Heavy meromyosin-binding filaments in the mitotic apparatus of mammalian cells

Hinkley, Robert E.; Telser, Alvin

doi:10.1016/0014-4827(74)90662-4

Cited by 46 publications

(17 citation statements)

References 12 publications

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“…By neither Sanger's methods nor our own is actin seen in appreciable amounts in the interzone fibers of anaphase cells. This result differs from the observations made with glycerination, HMM decoration, and electron microscopy where decorated filaments are found in the spindle midzone (4,13,14,17,18,20). The cause of this discrepancy is not clear.…”

Section: Discussioncontrasting

confidence: 89%

“…Actin has been observed before in mitotic spindles by HMM as an actin-specific marker and glycerination of the cells as preparative treatment (1,4,13,14,17,18,20,41). Recent work by Forer and Jackson (14) showing HMM decorated filaments in the spindles of endosperm tissue from the plant Haemanthus make it unlikely that cortical actin is the sole source of spindle actin, because these cells lack a defined, gelatinous cortex.…”

Section: Discussionmentioning

confidence: 96%

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A comparison of the distribution of actin and tubulin in the mammalian mitotic spindle as seen by indirect immunofluorescence.

Cande¹,

Lazarides²,

McIntosh³

1977

The Journal of Cell Biology

146

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Rabbit antibodies against actin and tubulin were used in an indirect immunofluorescence study of the structure of the mitotic spindle of PtK1 cells after lysis under conditions that preserve anaphase chromosome movement. During early prophase there is no antiactin staining associated with the mitotic centers, but by late prophase, as the spindle is beginning to form, a small ball of actin antigenicity is found beside the nucleus. After nuclear envelope breakdown, the actiactin stains the region around each mitotic center, and becomes organized into fibers that run between the chromosomes and the poles. Colchicine blocks this organization, but does not disrupt the staining at the poles. At metaphase the antiactin reveals a halo of ill-defined radius around each spindle pole and fibers that run from the poles to the metaphase plate. Antitubulin shows astral rays, fibers running from chromosomes to poles, and some fibers that run across the metaphase plate. At anaphase, there is a shortening of the antiactin-stained fibers, leaving a zone which is essentially free of actin-staining fluorescence between the separating chromosomes. Antitubulin stains the region between chromosomes and poles, but also reveals substantial fibers running through the zone between separating chromosomes. Cells fixed during cytokinesis show actin in the region of the cleavage furrow, while antitubulin reveals the fibrous spindle remnant that runs between daughter cells. These results suggest that actin is a component of the mammalian mitotic spindle, that the distribution of actin differs from that of tubulin and that the distributions of these two fibrous proteins change in different ways during anaphase.The extent of our ignorance about molecular mechanisms for the cellular forces that move chromosomes is demonstrated by the number of different, successful models for mitotic movements (2,10,12,23,31,34). In spite of this ignorance, several aspects of the process are reasonably well understood. There is general agreement that chromosomes are moved by forces acting on them at the kinetochore region (34). Since microtubules are the major fibrous element in the spindle, accounting for the majority (19) if not all of the spindle birefringence (42), and since microtubules 552

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Section: Discussioncontrasting

confidence: 89%

Section: Discussionmentioning

confidence: 96%

A comparison of the distribution of actin and tubulin in the mammalian mitotic spindle as seen by indirect immunofluorescence.

Cande¹,

Lazarides²,

McIntosh³

1977

The Journal of Cell Biology

146

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“…Actin-like microfilaments are found in the chromosome-to-pole regions and in quite large numbers near the poles (Figs. 11-14 and references 3, t5, 11,10,16,24,12). In addition, they are found in the interzone.…”

Section: Schloss Et Al F-s-1 For Microfilament Localizationmentioning

confidence: 99%

“…Several reports indicate that decorated microfilaments are seen in glycerinated spindles after the addition of HMM (3,15,11,16,24,12). Only one report exists showing that microfilaments are seen in glycerinated spindles of locust spermatocytes without addition of HMM (16).…”

Section: Schloss Et Al F-s-1 For Microfilament Localizationmentioning

confidence: 99%

Myosin subfragment binding for the localization of actin-like microfilaments in cultured cells. A light and electron microscope study.

