Isolation of microfilaments from Amoeba proteus

Morgan, Joan; Fyfe, Derek A.; Wolpert, Lewis

doi:10.1016/0014-4827(67)90299-6

Cited by 25 publications

(10 citation statements)

References 8 publications

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“…But the products obtained in the two cases are different; instead of the fibrils and tactoids of aggregated actin filaments induced in amoeba (20,23), warming of the low calcium extract of sea urchin egg cytoplasm causes the formation of dense gel composed of actin and two other proteins which is not temperature reversible. The fine structure, the chemical composition, and the behavior of the fibrils obtained with ATP or EDTA in amoeba can be compared more directly to those of the fibrils induced in the dissolved urchin gel material by the addition of ATP.…”

Section: Discussionmentioning

confidence: 95%

“…These changes did not yield polymerizable tubulin, but instead caused the polymerization of actin and two other cytoplasmic proteins. These preliminary observations were developed into a simple and rapid method for the isolation of actin which has little in common with the more conventional techniques of muscle biochemistry that have been used to prepare actin from the egg, and in several respects it relates more directly to observations (20,23) on the fibrillar motile system of amoeba. Since the presentation of this method in abstract form (15), it has been adapted to the polymerization of amoeba actin (22).…”

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confidence: 99%

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Preparation and purification of polymerized actin from sea urchin egg extracts.

Kane¹

1975

The Journal of Cell Biology

219

145

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Isotonic extracts of the soluble cytoplasmic proteins of sea urchin eggs, containing sufficient EGTA to reduce the calcium concentration to low levels, form a dense gel on warming to 35 40~ Although this procedure is similar to that used to polymerize tubulin from mammalian brain, sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows this gel to have actin as a major component and to contain no tubulin. If such extracts are dialyzed against dilute salt solution, they no longer respond to warming, but gelation will occur if they are supplemented with 1 mM ATP and 0.020 M KC1 before heating. Gelation is not temperature reversible, but the gelled material can be dissolved in 0.6-1 M J~C1 and these solutions contain F-actin filaments. These filaments slowly aggregate to microscopic, birefringent fibrils when l mM ATP is added to the solution, and this procedure provides a simple method for preparing purified actin. The supernate remaining after actin removal contains the other two components of the gel, proteins of approximately 58,000 and 220,000 mol wt. These two proteins plus actin recombine to form the original gel material when the ionic strength is reduced. This reaction is reversible at 0~ and no heating is required.Following the demonstration of an actin-like protein, similar in properties to muscle actin, in myxomycete plasmodium (9, 10), similar methods revealed the presence of a cytoplasmic actin in unfertilized sea urchin eggs. This sea urchin egg actin was first identified and separated through its combination with rabbit muscle myosin (8,18,19), and the protein was later prepared directly from egg extracts by salting out with ammonium sulfate (17). These preparative methods gave no information concerning the localization of the actin within the egg, but there is good evidence lor its presence in the cleavage furrow during cytokinesis, where it was seen first in the electron microscope as bundles of microfilaments (28,30,37). Actin has been localized in the cleavage furrow in a variety of other cell types ( 1,21,27,29,34) and this actin has been specifically labeled with heavy meromyosin (31). The investigations reported here were begun with the aim of determining whether the methods developed for the polymerization of tubulin to microtubules in mammalian brain (2, 39) could be used to prepare tubulin from the sea urchin egg. Marine eggs are an excellent source of isolated mitotic apparatuses for the investigation of the role of microtubules in chromosome movement, but up to this time only the interaction of mammalian brain tuhulin with such isolated mitotic apparatuses has been reported (12, 25). Our attempts to prepare tubulin from extracts of sea urchin eggs with the methods developed for mammalian material were unsuccessful, and a number of modifications of this method were made to adapt it to the

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Section: Discussionmentioning

confidence: 95%

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confidence: 99%

Preparation and purification of polymerized actin from sea urchin egg extracts.

Kane¹

1975

The Journal of Cell Biology

219

145

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“…We did manage to isolate cytoplasm that continued to move, but it was not until 1967, working with Joan Morgan, that we isolated the filaments (Morgan et al 1967).…”

Section: From Soil Mechanics To Cell Mechanicsmentioning

confidence: 99%

Perspective

Wolpert

2015

Annu. Rev. Cell Dev. Biol.

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I am a developmental biologist, but I started off as a civil engineer. I did some research on soil mechanics but decided to change to biology. A friend changed my life when he told me about the mechanics of cell division, on which I did my PhD at Kings College. I then worked on the morphogenesis of the sea urchin embryo and became interested in how embryos are patterned, and I proposed positional information as a basic mechanism. I was a professor at the Middlesex Hospital Medical School, where we concentrated on how the chick limb developed.

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“…With Chris I tried to isolate the elements responsible for movement, assuming they would be similar to those involved in the contraction of muscle. We did manage to isolate cytoplasm that continued to move, but it was not until 1967, working with Joan Morgan, that we isolated the filaments (Morgan et al 1967).…”

Section: From Soil Mechanics To Cell Mechanicsmentioning

confidence: 99%

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2015

Annu. Rev. Cell Dev. Biol.

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Isolation of microfilaments from Amoeba proteus

Cited by 25 publications

References 8 publications

Preparation and purification of polymerized actin from sea urchin egg extracts.

Preparation and purification of polymerized actin from sea urchin egg extracts.

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