Neurons require axonal transport to establish and maintain axons and synapses. Cytoplasmic dynein is the motor for microtubule-based fast transport of various membranous organelles, including mitochondria, endosomes, and viruses in the retrograde direction from the axon terminus to the cell body. Proper dynein function is essential for nerve growth, maintenance and regeneration, as shown by the recent findings that human and mouse mutants defective in dynein complex function result in motor neuron neurodegeneration. Dynein is a large multi subunit protein complex and very little is known about how it is regulated to accomplish its diverse functions in neurons. This proposal will focus on the roles of two subunits that are part of the dynein cargo binding domain and which define distinct populations of dynein, the intermediate chains and the DYNLT (Tctex1 and rp3) light chains. There are six isoforms of the intermediate chains in neurons and the first aims of this proposal concentrate on two, one is ubiquitous and sufficient for housekeeping functions in cells, the other intermediate chain is found primarily in neurons. We have shown that both are present in neurites, axons, and dendrites. We have prepared a stable neural cell line expressing the ubiquitous isoform tagged with GFP;demonstrated that it is incorporated into functional dynein complexes;and associated with membrane bounded organelles. The movement of the GFP-dynein can be imaged in living neurites and axons of cultured neurons. The cell line and cultured neurons will be used to test several models for dynein regulation, by analyzing the behavior of the dynein using live cell imaging coupled with RNAi and dominant negative manipulation of dynein interacting proteins. We have found that in PC12 cells, the ubiquitous dynein isoform is involved in neurotrophin receptor kinase (Trk) signaling endosome transport, while in cultured hippocampal neurons signaling endosomes are transported by the neuronal isoform. In this proposal we will build on these observations and use biochemical, molecular, and live cell imaging methods to identify the mechanisms that underlies the specific binding of signaling endosomes to dyneins with the different isoforms. The dynein light chains are also thought to directly mediate dynein binding to specific proteins. While several DYNLT1 (Tctex1) binding partners had been previously been identified, we found that Bub3, a spindle checkpoint protein is a specific binding partner of the related light chain DYNLT3 (rp3). In the third aim we will investigate the functional significance of DYNLT3 and this binding to mitosis, and identify the distinct domains of DYNLT3 that are responsible for the specific binding. We will also examine the functional significance of the binding of this light chain to filamin A. These studies will provide important insight into dynein function, the axonal transport of signaling endosomes, and the role of disrupted axonal transport to neurodegeneration.
Active transport of many proteins and cargoes by motor proteins is required for the health and proper function of neurons. Mutations in one motor, dynein, result in motor neurodegeneration. This project investigates 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|