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J. Cell Biol.,
Volume 141, Number 1, April 6, 1998 163-174
-Tubulin Form a Protein Complex and
Are Organized into a Novel Lattice at the Centrosome
¶
* Program in Molecular Medicine and Department of Cell Biology, University of Massachusetts Medical Center, Worcester,
Massachusetts 01655; Pericentrin and Worcester Foundation for Biomedical Research, Shrewsbury, Massachusetts 01545; § Department of
Radiation Oncology, University of California, San Francisco, California 94143-0806; Department of Biology, Carnegie Institute
of Washington, Baltimore, Maryland 21210; and ¶ Biomedical Imaging Group, University of Massachusetts Medical Center,
Worcester, Massachusetts 01655
-tubulin are integral centrosome proteins that play a role in microtubule nucleation and organization. In this study, we examined the
relationship between these proteins in the cytoplasm
and at the centrosome. In extracts prepared from Xenopus eggs, the proteins were part of a large complex as
demonstrated by sucrose gradient sedimentation, gel
filtration and coimmunoprecipitation analysis. The
pericentrin--tubulin complex was distinct from the
previously described -tubulin ring complex (-TuRC)
as purified -TuRC fractions did not contain detectable
pericentrin. When assembled at the centrosome, the
two proteins remained in close proximity as shown by
fluorescence resonance energy transfer. The three-
dimensional organization of the centrosome-associated fraction of these proteins was determined using an improved immunofluorescence method. This analysis revealed a novel reticular lattice that was conserved from
mammals to amphibians, and was organized independent of centrioles. The lattice changed dramatically
during the cell cycle, enlarging from G1 until mitosis,
then rapidly disassembling as cells exited mitosis. In
cells colabeled to detect centrosomes and nucleated microtubules, lattice elements appeared to contact the minus ends of nucleated microtubules. Our results indicate that pericentrin and -tubulin assemble into a
unique centrosome lattice that represents the higher-order organization of microtubule nucleating sites at
the centrosome.
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