RESUMO
Colorectal cancer (CRC) is a high prevalence adenocarcinoma with progressive increases in metastasis-related mortality, but the mechanisms governing the extracellular matrix (ECM) degradation important for metastasis in CRC are not well-defined. We investigated a functional relationship between vimentin (Vim) and myosin 10 (Myo10), and whether this relationship is associated with cancer progression. We tested the hypothesis that Vim regulates the aggregation of Myo10 at the tips of cell extensions, which increases membrane-type 1 matrix metalloproteinase (MT1-MMP)-associated local collagen proteolysis and ECM degradation. Analysis of CRC samples revealed colocalization of Vim with Myo10 and MT1-MMP in cell extensions adjacent to sites of collagen degradation, suggesting an association with local cell invasion. We analyzed cultured CRC cells and fibroblasts and found that Vim accelerates aggregation of Myo10 at cell tips, which increases the cell extension rate. Vim stabilizes the interaction of Myo10 with MT1-MMP, which in turn increases collagenolysis. Vim depletion reduced the aggregation of Myo10 at the cell extension tips and MT1-MMP-dependent collagenolysis. We propose that Vim interacts with Myo10, which in turn associates with MT1-MMP to facilitate the transport of these molecules to the termini of cell extensions and there enhance cancer invasion of soft connective tissues.
Assuntos
Neoplasias Colorretais , Metaloproteinase 14 da Matriz , Humanos , Metaloproteinase 14 da Matriz/metabolismo , Vimentina/metabolismo , Colágeno , MiosinasRESUMO
Vimentin is a structural protein that is required for mesenchymal cell migration and directly interacts with actin, ß1 integrin and paxillin. We examined how these interactions enable vimentin to regulate cell migration on collagen. In fibroblasts, depletion of vimentin increased talin-dependent activation of ß1 integrin by more than 2-fold. Loss of vimentin was associated with reduction of ß1 integrin clustering by 50% and inhibition of paxillin recruitment to focal adhesions by more than 60%, which was restored by vimentin expression. This reduction of paxillin was associated with 65% lower Cdc42 activation, a 60% reduction of cell extension formation and a greater than 35% decrease in cell migration on collagen. The activation of PAK1, a downstream effector of Cdc42, was required for vimentin phosphorylation and filament maturation. We propose that vimentin tunes cell migration through collagen by acting as an adaptor protein for focal adhesion proteins, thereby regulating ß1 integrin activation, resulting in well-organized, mature integrin clusters.This article has an associated First Person interview with the first author of the paper.
Assuntos
Colágeno , Integrina beta1 , Adesão Celular , Movimento Celular , Análise por Conglomerados , Integrina beta1/genética , Integrina beta1/metabolismo , Paxilina/genética , Paxilina/metabolismo , Vimentina/genética , Vimentina/metabolismoRESUMO
The intermediate filament protein vimentin is a widely used phenotypic marker for identifying cells of the mesenchymal linkage such as fibroblasts and myofibroblasts, but the full repertoire of vimentin's functional attributes has not been fully explored. Here we consider how vimentin, in addition to its contributions to mechanical stabilization of cell structure, also helps to control the assembly of cell adhesions and migration through collagen matrices. While the assembly and function of matrix adhesions are critical for the differentiation of myofibroblasts and many other types of adherent cells, a potential mechanism that explains how vimentin affects the recruitment and abundance of centrally important proteins in cell adhesions has been elusive. Here we review recent data indicating that vimentin plays a central regulatory role in the assembly of focal adhesions which form in response to the attachment to collagen. We show that in particular, vimentin is a key organizer of the ß1 integrin adhesive machinery, which affects cell migration through collagen. This review provides a comprehensive picture of the surprisingly broad array of processes and molecules with which vimentin interacts to affect cell function in the context of fibroblast and myofibroblast adhesion and migration on collagen.
Assuntos
Miofibroblastos , Cicatrização , Adesão Celular , Colágeno , Fibroblastos , Adesões Focais , VimentinaRESUMO
Tropomyosin and cofilin are involved in the regulation of actin filament dynamic polymerization and depolymerization. Binding of cofilin changes actin filaments structure, leading to their severing and depolymerization. Non-muscle tropomyosin isoforms were shown before to differentially regulate the activity of cofilin 1; products of TPM1 gene stabilized actin filaments, but products of TPM3 gene promoted cofilin-dependent severing and depolymerization. Here, conformational changes at the longitudinal and lateral interface between actin subunits resulting from tropomyosin and cofilin 1 binding were studied using skeletal actin and yeast wild type and mutant Q41C and S265C actins. Cross-linking of F-actin and fluorescence changes in F-actin labeled with acrylodan at Cys41 (in D-loop) or Cys265 (in H-loop) showed that tropomyosin isoforms differentially regulated cofilin-induced conformational rearrangements at longitudinal and lateral filament interfaces. Tryptic digestion of F-Mg-actin confirmed the differences between tropomyosin isoforms in their regulation of cofilin-dependent changes at actin-actin interfaces. Changes in the fluorescence of AEDANS attached to C-terminal Cys of actin, as well as FRET between Trp residues in actin subdomain 1 and AEDANS, did not show differences in the conformation of the C-terminal segment of F-actin in the presence of different tropomyosins ± cofilin 1. Therefore, actin's D- and H-loop are the sites involved in regulation of cofilin activity by tropomyosin isoforms.
