A FINE STRUCTURAL ANALYSIS OF CLEAVAGE INDUCTION AND FURROWING IN THE EGGS OF <i>ARBACIA PUNCTULATA </i>

Tilney, Lewis G.; Marsland, Douglas

doi:10.1083/jcb.42.1.170

Cited by 101 publications

(21 citation statements)

References 31 publications

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“…7) (Suzuki et al 1995). Compared to ultrastructural images of the contractile ring of animal cells (e.g., Schroeder 1968Schroeder , 1970Schroeder , 1972Tilney and Marsland 1969;Selman and Perry 1970;Szollosi 1970), the actin ring of C. caldarium RK-1 shows outstanding structural simplicity. Furthermore, distribution of actin filaments is limited to the area beneath the center of the cleavage furrow in C. caldarium RK-1, while actin filaments are widely distributed around the furrow in the traditional materials.…”

Section: Ultrastructure Of the Contractile Ringmentioning

confidence: 95%

A possible role for actin dots in the formation of the contractile ring in the ultra-micro algaCyanidium caldarium RK-1

et al. 1998

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Summary. In the primitive red alga Cyanidium caldarium RK-1, cytokinesis is controlled by a simple contractile ring, as in animal cells. To clarify the mechanism of formation of the contractile ring, we isolated actin genes and performed an immunocytological study. C. caldarium RK-1 has two actin genes encoding proteins with the same sequence of 377 amino acids. The primary structure indicated that the actin molecules of C. caldarium RK-1 are typical, despite the fact that the organism is considered to be phylogenetically primitive. We prepared antiserum against a C. caldarium RK-1 actin fusion protein and indirect immunofluorescence staining was performed. In interphase cells, many actin dots were observed in the cytoplasm but none at the future cleavage plane. Prior to cytokinesis, some of these dots appeared and became aligned along the equatorial plane. At the same time, a thin "immature" contractile ring was observed to appear to be formed by connection of the aligned actin dots. This immature contractile ring thickened to nearly its maximum size by the time cytokinesis began. The formation of the contractile ring seemed to be a result of de novo assembly of actin monomers, rather than a result of the accumulation and bundling of pre-existing actin filaments. During the constriction of the contractile ring, no actin dots were observed in the cytoplasm. These observations suggest that actin dots are responsible for the formation of the contractile ring, but are not necessary for its disintegration. Furthermore, immunogold localization specific for actin revealed at electron microscopy level that fine filaments running just beneath the cleavage furrow are, in fact, aetin filaments.

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Section: Ultrastructure Of the Contractile Ringmentioning

confidence: 95%

A possible role for actin dots in the formation of the contractile ring in the ultra-micro algaCyanidium caldarium RK-1

et al. 1998

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“…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).…”

mentioning

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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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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“…Their localization in cell types other than muscle has led to the speculation that they may function in certain types of contractile processes such as cleavage (1)(2)(3)(4), neural-tube formation (5), resorption of ascidian tadpole tails (6,7), certain types of cytoplasmic streaming (8), shortening of secondary mesenchymal processes during gastrulation (9), and pulsations of intestinal epithelial cell microvilli (10). Recent evidence in support of these speculations has come from experiments with cytochalasin B (11,12).…”

mentioning

confidence: 99%

Actin in the Brush-Border of Epithelial Cells of the Chicken Intestine

Tilney

Mooseker

1971

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

Self Cite

146

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The major soluble protein of the isolated brush-border of the intestinal epithelium has a molecular weight and net charge indistinguishable from those of skeletal-muscle actin, as determined by polyacrylamide gel electrophoresis. Furthermore, this protein, isolated from acetone powders of the purified brush-border, undergoes a G to F transformation in the presence of Mg++. The filaments have a substructure indistinguishable from muscle actin, as seen by the negative-staining technique, and bind heavy meromyosin with the arrowhead configuration characteristic of actin. The filaments in the microvilli of the intact brush-border also bind heavy meromyosin. Thus, actin seems to be present in intestinal epithelial cells.Intracellular filaments, about 5 nm in diameter, are now recognized to be common constituents of eucaryotic cells. Their localization in cell types other than muscle has led to the speculation that they may function in certain types of contractile processes such as cleavage (1-4), neural-tube formation (5), resorption of ascidian tadpole tails (6, 7), certain types of cytoplasmic streaming (8), shortening of secondary mesenchymal processes during gastrulation (9), and pulsations of intestinal epithelial cell microvilli (10). Recent evidence in support of these speculations has come from experiments with cytochalasin B (11,12). In man-y, but not all, of the above svstems, this compound affects the integrity of the filaments and the contractile function is lost.5-nm Filaments also occur in primitive cell types, such as the acellular slime mold, Physarum, and amoeba. The filaments in both of these organisms have close biochemical homology with skeletal-muscle actin (13)(14)(15)(16). Furthermore, the isolated filament protein of both systems has been shown to bind heavy meromyosin (HMM), with the arrowhead configuration characteristic of muscle actin (16)(17)(18) The chickens were starved for 1 day before they were killed. Before and after filtration with glass wool, the pellet was washed several extra times to remove the yellowish unconsolidated layer on top of the pellet. Purity was assessed by light microscopy (phase-contrast and Nomarski interference) and byr electron microscopy.Polyacrylamide Gel Electrophoresis. The molecular weights of proteins from the whole isolated brush-border and from proteins extracted from acetone powders of the brush-border were determined electrophoretically (24, 25) on 5% polyacrylamide gels that contained 0.1% sodium dodecyl sulfate (SDS) at pH 7.1. The purified brush-border, isolated brushborder actin, chick actin prepared from acetone powders (26), and protein standards were heated for 2 min at 1000C in 1.0% SDS-1.0% 2-mercaptoethanol-10 mM phosphate buffer (pH 7.1) before electrophoresis. The gels were calibrated with bovine serum albumin (molecular weight 68,000 and 136,000), catalase (molecular weight 60,000), chick actin (molecular weight 46,000), and pepsin (molecular weight 36,000). Bromphenol blue was used as a tracking dye.For the determination of net p...

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A FINE STRUCTURAL ANALYSIS OF CLEAVAGE INDUCTION AND FURROWING IN THE EGGS OF ARBACIA PUNCTULATA

Cited by 101 publications

References 31 publications

A possible role for actin dots in the formation of the contractile ring in the ultra-micro algaCyanidium caldarium RK-1

A possible role for actin dots in the formation of the contractile ring in the ultra-micro algaCyanidium caldarium RK-1

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

Actin in the Brush-Border of Epithelial Cells of the Chicken Intestine

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