GFP as a tool to analyze the organization, dynamics and function of nuclei and microtubules in Neurospora crassa
GFP as a tool to analyze the organization, dynamics and function of nuclei and microtubules in Neurospora crassa
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DOI：
10.1016/j.fgb.2004.06.008
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发表时间：
2004-10-01
影响因子：
3
通讯作者：
Read, ND
中科院分区：
文献类型：
--
作者：
Freitag, M;Hickey, PC;Read, ND
我们报告了一个通用的绿色荧光蛋白表达质粒的建设，并证明其在粗糙脉孢菌的效用。为了使细胞核和微管可视化，我们产生了sgfp与脉孢菌组蛋白H1（hH 1）和β-微管蛋白（Bml）的羧基末端融合。利用可诱导的脉孢菌ccg-1启动子实现了GFP融合蛋白的强表达。核和微管的组织和动态观察活的营养菌丝，发展中的子囊，子囊孢子的常规和共聚焦激光扫描荧光显微镜。活细胞中GFP融合蛋白的观察结果在很大程度上证实了先前通过用各种显微镜技术检查固定细胞所获得的结果。H1-GFP显示动态核形状。微管大多平行于生长轴排列在顶端室，但更随机地排列在亚顶端室。在萌发的大分生孢子中的β-微管蛋白-GFP的延时成像揭示了微管的聚合和解聚。在杂合杂交中，H1-GFP和β-微管蛋白-GFP表达被沉默，推测是通过减数分裂沉默。H1-GFP在巨大香蕉子囊孢子的共同细胞质中的hH 1（+）-sgfp（+）细胞核附近翻译，但它扩散到所有细胞核中，这是GFP融合蛋白实用性的另一个例证。(C)2004年爱思唯尔公司All rights reserved.
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. (C) 2004 Elsevier Inc. All rights reserved.