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GFP as a tool to analyze the organization, dynamics and function of nuclei and microtubules in Neurospora crassa.
Fungal Genet Biol 2004; 41(10):897-910FG

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+Show Affiliations

Institute of Molecular Biology and Department of Biology, University of Oregon, Eugene, OR 97403, USA. freitag@molbio.uoregon.eduNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

15341912

Citation

Freitag, Michael, et al. "GFP as a Tool to Analyze the Organization, Dynamics and Function of Nuclei and Microtubules in Neurospora Crassa." Fungal Genetics and Biology : FG & B, vol. 41, no. 10, 2004, pp. 897-910.
Freitag M, Hickey PC, Raju NB, et al. GFP as a tool to analyze the organization, dynamics and function of nuclei and microtubules in Neurospora crassa. Fungal Genet Biol. 2004;41(10):897-910.
Freitag, M., Hickey, P. C., Raju, N. B., Selker, E. U., & Read, N. D. (2004). GFP as a tool to analyze the organization, dynamics and function of nuclei and microtubules in Neurospora crassa. Fungal Genetics and Biology : FG & B, 41(10), pp. 897-910.
Freitag M, et al. GFP as a Tool to Analyze the Organization, Dynamics and Function of Nuclei and Microtubules in Neurospora Crassa. Fungal Genet Biol. 2004;41(10):897-910. PubMed PMID: 15341912.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - GFP as a tool to analyze the organization, dynamics and function of nuclei and microtubules in Neurospora crassa. AU - Freitag,Michael, AU - Hickey,Patrick C, AU - Raju,Namboori B, AU - Selker,Eric U, AU - Read,Nick D, PY - 2004/04/13/received PY - 2004/06/28/accepted PY - 2004/9/3/pubmed PY - 2004/11/17/medline PY - 2004/9/3/entrez SP - 897 EP - 910 JF - Fungal genetics and biology : FG & B JO - Fungal Genet. Biol. VL - 41 IS - 10 N2 - 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. SN - 1087-1845 UR - https://www.unboundmedicine.com/medline/citation/15341912/GFP_as_a_tool_to_analyze_the_organization_dynamics_and_function_of_nuclei_and_microtubules_in_Neurospora_crassa_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1087-1845(04)00095-7 DB - PRIME DP - Unbound Medicine ER -