Assembly properties of fluorescein-labeled tubulin in vitro before and after fluorescence bleaching.

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Abstract
Brain [bovine] tubulin was conjugated with dichlorotriazinyl-aminoflurorescein (DTAF) to form a visualizable complex for the study of tubulin dynamics in living cells. Several assays confirm the finding of Keith et al. that DTAF-tubulin polymerizes like control tubulin in vitro. The fluorescein moiety of the complex is readily bleached by the 488-nm line from an argon ion laser. When irradiations are performed over short times (< 1 s) and in the presence of 2 mM glutathione, a mixture of DTAF-tubulin and control protein (as occurs after microinjection of the fluorescent conjugate into cells) will retain full polymerization activity. Slow bleaching (.apprx. 5 min) or bleaching without glutathione promotes formation of covalent cross-links between neighboring polypeptides and kills the polymerization activity of DTAF-tubulin, including some molecules that are neither cross-linked nor bleached. Even under conditions that damage DTAF-tubulin, DTAF-microtubules are not destroyed by bleaching. They will continue to elongate by addition of DTAF-tubulin subunits to their free ends, and they neither bind nor exchange subunits along their lateral surfaces. Apparently DTAF-tubulin is a suitable analog for tubulin, both in studies of protein incorporation and for investigations of fluorescence redistribution after photobleaching.