γ-Tubulin participates in the formation of the midbody during cytokinesis in mammalian cells

Animal cells undergoing cytokinesis form an inter-cellular bridge containing two bundles of microtubules interdigitated at their plus ends, which constitute the midbody. Polyclonal antibodies raised against three specific amino acid sequences of gamma-tubulin (EEFATEGGDRKDV, NIIQGEADPTDVHKSL and EYHAATRPDYISWGTQEQ) specifically stained the centrosome in interphase, the spindle poles in all stages of mitosis, and the extremities of the midbody in mammalian cells (Potorous, human, Chinese hamster, mouse). This staining was prevented by the corresponding peptides, by Xenopus gamma-tubulin, but was not modified by purified alpha beta-tubulin heterodimer. An identical staining was obtained with affinity-purified antibodies against the carboxyl-terminal amino acid sequence of human gamma-tubulin. No gamma-tubulin could be detected in the interzone during anaphase and early telophase. Material containing gamma-tubulin first appeared in the two daughter cells on each side of the division plane in late telophase, and accumulated transiently at the minus ends of the two microtubule bundles constituting the midbody for one hour after metaphase. Micro-injection of gamma-tubulin antibodies into anaphase cells prevented the subsequent formation of the microtubule bundles between the two daughter cells. In contrast with previous views, these observations suggest that the microtubules constituting the midbody may be nucleated on special microtubule organizing centres, active during late telophase only, and assembled on each side of the dividing plane between the daughter cells.