Localization and function of calmodulin in live-cells of Aspergillus nidulans
Localization and function of calmodulin in live-cells of Aspergillus nidulans
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钙调蛋白在构巢曲霉活细胞中的定位和功能
DOI：
10.1016/j.fgb.2009.12.008
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发表时间：
2010-03-01
影响因子：
3
通讯作者：
Lu, Ling
中科院分区：
文献类型：
--
作者：
Chen, Shaochun;Song, Yiju;Lu, Ling
钙调素（CaM）是一种可逆结合Ca ~（2+）的小分子真核蛋白。对CaM在遗传上易处理的生物体中的定位的研究已经产生了对CaM功能的许多见解。在这里，我们描述了动态定位的构巢曲霉钙调素（AnCaM）在活细胞中，通过使用重组菌株与同源，单一的交叉插入的目标基因，将GFP融合拷贝下的诱导型alcA启动子和RFP-CaM整合下的天然凸轮启动子。我们发现，CaM融合的本地化是相当动态的整个菌丝，集中在活跃的生长部位在萌发，菌丝生长，胞质分裂和分生孢子。alcA启动子抑制的钙调素的耗竭诱导的显着异常的芽苗与肿胀的芽管。此外，高度集中的GFP-CaM在极端顶端的位置似乎决定了菌丝的方向。这些数据共同表明，钙调素是不断需要新的菌丝生长。与此相反，这种不断积累的顶点，GFP-CaM只是短暂地定位在隔膜网站在胞质分裂。值得注意的是，耗尽的钙调素引起的缺陷，隔膜形成完全阻塞，表明在隔膜网站的瞬时钙调素积累是必不可少的隔膜。此外，正常的本地化的钙调素在菌丝尖端需要存在的功能肌动蛋白细胞骨架和马达蛋白KipA，这是必不可少的定位Spitzenkorper。这是首次报道丝状真菌中通过位点特异性同源整合在活细胞中定位和功能的CaM。(C)2010年由Elsevier Inc.出版
Calmodulin (CaM) is a small, eukaryotic protein that reversibly binds Ca2+. Study of CaM localization in genetically tractable organisms has yielded many insights into CaM function. Here, we described the dynamic localization of Aspergillus nidulans CaM (AnCaM) in live-cells by using recombination strains with homologous, single cross-over insertions at the target gene which placed the GFP fused copy under the inducible alcA promoter and the RFP-CaM integration under the native cam promoter. We found that the localization of CaM fusion was quite dynamic throughout the hypha and was concentrated to the active growing sites during germination, hyphal growth, cytokinesis and conidiation. The depletion of CaM by alcA promoter repression induced the explicit abnormalities of germlings with the swollen germ tubes. In addition, the position of highly concentrated GFP-CaM in the extreme apex seemed to determine the hyphal orientation. These data collectively suggest that CaM is constantly required for new hyphal growth. In contrast to this constant accumulation at the apex, GFP-CaM was only transiently localized at septum sites during cytokinesis. Notably, depletion of CaM caused the defect of septation with a completely blocked septum formation indicating that the transient CaM accumulation at the septum site is essential for septation. Moreover, the normal localization of CaM at a hyphal tip required the presence of the functional actin cytoskeleton and the motor protein KipA, which is indispensable for positioning Spitzenkorper. This is the first report of CaM localization and function in live-cells by the site-specific homologous integration in filamentous fungi. (C) 2010 Published by Elsevier Inc.