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

¹ Duke University Medical Center

^* To whom correspondence should be addressed. E-mail: endow001{at}mc.duke.edu.

Submitted on April 3, 2008
Revised on April 21, 2008
Accepted on 2 June 2008

	Abstract

Fluorescence recovery after photobleaching (FRAP) 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 FRAP to analyze -tubulin binding to the mitotic spindle and centrosomes to determine the role of -tubulin in microtubule nucleation in the spindle. We find rapid -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 -tubulin is consistent with a major species existing in the cytoplasm as the less efficiently nucleating -tubulin small complex (TuSC) or -tubulin, rather than -tubulin ring complex (TuRC). The fluorescence recovery kinetics we observe implies that -tubulin functions by binding weakly to spindle microtubules. -tubulin may interact transiently with the spindle, nucleating microtubules very rapidly, differing from centrosomes, where -tubulin binds tightly to nucleate microtubules.

Key Words: Binding interactions, Centrosomes, Diffusion, FRAP, GFP, Microtubule nucleation