Schloss¹,

Milsted²,

Goldman³

1977

The Journal of Cell Biology

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Fluorescein-labeled heavy meromyosin subfragment-1 (F-S-l) has been purified by ion exchange chromatography and characterized in terms of its ability to bind specifically to actin. F-S-1 activates the Mg+ § triphosphatase activity of rabbit skeletal muscle actin and decorates actin as shown by negative stains and thin sections of rabbit actin and rat embryo cell microfilament bundles, respectively. Binding of F-S-1 to cellular structures is prevented by pyrophosphate and by competition with excess unlabeled S-1.The F-S-1 is used in light microscope studies to determine the distribution of actin-containing structures in interphase and mitotic rat embryo and rat kangaroo cells. Interphase cells display the familiar pattern of fluorescent stress fibers. Chromosome-to-pole fibers are fluorescent in mitotic cells.The glycerol extraction procedures employed provide an opportunity to examine cells prepared in an identical manner by light and electron microscopy. The latter technique reveals that actin-like microfilaments are identifiable in spindles of glycerinated cells before and after addition of S-1 or HMM. In some cases, microfilaments appear to be closely associated with spindle microtubules. Comparison of the light and electron microscope results aids in the evaluation of the fluorescent myosin fragment technique and provides further evidence for possible structural and functional roles of actin in the mitotic apparatus.KEY WORDS microfilaments HMM subfragment-1 cultured cells actin mitotic spindle fluorescence microscopy Determination of the distribution and organization of actin-like microfilaments in nonmuscle cells is an important approach to discerning the mechanisms of cellular motility (see references 40 and 41 for reviews). Microfilaments are identified in cells fixed and thin-sectioned for electron microscopy by their size (-6 nm diameter) and ability to bind the myosin fragments heavy meromyosin (HMM) and HMM subfragment-1 (S-l), resulting in "arrowhead" configurations or decorated complexes (26,27,18). This reaction is considered to be specific for actin, as HMM and S-1 do not decorate other fibrous structures in the cell (27,19,40 also been employed to study the overall distribution of structures thought to contain actin (5). However, the supramolecular organization of aco tin cannot be determined by these techniques because of the limits of resolution of the light microscope (22). Only a few attempts have been made to correlate directly information from light and electron microscope studies. For instance, the distribution of phase-dense stress fibers (5) and birefringent fibers seen with polarized light microscopy (17) has been shown to correspond to that of microfilament bundles in several cultured animal cells (20,22).Fluorescein-labeled HMM (F-HMM), S-1 (F-S-1), and antiactin antibody in indirect immunofluorescence studies have been shown to stain stress fibers in extensively spread cells which correspond to microfilament bundles (45,47,22,29,20). We have shown that comparison of light an...

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“…Most investigators have attempted to show that either actin or tubulin in the mitotic spindle is involved in the force production required for chromosomal movement (1)(2)(3)(4)(5). The evidence for and against each protein includes biochemical, electron microscopic, and fluorescence microscopic results.…”

mentioning

confidence: 99%

Differential staining of actin in metaphase spindles with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin and fluorescent DNase: is actin involved in chromosomal movement?

Barak¹,

Nothnagel²,

Demarco³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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The distribution and polymerization state ofactin in metaphase rat kangaroo cells was studied by fluorescence microscopy. Formaldehyde-fixed, acetone-extracted cells were labeled with either of two types of actin probes. The first, 7-nitrobenz-2-oxa-1,3-diazole-phallacidin, has high affinity for F actin and does not bind monomeric G actin. The second was a conjugate of DNase I labeled with either tetramethylrhodamine or fluorescein. DNase binds with high affinity to G actin and with lesser affinity to F actin. The polymerization state of actin was deduced by comparing the fluorescence distribution of the phallacidin derivative with that of the fluorescent DNase. The results indicate that the pole-to-chromosome region ofthe metaphase spindle contains G actin but little if any conventional F actin. F actin is found concentrated in a diffuse distribution outside the spindle region in metaphase cells and returns to the interzone area between the chromosomes by early telophase. These results exclude spindle models for chromosomal movement that require more than about five F actin filaments per chromosome, support the hypothesis that F actin is involved in force generation for cell cleavage, and are not inconsistent with the possibility that actin outside the spindle may be involved in chromosomal movement.The mechanism responsible for chromosomal movement during mitosis has not been established. Most investigators have attempted to show that either actin or tubulin in the mitotic spindle is involved in the force production required for chromosomal movement (1-5). The evidence for and against each protein includes biochemical, electron microscopic, and fluorescence microscopic results.Strong evidence exists that tubulin is a spindle component, although there remains some uncertainty regarding the amount present (5-7). Electron micrographs show microtubules inserted into chromosomal kinetochores, structures thought to be tubulin-nucleation centers (5). Each chromosome has one such site of attachment. This has led to speculation that tubulin participates directly in generating force for chromosomal movement (for review, see ref. 5).Biochemical evidence shows that actin is a component of nonmuscle cells (8). However, it is difficult to determine the subcellular location of actin by biochemical techniques. Some experiments have indicated the presence of actin in nuclear fractions (9, 10), but others have indicated that actin in these fractions may be a cytoplasmic contaminant. By using isolated nuclei, Comings and Harris found that actin and myosin constitute at most a very small fraction of nonhistone proteins (6).Electron microscopy has provided evidence for the existence ofspindle actin (11)(12)(13)(14). These experiments have identified actin by the appearance of characteristic arrowhead complexes formed from the binding of heavy meromyosin (HMM) or its nuclease S1 subfragment to actin filaments (15). Glycerination has been a standard step in the preparation ofthese specimens, and it has been suggested that c...

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Heavy meromyosin-binding filaments in the mitotic apparatus of mammalian cells

Cited by 46 publications

References 12 publications

A comparison of the distribution of actin and tubulin in the mammalian mitotic spindle as seen by indirect immunofluorescence.

A comparison of the distribution of actin and tubulin in the mammalian mitotic spindle as seen by indirect immunofluorescence.

Myosin subfragment binding for the localization of actin-like microfilaments in cultured cells. A light and electron microscope study.

Differential staining of actin in metaphase spindles with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin and fluorescent DNase: is actin involved in chromosomal movement?

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