K. Kevin Pfister
Primary AppointmentAssociate 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
Structure, Function, and Regulation of Cytoplasmic Dynein: Its Role in Intracellular Microtubule-based Transport, Membrane Bounded Organelle Trafficking, Axonal Transport and Mitosis
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 and neurofilaments,
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.