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J. Cell Biol., Volume 146, Number 3, August 9, 1999 585-596
Copyright © 1999 by The Rockefeller University Press.

The Sudden Recruitment of {gamma}-Tubulin to the Centrosome at the Onset of Mitosis and its Dynamic Exchange Throughout the Cell Cycle, Do Not Require Microtubules

Alexey Khodjakova,b and Conly L. Riedera,b
a Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, Albany, New York 12201-0509
b Department of Biomedical Sciences, State University of New York, Albany, New York 12222

Correspondence to: Alexey Khodjakov, Division of Molecular Medicine, Wadsworth Center, P.O. Box 509, Albany, New York 12201-0509. Tel:(518) 486-5339 Fax:(518) 486-4901 E-mail:khodj{at}wadsworth.org.

{gamma}-Tubulin is a centrosomal component involved in microtubule nucleation. To determine how this molecule behaves during the cell cycle, we have established several vertebrate somatic cell lines that constitutively express a {gamma}-tubulin/green fluorescent protein fusion protein. Near simultaneous fluorescence and DIC light microscopy reveals that the amount of {gamma}-tubulin associated with the centrosome remains relatively constant throughout interphase, suddenly increases during prophase, and then decreases to interphase levels as the cell exits mitosis. This mitosis-specific recruitment of {gamma}-tubulin does not require microtubules. Fluorescence recovery after photobleaching (FRAP) studies reveal that the centrosome possesses two populations of {gamma}-tubulin: one that turns over rapidly and another that is more tightly bound. The dynamic exchange of centrosome-associated {gamma}-tubulin occurs throughout the cell cycle, including mitosis, and it does not require microtubules. These data are the first to characterize the dynamics of centrosome-associated {gamma}-tubulin in vertebrate cells in vivo and to demonstrate the microtubule-independent nature of these dynamics. They reveal that the additional {gamma}-tubulin required for spindle formation does not accumulate progressively at the centrosome during interphase. Rather, at the onset of mitosis, the centrosome suddenly gains the ability to bind greater than three times the amount of {gamma}-tubulin than during interphase.

Key Words: centrosome, mitosis, {gamma}-tubulin, green fluorescent protein, microtubules


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