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

Abstract

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

Cytochalasin B inhibits actin-related gelation of HeLa cell extracts.

“…Gelation of cytoplasmic extracts has been related to actin (23) or interactions of actin and other proteins (13,35,37) in at least four different systems. In the macrophage system (35), gelation is also inhibited by >1 /xM CB* (11).…”

“…Recently, several laboratories have discovered that gelation of suitable cell extracts can be related to actin (23), actin and a high molecular weight protein (HMWP) (35, 37) or actin, a HMWP, and a third protein (13). Knowing that CB disrupts the contractile ring (which contains actin) (28,29) and binds to both high and low affinity sites in HeLa cells (15), it seemed of interest to test whether CB affected the formation and composition of gel in HeLa cell extracts.…”

Isolation of a high molecular weight actin-binding protein from baby hamster kidney (BHK-21) cells.

“…HMWPs that interact with actin have been implicated in cytoplasmic consistency changes (3,10,11,(13)(14)(15)19). Macrophage ABP and smooth muscle filamin have been isolated and appear to be similar (7)(8)(9)(10).…”

“…Because the ratios of actin to myosin are an order of magnitude higher in nonmuscle cells than in muscle cells (1, 2), functional interactions of actin with proteins other than those involved in the regulation of actomyosin ATPase have also been proposed. Kane showed that actin in sea urchin egg extracts gelled upon warming to 370C (3). Two polypeptides, of molecular weight 58,000 and 220,000, appeared to be involved in the process.…”

Fascin Over Expression is Associated with Dysplastic Changes in Sinonasal Inverted Papillomas: A Study of 47 Cases

“…Fascin was first identified in sea-urchin egg extracts [14] and was identified in human HeLa cells in 1985 [15]. It is one of the actin cross-linking binding proteins required for the formation of actin-based cell-surface protrusions and increased cell motility in various transformed epithelial cells [16,17].…”