GSBS Dissertations and Theses

Title

Pericentrin and Gamma Tubulin Form a Novel Lattice and a Protein Complex that is an Essential Unit of Centrosome Assembly: a Dissertation

Approval Date

December 1999

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Molecular and Cellular Biology

Subjects

Microtubule-Associated Proteins; Tubulin; Centrosome; Academic Dissertations

Abstract

Pericentrin and γ-tubulin are two resident centrosome proteins that are involved in microtubule nucleation and organization. When cytosolic extracts of Xenopus eggs were analyzed on sucrose gradients and gel filtration, the two proteins comigrated on gradients and co-eluted from the column. Immunodepletion of γ-tubulin removed all of the soluble pericentrin. The complex of the two proteins was estimated to be ~3-5 megaDaltons (MD), consisting of a pericentrin complex of ~20S and a γ-tubulin complex of ~25S, presumably the γ-TURC (~2 MD). When analyzed at the centrosome by enhanced deconvolution immunofluorescence the two proteins colocalized within a novel ring-like lattice structure, unlike other centrosome proteins analyzed, and were sufficiently close to generate FRET. The levels of the two proteins increased through the cell cycle, peaking at metaphase, and these changes were accompanied by structural changes in the lattice. Nucleated microtubules appeared to contact lattice elements throughout the centrosome. Inhibition of pericentrin function diminished assembly of γ-tubulin onto centrosomes, as did microtubule depolymerization and inhibition of dynein funciton. Separate fractions of the two proteins showed that pericentrin was required in the form a ~20S complex to bind γ-tubulin and for γ-tubulin assembly and microtubule nucleation. Overexpressed and purified pericentrin from cells eluted as a single polypeptide and was not competent to bind γ-tubulin. These results show that pericentrin in the context of a ~20S complex functions to assemble γ-tubulin into the centrosome lattice, and suggests that the pericentrin complex associated with the γ-TURC consists of an essential unit for centrosome formation.

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