Actin associated with membranes from 3T3 mouse fibroblast and HeLa cells.

Abstract

A protein component of membranes isolated from 3T3 mouse fibroblasts and HeLa cells has been identified as actin by peptide mapping. Extensive but apparently not total coincidence was found between the peptide maps of these two nonmuscle membrane-associated actins compared to chick skeletal muscle actin. Between 2 and 4% of the total membrane protein appears in the actin band on sodium dodecyl sulfate polyacrylamide gels of 3T3 membranes while about 4% of the membrane protein appears as the actin band from HeLa membranes. These values represent approximately the same proportion of actin to total protein found in the cell homogenates. Treatment of intact cells with levels of cytochalasin B sufficient to cause pronounced morphological changes did not change the amount of actin associated with the membrane in either 3T3 or HeLa cells. However, incubation of isolated membranes under conditions favoring conversion of actin from filamentous to monomeric form resulted in dissociation of approximately 80 and 60% of the actin from 3T3 and HeLa membranes, respectively. Thus, approximately 20% of 3T3 membrane actin and 40% of HeLa membrane actin remained associated with the membrane even under actin depolymerizing conditions.Actin has now been identified as a component of essentially all eukaryotic animal cells in which it has been sought. In addition, myosin-and tropomyosin-like proteins have been identified in many of these nonmuscle cells. (An extensive review on the subject of contractile proteins of nonmuscle cells has recently appeared [29].) In several electron microscope preparations, apparent contact between plasma membrane and actin filaments (or thin filaments with the dimensions of actin) has been observed in situ (24,36,42,44,45) or in isolated plasma membranes (28). Biochemical evidence has also been presented that actin is associated with membranes of synaptosomes (3). It is, therefore, likely that actin filaments function at least in part via attachment to the plasma membrane of cells. Furthermore, control of the membrane-actin association seems a likely point for regulation of actin function as an effector of diverse cellular movements and morphological changes. We have, therefore, chosen as the initial phase of studies on the mechanisms of cellular movements to try to identify actin in membranes from 3T3 mouse fibroblasts and HeLa cells.

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

“…The plasma membrane fraction of HeLa cells contains a concentrated form of HMWP (11,35), and actin-binding protein has been reported to be present in macrophage plasma membranes (21) and the cortical blebs released from macrophages by sonication (6). The recent observation of actinbinding protein by immunofluorescence techniques on phagolysosomes of polymorphonuclear leukocytes (3) suggests that it (or HMWP) is located on the inner surface of the plasma membrane.…”

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

“…The present observations, showing a concomitant inhibition of gelation and specific disaggregation of both actin and HMWP by CB, provide additional evidence for the specific role of actin and HMWP in gelation of cytoplasmic extracts. We previously reported that actin is present and H M W P is concentrated in the plasma membrane fraction of HeLa cells (10), and that both actin and the H M W P can be enriched in a precipitate 1.2 fraction of a suitable extract of the plasma mem- 1.0 brane (37). These results indicate that actin and _g H M W P from plasma membrane or whole cell ~ o.a extracts undergo similar interactions.…”

“…HeLa cells, grown as described (10,37), were collected at room temperature by low-speed centrifugation and washed once with 20 vol of cold 0.1 M NaCl. All subsequent operations were carried out at 0-4~ Cells were suspended in 2 vol (based on the packed cell volume before washing) of 0.…”

The polymerization of actin: II. how nonfilamentous actin becomes nonrandomly distributed in sperm: evidence for the association of this actin with membranes

“…Thus, the interwoven network that has been so elegantly described by Buckley and Porter (4) in preparations of tissue culture cells that were fixed, critically point dried, and examined with high voltage microscopy may in fact be actin and its associated proteins. It may also be no coincidence that when the plasma membrane fraction of cultured cells, namely HeLa cells, is run by SDS-gel electrophoresis, not only is actin present (8), but also there is a pair of bands whose molecular weights resemble that of the proteins associated with the actin in sperm. Similar proteins are present in myoblasts, chrondroblasts (my unpublished observations), and human erythrocytes (40).…”