Pericentrin and γ-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–γ-tubulin complex was distinct from the previously described γ-tubulin ring complex (γ-TuRC) as purified γ-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 γ-tubulin assemble into a unique centrosome lattice that represents the higher-order organization of microtubule nucleating sites at the centrosome.
Pericentrin and γ-Tubulin Form a Protein Complex and Are Organized into a Novel Lattice at the Centrosome
Dr. F.S. Fay died on March 18, 1997.
We dedicate this manuscript to the memory of Fredric S. Fay who served as an inspiration for this work and a spirited friend.
Special thanks to D. Mazia whose excitement and advice about centrosomes were invaluable during the course of this work. We also thank the following individuals for their assistance: D. Schmidt (UMMC) for FRET analysis; L. Boyer for gel filtration; R. Craig (UMMC) and colleagues for rapid freeze; J. Gosslin (UMMC) for mouse oocytes; J. Burkhardt (UCSF) for monoclonal antibody production; L. Lifschitz, K. Fogarty, and D. Bowman (UMMC) for image analysis; T. Stearns (Stanford University, Palo Alto, CA), P. Draber (Academy of Sciences of the Czech Republic), R. Vallee (Worcester Foundation, Shrewsbury, MA), J. Salisbury (Mayo Clinic, Rochester, MN) for antibodies, B. Oakley for γ-tubulin clones; and R. Tsein (University of California, San Diego, CA) for GFP constructs. For discussions and comments on the manuscript we thank M. Kirschner, R. King, R. Vallee, R. Davis, and A. Pereira.
S.J. Doxsey is a recipient of an Established Investigator Award from the American Heart Association (96-276). This work was supported by grants from the American Cancer Society (IRG-203) to S.J. Doxsey, from the National Institutes of Health (RO1GM51994 and RO1 RR09799-01A1) to S.J. Doxsey and W. Carrington, respectively, and from the National Science Foundation (BIR-9200027 and DBI-9724611) to F.S. Fay and W. Carrington, respectively.
Address all correspondence to Stephen J. Doxsey, Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester, MA 01655. Tel.: (508) 856-1613. Fax: (508) 856-4289. E-mail: stephen.doxsey @ummed.edu
Jason B. Dictenberg, Wendy Zimmerman, Cynthia A. Sparks, Aaron Young, Charles Vidair, Yixian Zheng, Walter Carrington, Fredric S. Fay, Stephen J. Doxsey; Pericentrin and γ-Tubulin Form a Protein Complex and Are Organized into a Novel Lattice at the Centrosome . J Cell Biol 6 April 1998; 141 (1): 163–174. doi: https://doi.org/10.1083/jcb.141.1.163
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