Bergs Anna, Ishitsuka Yuji, Evangelinos Minoas, Nienhaus G U, Takeshita Norio
Department of Microbiology, Institute for Applied Bioscience, Karlsruhe Institute of Technology Karlsruhe, Germany.
Institute of Applied Physics, Karlsruhe Institute of Technology Karlsruhe, Germany.
Front Microbiol. 2016 May 9;7:682. doi: 10.3389/fmicb.2016.00682. eCollection 2016.
Highly polarized growth of filamentous fungi requires a continuous supply of proteins and lipids to the hyphal tip. This transport is managed by vesicle trafficking via the actin and microtubule cytoskeletons and their associated motor proteins. Particularly, actin cables originating from the hyphal tip are essential for hyphal growth. Although, specific marker proteins have been developed to visualize actin cables in filamentous fungi, the exact organization and dynamics of actin cables has remained elusive. Here, we observed actin cables using tropomyosin (TpmA) and Lifeact fused to fluorescent proteins in living Aspergillus nidulans hyphae and studied the dynamics and regulation. GFP tagged TpmA visualized dynamic actin cables formed from the hyphal tip with cycles of elongation and shrinkage. The elongation and shrinkage rates of actin cables were similar and approximately 0.6 μm/s. Comparison of actin markers revealed that high concentrations of Lifeact reduced actin dynamics. Simultaneous visualization of actin cables and microtubules suggests temporally and spatially coordinated polymerization and depolymerization between the two cytoskeletons. Our results provide new insights into the molecular mechanism of ordered polarized growth regulated by actin cables and microtubules.
丝状真菌的高度极化生长需要向菌丝尖端持续供应蛋白质和脂质。这种运输由通过肌动蛋白和微管细胞骨架及其相关运动蛋白进行的囊泡运输来管理。特别是,源自菌丝尖端的肌动蛋白束对菌丝生长至关重要。尽管已经开发出特定的标记蛋白来可视化丝状真菌中的肌动蛋白束,但肌动蛋白束的确切组织和动态仍然难以捉摸。在这里,我们在活的构巢曲霉菌丝中使用与荧光蛋白融合的原肌球蛋白(TpmA)和Lifeact观察肌动蛋白束,并研究其动态和调控。绿色荧光蛋白标记的TpmA可视化了从菌丝尖端形成的动态肌动蛋白束,其具有伸长和收缩周期。肌动蛋白束的伸长和收缩速率相似,约为0.6μm/s。肌动蛋白标记物的比较表明,高浓度的Lifeact降低了肌动蛋白的动态性。肌动蛋白束和微管的同时可视化表明,两个细胞骨架之间在时间和空间上协调聚合和解聚。我们的结果为肌动蛋白束和微管调节有序极化生长的分子机制提供了新见解。