title: 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 beta-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 beta-tubulin-GFP in germinating macroconidia revealed polymerization and depolymerization of microtubules. In heterozygous crosses, H1-GFP and beta-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.
authors.affiliation: Institute of Molecular Biology and Department of Biology, University of Oregon, Eugene, OR 97403, USA. freitag@molbio.uoregon.edu
authors.forename: Michael
authors.initials: M
authors.lastname: Freitag
authors.forename: Patrick C
authors.initials: PC
authors.lastname: Hickey
authors.forename: Namboori B
authors.initials: NB
authors.lastname: Raju
authors.forename: Eric U
authors.initials: EU
authors.lastname: Selker
authors.forename: Nick D
authors.initials: ND
authors.lastname: Read
mesh_terms: D019677:Artificial Gene Fusion; D002467:Cell Nucleus / Q000378:metabolism*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006657:Histones / Q000235:genetics / Q000378:metabolism; D025301:Hyphae / Q000737:chemistry / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008870:Microtubules / Q000378:metabolism*; D009492:Neurospora crassa / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013172:Spores, Fungal / Q000737:chemistry / Q000378:metabolism; D013194:Staining and Labeling; D014404:Tubulin / Q000235:genetics / Q000378:metabolism
chemical_list: D006657:Histones; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D049452:Green Fluorescent Proteins