A 45,000-mol-wt Protein from Unfertilized Sea Urchin Eggs Severs Actin Filaments in a Calcium-dependent Manner and Increases the Concentration of Nonfilamentous Actin Steady-state

A 45,000-mol-wt protein has been purified from unfertilized sea urchin (StrongyIocentrotus purpuratus) eggs. The isolation scheme includes DEAE cellulose ion-exchange chromatography, gel filtration, and hydroxylapatite chromatography. The homogeneity of the isolated protein is >90% by SDS PAGE. The 45,000-mol-wt protein reduces the viscosity of actin filaments in a Ca2+-dependent manner. The free calcium concentration required for the activity of this protein is in the micromolar range. Electron microscopic studies reveal that the formation of short filaments parallels the decrease in viscosity. Energy transfer and sedimentation experiments indicate a net disassembly of actin filaments and an increase in the steadystate nonfilamentous actin concentration in the presence of Ca 2÷ ions and the 45,000-mol-wt protein. The increase in the steady-state nonfilamentous actin concentration is proportional to the amount of 45,000-mol-wt protein added. The actin molecules disassembled by the addition of the 45,000-mol-wt protein are capable of polymerization. The organization of the contractile proteins in nonmuscle cells is a subject of great interest in biological research. A number of proteins that interact with actin in several different modes and alter the assembly of purified actin have been isolated from a wide variety of cells (for reviews see references 9, 20, 32, 46). These proteins are believed to play a regulatory role in the cytoskeletal changes that occur during cell locomotion, secretion, cytoplasmic streaming, and cytokinesis. Sea urchin eggs provide an excellent system to study the molecular mechanism of actin assembly because it is easy to obtain a large homogenous population undergoing a synchronized change in cytoskeleton arrangement upon fertilization (8, 10). In unfertilized sea urchin eggs of S. purpuratus, there is an actin concentration o f 3 mg/ml and -40% of this actin appears to be monomeric (24). Otto et al. (26) reported that in Tripneustes gratilla, a Hawaiian sea urchin, ~90% of the actin is nonsedimentable at 100,000 g for 60 min in an isotonic extract of unfertilized eggs. An identical extract of fertilized T. gratilla eggs has 60-65% nonsedimentable actin. The state of the actin in the cortex of sea urchin eggs changes dramatically upon fertilization. The numerous short microvilli that cover the surface of the unfertilized egg elongate greatly (33, 34), and each microvillus contains a bundle of actin filaments (6). Upon fertilization there is also a rapid Na + influx, an increase in intracellular free Ca 2÷ concentration, and a pH increase (for reviews, see references 11, 12, 41). The elongation and formation of actin filament bundles have been suggested to be Ca 2+ and pH regulated (1). In that the in vitro steady-state, nonfilamentous concentration of purified sea urchin egg actin is 0.02-0.05 mg/ml (24), and the concentration of total actin in unfertilized eggs is much greater, the regulation of the state of actin assembly may involve accessory proteins. Several actin-binding proteins have been isolated from eggs. Fascin, a 58,000-mol-wt protein, has been shown to crosslink actin filaments and form bundles (5). Immunofluorescent antibody staining has located fascin in the fertilized egg cortex (26) and in the pseudopods of sea urchin coelomocytes (25). Fascin cross-linked actin-filament bundles prepared in vitro have a transverse-stripe pattern of 1 l-nm axial periodicity, which is similar to that observed in sea urchin microvilli in situ (6, 37). These needle-shaped bundles form a three-dimensional gel when a 220,000-mol-wt egg protein is added (5). The isolations of fascin and the 220,000-mol-wt protein start "[-HE JOURNAL OF CELL BIOLOGY • VOLUME 99 SEPTEMBER 1984 844-851 844 © The Rockefeller University Press • 0021-9525/84/0910844108 $1.00 on F ebuary 0, 2013 jcb.rress.org D ow nladed fom Published September 1, 1984