Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada, L8N 3Z5.
J Cell Sci. 2012 Sep 1;125(Pt 17):3977-88. doi: 10.1242/jcs.097667. Epub 2012 May 23.
Cofilin protein is involved in regulating the actin cytoskeleton during typical steady state conditions, as well as during cell stress conditions where cofilin saturates F-actin, forming cofilin-actin rods. Cofilin can enter the nucleus through an active nuclear localization signal (NLS), accumulating in nuclear actin rods during stress. Here, we characterize the active nuclear export of cofilin through a leptomycin-B-sensitive, CRM1-dependent, nuclear export signal (NES). We also redefine the NLS of cofilin as a bipartite NLS, with an additional basic epitope required for nuclear localization. Using fluorescence lifetime imaging microscopy (FLIM) and Förster resonant energy transfer (FRET) between cofilin moieties and actin, as well as automated image analysis in live cells, we have defined subtle mutations in the cofilin NLS that allow cofilin to bind actin in vivo and affect cofilin dynamics during stress. We further define the requirement of cofilin-actin rod formation in a system of cell stress by temporal live-cell imaging. We propose that cofilin nuclear shuttling is critical for the cofilin-actin rod stress response with cofilin dynamically communicating between the nucleus and cytoplasm during cell stress.
丝状肌动蛋白（actin）细胞骨架调节蛋白肌动蛋白结合蛋白（cofilin）在典型的稳定状态条件下以及细胞应激条件下（此时肌动蛋白饱和，形成肌动蛋白丝）参与调节肌动蛋白细胞骨架。肌动蛋白结合蛋白可通过活性核定位信号（NLS）进入细胞核，并在应激时积聚在核中的肌动蛋白丝中。在这里，我们通过莱普霉素-B 敏感的 CRM1 依赖性核输出信号（NES）来描述肌动蛋白结合蛋白的主动核输出。我们还将肌动蛋白结合蛋白的 NLS 重新定义为具有核定位所需的附加碱性表位的二部分 NLS。使用荧光寿命成像显微镜（FLIM）和肌动蛋白之间肌动蛋白结合蛋白部分之间的Förster 共振能量转移（FRET）以及活细胞中的自动图像分析，我们已经定义了肌动蛋白结合蛋白 NLS 中的细微突变，使肌动蛋白结合蛋白能够在体内与肌动蛋白结合并影响应激过程中的肌动蛋白结合蛋白动力学。我们通过时间分辨活细胞成像进一步定义了细胞应激系统中肌动蛋白结合蛋白丝形成的要求。我们提出，肌动蛋白结合蛋白核穿梭对于肌动蛋白结合蛋白-肌动蛋白丝应激反应至关重要，在细胞应激过程中，肌动蛋白结合蛋白在核和细胞质之间动态通讯。
