GFP as a tool to analyze the organization, dynamics and function of nuclei and microtubules in Neurospora crassa

Abstract

We report the construction of a versatile GFP expression plasmid and demonstrate its utility in Neurospora crassa. To visualize nuclei and microtubules, we generated carboxy-terminal fusions of sgfp to Neurospora histone H1 ( hH1) and β-tubulin ( Bml). Strong expression of GFP fusion proteins was achieved with the inducible Neurospora ccg-1 promoter. Nuclear and microtubule organization and dynamics were observed in live vegetative hyphae, developing asci, and ascospores by conventional and confocal laser scanning fluorescence microscopy. Observations of GFP fusion proteins in live cells largely confirmed previous results obtained by examination of fixed cells with various microscopic techniques. H1-GFP revealed dynamic nuclear shapes. Microtubules were mostly aligned parallel to the growth axis in apical compartments but more randomly arranged in sub-apical compartments. Time-lapse imaging of β-tubulin-GFP in germinating macroconidia revealed polymerization and depolymerization of microtubules. In heterozygous crosses, H1-GFP and β-tubulin-GFP expression was silenced, presumably by meiotic silencing. H1-GFP was translated in the vicinity of hH1 +- sgfp + nuclei in the common cytoplasm of giant Banana ascospores, but it diffused into all nuclei, another illustration of the utility of GFP fusion proteins.