CENTROSOMAL COMPONENTS IMMUNOLOGICALLY RELATED TO TEKTINS FROM CILIARY AND FLAGELLAR MICROTUBULES

Centrosomes are critical for the nucleation and organization of the mi crotubule cytoskeleton during both interphase and cell division. Using antibodies raised against sea urchin sperm flagellar microtubule prot eins, we characterize here the presence and behavior of certain compon ents associated with centrosomes of the surf clam Spisula solidissima and cultured mammalian cells. A Sarkosyl detergent-resistant fraction of axonemal microtubules was isolated from sea urchin sperm flagella a nd used to produce monoclonal antibodies, 16 of which were specific- o r cross-specific for the major polypeptides associated with this micro tubule fraction: tektins A, B and C, acetylated alpha-tubulin, and 77 and 83 kDa polypeptides. By 2-D isoelectric focussing/SDS polyacrylami de gel electrophoresis the tektins separate into several polypeptide s pots. Identical spots were recognized by monoclonal and polyclonal ant ibodies against a given tektin, indicating that the different polypept ide spots are isoforms or modified versions of the same protein. Four independently derived monoclonal anti-tektins were found to stain cent rosomes of S. solidissima oocytes and CHO and HeLa cells, by immunoflu orescence microscopy. In particular, the centrosome staining of one mo noclonal antibody specific for tektin B (tekB3) was cell-cycle-depende nt for CHO cells, i.e. staining was observed only from early prometaph ase until late anaphase, By immune-electron microscopy tekB3 specifica lly labeled material surrounding the centrosome, whereas a polyclonal anti-tektin B recognized centrioles as well as the centrosomal materia l throughout the cell cycle. Finally, by immunoblot analysis tekB3 sta ined polypeptides of 48-50 kDa in isolated spindles and centrosomes fr om CHO cells.