Actin polymerization and interaction with other proteins in temperature-induced gelation of sea urchin egg extracts.

Abstract

The gel which forms on warming the extracts of the cytoplasmic proteins of sea urchin eggs has been separated into two fractions, one containing F-actin and the other containing two proteins of 58,000 and 220,000 mol wt. When combined in 0.1 M KCI, even at 0~ these components will form gel material identical to that formed by warming extracts. This gel is a network of laterally aggregated F-actin filaments which are in register and which display a complex cross-banding pattern generated by the presence of the other two proteins. Low concentrations of calcium block the assembly of these proteins to form this complex structure, which may play some cytoskeletal role in the cytoplasm.This association of F-actin with the other proteins to form a gel is very likely the last step of the process occurring in warmed extracts. At low temperatures, gelation of extracts is limited by the relative absence of F-actin, as demonstrated by the inability to sediment it at 100,000 g and also by the fact that gelation occurs immediately if exogenous Foactin is added to cold extracts. The transformation of the G-actin present in the extract to the F-form is apparently repressed at low temperatures. This is shown directly by the failure of added G-actin to polymerize at low temperatures in the presence of extract. These observations resemble those which have been reported on preparations from amoeboid cells and may be significant in the involvement of actin and these other proteins in cell division and later developmental processes.In a previous publication (10) it was demonstrated that warming an extract of the soluble cytoplasmic proteins of the sea urchin egg caused the formation of a gel in the solution, visible in the light microscope as a net of birefringent threads. The extraction medium contained sufficient glycerol to render it isotonic, EGTA to chelate the cell calcium, and buffer to control the pH during homogenization. Dialysis of the extract to remove all small molecular weight components reduced or eliminated this gelation on warming, but gelation could be restored by the addition of ATP and KC1. Gelation was not temperature reversible, as the gels were stable at 0~ once formed, but the material was soluble in high salt (0.5-1 M) solution after centrifugation from the extract. Subsequent reduction of the salt concentration of this dissolved gel solution to approx. 0.1 M by dilution or dialysis caused the reappearance of the gel, a process which does not require warming and which will occur at 0~ Sodium dodecyl sulfate (SDS) gel electrophoresis showed three major components to be present in this dissolved gel: a protein which co-migrated 704

Effects of myosin and heavy meromyosin on actin-related gelation of HeLa cell extracts.

“…While jelled extracts of Acanthamoeba (22,23), macrophages (28,29), and leukemic cells (4) shrink spontaneously, neither jelled extracts of sea urchin eggs (15,16) nor those of HeLa cells (this report) will do so. Why is a myosin supplement necessary for HeLa cell extracts to contract?…”

“…It should be pointed out, however, that the electrophoretic patterns of even the most highly purified gels (from cushions) are still somewhat complicated, so it is not yet possible to decide whether other proteins, which could resemble the intermediate molecular weight protein found in gels formed by sea urchin egg extracts (15,16), or the highly potent, low molecular weight gelation factors reported for Acanthamoeba (20), also participate in gelation.…”

“…Thus, the simplest explanation for the myosin requirement is that the myosin concentration of HeLa cell extract is too low to support shrinkage. (The myosin content of sea urchin egg extract cannot be estimated from available data [15,16].) Although quantitative comparisons of the composition of these systems will be required to prove this explanation, it is of interest to note that the lowest concentration of myosin used in these experiments (0.14 mg/ml, Table I) did induce shrinkage and that it is close to the estimated concentration of myosin (0.13 mg/ml) in Acanthamoeba extracts, which shrink endogenously after gelation (22).…”

“…In common with Acantharnoeba (22, 23), macrophages (28,29), leukemic cells (4), sea urchin eggs (15,16), and giant amebas (31), suitable extracts of HeLa cells can form a gel. In all cases, gelation can be related to a polymerization of actin induced by warming, but the generality of the role of the other proteins which have been isolated with gels is less well established.…”

“…The ability of cytoplasmic extracts to jell was recognized more recently, and it has been related to actin (22), actin and a high molecular weight actin-binding protein (4,29,34,35), actin and two other proteins (15,16), or actin and several low molecular weight proteins (20). The gels formed by three of these cytoplasmic extracts can shrink spontaneously (4,22,29), and the incorporation of myosin into the shrunken gels has suggested that interactions of actin and myosin induce gel shrinkage.…”