Mitosis-specific anchoring of gamma tubulin complexes by pericentrin controls spindle organization and mitotic entry.
Microtubule nucleation is the best known function of centrosomes. Centrosomal microtubule nucleation is mediated primarily by gamma tubulin ring complexes (gamma TuRCs). However, little is known about the molecules that anchor these complexes to centrosomes. In this study, we show that the centrosomal coiled-coil protein pericentrin anchors gamma TuRCs at spindle poles through an interaction with gamma tubulin complex proteins 2 and 3 (GCP2/3). Pericentrin silencing by small interfering RNAs in somatic cells disrupted gamma tubulin localization and spindle organization in mitosis but had no effect on gamma tubulin localization or microtubule organization in interphase cells. Similarly, overexpression of the GCP2/3 binding domain of pericentrin disrupted the endogenous pericentrin-gamma TuRC interaction and perturbed astral microtubules and spindle bipolarity. When added to Xenopus mitotic extracts, this domain uncoupled gamma TuRCs from centrosomes, inhibited microtubule aster assembly, and induced rapid disassembly of preassembled asters. All phenotypes were significantly reduced in a pericentrin mutant with diminished GCP2/3 binding and were specific for mitotic centrosomal asters as we observed little effect on interphase asters or on asters assembled by the Ran-mediated centrosome-independent pathway. Additionally, pericentrin silencing or overexpression induced G2/antephase arrest followed by apoptosis in many but not all cell types. We conclude that pericentrin anchoring of gamma tubulin complexes at centrosomes in mitotic cells is required for proper spindle organization and that loss of this anchoring mechanism elicits a checkpoint response that prevents mitotic entry and triggers apoptotic cell death.
Zimmerman
Wendy C
WC
Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Sillibourne
James
J
Rosa
Jack
J
Doxsey
Stephen J
SJ
D000818:Animals; D000941:Antigens / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D017209:Apoptosis / Q000235:genetics; D002460:Cell Line; D018385:Centrosome / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008938:Mitosis / Q000235:genetics / Q000502:physiology*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D008941:Spindle Apparatus / Q000235:genetics / Q000502:physiology / Q000648:ultrastructure*; D014404:Tubulin / Q000378:metabolism*
D000941:Antigens; D008869:Microtubule-Associated Proteins; D034741:RNA, Small Interfering; C112305:TUBGCP2 protein, human; C112306:TUBGCP3 protein, human; D014404:Tubulin; C086178:pericentrin