The disassembly and reassembly of functional centrosomes in vitro.
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, gamma-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, gamma-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.
Department of Biochemistry, Cell, and Molecular Biology, University of Kansas, Lawrence, KS 66045, USA.
B J
BJ
Schnackenberg
A
A
Khodjakov
C L
CL
Rieder
R E
RE
Palazzo
D000595:Amino Acid Sequence; D000818:Animals; D049872:Bivalvia / Q000648:ultrastructure*; D018385:Centrosome; D008868:Microtubule Proteins; D008969:Molecular Sequence Data; D009865:Oocytes / Q000648:ultrastructure
D008868:Microtubule Proteins