Actomyosin Stress Fibers Research Articles

Intestinal epithelial cell migration is regulated by substance P

Abstract Introduction: Intestinal epithelial cells reseal mucosal wounds through migration. This process of mucosal restitution is vital for many intestinal functions. Recent evidence implicates a role for Substance P (SP), a common neurotransmitter in the neurons of the intestinal mucosa, in the maintenance of gastrointestinal mucosal integrity. We have previously demonstrated that SP increases migration. We hypothesized that this enhanced migration was dependent on specific SP receptor-mediated actions that involved increased calcium flux and cytoskeletal arrangements. Methods: Studies were performed upon cultured IEC-6 cells. Calcium levels were measured by fura-2 imaging. Stress-fiber staining was measured using specific antibodies staining. Migration was directly measured as cells per millimeter over a 6 hour period. Results: Exposure of cells to SP (10-9 M) after wounding increased migration (from 50 + 6 to 73 + 6 cells/mm). Co-incubation with the SP receptor (NK1 receptor subtype) antagonist completely blocked this response. Exposure of cells to SP demonstrated dose-dependent elevation of cytosolic Ca+2 levels at physiologic levels (10-9 to 10–12M). This Ca+2 response was blocked completely NK1 receptor antagonism at a dose of 100mcM. SP increased actomyosin stress fiber staining by 12% versus control cells. Conclusions: These results indicate that for intestinal epithelial cells, Substance P stimulates Ca+2 release, stress fiber formation, and cell migration.

Membrane and acto-myosin tension promote clustering of adhesion proteins.

Physicists have studied the aggregation of adhesive proteins, giving a central role to the elastic properties of membranes, whereas cell biologists have put the emphasis on the cytoskeleton. However, there is a dramatic lack of experimental studies probing both contributions on cellular systems. Here, we tested both mechanisms on living cells. We compared, for the same cell line, the growth of cadherin-GFP patterns on recombinant cadherin-coated surfaces, with the growth of vinculin-GFP patterns on extracellular matrix protein-coated surfaces by using evanescent wave microscopy. In our setup, cadherins are not linked to actin, whereas vinculins are. This property allows us to compare formation of clusters with proteins linked or not to the cytoskeleton and thus study the role of membrane versus cytoskeleton in protein aggregation. Strikingly, the motifs we obtained on both surfaces share common features: they are both elongated and located at the cell edges. We showed that a local force application can impose this symmetry breaking in both cases. However, the origin of the force is different as demonstrated by drug treatment (butanedione monoxime) and hypotonic swelling. Cadherins aggregate when membrane tension is increased, whereas vinculins (cytoplasmic proteins of focal contacts) aggregate when acto-myosin stress fibers are pulling. We propose a mechanism by which membrane tension is localized at cell edges, imposing flattening of membrane and enabling aggregation of cadherins by diffusion. In contrast, cytoplasmic proteins of focal contacts aggregate by opening cryptic sites in focal contacts under acto-myosin contractility.

Borg/Septin Interactions and the Assembly of Mammalian Septin Heterodimers, Trimers, and Filaments

Septins constitute a family of guanine nucleotide-binding proteins that were first discovered in the yeast Saccharomyces cerevisiae but are also present in many other eukaryotes. In yeast they congregate at the bud neck and are required for cell division. Their function in metazoan cells is uncertain, but they have been implicated in exocytosis and cytokinesis. Septins have been purified from cells as hetero-oligomeric filaments, but their mechanism of assembly is unknown. Further studies have been limited by the difficulty in expressing functional septin proteins in bacteria. We now show that stable, soluble septin heterodimers can be produced by co-expression from bicistronic vectors in bacteria and that the co-expression of three septins results in their assembly into filaments. Pre-assembled dimers and trimers bind guanine nucleotide and show a slow GTPase activity. The assembly of a heterodimer from monomers in vitro is accompanied by GTP hydrolysis. Borg3, a downstream effector of the Cdc42 GTPase, binds specifically to a septin heterodimer composed of Sept6 and Sept7 and to the Sept2/6/7 trimer, but not to septin monomers or to other heterodimers. Septins associate through their C-terminal coiled-coil domains, and Borg3 appears to recognize the interface between these domains in Sept6 and Sept7.

PKC ε is associated with myosin IIA and actin in fibroblasts

Proteins coimmunoprecipitating with protein kinase C (PKC) ε in fibroblasts were identified through matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI TOF m/s). This method identified myosin IIA in PKC ε immunoprecipitates, as well as known PKC ε binding proteins, actin, β'Cop and cytokeratin. Myosin is not a substrate for PKC ε. Immunofluorescence analysis showed that PKC ε is colocalised with actin and myosin in actomyosin stress fibers in fibroblasts. Inhibitors of PKC and myosin ATPase activity, as well as microfilament-disrupting drugs, all inhibited spreading of fibroblasts after passage, suggesting a role for a PKC ε–actin–myosin complex in cell spreading.

RhoA inactivation enhances endothelial barrier function.

The modulation of endothelial barrier function is thought to be a function of contractile tension mediated by the cell cytoskeleton, which consists of actomyosin stress fibers (SF) linked to focal adhesions (FA). We tested this hypothesis by dissociating SF/FA with Clostridium botulinum exoenzyme C3 transferase (C3), an inhibitor of the small GTP-binding protein RhoA. Bovine pulmonary artery endothelial cell (EC) monolayers given C3, C3 + thrombin, thrombin, or no treatment were examined using a size-selective permeability assay and quantitative digital imaging measurements of SF/FA. C3 treatment disassembled SF/FA, stimulated diffuse myosin II immunostaining, and reduced the phosphotyrosine (PY) content of paxillin and 130- to 140-kDa proteins that included p125(FAK). C3-treated monolayers displayed a 60-85% decline in F-actin content and a 170-300% increase in EC surface area with enhanced endothelial barrier function. This activity correlated with reorganization of F-actin and PY protein(s) to beta-catenin-containing cell-cell junctions. Because C3 prevented the thrombin-induced formation of myosin ribbons, SF/FA, and the increased PY content of proteins, these characteristics were Rho dependent. Our data show that C3 inhibition of Rho proteins leads to cAMP-like characteristics of reduced SF/FA and enhanced endothelial barrier function.

A fine-structural analysis of normal and modulated cells in myogenic cultures

It has formerly been reported that muscle and nonmuscle progenitor cells in clonal culture could be distinguished from one another on the basis of their distinctive morphologies. In light of recent studies critical of this interpretation, this investigation was concerned with identifying whether one could truly discriminate between myoblasts and fibroblast-like cells. The results reveal that not only is cell morphology a reliable criterion, but myoblasts and fibroblasts can also be distinguished on the basis of their fine-structural characteristics. Cytologically, spindle-shaped myoblasts exhibit an unusually dense concentration of free ribosomes and polysomes, a Golgi of variable dimensions, and a paucity of rough endoplasmic reticulum (RER). Flattened, stellate fibroblasts show an extensive elaboration of RER, multiple Golgi complexes, actomyosin stress fibers, and dense accumulations of intermediate (10 nm) filaments. A third cell type, the mesenchyme-like cell, exhibits the morphology of the fibroblast and has a myoblast-like cytology. The morphological and ultrastructural characteristics of myoblasts are dependent upon culture conditions. In less than optimum culture environments, myoblast morphology, cytology, and differentiation can be reversibly altered or “modulated.” Myoblast modulations induced by both bromodeoxyuridine and by culture conditions which simulate an ischemic environment are analyzed. Myoblast modulation and its implication to muscular dystrophy are discussed.