We are upgrading the repository! A content freeze is in effect until December 6, 2024. New submissions or changes to existing items will not be allowed during this period. All content already published will remain publicly available for searching and downloading. Updates will be posted in the Website Upgrade 2024 FAQ in the sidebar Help menu. Reach out to escholarship@umassmed.edu with any questions.
Association of Pericentrin with the γ Tubulin Ring Complex: a Dissertation
Authors
Zimmerman, Wendy CherieFaculty Advisor
Stephen Doxsey, Ph.D.Academic Program
Biochemistry and Molecular PharmacologyUMass Chan Affiliations
Molecular MedicineDocument Type
Doctoral DissertationPublication Date
2004-06-03Keywords
AntigensCentrosome
Microtubule-Associated Proteins
Microtubules
Tubulin
Xenopus Proteins
Amino Acids, Peptides, and Proteins
Biological Factors
Cells
Macromolecular Substances
Metadata
Show full item recordAbstract
Pericentrin is a molecular scaffold protein. It anchors protein kinases, (PKB, (Purohit, personal communication), PKC, (Chen et al., 2004), PKA Diviani et al., 2000), the γ tubulin ring complex, (γ TuRC) (Zimmerman et al., 2004), and possibly dynein (Purohit et al., 1999) to the spindle pole. The γ TuRC is a ~ 2 MDa complex which binds the minus ends of microtubules and nucleates microtubules in vitro, (Zheng et al., 1995). Prior to this work, nothing was known about the association of the γTuRC with pericentrin. Herein I report the biochemical identification of a large protein complex in Xenopus extracts containing pericentrin, the γ TuRC, and other as yet unidentified proteins. Immunodepletion of γ tubulin results in co-depletion of pericentrin, indicating that virtually all the pericentrin in a Xenopus extract is associated with γ tubulin. However, pericentrin is not a member of the, γ TuRC, since isolated γ TuRCs do not contain pericentrin. The association of pericentrin with the γ TuRC is readily disrupted, resulting in two separable complexes, a small pericentrin containing complex of approximately 740 KDa and the the γ TuRC, 1.9 MDa in Xenopus. Co overexpression/ coimmunoprecipitation and yeast two hybrid studies demonstrate that pericentrin binds the γTuRC through interactions with both GCP2 and GCP3. When added to Xenopus mitotic extracts, the GCP2/3 binding domain uncoupled γ TuRCs from centrosomes, inhibited microtubule aster assembly and induced rapid disassembly of pre-assembled asters. All phenotypes were significantly reduced in a pericentrin mutant with diminished GCP2/3 binding, and were specific for mitotic centro somal asters as I observed little effect on interphase asters or on asters assembled by the Ran-mediated centrosome-independent pathway. Overexpression of the GCP2/3 binding domain of pericentrin in somatic cells perturbed mitotic astral microtubules and spindle bipolarity. Likewise pericentrin silencing by small interfering RNAs in somatic cells disrupted γ tubulin localization and spindle organization in mitosis but had no effect on γ tubulin localization or microtubule organization in interphase cells. Pericentrin silencing or overexpression induced G2/antephase arrest followed by apoptosis in many but not all cell types. I conclude that pericentrin anchoring of γ 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. Additionally, I provide functional and in vitro evidence to suggest that the larger pericentrin isoform (pericentrin B/ Kendrin) is not functionally homologous to pericentrin/pericentrin A in regard to it's interaction with the γ TuRC.DOI
10.13028/7rkg-kp35Permanent Link to this Item
http://hdl.handle.net/20.500.14038/31417Notes
Some images did not scan well. Please consult original document. Pages iii-x are duplicated in original publication.
Rights
Copyright is held by the author, with all rights reserved.ae974a485f413a2113503eed53cd6c53
10.13028/7rkg-kp35