XENOPUS-LAEVIS ACTIN-DEPOLYMERIZING FACTOR COFILIN - A PHOSPHORYLATION-REGULATED PROTEIN ESSENTIAL FOR DEVELOPMENT

Two cDNAs, isolated from a Xenopus laevis embryonic library, encode pr oteins of 168 amino acids, both of which are 77% identical to chick co filin and 66% identical to chick actin-depolymerizing factor (ADF), tw o structurally and functionally related proteins. These Xenopus ADF/co filins (XACs) differ from each other in 12 residues spread throughout the sequence but do not differ in charge. Purified GST-fusion proteins have pH-dependent actin-depolymerizing and F-actin-binding activities similar to chick ADF and cofilin. Similarities in the developmental a nd tissue specific expression, embryonic localization, and in the cDNA sequences of the noncoding regions, suggest that the two XACs arise f rom allelic variants of the pseudotetraploid X. laevis. Immunofluoresc ence localization of XAC in oocyte sections with an XAC-specific monoc lonal antibody shows it to be diffuse in the cortical cytoplasm. After fertilization, increased immunostaining is observed in two regions: a long the membrane, particularly that of the vegetal hemisphere, and at the interface between the cortical and animal hemisphere cytoplasm. T he cleavage furrow and the mid-body structure are stained at the end o f first cleavage. Neuroectoderm derived tissues, notochord, somites, a nd epidermis stain heavily either continuously or transiently from sta ges 18-34. A phosphorylated form of XAC (pXAC) was identified by 2D We stern blotting, and it is the only species found in oocytes. Dephospho rylation of >60% of the pXAC occurs within 30 min after fertilization. Injection of one blastomere at the 2 cell stage, either with constitu tively active XAC or with an XAC inhibitory antibody, blocked cleavage of only the injected blastomere in a concentration-dependent manner w ithout inhibiting nuclear division. The cleavage furrow of eggs inject ed with constitutively active XAC completely regressed. Blastomeres in jected with neutralized antibody developed normally. These results sug gest that XAC is necessary for cytokinesis and that its activity must be properly regulated for cleavage to occur.