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.
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