Immunofluorescence of the microtubular skeleton in growing and drug-treated yeast protoplasts

The microtubular system in growing protoplasts of Saccharomyces uvarum was visualized by immunofluorescence using the monoclonal antitubulin antibody TU 01. We confirmed the coexistence of regular spindle configuration and extensive cytoplasmic networks in growing protoplasts and also observed a distinct distortion of cytoplasmic microtubules in association with wall removal. After a short period for recovery of protoplasts in nutrient medium a restitution of cytoplasmic microtubules and their resumed contact with the protoplast surface was observed. Treatment of growing protoplasts with nocodazole resulted in the disappearance of spindle and cytoplasmic microtubules in the relevant fraction of the protoplast population. In carbendazime (MBC)-arrested protoplasts spindle microtubules were absent but cytoplasmic microtubules associated with spindle pole bodies were clearly visible. Microtubule reassembly on spindle pole bodies occurred within 30 min after washing out nocodazole as well as carbendazime. The approach using protoplasts suggests a simple way in which the differential effect of antimicrotubule agents can be experimentally tested and the microtubule organizing activity of yeast protoplasts visualized at the population level.