Actin Cytoskeleton of Spread Fibroblasts Appears to Assemble at the Cell Edges

J. Cell Sd. 82, 235-248 (1986) 235 Printed in Great Britain © The Company of Biologists Limited 1986 ACTIN CYTOSKELETON OF SPREAD FIBROBLASTS APPEARS TO ASSEMBLE AT THE CELL EDGES TATJANA M. SVITKINA, ALEXANDER A. NEYFAKH, JR Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, Moscow 119899, USSR AND ALEXANDER D. BERSHADSKY All-Union Cancer Research Center, Academy of Medical Sciences, Moscow 115478, USSR SUMMARY The action of metabolic inhibitors on actin cytoskeleton of cultured quail embryo fibroblasts has been studied using electron microscopy of platinum replicas and immunofluorescence microscopy. Sodium azide as well as other inhibitors (oligomycin and dinitrophenol) caused the disassembly of all types of actin structures: actin meshwork at the cell active edges, microfilament sheath underlying the cell surface, and microfilament bundles. Studying the time- and dose-dependence of the destruction process we have found that the active edge meshwork and microfilament sheath are much more labile than microfilament bundles. After the removal of metabolic inhibitors actin cytoskeleton restoration begins at the cell edges. The first sign of this process is the formation of actin meshwork along the whole cell perimeter (l-10min of recovery). Sometimes fragments of this meshwork bend upwards forming ruffles. Later (10-20 min of recovery) the microfilament sheath appears at the cell periphery as a narrow band. The sheath seems to be formed from the edge meshwork, since ruffles in the process of transformation to sheath could be seen. During the following restoration the microfilament sheath gradually expands towards the cell centre. The last step of actin cytoskeleton restoration (60—120 min of recovery) is the formation of bundles. We suggest that the actin in spread fibroblasts .polymerizes predominantly at the cell edges and then moves centripetally, forming the microfilament sheath and bundles. INTRODUCTION The actin cytoskeleton of non-muscle cells is a highly dynamic and complex system. In cultured fibroblasts actin microfilaments form several morphologically and biochemically different structures of which microfilament bundles are the most obvious and well characterized (see review by Byers et al. 1984). In addition, actin forms a fine meshwork at the cell's leading edges (Small et al. 1978). The third type of actin structure is the microfilament sheath underlying the upper surface of spread fibroblasts. The sheath is clearly seen in detergent-extracted cells by electron microscopy of platinum replicas (Svitkina et al. 1984). The mechanism of formation of these three cytoskeletal structures and their relationships are unclear. Since actin structures are highly dynamic, the promising approach for studying these problems is the investigation of the processes of destruction and the following restoration of the actin cytoskeleton in living cell. Cytochalasins are well-known agents causing actin destruction (Weber et al. 1976; Key words: actin cytoskeleton, fibroblasts, metabolic inhibitors, platinum replicas. 236 T. M. Svitkina, A. A. Neyfakh, jfr and A. D. Bershadsky Schliwa, 1982). However, cytochalasin-induced actin depolymerization is accom- panied by a dramatic change in cell shape (Sanger & Holtzer, 1972). In this study we used metabolic inhibitors, particularly NaN3, for the dis- integration of actin cytoskeleton. We have shown that metabolic inhibitors destroy microfilament bundles, while the cell morphology changes very slightly (Bershadsky etal. 1980). Here we show that all actin-containing structures of cultured quail embryo fibroblasts are sensitive to metabolic inhibitors. The restoration of actin cytoskeleton after the removal of inhibitors begins at the cell periphery. In a few minutes the actin edge meshwork is formed. Then the microfilament sheath appears near the cell edge and gradually covers the central parts of the cytoplasm. We suppose that under normal conditions actin polymerization occurs also at the cell edges and then this polymerized actin moves centripetally, forming the microfilament sheath and possibly microfilament bundles. MATERIALS AND METHODS Cells Quail embryo fibroblast culture was obtained by trypsinization of 11- to 13-day-old embryos. Cells were cultivated in 199 medium containing 0-25 % lactalbumin hydrolysate, 5 % tryptose phosphate broth and 5% bovine serum. Secondary cultures were seeded onto 5mmX5mm coverslips at low density and incubated for 48-72h at 37°C. After washing with phosphate- buffered saline (PBS), cells were treated with sodium azide (Sigma), oligomycin (Serva) or dinitrophenol (Sigma) in Dulbecco's salt solution at 37°C. Before fixation, control and drug-treated cell cultures were extracted with 1 % Triton X-100 in buffer M (50mM-imidazole, 50mM-KCl, 0-5mM-MgCl2, OlmM-EDTA, 1 mM-EGTA, 1 mM-2-mercaptoethanol, pH6-8) containing 4% polyethyleneglycol (Mr 40 000) for 5 min at room temperature. Immunofluorescence Triton-extracted cells were rinsed with buffer M and fixed with 4% formaldehyde in PBS for 20 min or more. Actin antibody and the procedure for cell staining have been described (Bershadsky et al. 1980). For myosin staining monospecific rabbit antibody to calf spleen myosin was used. Its specificity was tested by Western blotting. The isolation and purification of myosin, as well as affinity chromatography of immune serum, were performed by Dr F. K. Gioeva. Electron microscopy Details of the platinum-replica technique have been described (Svitkina etal. 1984). Briefly, Triton-extracted cells were rinsed with buffer M and fixed with 2% glutaraldehyde in 0-1M- sodium cacodylate buffer (pH 7-2-7-4) for 20 min or more. Then specimens were dehydrated in graded acetones, treated with 0-1 % uranyl acetate in acetone for 20 min, washed in two changes of pure acetone and critical point dried in a Balzers device. Dried cytoskeletons were rotary shadowed with Pt-C in a BAE 080 T apparatus (Balzers) at an angle of 45 °. The thickness of the platinum layer as determined by the method of Heuser (1983) was 2nm. The replicas were strengthened with carbon shadowing at an angle of 90°. Coverslips were removed with HF. Cell residues were dissolved with 30% aqueous C1O3. Replicas were examined in a Jeol 100C or Philips EM400 electron microscope at 80 kV. Negatives were photographically reversed before printing. ATP measurement Subconfluent cultures of fibroblasts on 35 mm Petri dishes (3X105 cells/dish) were treated as described in Fig. 9, washed with Dulbecco's salt solution and extracted with ice-cold 3 % tri- chloroacetic acid for 3 min. Samples of the extract were diluted 500-fold with 0-1 M-Tris—acetate Assembly ofactin cytoskeleton 237 buffer (pH7-7S) containing 2mM-EDTA, and frozen. ATP measurement was performed by the luciferin-luciferase assay using ATP Monitoring Reagent (LKB) and PICO ATP Luminometer (Jobin Yvon, France). RESULTS The actin cytoskeleton of control cells In order to study the actin cytoskeleton structure of quail embryo fibroblasts we have exploited two techniques: electron microscopy of platinum replicas of detergent-extracted cells, and immunofluorescence microscopy of the cells stained with actin and myosin antibodies. Previously we used the platinum replica technique for the study of the cytoskeleton of mouse embryo fibroblasts (Svitkina et al. 1984). The structure of the cytoskeleton of quail fibroblasts is principally the same. The central part of a quail cell is covered with a dense planar sheath of microfilaments (Fig. 1). Local parallel orientation of microfilaments is a characteristic feature of the sheath (Fig. 2). The peripheral lamellar parts of the cells are not covered by the sheath and distal parts of microfilament bundles terminating in adhesion plaques can be seen there (Fig. 1). These sheath-free lamellar regions are narrow in comparison with similar regions of mouse fibroblasts cytoskeleton. At the active edges of lamellae dense planar meshworks of microfilaments are visible (Fig. 1). Immunofluorescence with actin antibody revealed three types of actin-containing structures (Fig. 3). Numerous bright straight lines crossing the cell clearly cor- respond to microfilament bundles. Bright fluorescence at the active cell edges corresponds to an active edge actin meshwork. Diffuse fluorescence all over the cell most probably represents the cortical microfilament sheath. Myosin antibody intensively stained microfilament bundles and the microfilament sheath, while active edges were unstained (Fig. 4), confirming the previous data (Hegeness et al. 1977). Destruction of actin cytoskeleton by metabolic inhibitors The actin cytoskeleton of quail fibroblasts was dramatically destroyed after treatment of cells for 1 h with metabolic inhibitors in buffered saline without an energy source (Dulbecco's solution). Metabolic inhibitors acting by different mech- anisms, such as the cytochrome oxidase inhibitor sodium azide (10—20 mM), the H+- ATPase inhibitor oligomycin (1/iM) and the oxidative phosphorylation uncoupler 2,4-dinitrophenol (0-3 mM) had similar effects. Staining of the active edge and microfilament sheath with actin antibody completely disappeared. Microfilament bundles became fragmented and their remnants became randomly distributed in the cytoplasm (Fig. 5). Myosin antibody also stained only the remnants of the bundles in drug-treated cells (Fig. 6). Electron microscopy shows the absence of active edge actin meshwork and microfilament sheath. The fragments of the microfilament bundles are interspersed with

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