Assuntos
Citoesqueleto de Actina/química , Actinas/química , Cofilina 1/química , Tropomiosina/química , Animais , Citoesqueleto/química , Humanos , Camundongos , Modelos Moleculares , Mutação , Polimerização , Ligação Proteica , Domínios Proteicos , Isoformas de Proteínas , Coelhos , Saccharomyces cerevisiae , Tripsina/químicaRESUMO
The migration of cells through constricting spaces or along fibrous tracks in tissues is important for many biological processes and depends on the mechanical properties of a cytoskeleton made up of three different filaments: F-actin, microtubules, and intermediate filaments. The signaling pathways and cytoskeletal structures that control cell motility on 2D are often very different from those that control motility in 3D. Previous studies have shown that intermediate filaments can promote actin-driven protrusions at the cell edge, but have little effect on overall motility of cells on flat surfaces. They are however important for cells to maintain resistance to repeated compressive stresses that are expected to occur in vivo. Using mouse embryonic fibroblasts derived from wild-type and vimentin-null mice, it is found that loss of vimentin increases motility in 3D microchannels even though on flat surfaces it has the opposite effect. Atomic force microscopy and traction force microscopy experiments reveal that vimentin enhances perinuclear cell stiffness while maintaining the same level of acto-myosin contractility in cells. A minimal model in which a perinuclear vimentin cage constricts along with the nucleus during motility through confining spaces, providing mechanical resistance against large strains that could damage the structural integrity of cells, is proposed.
Assuntos
Movimento Celular , Vimentina/deficiência , Animais , Fenômenos Biomecânicos , Capilares/efeitos dos fármacos , Colágeno/farmacologia , Citoesqueleto/metabolismo , Hidrogéis/farmacologia , Camundongos , Miosina Tipo II/metabolismo , Células NIH 3T3 , Vimentina/metabolismoRESUMO
Vimentin expression contributes to cellular mechanoprotection and is a widely recognized marker of fibroblasts and of epithelial-mesenchymal transition. But it is not understood how vimentin affects signaling that controls cell migration and extracellular matrix (ECM) remodeling. Recent data indicate that vimentin controls collagen deposition and ECM structure by regulating contractile force application to the ECM and through post-transcriptional regulation of ECM related genes. Binding of cells to the ECM promotes the association of vimentin with cytoplasmic domains of adhesion receptors such as integrins. After initial adhesion, cell-generated, myosin-dependent forces and signals that impact vimentin structure can affect cell migration. Post-translational modifications of vimentin determine its adaptor functions, including binding to cell adhesion proteins like paxillin and talin. Accordingly, vimentin regulates the growth, maturation and adhesive strength of integrin-dependent adhesions, which enables cells to tune their attachment to collagen, regulate the formation of cell extensions and control cell migration through connective tissues. Thus, vimentin tunes signaling cascades that regulate cell migration and ECM remodeling. Here we consider how specific properties of vimentin serve to control cell attachment to the underlying ECM and to regulate mesenchymal cell migration and remodeling of the ECM by resident fibroblasts.
RESUMO
The formation of extensions in cell migration requires tightly coordinated reorganization of all three cytoskeletal polymers but the mechanisms by which intermediate filament networks interact with actin to generate extensions are not well-defined. We examined interactions of the actin binding protein filamin A (FLNA) with vimentin in extension formation by fibroblasts. Knockdown (KD) of vimentin in fibroblasts reduced the lengths of cell extensions by 50% (p < 0.001). After cell binding to fibronectin, there was a time-dependent increase of phosphorylation of serine 39, 56 and 72 in vimentin, which was associated with vimentin filament assembly. Of the FLNA-interacting kinases that could phosphorylate vimentin, we focused on PAK1, which we found by reciprocal immunoprecipitation associated with FLNA. Enzyme inhibitor studies and siRNA KD demonstrated that PAK1 was required for vimentin phosphorylation and formation of cell extensions. In sedimentation assays, vimentin was exclusively detected in the insoluble pellet fraction of cells expressing FLNA while in FLNA KD cells there was increased vimentin in the supernatants of FLN KD cells. Compared with wild type, FLNA KD cells showed loss of phosphorylation of serine 56 and 72 in vimentin and reduced numbers and lengths of cell extensions by >4-fold. We suggest that the association of PAK1 with FLNA enables vimentin phosphorylation and filament assembly, which are important in the development and stabilization of cell extensions during cell migration.