusion efficiency and coincident cell migration without altering essential parameters of lamellipodia, including their maximal rate of forward advancement and actin polymerization. We also confirmed lamellipodia and migration phenotypes with CRISPR/Cas9-mediated Lpd knockout Rat2 fibroblasts, excluding cell type-specific effects. Moreover, computer-aided analysis of cell-edge morphodynamics on B16-F1 cell lamellipodia revealed that loss of Lpd correlates with reduced temporal protrusion maintenance as a prerequisite of nascent adhesion formation. We conclude that Lpd optimizes protrusion and nascent adhesion formation by counteracting frequent, chaotic retraction and membrane ruffling.This article has an associated First Person interview with the first author of the paper.


Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina , Seudópodos , Citoesqueleto de Actina , Complejo 2-3 Proteico Relacionado con la Actina/genética , Actinas/genética , Adhesión Celular , Movimiento Celular
2.
Proc Natl Acad Sci U S A ; 116(9): 3594-3603, 2019 02 26.
Artículo en Inglés | MEDLINE | ID: mdl-30808751

RESUMEN

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.


Asunto(s)
Citoesqueleto de Actina/genética , Proteínas Portadoras/genética , Proteínas Contráctiles/genética , Melanoma Experimental/genética , Citoesqueleto de Actina/química , Actinas/genética , Animales , Sistemas CRISPR-Cas , Movimiento Celular/genética , Polaridad Celular/genética , Proteínas Contráctiles/química , Dictyostelium/genética , Modelos Animales de Enfermedad , Forminas , Humanos , Melanoma Experimental/patología , Ratones , Microscopía Electrónica , Contracción Muscular/genética , Proteína de Unión al GTP rhoA/química , Proteína de Unión al GTP rhoA/genética
3.
Front Cell Dev Biol ; 9: 634708, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33598464

RESUMEN

The Arp2/3 complex generates branched actin filament networks operating in cell edge protrusion and vesicle trafficking. Here we employ a conditional knockout mouse model permitting tissue- or cell-type specific deletion of the murine Actr3 gene (encoding Arp3). A functional Actr3 gene appeared essential for fibroblast viability and growth. Thus, we developed cell lines for exploring the consequences of acute, tamoxifen-induced Actr3 deletion causing near-complete loss of functional Arp2/3 complex expression as well as abolished lamellipodia formation and membrane ruffling, as expected. Interestingly, Arp3-depleted cells displayed enhanced rather than reduced cell spreading, employing numerous filopodia, and showed little defects in the rates of random cell migration. However, both exploration of new space by individual cells and collective migration were clearly compromised by the incapability to efficiently maintain directionality of migration, while the principal ability to chemotax was only moderately affected. Examination of actin remodeling at the cell periphery revealed reduced actin turnover rates in Arp2/3-deficient cells, clearly deviating from previous sequestration approaches. Most surprisingly, induced removal of Arp2/3 complexes reproducibly increased FMNL formin expression, which correlated with the explosive induction of filopodia formation. Our results thus highlight both direct and indirect effects of acute Arp2/3 complex removal on actin cytoskeleton regulation.

4.
Mol Biol Cell ; 28(10): 1311-1325, 2017 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-28331069

RESUMEN

Cell migration and cell-cell communication involve the protrusion of actin-rich cell surface projections such as lamellipodia and filopodia. Lamellipodia are networks of actin filaments generated and turned over by filament branching through the Arp2/3 complex. Inhibition of branching is commonly agreed to eliminate formation and maintenance of lamellipodial actin networks, but the regulation of nucleation or elongation of Arp2/3-independent filament populations within the network by, for example, formins or Ena/VASP family members and its influence on the effectiveness of protrusion have been unclear. Here we analyzed the effects of a set of distinct formin fragments and VASP on site-specific, lamellipodial versus cytosolic actin assembly and resulting consequences on protrusion. Surprisingly, expression of formin variants but not VASP reduced lamellipodial protrusion in B16-F1 cells, albeit to variable extents. The rates of actin network polymerization followed a similar trend. Unexpectedly, the degree of inhibition of both parameters depended on the extent of cytosolic but not lamellipodial actin assembly. Indeed, excess cytosolic actin assembly prevented actin monomer from rapid translocation to and efficient incorporation into lamellipodia. Thus, as opposed to sole regulation by actin polymerases operating at their tips, the protrusion efficiency of lamellipodia is determined by a finely tuned balance between lamellipodial and cytosolic actin assembly.


Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Seudópodos/fisiología , Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Animales , Movimiento Celular/fisiología , Citoesqueleto/metabolismo , Citosol/metabolismo , Humanos , Seudópodos/metabolismo
5.
Nat Commun ; 8: 14832, 2017 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-28327544

RESUMEN

Migration frequently involves Rac-mediated protrusion of lamellipodia, formed by Arp2/3 complex-dependent branching thought to be crucial for force generation and stability of these networks. The formins FMNL2 and FMNL3 are Cdc42 effectors targeting to the lamellipodium tip and shown here to nucleate and elongate actin filaments with complementary activities in vitro. In migrating B16-F1 melanoma cells, both formins contribute to the velocity of lamellipodium protrusion. Loss of FMNL2/3 function in melanoma cells and fibroblasts reduces lamellipodial width, actin filament density and -bundling, without changing patterns of Arp2/3 complex incorporation. Strikingly, in melanoma cells, FMNL2/3 gene inactivation almost completely abolishes protrusion forces exerted by lamellipodia and modifies their ultrastructural organization. Consistently, CRISPR/Cas-mediated depletion of FMNL2/3 in fibroblasts reduces both migration and capability of cells to move against viscous media. Together, we conclude that force generation in lamellipodia strongly depends on FMNL formin activity, operating in addition to Arp2/3 complex-dependent filament branching.


Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas/metabolismo , Seudópodos/metabolismo , Animales , Fenómenos Biomecánicos , Sistemas CRISPR-Cas/genética , Movimiento Celular , Fibroblastos/metabolismo , Forminas , Técnicas de Silenciamiento del Gen , Melanoma Experimental/patología , Ratones , Ratones Noqueados , Modelos Biológicos , Células 3T3 NIH , Fenotipo , Polimerizacion , Seudópodos/ultraestructura , Interferencia de ARN
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA