A Study of the Assembly Mechanism of Pericentrin and γ Tubulin onto the Centrosome in Mammalian Cells: A Dissertation

Abstract

The mechanism for centrosome assembly in somatic cells has previously been proposed to be microtubule independent. Studies presented in this dissertation demonstrate that in somatic cells pericentrin and γ tubulin, two paradigm centrosome proteins, assemble onto the centrosome in a microtubule, and dynein/dynactin dependent manner. High resolution, three-dimensional, time-lapse digital imaging of pericentrin-GFP labeled centrosomes has revealed tiny particles that move vectorally towards the centrosome at rates exceeding 1μm/second. These pericentrin-GFP particles contain γ tubulin and are not readily visible by standard two-dimensional digital imaging microscopy. Further studies have shown that dynein colocalizes with tiny particles of endogenous pericentrin outside of the centrosome which may reflect assembly intermediates in transit towards the centrosome. Furthermore, when dynein function is disrupted in G1 cells by nocodazole treatment, dynamitin overexpression, or dynein IC antibody (70.1) injection, assembly of pericentrin and γ tubulin onto the centrosome throughout the cell cycle is greatly reduced. Moreover, microtubule co-sedimentation studies have demonstrated that pericentrin associates with microtubules in vitro and is dependent on functional dynein/dynactin. Together these data strongly suggest that pericentrin and γ tubulin are novel cargoes of the dynein/dynactin motor complex which transports these proteins -and likely other components of the 3MDa nucleating complex (Dictenberg et al., 1998)- to the centrosome via rnicrotubules.