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Originally published as Biophys J. BioFAST on June 20, 2008.
doi:10.1529/biophysj.108.134593
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Biophysical Journal 95:3048-3058 (2008)
© 2008 The Biophysical Society

Fluorescence Recovery Kinetic Analysis of {gamma}-Tubulin Binding to the Mitotic Spindle

Mark A. Hallen, Jianghai Ho, Christine D. Yankel and Sharyn A. Endow

Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710

Correspondence: Address reprint requests to Sharyn A. Endow, Tel.: 919-684-4311; Fax: 919-681-9929; E-mail: endow001{at}mc.duke.edu.

Fluorescence recovery after photobleaching has been widely used to study dynamic processes in the cell, but less frequently to analyze binding interactions and extract binding constants. Here we use it to analyze {gamma}-tubulin binding to the mitotic spindle and centrosomes to determine the role of {gamma}-tubulin in microtubule nucleation in the spindle. We find rapid {gamma}-tubulin turnover in mitotic spindles of Drosophila early embryos, characterized by diffusional interactions and weak binding, differing from centrosomes with tight binding interactions. The diffusion coefficient of {gamma}-tubulin is consistent with a major species existing in the cytoplasm as the less efficiently nucleating {gamma}-tubulin small complex ({gamma}TuSC) or {gamma}-tubulin, rather than {gamma}-tubulin ring complex ({gamma}TuRC). The fluorescence recovery kinetics we observe implies that {gamma}-tubulin functions by binding weakly to spindle microtubules. {gamma}-Tubulin may interact transiently with the spindle, nucleating microtubules very rapidly, differing from centrosomes, where {gamma}-tubulin binds tightly to nucleate microtubules.




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