Fluorescent antibody localization of myosin in the cytoplasm, cleavage furrow, and mitotic spindle of human cells.

Self Cite

218

101

We documented the activity of cultured cells on time-lapse videotapes and then stained these identified cells with antibodies to actin and myosin. This experimental approach enabled us to directly correlate cellular activity with the distribution of cytoplasmic actin and myosin. When trypsinized HeLa cells spread onto a glass surface, the cortical cytoplasm was the most actively motile and random, bleb-like extensions (0.5-4.0 micrometer wide, 2-5 micrometer long) occurred over the entire surface until the cells started to spread. During spreading, ruffling membranes were found at the cell perimeter. The actin staining was found alone in the surface blebs and ruffles and together with myosin staining in the cortical cytoplasm at the bases of the blebs and ruffles. In well-spread, stationary HeLa cells most of the actin and myosin was found in stress fibers but there was also diffuse antiactin fluorescence in areas of motile cytoplasm such as leading lamellae and ruffling membranes. Similarly, all 22 of the rapidly translocating embryonic chick cells had only diffuse actin staining. Between these extremes were slow-moving HeLa cells, which had combinations of diffuse and fibrous antiactin and antimyosin staining. These results suggest that large actomyosin filament bundles are associated with nonmotile cytoplasm and that actively motile cytoplasm has a more diffuse distribution of these proteins.

Section: Fixed Cells Cell Fixationmentioning

confidence: 51%

Relation between cell activity and the distribution of cytoplasmic actin and myosin.

Herman¹,

Crisona²,

Pollard³

1981

Self Cite

218

101

“…Why is a myosin supplement necessary for HeLa cell extracts to contract? Myosin is undoubtedly present in HeLa cells, having been identified by means of a highly purified antimyosin antibody (10). Furthermore, the 100,000 g supernatant fraction of HeLa cells lysed in 0.01 M NaC1, 0.0015 M MgC12, and 0.01 M Tris, pH 7.2, and then supplemented with 0.5 M KCI, contained a polypeptide which comigrated with myosin heavy chain, and which coprecipitated with myosin added to such extracts and precipitated by dialysis to 0.05 M KC1 (author's unpublished observations).…”

Section: Discussionmentioning

confidence: 99%

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

Weihing¹

1977

The gelation induced by warming (to 25~ the 100,000 g supernatant fraction (extract) of HeLa cells lysed in a buffer containing sucrose, ATP, DTE, EGTA, imidazole, and Triton X-100 was studied in the presence of myosin and heavy meromyosin (HMM). Myosin mixed with extract induces shrinkage of the gel, but jelled extract or myosin alone does not shrink. In the concentration range, 0.14-1.04 mg/ml of myosin, the degree of shrinkage is roughly proportional to the concentration of myosin. Supplemental MgClz also promotes shrinkage. HMM (0.4-0.8 mg/ml) can inhibit gel formation by extract in tubes or floated on a sucrose cushion. Gel electrophoresis of gels shrunken by added myosin or electrophoresis of the proteins which can be sedimented from extract after incubation in the presence of HMM indicate that both myosin and HMM interfere with the changes in sedimentability of the high molecular weight protein (HMWP) thought to participate (together with actin) in gel formation in HeLa cell extracts (R. R. It has been recognized since 1960 that the cytoplasm of giant amebas can carry out lifelike movements after release from the cell (2). 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. These newer observations on gelation and shrinkage seem to be related to the older observations on consistency changes and movement of ameba extracts by the observation of a gel-like consistency for the ameba cytoplasm unThE JOURNAL OF CELL BIOLOGY 9 VOLUME 75, 1977 9 pages 95-103 95 on

“…Our data are consistent with the observation that ATP is not required for chromosomal fiber shortening in permeabilized cell models (4). Yet myosin and actin seem to be present in the mitotic spindle in background or even above background concentrations (2,10,11,43). As techniques for the fixation and visualization of these proteins improve, their presence as relevant spindle componems may or may not be confirmed.…”

Section: Ki[hart ~T M Myosin ~S Not Involved In Chromosome Movementmentioning

confidence: 99%

“…Protein concentration was determined by optical density (10) or was estimated by the method of Lowry et al (30) using bovine serum albumin as a standard. Protein concentrations of the various y-globulin solutions used for microinjection were as follows: antimyosin 55 mg/ml; nonimmune y-globulin 55 mg/ml, absorbed antimyosin 47 mg/ml, buffer-absorbed y-globulin, 55 mg/ml; and fluoresccin-labeled y-globulin, 55 mg/mL…”

Section: Microinjectionmentioning

confidence: 99%

Evidence that myosin does not contribute to force production in chromosome movement.

Kiehart¹,

Mabuchi²,

Inoue³

1982

121

Antibody against cytoplasmic myosin, when microinjected into actively dividing cells, provides a physiological test for the role of actin and myosin in chromosome movement. Anti-Asterias egg myosin, characterized by Mabuchi and Okuno (1977, J. Cell Biol., 74:251), completely and specifically inhibits the actin activated Mg++-ATPase of myosin in vitro and, when microinjected, inhibits cytokinesis in vivo. Here, we demonstrate that microinjected antibody has no observable effect on the rate or extent of anaphase chromosome movements. Neither central spindle elongation nor chromosomal fiber shortening is affected by doses up to eightfold higher than those required to uniformly inhibit cytokinesis in all injected cells. We calculate that such doses are sufficient to completely inhibit myosin ATPase activity in these cells.Cells injected with buffer alone, with myosin-absorbed antibody, or with nonimmune yglobulin, proceed normally through both mitosis and cytokinesis. Control y-globulin, labeled with fluorescein, diffuses to homogeneity throughout the cytoplasm in 2-4 rain and remains uniformly distributed. Antibody is not excluded from the spindle region. Prometaphase chromosome movements, fertilization, pronuclear migration, and pronuclear fusion are also unaffected by microinjected antimyosin.These experiments demonstrate that antimyosin blocks the actomyosin interaction thought to be responsible for force production in cytokinesis but has no effect on mitotic or meiotic chromosome motion. They provide direct physiological evidence that myosin is not involved in force production for chromosome movement.Anaphase chromosome movement in eucaryotes is usually the result of two distinct motions: the chromosomal fibers shorten as the chromosomes move toward the spindle poles, and the central spindle elongates as the poles move apart. These motions, which together insure the appropriate segregation of the daughter chromosomes during cell division, are likely the result of different force-producing mechanisms (4,5, 19,44,47). Because the spindle is labile, its ultrastructure is complex, and the actual force required to move the chromosomes is small (42, 54), a comprehensive catalog of the molecules responsible for force production in anaphase movement is not available. As a result, various theories that attempt to explain chromosomal fiber shortening and central spindle elongation have included virtually every known biological mechanochemical transducing system.