Spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching.

Abstract

The rate of exchange of tubulin that is incorporated into spindle microtubules with dimeric tubulin in the cytoplasm was measured in sea urchin eggs by studying fluorescence redistribution after photobleaching (FRAP). Dichlorotriazinyl amino fluorescein (DTAF) was used to label bovine brain tubulin. DTAF-tubulin was injected into fertilized eggs of L. variegatus and allowed to equilibrate with the endogenous tubulin pool. Fluorescent spindles formed at the same time that spindles were seen in control eggs, and the injected embryos preceeded through many cycles of division on schedule, suggesting that DTAF-tubulin is a good analog of tubulin in vivo. A microbeam of argon laser light was used to bleach parts of the fluorescent spindles, and FRAP was recorded with a sensitive video camera. Laser bleaching did not affect spindle structure, as seen with polarization optics, nor spindle function, as seen by rate of progress through mitosis, even when one spindle was bleached several times in a single cell cycle. Video image analysis was used to measure the rate of FRAP and to obtain a low resolution view of the flurorescence redistribution process. The half-time for spindle FRAP is .apprx. 19 s, even when an entire half-spindle is bleached. Complete exchange of tubulin in nonkinetochore spindle and astral microtubules appeared to occur within 60-80 s at steady state. This rate is too fast to be explained by a simple microtubule end-dependent exchanges of tubulin. Efficient microtubule treadmilling would be fast enough, but with current techniques there was no evidence for movement of the bleached spot during recovery, which would be expected on the basis of Margolis and Wilson''s model-fluorescence recovers uniformly. Microtubules may be depolymerizing and repolymerizing rapidly and asynchronously throughout the spindle and aster, but the FRAP data are most compatible with a rapid exchange of tubulin subunits all along the entire lengths of nonkinetochore spindle and astral microtubules.