Immunocytochemical evidence for centrosomal phosphoproteins in mitotic sea urchin eggs

Abstract

The change in distribution of centrosomal phosphoproteins was examined in sea urchin eggs from fertilization to the first cleavage by immunofluorescence staining with the anti-phosphoprotein antibodies, MPM-1 and MPM-2. The antibodies reacted with female pronuclei in unfertilized eggs as well as centriolar complexes located at the base of sperm flagella. After insemination, male and female pronuclei fused together to form a zygotic nucleus which was visualized by staining of fertilized eggs with the antiphosphoprotein antibodies. No major change in staining pattern was detected in extracted whole eggs until mitosis. As the fertilized eggs approached mitosis, however, the antigens started to redistribute from nuclei to the perinuclear position where the mitotic centrosomes were located. Detailed immunofluorescence observation of isolated spindles revealed that the phosphoantigens were retained in isolated structures. A major 225 kd polypeptide was recognized by the antibodies, suggesting that the 225 kd protein is a phosphocomponent of centrosomes. The area recognized by the antibody in mitotic poles enlarged with the progress of mitosis, suggesting that the antigens were apparently localized in the centrosphere. Centrospheres prepared from isolated spindles by salt extraction strongly reacted with the antibodies. One or two bright dots, which may represent centrioles, were visible in the isolated centrosphere. At the end of mitosis, the antigens again appeared in the newly formed daughter nuclei. Centriole-containing cytasters and centriole-free monasters were parthenogenetically induced in unfertilized eggs (Kuriyama and Borisy, (1983) J. Cell Sci. 61: 175-189). The antibodies stained centers of both the asters whether they contained centrioles or not, indicating that the antibodies recognizes the components of the pericentriolar material.