University of Virginia Health System

Email Address: kkp9w@virginia.edu

Dynein Function in Neurons and Axonal Transport.
In the classic paradigm for the fast axonal transport of membrane bounded organelles, cytoplasmic dynein is the motor for retrograde membrane bounded organelle traffic from the axon terminal to the cell body, while members of the kinesin family move membrane bounded organelles toward the axonal tip in anterograde transport. Dynein is first synthesized in the cell body and then transported in the anterograde direction and after reaching the terminal it becomes the motor for retrograde transport. We have identified two distinct pools of dynein travelling transported in the anterograde direction and we can distinguish these two pools by their intermediate chain subunits. One pool is traveling at a fast speed associated with membrane bounded organelles. The other is traveling much slower and is associated with the cytoskeletal filaments, actin, microtubules, and neurofilaments. This raises the exciting prospect that dynein has roles in the transport of cytoskeletal filaments in the axon. We are using various molecular, immunocytochemical, and live cell imaging approaches with GFP-tagged versions of the intermediate chains to identify and characterize the different dynein populations in axons. We are utilizing biochemical and molecular methods to analyze the proteins associated with the different pools of dynein. We also have evidence for the differential phosphorylation of the dynein intermediate chain subunits in axons and are characterizing the effects of this phosphorylation on the functional properties of dynein in vitro and in vivo.

We have made cell lines with the stable expression of intermediate chain-GFP fusion proteins and are using them to characterize dynein movement in vivo. We have found that, in PC12 cell neurite processes, puncta containing dynein move in both the anterograde and retrograde directions (Myers et al., 2004, American Society for Cell Biology Annual Meeting Abstracts). We have also found that dynein is enriched in and associated with microtubules in the central and peripheral regions of the growth cones and with actin in the peripheral region of cultured hippocampal neurons.

The Role of Light Chains in Dynein Function: Dynein Involvement in Spindle Check Point Protein Transport.
We have identified three families of cytoplasmic dynein light chains and found that light chain dimers bind to the intermediate chains. To investigate the role of different light chains in dynein binding to specific protein cargo we are concentrating on the two members of one light chain family, Tctex1 and rp3. We have found that rp3 binds to full length human Bub3 in yeast two-hybrid and in vitro binding assays. Furthermore the entire dynein complex co-pellets with Bub3 in GST-pull down experiments. Immunofluorescence analysis reveals that ectopically expressed rp3 co-localizes with Bub3 at kinetochores in LLCPK cells during prometaphase (Lo et al, 2004, American Society for Cell Biology Annual Meeting Abstracts). We are continuing our analysis of the role of this light chain and dynein in spindle check pint inactivation. Several other candidate rp3 binding proteins have been identified and are being investigated. The specificity of the interactions of the various light chain isoforms with the various intermediate chain isoforms is being analyzed.

Virus Transport
In collaboration with Dr. Phillip Leopold at Weill Medical College of Cornell, we are investigating the interaction of cytoplasmic dynein with Adenovirus and are seeking to identify the viral and dynein subunits that interact during dynein mediated transport of the virus along microtubules to the nucleus.