Assembly properties of fluorescein-labeled tubulin in vitro before and after fluorescence bleaching.
Brain tubulin has been conjugated with dichlorotriazinyl-aminofluorescein (DTAF) to form a visualizable complex for the study of tubulin dynamics in living cells. By using several assays we confirm the finding of Keith et al. (Keith, C. H., J. R. Feramisco, and M. Shelanski, 1981, J. Cell Biol., 88:234-240) 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 (less than 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 living cells) will retain full polymerization activity. Slow bleaching (approximately 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, however, 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. These results suggest that DTAF-tubulin is a suitable analog for tubulin, both in studies of protein incorporation and for investigations of fluorescence redistribution after photobleaching.
D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D005407:Flagella / Q000648:ultrastructure; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000494:pharmacology*; D007700:Kinetics; D007834:Lasers; D046911:Macromolecular Substances; D008870:Microtubules / Q000648:ultrastructure; D012617:Sea Urchins; D013861:Thiocyanates; D014404:Tubulin / Q000378:metabolism* / Q000528:radiation effects
D005452:Fluoresceins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D013861:Thiocyanates; D014404:Tubulin; C039083:5-((4,6-dichloro-1,3,5-triazin-2-yl)amino)fluorescein; D016650:Fluorescein-5-isothiocyanate