In our opinion, all of the phenomena that are inhibited by cytochalasin can be thought of as resulting from contractile activity of cellular organelles. Smooth muscle contraction, clot retraction, beat of heart cells, and shortening of the tadpole tail are all cases in which no argument of substance for alternative causes can be offered. The morphogenetic processes in epithelia, contractile ring function during cytokinesis, migration of cells on a substratum, and streaming in plant cells can be explained most simply on the basis of contractility being the causal event in each process. The many similarities between the latter cases and the former ones in which contraction is certain argue for that conclusion. For instance, platelets probably contract, possess a microfilament network, and behave like undulating membrane organelles. Migrating cells possess undulating membranes and contain a similar network. It is very likely, therefore, that their network is also contractile. In all of the cases that have been examined so far, microfilaments of some type are observed in the cells; furthermore, those filaments are at points where contractility could cause the respective phenomenon. The correlations from the cytochalasin experiments greatly strengthen the case; microfilaments are present in control and "recovered" cells and respective biological phenomena take place in such cells; microfilaments are absent or altered in treated cells and the phenomena do not occur. The evidence seems overwhelming that microfilaments are the contractile machinery of nonmuscle cells. The argument is further strengthened if we reconsider the list of processes insensitive to cytochalasin (Table 2). Microtubules and their sidearms, plasma membrane, or synthetic machinery of cells are presumed to be responsible for such processes, and colchicine, membrane-active drugs, or inhibitors of protein synthesis are effective at inhibiting the respective phenomena. These chemical agents would not necessarily be expected to affect contractile apparatuses over short periods of time, they either do not or only secondarily interfere with the processes sensitive to cytochalasin (Table 1). It is particularly noteworthy in this context that microtubules are classed as being insensitive to cytochalasin and so are not considered as members of the "contractile microfilament" family. The overall conclusion is that a broad spectrum of cellular and developmental processes are caused by contractile apparatuses that have at least the common feature of being sensitive to cytochalasin. Schroeder's important insight (3) has, then, led to the use of cytochalasin as a diagnostic tool for such contracile activity: the prediction is that sensitivity to the drug implies presence of some type of contractile microfilament system. Only further work will define the limits of confidence to be placed upon such diagnoses. The basis of contraction in microfilament systems is still hypothetical. Contraction of glycerol-extracted cells in response to adenosine triphosphate (53), ex...

A new high-molecular-weight protein, named filamin, was isolated from chicken gizzard. In chicken gizzard, filamin is present in an amount approximately 30-40% of that of myosin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of highly purified filamin revealed a single polypeptide of about 250,000 daltons. Rabbit antibody directed against purified chicken gizzard filamin did not crossreact with myosin purified from the same source. By the use of microcomplement fixation and indirect immunofluorescent staining with antibodies to chicken gizzard filamin, an antigenically similar or identical protein was found to be widely distributed both in other organs of the chicken and in cultured cells of other species, but not in chicken skeletal muscle. In cultured cells, filamin was found largely to be arranged as a filamentous array very similar to that found for myosin. These data imply that filamin is a widely occurring and chemically conserved component of filaments in smooth muscle and non-muscle cells.A variety of contractile proteins, such as myosin, actin, and tropomyosin, have been isolated from many non-muscle cells (for review see ref. 1). Proposals have been made that these proteins play a role in shape change, motility, and other mechanochemical activities of cells. The molecular mechanisms for these activities are, however, largely undetermined.We have previously reported that a human smooth muscle myosin-like component was found in several human nonmuscle cells. This smooth muscle myosin-like component was found partly associated with the cytoplasmic surface of the plasma membrane of human WI38 cells (2). These observations have led us to investigate further the molecular and immunological properties of contractile proteins from smooth muscles.During the isolation of myosin from chicken gizzard smooth muscle, we observed a major high-molecular-weight protein not previously identified. We have obtained a homogeneous preparation of this protein and a high titer monospecific rabbit antiserum against it. Further chemical and immunochemical studies showed that this protein is distinct from chicken gizzard myosin. However, by an indirect immunofluorescent staining technique, we have found that this protein forms part of the intracellular filamentous structure in a variety of cells from different species. The filamentous staining patterns are very similar to those of smooth musclelike myosins of the same cell type. Because of its structural location and apparent ubiquity, we have named this new high-molecular-weight protein "filamin". This report is a preliminary survey of the properties of filamin; further details will be published elsewhere* t MATERIALS AND METHODS Purification of Chicken Gizzard Filamin and Myosin*. The detailed procedures will be published elsewhere. Briefly, both filamin and myosin were extracted by homogenizing freshly prepared chicken gizzard smooth muscle in a high salt buffer. Myosin precipitated and filamin remained soluble when the clarified extract was dialyzed again...

