Intracellular localization of the high molecular weight microtubule accessory protein by indirect immunofluorescence.

Microtubules purified by successive cycles of po- lymerization and depolymerization contain, in ad- dition to tubulin, a series of accessory proteins. When tubulin is separated from these accessory proteins by ion exchange chromatography, it will no longer assemble into microtubules under stan- dard in vitro polymerizing conditions (buffered aqueous solutions, neutral pH, 1 mM Mg). Fur- thermore, it has been shown that two of these nontubulin proteins, the high molecular weight protein (HMW protein) (11, 14, 18) and tau protein (19), are necessary and sufficient for both the initiation and the elongation process of micro- tubule assembly in vitro (18, 22). They also co- purify with tubulin through successive cycles of polymerization in a constant stoichiometric ratio (2, 15). We have studied the relationship between these microtubule accessory proteins and microtubules in vivo by examining their distribution by the indirect immunofluorescence technique. In a pre- vious report (4), we showed that tau protein is associated with microtubules in intact cells. In the present study, we examine the relationship be- tween HMW protein and microtubules and dem- onstrate that precipitating antisera prepared in rabbits to electrophoretically purified HMW pro- tein give specific immunofluorescent staining of microtubules in rat glial ceils. As a result of these studies, we conclude that HMW protein is associ- ated with microtubules in glial ceils. THE JOUItNALOF CELLBIOLO6Y9VOLUME76, 1978 9pages 781-786 781 Preparation of Antisera Porcine brain microtubules were purifies by two cycles of polymerization and depolymerization using a modifi- cation of the method of Shelanski et al. (17) and Weingarten et ai. (20), and tubulin was separated from accessory proteins by phosphocellulose chromatography (19). Tubulin and HMW protein were farther purified by electrophoretic elution from polyacrylamide gels con- taining 0.1% SDS as previously described (4). Tau protein was purified by the method of Cleveland et al. (2) and further purified by elution from SDS-containing polyacrylamide gels. Antisera to tubulin and HMW protein were prepared as follows: Female New Zealand White rabbits were injected subcutaneously with 250/xg of protein in com- plete Freund's Adjuvant and a second time 20 days later. Another injection of 500/~g of protein in incom- plete Freund's Adjuvant was given 15 days after the second injection, and the rabbits were bled 2 wk after this last injection. Preimmune sera were obtained from the same animals before immunization. lmmunodiffusion Tests Antisera were analyzed by Ouchterlony's double dif- fusion test, with 1% agarose in phosphate-buffered saline (PBS) at room temperature. For absorption exper- iments, electrophoretically purified antigen at a concen- tration of 1 mg/ml was added to the antiserum well, and allowed to diffuse for 10 min before application of the antiserum. Ce//s For immunofluorescence, rat glial cells strain C6 (American Type Culture Collection, Rockville, Md.) were grown on 22-mm glass cover slips in alpha mini- mum essential medium supplemented with 10% fetal calf serum. In some experiments, culture medium was removed and replaced with the same medium containing Colcemid at 0.5/~g/ml for 60 min before fixation of the cells. Cells grown on cover slips were rinsed twice in PBS, pH 7.0, and then fixed for 4 min in methanol, and 2 min in acetone, both at -20"C (16). Cells were then treated with one of the antisera at a dilution of 1:30 (in PBS), followed by fluorescein conjugated goat IgG to rabbit IgG (Hyland Diagnostics Div., Travenol Laboratories, Inc., Costa Mesa, Calif.) diluted 1:5 (4). Cells were examined with a Zeiss Photomicroscope II equipped with epifluoreseence optics and photographed on Ilford FP-4 film developed in Diafine. The antisera were absorbed with antigens as previously described (4).