Isolation of cleavage furrows from eggs of regular sea urchins and identification of furrow-specific proteins

We have developed a method for the isolation of cleavage furrows from dividing sea urchin eggs, which is applicable to various sea urchin species, The new method differs from that used for isolating cleavage furrows from sand dollar Clypeaster japonicus eggs [Yonemura, S., Mabuchi, I., and Tsukita, S. (1991) J. Cell Sci. 100, 73-84] in the type and concentration of detergent included in the isolation medium, the temperature during the treatment of dividing eggs with the isolation medium, and the centrifugation conditions. The contractile ring was included in the isolated cleavage furrows, as seen on rhodamine-phalloidin staining of actin filaments. When the furrows were isolated with the isolation medium containing both NaF and beta-glycerophosphate, which are potent protein phosphatase inhibitors, the isolated furrows were found to be accompanied by the mitotic apparatus, When the isolation was carried out in the absence of both NaF and beta-glycerophosphate, cleavage furrows without the mitotic apparatus were obtained. The development of a method of isolation of cleavage furrows from regular sea urchin eggs enabled us to compare protein constituents among furrows from different sea urchin and sand dollar species. We found that 32, 36, and 51 kDa proteins were concentrated in common in the cleavage furrows isolated from eggs of the sand dollars, C. japonicus and Scaphechinus mirabilis, and the sea urchins, Hemicentrotus pulcherrimus and Strongylocentrotus nudus, on two-dimensional gel electrophoreses.