K. Kevin Pfister
- BA, University of Virginia
- PhD, Princeton University
- Postdoc, University of California, Berkeley
- Postdoc, University of Texas Southwestern Medical Center
- Associate Professor, Cell Biology
- Phone: 434-924-1912
- Email: email@example.com
Regulation of Cytoplasmic Dynein for Membrane Bounded Organelle Trafficking in Axons
My lab is studying mechanisms underlying microtubule-based intracellular transport,specifically the structure, function, and regulation of the motor protein cytoplasmic dynein. This large protein complex is composed of six distinct subunits and is the protein responsible for most intracellular movement toward the minus ends of microtubules. Cytoplasmic dynein has many important roles. It transports various membrane bounded organelles, including endosomes, lysosomes, and mitochondria. It is involved in the positioning of the centrosome, the nucleus, and the Golgi. Cytoplasmic dynein is also responsible for virus transport to the nucleus, retrograde axonal transport, and microtubule and neurofilament transport. It is involved in spindle assembly, kinetochore function, and spindle pole separation during mitosis. The focus of the lab is how cells regulate cytoplasmic dynein to accomplish these various functions. We have recently identified three families of dynein light chains, and multiple alternative splicing and phospho-isoforms of the intermediate chains, and are characterizing the roles of these polypeptides in dynein regulation and cargo binding. We are concentrating on the regulation of the axonal transport of membrane bounded organelles, microtubules andneurofilaments, and the role of the Tctex1 family of light chains in specific dynein function.
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.
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.
- Blasier K, Humsi M, Ha J, Ross M, Smiley W, Inamdar N, Mitchell D, Lo K, Pfister K. Live cell imaging reveals differential modifications to cytoplasmic dynein properties by phospho- and dephosphomimic mutations of the intermediate chain 2C S84. Journal of neuroscience research. 2014;92(9): 1143-54. PMID: 24798412 | PMCID: PMC4107179
- Mitchell D, Blasier K, Jeffery E, Ross M, Pullikuth A, Suo D, Park J, Smiley W, Lo K, Shabanowitz J, Deppmann C, Trinidad J, Hunt D, Catling A, Pfister K. Trk activation of the ERK1/2 kinase pathway stimulates intermediate chain phosphorylation and recruits cytoplasmic dynein to signaling endosomes for retrograde axonal transport. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2012;32(44): 15495-510. PMID: 23115187 | PMCID: PMC3500848
- Ha J, Lo K, Myers K, Carr T, Humsi M, Rasoul B, Segal R, Pfister K. A neuron-specific cytoplasmic dynein isoform preferentially transports TrkB signaling endosomes. The Journal of cell biology. 2008;181(6): 1027-39. PMID: 18559670 | PMCID: PMC2426944
- Lo K, Kogoy J, Pfister K. The DYNLT3 light chain directly links cytoplasmic dynein to a spindle checkpoint protein, Bub3. The Journal of biological chemistry. 2007;282(15): 11205-12. PMID: 17289665
- Lo K, Kan H, Pfister K. Identification of a novel region of the cytoplasmic Dynein intermediate chain important for dimerization in the absence of the light chains. The Journal of biological chemistry. 2006;281(14): 9552-9. PMID: 16452477