Department of Biochemistry and Molecular Biology, the Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA.
Cytoskeleton (Hoboken). 2013 Apr;70(4):215-27. doi: 10.1002/cm.21102. Epub 2013 Mar 21.
Cytoplasmic dynein plays important roles in mitosis and the intracellular transport of organelles, proteins, and mRNAs. Dynein function is particularly critical for survival of neurons, as mutations in dynein are linked to neurodegenerative diseases. Dynein function is also implicated in neuronal regeneration, driving the active transport of signaling molecules following injury of peripheral neurons. To enhance our understanding of dynein function and regulation in neurons, we established a novel knock-in mouse line in which the neuron-specific cytoplasmic dynein 1 intermediate chain 1 (IC-1) is tagged with both GFP and a 3xFLAG tag at its C-terminus. The fusion gene is under the control of IC-1's endogenous promoter and is integrated at the endogenous locus of the IC-1-encoding gene Dync1i1. The IC-1-GFP-3xFLAG fusion protein is incorporated into the endogenous dynein complex, and movements of GFP-labeled dynein expressed at endogenous levels can be observed in cultured neurons for the first time. The knock-in mouse line also allows isolation and analysis of dynein-bound proteins specifically from neurons. Using this mouse line we have found proteins, including 14-3-3 zeta, which physically interact with dynein upon injury of the brain cortex. Thus, we have created a useful tool for studying dynein function in the central nervous system under normal and pathologic conditions.
细胞质动力蛋白在有丝分裂和细胞器、蛋白质和 mRNAs 的细胞内运输中发挥着重要作用。动力蛋白的功能对神经元的存活尤为关键，因为动力蛋白的突变与神经退行性疾病有关。动力蛋白的功能也与神经元再生有关，它驱动着周围神经元损伤后信号分子的主动运输。为了增强我们对神经元中动力蛋白功能和调节的理解，我们建立了一种新型的基因敲入小鼠品系，其中神经元特异性细胞质动力蛋白 1 中间链 1（IC-1）在其 C 末端被 GFP 和 3xFLAG 标签双重标记。融合基因受 IC-1 内源性启动子的控制，并整合到 IC-1 编码基因 Dync1i1 的内源性基因座上。IC-1-GFP-3xFLAG 融合蛋白被整合到内源性动力蛋白复合物中，并且可以首次在培养的神经元中观察到以内源性水平表达的 GFP 标记的动力蛋白的运动。该基因敲入小鼠品系还允许从神经元中特异性分离和分析与动力蛋白结合的蛋白质。使用这种小鼠品系，我们发现了一些蛋白质，包括 14-3-3 zeta，它们在大脑皮层损伤时与动力蛋白发生物理相互作用。因此，我们创建了一个有用的工具，用于研究正常和病理条件下中枢神经系统中的动力蛋白功能。
