Fluorescence recovery kinetic analysis of gamma-tubulin binding to the mitotic spindle.
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 (gammaTuSC) or gamma-tubulin, rather than gamma-tubulin ring complex (gammaTuRC). 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.
Hallen
Mark A
MA
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Ho
Jianghai
J
Yankel
Christine D
CD
Endow
Sharyn A
SA
D000818:Animals; D018385:Centrosome / Q000378:metabolism; D004058:Diffusion; D004330:Drosophila / Q000166:cytology / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D007700:Kinetics; D008870:Microtubules / Q000378:metabolism; D011485:Protein Binding; D008941:Spindle Apparatus / Q000378:metabolism*; D013997:Time Factors; D014404:Tubulin / Q000378:metabolism*
D014404:Tubulin