Association of anti-dynein-1 cross-reactive antigen with the mitotic spindle of mammalian cells.

Two anti-dynein-1 antibodies were affinity-purified from rabbit antiserum produced by immunization with dynein-1 from sea urchin sperms. One was prepared with dynein-1 as the ligand and the other with the A heavy chains of dynein-1 as the ligand. In Western blots of axonemal proteins, the former antibody reacted with the A heavy chains of dynein-1 as well as with several other smaller polypeptides, whereas the latter bound almost exclusively to the A heavy chains. PtK2 cells stained by indirect immunofluorescence with either of these antidyneins had identical fluorescence patterns. The interphase cell showed rather diffuse and weak fluorescence in its nucleus and perinuclear cytoplasm. Its primary cilium and its centrioles also fluoresced. During prophase and prometaphase, a more intense fluorescence was present in the asters and developing spindle. During metaphase and anaphase the half-spindles fluoresced intensely in a fibrous pattern that corresponded to that of the spindle fibers, showing less intense fluorescence in the anaphase interzone. In telophase and early interphase, the intercellular bridge on each side of the midbody also was stained. These results are evidence that dynein-1, specifically the A heavy chains and/or a related antigen, is densely packed in the mitotic spindle of PtK2 cells.