Research Summary
My lab is interested in a
variety of biological phenomena involving motor proteins, with a
major emphasis on cytoplasmic dynein. Cytoplasmic dynein was
initially described as the motor for retrograde axonal transport,
but it is now known to have important functions in mitosis, cell
migration, growth cone motility, virus transport, and many other
aspects of neuronal and nonneuronal cell behavior, many of which are
under investigation in the lab.
One project involves the role
of cytoplasmic dynein in the human brain developmental disease
lissencephaly. This condition arises from mutations in the novel
dynein regulator, LIS1. We find that LIS1 and cytoplasmic dynein are
required for fibroblast migration and growth cone extension. Using
in utero electroporation into embryonic rat brain we have also
performed RNAi for LIS1, cytoplasmic dynein, myosin II, kinesin, and
additional factors in neural progenitor cells. By live imaging of
brain slices we can directly monitor effects on neuronal
progenitor/stem cell division, differentiation, and migration. We
can also introduce markers for microtubules, centrosomes, nuclei,
and other subcellular structures into neural progenitor cells (Fig
1; 2), leading to exciting new insights into the mechanisms
responsible for neuronal migration.
We have also
investigated the role of LIS1 and of the LIS1- and
dynein-interacting proteins NudE and NudEL (Nde1 and Ndel1) in
cytoplasmic dynein motor function using protein biochemistry and
single molecule techniques. Our results (with the S. Gross lab at UC
Irvine) indicate that NudE, LIS1 and dynein form a supercomplex
which exhibits markedly persistent force production under load. We
are currently evaluating the biological implications of this
behavior, and examining the effect of other disease genes and
mutations on dynein mechanochemistry.
We have recently found
that cytoplasmic dynein is required for end-on attachment of
microtubules to kinetochores in mitotic cells (Fig. 3), in addition
to its role in removing mitotic checkpoint proteins from these sites
and in poleward chromosome movement. We plan to explore how the
diverse array of known and unknown dynein interacting proteins
regulate kinetochore behavior and chromosome segregation.
We
are also exploring how cytoplasmic dynein may be differentially
recruited to vesicular vs. pathogenic forms of cargo. We find a
direct interaction between dynein and adenovirus through a specific
capsid protein, and we are exploring the therapeutic and
evolutionary significance of these results. We are also trying to
define the considerably more complex and regulated mechanisms for
recruitment of cytoplasmic dynein to nuclei and other membranous
organelles.
Fig. 1: Neuronal migration in live rat brain slices. Centrosome moves continuously, followed by very discontinuous nuclear movements. LIS1 RNAi (right) inhibits centrosome movement, and arrests movement of nucleus (as it moves out of focal plane). Centrosomes labeled with RFP centrin (shown in green); nucleus labeled with histone H1 (shown in red). From Tsai, J.-W., et al., 2007
Fig. 2: Behavior of neuronal precursor cell microtubules in live brain slices. Plus ends of growing microtubules are labeled with GFP-EB3 (green); centrosomes with RFP centrin (red). Movie at left focuses on microtubules in migratory process; movie at right includes microtubules in cell body region. From Tsai, J.-W., et al., 2007.
Fig. 3: Metaphase kinetochores in control (left) and cytoplasmic dynein defective (right) HeLa cells. Dynein inhibition disrupts normal oscillations of paired kinetochores, consistent with abnormal microtubule attachment. Kinetochores labeled with GFP-CENP-A; cells were arrested in metaphase with Mg132. (From Varma, D., et al., 2008).
