An improved method for fixing amoebae for electron microscopy

Bhowmick, Dilip Kumar; Wohlfarth‐Bottermann, K. E.

doi:10.1016/0014-4827(65)90258-2

Experimental Cell Research

1965

DOI: 10.1016/0014-4827(65)90258-2

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An improved method for fixing amoebae for electron microscopy

Dilip Kumar Bhowmick

K. E. Wohlfarth‐Bottermann

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1966

1975

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Cited by 20 publications

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“…Several The dimensions of the thick filaments produced in vitro are the same as those of thick filaments reported in fixed and sectioned ameboid cells of various species (13,14,3,9), as well as in an Amoeba proteus cytoplasm fraction, (22,19), and in platelets after certain treatments (23). Similar filaments have also been found in Physarum undergoing spherule formation (12).…”

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confidence: 63%

Filament Formation by Purified Physarum Myosin

Nachmias

1972

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

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Physarum myosin can be separated from actomyosin by ultracentrifugation, and purified by gel filtration. Unlike actomyosin, myosin is soluble in 0.05 M KCI in the pH range of 6-7. However, in the absence of actin, the slime mold myosin can be precipitated in 0.05 M KCI by the addition of millimolar concentrations of CaCi2. The precipitates consist of aggregated, short bipolar filaments. Magnesium has a similar effect, but results in the precipitation of more loosely packed aggregates.The length of the compact filaments is 0.45 ism; thus, predominantly tail-to-tail, but also some head-to-tail, interactions occur under these conditions. Since the size and shape of these thick filaments are close to those seen in fixed and sectioned ameboid cells and in platelets, all of these filaments are probably composed of myosins.Actomyosin has been prepared from plasmodia of Physarum polycephalum by extraction at high ionic strength and alkaline pH, followed by precipitation at low ionic strength (7,5,16,17). A chelating agent must be present in the first step to obtain a precipitate that redissolves; the procedure can then be repeated to yield three-or four-times precipitated actomyosin. The success of the procedure depends upon the fact that the plasmodial actomyosin has solubility properties similar to those of muscle actomyosins. On the other hand, the Physarum myosin, which can be separated from the actin component by additional procedures, is strikingly different in its solubility properties from muscle myosins. Unlike, for example, vertebrate striated muscle myosin, which readily forms filaments at 0.1 M KCl with S values in the range of 70-310 (10, 11, 20), Physarum myosin remains soluble even in 0.05 M KCl, and no filaments are visible by electron microscopy despite variation of several parameters (15). Myosin purified by two different approaches forms only small soluble aggregates, with S values of 8-15, at low ionic strengths as compared to S values of 6 at high ionic strength (1, 5, 6).However, the solubility of Physarum myosin is not, apparently, due to any marked difference in shape factors as compared to vertebrate muscle myosins. Physarum and vertebrate striated muscle myosin behave similarly on an Agarose 4B column (ref 1. and unpublished observations in this laboratory) and in an analytical ultracentrifuge (5, 6, 1).Both Physarum myosin-enriched actomyosin upon negativestaining (17) and metal-shadowed Physarum myosin (6) show the existence of an extended, or "tail," portion of the molecule that is at least 120 nm long.Therefore, the fact that the slime mold myosin does not form filaments at low ionic strength may be due to a charge 2011 distribution along the extended part of the molecule different from that characteristic of muscle myosins.In support of this view, we report here that both partially purified Physarum myosin and myosin purified by gel filtration are capable of forming short filaments at low ionic strengths, provided that calcium salts are present.Partially purified Physarum myosin wa...

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supporting

confidence: 63%

Filament Formation by Purified Physarum Myosin

Nachmias

1972

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

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Microfibrillar structures in the nucleus and cytoplasm of amoeba proteus (1)

Leeson

Bhatnagar

1975

J. Exp. Zool.

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Structure of synthetic and native myosin filaments from Amoeba proteus

D’Haese

Hinssen

1974

Cell Tissue Res.

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An improved method for fixing amoebae for electron microscopy

Cited by 20 publications

References 12 publications

Filament Formation by Purified Physarum Myosin

Filament Formation by Purified Physarum Myosin

Microfibrillar structures in the nucleus and cytoplasm of amoeba proteus (1)

Structure of synthetic and native myosin filaments from Amoeba proteus

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