In vitro microtubule-nucleating activity of spindle pole bodies in fission yeast Schizosaccharomyces pombe: cell cycle-dependent activation in xenopus cell-free extracts.
The spindle pole body (SPB) is the equivalent of the centrosome in fission yeast. In vivo it nucleates microtubules (MTs) during mitosis, but, unlike animal centrosomes, does not act as a microtubule organizing center (MTOC) during interphase. We have studied the MT-nucleating activity of SPBs in vitro and have found that SPBs in permeabilized cells retain in vivo characteristics. SPBs in cells permeabilized during mitosis can nucleate MTs, and are recognized by two antibodies: anti-gamma-tubulin and MPM-2 which recognizes phosphoepitopes. SPBs in cells permeabilized during interphase cannot nucleate MTs and are only recognized by anti-gamma-tubulin. Interphase SPBs which cannot nucleate can be converted to a nucleation competent state by incubation in cytostatic factor (CSF)-arrested Xenopus egg extracts. After incubation, they are recognized by MPM-2, and can nucleate MTs. The conversion does not occur in Xenopus interphase extract, but occurs in Xenopus interphase extract driven into mitosis by preincubation with exogenous cyclin B. The conversion is ATP dependent and inhibited by protein kinase inhibitors and alkaline phosphatase. Purified, active, cdc2 kinase/cyclin B complex in itself is not effective for activation of MT nucleation, although some interphase SPBs are now stained with MPM-2. These results suggest that the ability of SPBs in vitro to nucleate MTs after exposure to CSF-arrested extracts is activated through a downstream pathway which is regulated by cdc2 kinase.
Masuda
H
H
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Sevik
M
M
Cande
W Z
WZ
D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002474:Cell-Free System; D016213:Cyclins / Q000378:metabolism; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism*; D008938:Mitosis / Q000502:physiology*; D012568:Schizosaccharomyces / Q000502:physiology*; D008941:Spindle Apparatus / Q000378:metabolism*; D014981:Xenopus / Q000378:metabolism
D016213:Cyclins; D016203:CDC2 Protein Kinase