RECRUITMENT OF ANTIGENIC GAMMA-TUBULIN DURING MITOSIS IN ANIMAL-CELLS- PRESENCE OF GAMMA-TUBULIN IN THE MITOTIC SPINDLE

It has been claimed repeatedly that gamma-tubulin is exclusively local ized at the spindle poles in mitotic animal cells, where it plays a ro le in microtubule nucleation. In addition to this localization, we hav e observed a gamma-tubulin-specific staining of the mitotic spindle in several animal cells (human, kangaroo rat, mouse, Chinese hamster, Xe nopus and Drosophila) using five polyclonal antibodies raised against unique gamma-tubulin sequences and four different fixation protocols. In HeLa and PtK2 cells, gamma-tubulin was detected in the mitotic spin dle from late prometaphase to telophase. In contrast, in other cell ty pes, it was detected in metaphase only. In all cases we failed to dete ct gamma-tubulin in the short aster microtubules at the spindle poles. Electron microscopic observation revealed that at least part of the g amma-tubulin localized on the surface of spindle microtubules with a p referential distribution along kinetochore microtubules. In HeLa cells , the amount of antigenic gamma-tubulin was fairly constant in the spi ndle poles during mitosis from prometaphase to telophase. In contrast, gamma-tubulin appeared in the mitotic spindles in prometaphase. The a mount of gamma-tubulin decreased in telophase, where it relocalized in the interzone. In metaphase cells about 15-25% of the total fluoresce nce was localized at the spindle poles, while 75-85% of the fluorescen ce was distributed over the rest of the spindle. These results suggest that the localization and timing of gamma-tubulin during the cell cyc le is highly regulated and that its physiological role could be more c omplex and diverse than initially assumed.