Effects of small doses of cytochalasins on fibroblasts: preferential changes of active edges and focal contacts.

Abstract

The effects of low doses of cytochalasin B (2 ,ug/ ml) and cytochalasin D (0.2 lag/ml) on the spreading of normal mouse fibroblasts in culture were investigated to find out which components of cell-substrate interactions are most sensitive to alterations of the state of actin cytoskeleton. Cytochalasin B disorganized the cortical layer of actin microfilaments and caused partial or complete disappearance of microfilament bundles; focal contacts with the substrate seen by interference-reflection microscopy also disappeared. Diffuse close contacts were apparently insensitive to cytochalasin B. Low doses of cytochalasin B did not inhibit the outgrowth and maintenance of lamellas at the cell periphery. However, in contrast to controls, these lamellas had no distal zones with convex outer edges and ruffles at the upper surfaces. The disappearance of these ruffling active edges was accompanied by loss of the ability to clear the surface of the lamellas from the concanavalin A receptors crosslinked by the corresponding ligand. The effects of cytochalasin D were similar to those of cytochalasin B. Thus, ruffling active edges and focal contacts can be regarded as specialized parts of lamellas with increased sensitivity to cytochalasins; the presence of ruffling active edges is essential for the initiation of centripetal movement of the patches of crosslinked surface receptors.Substrate-spread fibroblasts and epithelial cells have a special structure, the lamellar cytoplasm, at their periphery. Characteristic features of the lamellar cytoplasm. include a flattened shape, the absence of large organelles, the presence of numerous focal contacts with the substrate at the lower surface, and a distinctive ability of the surface to clear itself from patched membrane receptors (see review in ref. 1). The zones of active edges are usually located at the distal parts of the lamellar cytoplasm; these are the zones where pseudopods, such as lamellopodia and ruffles, are formed. A study of spreading in medium containing cytochalasin B (CB) at 10 ,ug/ml suggested that formation of the lamellar cytoplasm can be regarded as a combination of two functionally different processes-CB-resistant rudimentary pseudopodial reactions leading to the extension and attachment of arborized outgrowths and CB-sensitive lamellization of these outgrowths leading to their transformation into flattened wide lamellas (2). Experiments presented in this paper show that lamellization, in its turn, can be subdivided into components that have different sensitivities to CB: a low concentration of CB (2 ,ug/ml) prevents the formation of ruffling active edges but does not inhibit the outgrowth of lamellas that lack this specialized distal part. MATERIALS AND METHODSMouse embryo fibroblasts were cultured in a 1:1 mixture of Eagle's medium and 0.5% lactalbumin hydrolysate/10% bovine serum. CB (Serva, Heidelberg, Federal Republic of Germany) was added to this medium at 2 pig/ml (low-CB medium) or 10 jkg/ml (high-CB medium). Cytochalasin D (Sigma) was used at 0...

A Cell-free System to Study Regulation of Focal Adhesions and of the Connected Actin Cytoskeleton

“…Contractility is important for actin stress fibers and focal adhesion formation (Chrzanowska-Wodnicka and Burridge, 1996), and stress fibers are essential for the maintenance of focal adhesions in intact cells (Domnina et al, 1982). Incubation of VPMs at 37°C leads to a drastic loss of components from the focal contacts of a major fraction of VPMs, indicating that a detectable presence of these proteins at the membrane is not required for integrin redistribution under cell-free conditions.…”

What structures, besides adhesions, prevent spread cells from rounding up?

“…Cytochalasin B [Domina et al, 1982;Yahara et al, 19821 and ATP depletion [Bershadsky et al, 1980;Svitkina et al, 1986;Glascott et al, 19871 are both treatments that are known to effect the state of actin polymerization. Therefore, we incubated 3T3 cells with cytochalasin B, or depleted their ATP with sodium azide ( 5 mM) and 2-deoxy-D-glucose (4.5 mg/ml) in glucosedeficient DME with 10% calf serum, for up to 90 minutes.…”

Regulation and drug insensitivity of f‐actin association with adhesion areas of transformed cells

“…3b). Although CB is known to induce abnormal forms of actin (Domnina et al, 1982) such as F-actin in "needles" and blebs (Fig. 3a), it does not induce F-actin aggregates in NRK cells.…”

“…Information on the regulation of such cell structures by any of these nucleotides or drugs is meager. Movement may well be associated with these CB-resistant SAG or MAG structures, because the only known CB-resistant structures in normal cells are the moving lamellas at the cell periphery (Domnina et al, 1982). Knowing these characteristics about F-actin-membrane structures may aid in identifying the causes of uninhibited growth, as well as adhesion and movement abnormalities in tumor cells.…”