Cytochalasin B inhibits actin-related gelation of HeLa cell extracts.

Abstract

When the 100,000 g supernatant fraction (extract) of HeLa cells lysed in a buffer containing sucrose, ATP, DTE, EGTA, imidazole, and Triton X-100 is incubated at 25 degrees C, it gels, and actin and a HMWP are progressively enriched in the extract and in gel isolated from extract. CB (greater than or equal to 0.25 muM) inhibits gelation and specifically lowers the concentrations of actin and the HMWP in the fraction which sediments at 100,000 g after incubation. These results indicate that actin and HMWP are partly disaggregated by cytochalasin treatment, and thus that their aggregation is related gelation. Inasmuch as previous results showed that actin is present and HMWP is enriched in the plasma membrane fraction of HeLa cells, the results also point to a possible relation between plasma membrane-associated gel and in vivo effects of CB.

Effects of myosin and heavy meromyosin on actin-related gelation of HeLa cell extracts.

“…Since that time, studies on fractionation and ultrastructure of the cytoplasm and on the Ca ++ and ATP requirements for movement and consistency changes (24,30,31,32) have shown that rapid changes in the organization and interactions of actin and myosin most likely provide the molecular machinery for these changes. Recently, the studies on control of movement and consistency change by Ca ++ and ATP have been extended to cultured mammalian cells (14).The ability of cytoplasmic extracts to jell was recognized more recently, and it has been related to actin (22), actin and a high molecular weight actin-binding protein (4,29,34,35), actin and two other proteins (15, 16), or actin and several low molecular weight proteins (20). The gels formed by three of these cytoplasmic extracts can shrink spontaneously (4,22,29), and the incorporation of myosin into the shrunken gels has suggested that interactions of actin and myosin induce gel shrinkage.…”

“…The ability of cytoplasmic extracts to jell was recognized more recently, and it has been related to actin (22), actin and a high molecular weight actin-binding protein (4,29,34,35), actin and two other proteins (15,16), or actin and several low molecular weight proteins (20). The gels formed by three of these cytoplasmic extracts can shrink spontaneously (4,22,29), and the incorporation of myosin into the shrunken gels has suggested that interactions of actin and myosin induce gel shrinkage.…”

Isolation of a high molecular weight actin-binding protein from baby hamster kidney (BHK-21) cells.

“…HMWPs that interact with actin have been implicated in cytoplasmic consistency changes (3,10,11,(13)(14)(15)19). Macrophage ABP and smooth muscle filamin have been isolated and appear to be similar (7)(8)(9)(10).…”

“…The present investigation has made possible the comparison of HMWP isolated from cultured fibroblasts with ABP and filamin. We have adopted the general term "HMWP" (19) to avoid additional terminology; precise comparison of the three proteins awaits amino acid sequence determination to unequivocally demonstrate their proposed identity (9,31).…”

“…Actinbinding protein (ABP) from macrophages (5) and leukocytes (6) and filamin from smooth muscle (7-9) have similar, but not identical, physical and biological characteristics, such as size (two identical 250,000-dalton subunits), amino acid composition, and their ability to participate with purified actin in a gelation reaction (5-11). Electrophoretic or immunological analyses have resulted in identification of proteins similar to ABP and filamin in extracts of many other cells (12-17), including cultured mammalian cells (18)(19)(20). In addition to the high molecular weight actin crosslinkers, several low molecular weight gelation factors have been isolated from Acanthamoeba (21).…”