A permeabilized cell model for studying cell division: a comparison of anaphase chromosome movement and cleavage furrow constriction in lysed PtK1 cells.

Cande, W. Zacheus; McDonald, Kent L.; Meeusen, Ronald L.

doi:10.1083/jcb.88.3.618

Cited by 71 publications

(39 citation statements)

References 35 publications

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“…It is corroborated by the failure of Nethylmaleimide-modified myosin subfragment 1, cytochalasin and antimyosin, agents reported to block actomyosin function, to inhibit chromosome movements in permeabillzed cell models (6,39,51). Our data are consistent with the observation that ATP is not required for chromosomal fiber shortening in permeabilized cell models (4).…”

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

confidence: 88%

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

Kiehart¹,

Mabuchi²,

Inoue³

1982

The Journal of Cell Biology

122

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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.

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

confidence: 88%

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

Kiehart¹,

Mabuchi²,

Inoue³

1982

The Journal of Cell Biology

122

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“…However, it has not been determined whether these areas represent holes in the membrane or just regions of the membrane oblique to the plane of the thin sections. The relative intactness of the permeabilized cells prepared by the methods described here is in contrast to that found with other permeabilization procedures (3,5), which appeared more destructive. Dye-labeled immunoglobulin molecules were used to estimate the size of the holes in the plasma membrane that were generated by saponin.…”

Section: Resultscontrasting

confidence: 65%

“…3 H-labeled leucine, lysine, phenylalanine, proline, and tyrosine. All protein synthesis assays were performed at 28°C.…”

Section: Methodsmentioning

confidence: 99%

Organization of Mammalian Cytoplasm

Hudder

Nathanson

Deutscher

2003

Molecular and Cellular Biology

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Although the role of macromolecular interactions in cell function has attracted considerable attention, important questions about the organization of cells remain. To help clarify this situation, we used a simple protocol that measures macromolecule release after gentle permeabilization for the examination of the status of endogenous macromolecules. Treatment of Chinese hamster ovary cells with saponin under carefully controlled conditions allowed entry of molecules of at least 800 kDa; however, there were minimal effects on internal cellular architecture and protein synthesis remained at levels comparable to those seen with intact cells. Most importantly, total cellular protein and RNA were released from these cells extremely slowly. The release of actin-binding proteins and a variety of individual cytoplasmic proteins mirrored that of total protein, while marker proteins from subcellular compartments were not released. In contrast, glycolytic enzymes leaked rapidly, indicating that cells contain at least two distinct populations of cytoplasmic proteins. Addition of microfilament-disrupting agents led to rapid and extensive release of cytoplasmic macromolecules and a dramatic reduction in protein synthesis. These observations support the conclusion that mammalian cells behave as highly organized, macromolecular assemblies (dependent on the actin cytoskeleton) in which endogenous macromolecules normally are not free to diffuse over large distances.Tremendous progress has been made in our understanding of cell function. For the most part, this has been accomplished through the use of a traditional reductionist approach in which individual cellular components are identified and isolated and their cellular roles are reconstructed on the basis of their functions in vitro. While such an approach has proven to be highly successful, especially for determining the "players" in cell metabolism, it falls short in explaining how these components actually function within the cell. In fact, in many cases, particularly those involving complex cellular processes, it often has not been possible to recreate the efficiency of cellular reactions in vitro. Understanding what accounts for such differences in efficiency is essential if we are to explain cellular function in its entirety.In recent years, considerable attention has focused on the importance of macromolecular interactions in cell function (see, e.g., reference 10) and on the fact that enzymes contributing to complex processes often are bound to each other and that intermediates in the process may be channeled (see, e.g., references 6 and 16 and the review in reference 19). As a consequence of such organization, processes within cells may be able to proceed much more efficiently than those carried out by the same enzymes dispersed in solution in vitro. Thus, important questions that remain to be answered are (i) how extensive is cellular organization, (ii) what cellular components are responsible for maintaining it, and (iii) are macromolecular interactions confined to i...

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“…Taxol-treated MTs are stabilized through either a lowered rate of dissociation (52, 61, 65) or a decreased rate of steady-state tubulin flux (66). All work on taxol, except that of Wolniak et al (69), has been done on animal cells, and the data on mitosis in progress are still fragmentary (14,15,16). In animal culture cells, taxol treatment results in the formation of abundant and unusually arranged MTs (2,15,16,45,60,63).…”

mentioning

confidence: 99%

Action of taxol on mitosis: modification of microtubule arrangements and function of the mitotic spindle in Haemanthus endosperm.

Molè-Bajer¹,

Bajer²

1983

The Journal of Cell Biology

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We have studied the effect of taxol on mitosis in Haemanthus endosperm. ImmunoGold Stain (IGS), a new immunocytochemical method (17), was used to visualize microtubules (MTs) in the light microscope. Observations on MT arrangements were correlated with studies in vivo. Chromosome movements are affected in all stages of mitosis which progresses over at least 104 range of taxol concentrations. The three most characteristic effects on MTs are: (a) enhancement of the lateral associations between MTs, seen especially during the reorganization of the polar region of the spindle, (b) promotion of MT assembly, leading to the formation of additional MTs in the spindle and MT arrays in the cytoplasm, and (c) an increase in MT stability, demonstrated in their increased cold resistance. In this report, the emphasis is on the primary, immediate effects, occurring in the first 30 min of taxol action.Effects are detected after a few mins, are reversible, and are concentration/time dependent. The spindle and phragmoplast are remarkably modified due to the enhancement of lateral associations of MTs and the formation of abundant nonkinetochore and polar, asterlike MTs. The equatorial region of the interzone in anaphase may be entirely depleted of MTs, and the spindle may break perpendicular to the spindle axis. Mitosis is completed in these conditions, providing evidence for the motile autonomy of each half-spindle. Trailing chromosome arms in anaphase are often stretched and broken. Chromosome fragments are transported away from the polar regions, i.e., in the direction opposite to that expected (5, 6). This supplies the first direct evidence of pushing by elongating MTs in an anastral higher plant spindle. These observations draw attention to the relation between the lateral association of MT ends to assembly/disassembly and to the role of such an interaction in spindle function and organization.Taxol, a low molecular weight microcyclic alkaloid, was isolated from the bark of the western yew (Taxus brevifolia) by Wani et al. in 1971 (68). It is a potent drug which lowers the critical concentration of tubulin subunits which are required for the polymerization of MTs and shortens the lag time for microtubule (MT) assembly in vitro (59). Taxol binds specifically and reversibly to polymerized tubulin, i.e., MTs, and affects the allosteric transition of the receptor from the dimeric to polymeric state (52). Taxol-treated MTs are stabilized through either a lowered rate of dissociation (52, 61, 65) or a decreased rate of steady-state tubulin flux (66). All work on taxol, except that of Wolniak et al. (69), has been done on animal cells, and the data on mitosis in progress are still fragmentary (14,15,16). In animal culture cells, taxol treatment results in the formation of abundant and unusually arranged MTs (2,15,16,45,60,63). In such exponentially growing cultures, the cell cycle (in G2) and/or mitosis is inhibited by taxol (1,16,58,60). The short duration of mitosis and the small spindle make it difficult to study the ini...

show abstract

A permeabilized cell model for studying cell division: a comparison of anaphase chromosome movement and cleavage furrow constriction in lysed PtK1 cells.

Cited by 71 publications

References 35 publications

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

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

Organization of Mammalian Cytoplasm

Action of taxol on mitosis: modification of microtubule arrangements and function of the mitotic spindle in Haemanthus endosperm.

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