Mass Isolation of Cleavage Furrows from Dividing Sea Urchin Eggs

To develop a mass isolation procedure for the cleavage furrow from synchronized sea urchin eggs, we compared the stability of the cleavage furrow with that of the rest of the cortex (polar-region cortex) and the inner cytoplasm under various conditions using the rhodamine-phalloidin staining method. As a result, to remove the polar-region cortex and leave the cleavage furrow intact, it became clear that the type and concentration of detergent, the pH and Ca concentration of the isolation solution and the temperature were of critical importance, and that 0.04-0.1 % Nonidet P-40, pH 7.0-7.5, low calcium ion concentration and room temperature were optimal conditions. To solubilize the inner cytoplasm to release intact cleavage furrows, two factors, osmotic pressure and sea urchin species, were found to be important: 0.16 M glucose (or sucrose) was optimal, and we found Clypeaster japonicus to be the most appropriate. A shearing force, by gentle pipetting, was also required for furrow isolation. Taking these results into consideration, we have succeeded in developing a mass isolation procedure for cleavage furrow from C. japonicus. A total of 20–50 μg of protein of isolated cleavage furrow was recovered from 1 ml of packed dividing eggs. The structural integrity of the isolated cleavage furrow was well maintained and it was characterized by remnants of plasma membranes, actin filament meshwork including a contractile ring, and cytoplasmic vacuoles. Although the isolated furrow contained myosin II molecules, it showed no capability of in vitro reactivation.