The disassembly and reassembly of functional centrosomes in vitro

Academic Article

Abstract

  • Animal cells contain a single centrosome that nucleates and organizes a polarized array of microtubules which functions in many cellular processes. In most cells the centrosome is composed of two centrioles surrounded by an ill-defined 'cloud' of pericentriolar material. Recently, γ-tubulin- containing 25-nm diameter ring structures have been identified as likely microtubule nucleation sites within the pericentriolar material of isolated centrosomes. Here we demonstrate that when Spisula centrosomes are extracted with 1.0 M KI they lose their microtubule nucleation potential and appear by three-dimensional electron microscopy as a complex lattice, built from 12- to 15-nm thick elementary fiber(s), that lack centrioles and 25-nm rings. Importantly, when these remnants are incubated in extracts prepared from Spisula oocytes they recover their 25-nm rings, γ-tubulin, and microtubule nucleation potential. This recovery process occurs in the absence of microtubules, divalent cations, and nucleotides. Thus, in animals the centrosome is structurally organized around a KI-insoluble filament-based 'centromatrix' that serves as a scaffold to which those proteins required for microtubule nucleation bind, either directly or indirectly, in a divalent cation and nucleotide independent manner.
  • Author List

  • Schnackenberg BJ; Khodjakov A; Rieder CL; Palazzo RE
  • Start Page

  • 9295
  • End Page

  • 9300
  • Volume

  • 95