Neurons require axonal transport to establish and maintain axons and synapses. Cytoplasmic dynein is the motor for microtubule-based transport of various membranous organelles, including mitochondria, signaling endosomes, and viruses, in the retrograde direction from the axon terminus to the cell body. Mutations in dynein and its accessory proteins in humans and mice demonstrate that proper dynein function is essential for nerve growth, maintenance, regeneration, and survival. Defects in dynein based axonal transport have been observed in a wide range of neurodegenerative diseases including Alzheimer's disease and Motor Neuron Disease. Dynein is a large multi subunit protein complex. The intermediate chain (IC) plays a key role in the complex as it interacts with the other 5 subunits and with two known regulators of dynein activity. There are two IC genes in vertebrates and neuron specific isoforms of the IC. Little is known about how dynein binds to organelles during axonal transport. While dynactin complex is thought to be the most important binding protein there is evidence that an additional component is also involved in dynein binding to Trk containing signaling endosomes in axons. We found that phosphorylation of a newly identified conserved IC site is required for dynein binding to signaling endosomes. This phosphorylation does not modulate dynein binding to dynactin. We will use 2D difference gel electrophoresis to identify the component of Trk signaling endosome that interacts with the phospho-IC and then characterize its role in recruiting dynein to signaling endosomes. While the phosphorylation on the new IC site is important for dynein binding to signaling endosomes, our data indicate that it is not necessary for dynein binding to mitochondria or other organelles. This provides evidence for another mechanism for dynein binding to cargo. We will identify and characterize the other binding mechanisms. We will investigate the role of a second IC phosphorylation site that may also be important for dynein binding to endosomes. Dynein is transported into the axon in association with organelles moving in fast anterograde transport. We will use biochemical and live cell imaging approaches to identify and characterize the mechanism dynein uses to bind to organelles that primarily move in the anterograde direction. These studies will lead to a new model for dynein binding to organelles and important insights into the axonal transport of signaling endosomes which is essential for the function of the nervous system.
The health, survival and proper function of neurons requires the active transport of many organelles and other cargoes by motor proteins. Mutations in the motor dynein result in neurodegeneration. This project explores a new paradigm for how this motor attaches to cargo.
|Pfister, K Kevin (2015) Distinct functional roles of cytoplasmic dynein defined by the intermediate chain isoforms. Exp Cell Res 334:54-60|
|Blasier, Kiev R; Humsi, Michael K; Ha, Junghoon et al. (2014) Live cell imaging reveals differential modifications to cytoplasmic dynein properties by phospho- and dephosphomimic mutations of the intermediate chain 2C S84. J Neurosci Res 92:1143-54|
|Zhang, Jun; Twelvetrees, Alison E; Lazarus, Jacob E et al. (2013) Establishing a novel knock-in mouse line for studying neuronal cytoplasmic dynein under normal and pathologic conditions. Cytoskeleton (Hoboken) 70:215-27|
|Mitchell, David J; Blasier, Kiev R; Jeffery, Erin D et al. (2012) Trk activation of the ERK1/2 kinase pathway stimulates intermediate chain phosphorylation and recruits cytoplasmic dynein to signaling endosomes for retrograde axonal transport. J Neurosci 32:15495-510|