Edinburgh Napier University

  To investigate changes at the centrosome during the cell cycle,
we analyzed the composition of the pericentriolar material
from unsynchronized and S-phase-arrested cells by gel
electrophoresis and mass spectrometry. We identified HCA66,
a protein that localizes to the centrosome from S-phase to mitosis
and to the nucleolus throughout interphase. Silencing of HCA66
expression resulted in failure of centrosome duplication
and in the formation of monopolar spindles, reminiscent
of the phenotype observed after γ-tubulin silencing.
Immunofluorescence microscopy showed that proteins of the
γ-tubulin ring complex were absent from the centrosome in
these monopolar spindles. Immunoblotting revealed reduced
protein levels of all components of the γ-tubulin small complex
(γ-tubulin, GCP2, and GCP3) in HCA66-depleted cells. By
contrast, the levels of γ-tubulin ring complex proteins such as
GCP4 and GCP-WD/NEDD1 were unaffected. We propose that
HCA66 is a novel regulator of γ-tubulin function that plays a
role in stabilizing components of the γ-tubulin small complex,
which is in turn essential for assembling the larger γ-tubulin
ring complex.