The use of the fluorescence photobleaching recovery technique to study the self-assembly of tubulin

✍ Scribed by Tsutomu Arakawa; Carl Frieden

• Publisher: Elsevier Science
• Year: 1985
• Tongue: English
• Weight: 836 KB
• Volume: 146
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(85)90407-5

✦ Synopsis

Fhrorescently labeled microtubule-associated proteins or poly-r;lysine (13,000 MW) were prepared by reaction with fluorescein isothiocyanate. The labeled compounds were used as probes of the assembly of calf brain tubulin using fluorescence photobleaching recovery techniques which allow measurement of the diffusion coefficient and percentage mobility of the fluorescent probe. When unfractionated tub&in (defined as material containing tubulin and microtubule-associated proteins) was polymerized at room temperature or 37°C either probe could be incorporated into microtubules, since the observed diffusion coefficient (-1.7 X lo-* cm'/s) was much slower than that for either probe free in solution. The microtubules formed in the presence of labeled microtubule-aviated proteins were free to diffuse while those formed jn the presence of labeled polylysine were partially immobilized. Thus the fluorescence photobleaching recovery technique can be used to measure crosslinking of microtubules as well as assembly or interactions with other structures. When unfractionated tubulin was incubated with labeled polylysine in the presence of Ca" at room temperature, the observed diffusion coefficient (-5. I X lo-* cm'/s) probably represents the formation of rings of tubulin. The effect of mild and vigorous shearing, of cholchicine, and of different MgZ+ concentrations on the properties of the system were examined. 0 1985 Academic PW. IIIC.