Pfister, K. Kevin

K. Kevin Pfister

K. Kevin Pfister

Primary Appointment

Associate Professor, Cell Biology


  • BA, Biology, University of Virginia
  • PhD, Biology, Princeton University
  • Postdoc, Cell Biology-Mitosis, University of California, Berkeley
  • Postdoc, Neuroscience, University of Texas Southwestern Medical Center

Contact Information

Jordan Hall
Charlottesville, VA 22908
Telephone: 434-924-1912

Research Interests

Structure, Function, and Regulation of Cytoplasmic Dynein: Its Role in Intracellular Microtubule-based Transport, Membrane Bounded Organelle Trafficking, Axonal Transport and Mitosis

Research Description

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.

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.

Selected Publications