The surface distributions of three different membrane integral proteins, #2-microglobulin (part of the histocompatibility antigen complex), aminopeptidase (a-aminoacyl-peptide hydrolase; EC 3.4.11.2), and the Na+,K+-ATPase (ATP phosphohydrolase; EC 3.6. The idea that cell surface molecules can interact with intracellular cytoskeletal proteins has been widely entertained (for reviews see refs. 1-3). Until recently, however, the primary evidence for this has been indirect, based on the effects of drugs such as cytochalasin B and colchicine on the lateral mobilities of cell surface molecules. Direct evidence for such transmembrane linkages, however, was provided by Ash and Singer (4). generality, the molecular mechanisms, and the significance of such transmembrane linkages. In this paper, we have extended our experiments to several independent integral membrane proteins on the surfaces of human fibroblasts, including j32-microglobulin (5) and the enzymes aminopeptidase (a-aminoacyl-peptide hydrolase;EC3.4.11.2) (6) and Na+, K+-ATPase (ATP phosphohydrolase; EC 3.6.1.3) (7). Antibodies specific for these proteins were used to induce their clustering and to observe their surface distributions. In each case, a similar transmembrane linkage was induced. Furthermore, from separate studies* (8) Antibodies and Staining Reagents. The primary antibodies to membrane proteins were goat antibodies against rabbit intestine aminopeptidase purified by affinity chromatography (H. Feracci and S. Maroux, unpublished), IgG of bovine antibodies against human 132-microglobulin purified by DEAEcellulose chromatography (9), and IgG of rabbit antibodies against dog kidney Na+,K+-ATPase holoenzyme purified by DEAE-cellulose chromatography (7). These reagents were generous gifts of S.

Bundles of microfilaments very similar in appearance to actin are present in cytoplasmic suspensions obtained from Nitella flexilis. The microfilaments bind rabbit heavy meromyosin in arrowhead arrays similar to those produced on muscle actin. The arrowheads are removed with ATP. The results provide evidence that actin is present in green plants, probably in the form of microfilaments thought to be involved in cytoplasmic streaming.

In the course of studies on the ultrastructural localization and functions of contractile proteins in nonmuscle cells, we have found it useful to adapt the avidin-biotin complex technique introduced by Heitzmann and Richards (7) to the specific labeling of actin and myosin components in these cells. These authors modified cell surfaces by covalent attachment of biotinyl residues, and then visualized these residues in electron microscopy by staining with a ferritin-avidin conjugate, thereby making use of the extraordinarily high binding affinity of biotin for avidin (5). We have used this general procedure to develop two specific fluorescence staining procedures, one for intracellular actin, the other for myosin. The actin procedure involves the successive reaction with biotin-labeled heavy meromyosin (HMM) followed by fluorescein-labeled avidin, which in our hands has given results superior to those obtained with the use of direct fluorescein-labeled HMM as described by Sanger (11). The myosin labeling procedure involves the successive reaction with biotin-labeled antimyosin antibody followed by fluorescein-labeled avidin, which provides an alternative and comparably effective staining procedure to the usual indirect immunofluorescence technique. MATERIALS AND METHODS Muscle ProteinsActin was purified from acetone powders of rabbit back muscle by the method of Spudich and Watt (14). HMM was prepared from rabbit skeletal muscle myosin (15) by a limited tryptic digestion as described by Lowey et al. (10). These proteins were stored in 50% glycerol at -20~ Rabbit myofibrils were prepared by the procedure of Zak et ai. (18). Biotinyl-N-Hydroxysuccinimide Ester ( BOSu)This reagent was synthesized by the method of Heitzmann and Richards (7), and was stored over dessicant at 4~ It was found to be stable for at least 12 mo. Radioactive BOSu (6.4 • 104 cpm/~mol) was prepared by adding [~4C]biotin (Amersham/Searle Corp., Arlington Heights, I11.) to the biotin used for the synthesis. Biotin-Labeled HMM4 mg of fibrous actin and 4 mg of HMM were dialyzed separately overnight against 0.15 M KC1 in 0.01 M potassium phosphate buffer, pH 7.5 (PBK). The two protein solutions were combined, so as to protect the actin-binding site of HMM during the chemical modification (11). The volume was adjusted to 7.5 ml with PBK, and this solution was brought to room temperature. The pH of the protein solution was then raised to 8.8 by the addition of 2.5 ml of a buffer containing 0.2 M NaHCO.~ and 0.15 M KCI, pH 8.8. The reaction was initiated by adding 5 ~1 (preparation A) or 8/~1 (preparation B) of a freshly prepared solution of BOSu (12 mg/ml) in dimethylformamide. The solution was mixed gently and allowed to react for 5 rain with periodic gentle stirring. The reaction was then quenched by the addition of 50 pl of 1 M NH4CI (pH 6). The modified acto-HMM was pelleted by centrifugation for 1 h at 180,000 g. This pellet was recovered, suspended in 2 ml of PBK containing sodium pyrophosphate (0.01 M) and MgCI~ (0.002 M) (to dissociate the ac...