Tubulin in sea urchin embryonic cilia: post-translational modifications during regeneration

Tubulin is the major protein found in the membrane/periaxonemal matrix fraction of mature sea urchin embryonic cilia but its distribution and possible function during ciliary assembly are unknown. Hypertonic salt may be used to deciliate the embryos, allowing synchronous regrowth of cilia and subsequent deciliation of the regenerating embryos at various times. During the earliest stages of regeneration, the amounts of tubulin in the axoneme and membrane/matrix fractions are nearly equal, but the proportion of tubulin in the axoneme fraction increases coincident with the quasi-linear growth phase while the membrane/matrix tubulin remains constant. Antibodies to tyrosinated and detyrosinated alpha-tubulin show that both the membrane/matrix and axonemal tubulin fractions are primarily unmodified (i.e. tyrosinated) at the earliest stages of regeneration but are progressively and equally detyrosinated coincident with regeneration, approaching a final level of 50% C-terminal Glu. A monoclonal antibody to acetylated alpha-tubulin reveals that both tubulin fractions are equally and maximally acetylated at relatively early stages of regeneration. In contrast, three-times-repolymerized tubulin from either unfertilized eggs or midgastrula embryos is primarily tyrosinated (greater than 97%) and not detectably acetylated. These data suggest that membrane/matrix tubulin is a precursor to axonemal tubulin and that acetylation and detyrosination may be involved in partitioning tubulin among cytoplasmic, ciliary membrane/matrix, and 9 + 2 compartments.