JCB logo
Accuri Cytometers
  Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents
This Article
Right arrow Full Text
Right arrow Full Text (PDF, 593K)
Right arrow PPT slides of all figures
Right arrow Alert me when this article is cited
Right arrow Citation Map
Services
Right arrow Email this article
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new content in the JCB
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dictenberg, J. B.
Right arrow Articles by Doxsey, S. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dictenberg, J. B.
Right arrow Articles by Doxsey, S. J.
Right arrowPubmed/NCBI databases
*Substance via MeSH
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Facebook   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

© The Rockefeller University Press, 0021-9525/1998//163 $5.00
The Journal of Cell Biology, Volume 141, Number 1, , 1998 163-174

Regular Articles

Pericentrin and {gamma}-Tubulin Form a Protein Complex and Are Organized into a Novel Lattice at the Centrosome



Jason B. Dictenberg*, Wendy Zimmerman*, Cynthia A. Sparks{ddagger}, Aaron Young*, Charles Vidair§, Yixian Zheng||, Walter Carrington, Fredric S. Fay{dagger},¶, and Stephen J. Doxsey*

* Program in Molecular Medicine and Department of Cell Biology, University of Massachusetts Medical Center, Worcester, Massachusetts 01655; {ddagger} 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

Pericentrin and {gamma}-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–{gamma}-tubulin complex was distinct from the previously described {gamma}-tubulin ring complex ({gamma}-TuRC) as purified {gamma}-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 {gamma}-tubulin assemble into a unique centrosome lattice that represents the higher-order organization of microtubule nucleating sites at the centrosome.

Abbreviations used in this paper: CCD, charge-coupled device; GFP, green fluorescent protein; FRET, fluorescence resonance energy transfer; {gamma}-TuRC, {gamma}-tubulin ring complex; MTOC, microtubule organizing center.

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 {gamma}-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


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Facebook Facebook   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?


This article has been cited by other articles:



  Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents