3 MICROTUBULE-ORGANIZING CENTERS ARE REQUIRED FOR ASCUS GROWTH AND SPORULATION IN THE FUNGUS SORDARIA-MACROSPORA

The microtubule system of the Sordaria macrospora ascus was examined by antitubulin immunofluorescence, without the removal of the cell wall. The complex cytoskeleton revealed three possible microtubule-organizing centers (MTOCs): the spindle pole body (SPB), the nuclear envelope, and an apical organizing center. MPM-2, a mitotic phosphoprotein antibody which reacts with MTOCs, stained the apical center in a developmentally specific manner, and the nuclear envelope and SPB in a cell cycle-dependent fashion. Nocodazole was used in both high (10-15-mu-g/ml) and low (0.5-mu-g/ml) concentrations to depolymerize the networks and reveal their points of origin and recovery. The apical center was active from prophase I to the end of first meiosis. The nuclear envelope was the site of microtubule nucleation in early prophase and at the telophase/interphase transition, while SPBs were active in both nuclear division and sporulation. Mutant strains deficient in sporulation and with aberrant morphology were analyzed by antitubulin and MPM-2 immunofluorescence. Shape mutants showed abnormal or absent apical organizing centers and abnormal cortical microtubule patterns, indicating a possible role for the cortical network in the establishment and maintenance of ascus shape.