Localization of the sea urchin Spec3 protein to cilia and Golgi complexes of embryonic ectoderm cells.

Abstract

Expression of the Spec3 gene of Strongylocentrotus purpuratus is associated with ectodermal ciliogenesis. An antiserum was raised against the amino terminus of the deduced Spec3 amino acid sequence and used for immunofluorescent staining. Cilia and an apical structure at the base of the stained cilium of each ectodermal cell stained intensely in gastrula and later stage embryos. Microtubule-depolymerizing agents dispersed the concentrated spot of apical staining, suggesting a localization of Spec3 antigen to the Golgi complex. Immunogold electron microscopy confirmed the localization of Spec3 antigen on cilia and in the Golgi complex. Spec3 antigen showed a diffuse punctate staining pattern in the ectodermal cytoplasm of hatching blastula when Spec3 transcripts are most prevalent, suggesting that after synthesis, Spec3 is sequestered in the Golgi complex before appearing on cilia. Whereas the predicted Mr of the Spec3 protein is 21,600, immunoblotting with S. purpuratus proteins indicated that a Spec3 antigen was concentrated in cilia and migrated as an SDS-resistant aggregate of Mr approximately 350,000. Spec3 is also concentrated in cilia of Lytechinus pictus but the protein migrated with an Mr approximately 23,000 in this species. The S. purpuratus Spec3 antigen remains associated with the ciliary axoneme after extraction of membrane proteins.