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. Our recent work has implicated kinesin-3 and cytoplasmic dynein in basal vs. apical nuclear migration (INM) in radial progenitor cells. We have also obtained evidence for the role of nuclear pore proteins in G2-specific recruitment of dynein to the nuclear envelope in these cells, and we are interested in further exploring mechanisms of cell cycle control.
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 have recently found that NudE-LIS1 competes with the dynein processivity factor dynactin, suggesting a mechanism for adapting dynein to high- vs. low-load transport. In support of this possibility we find LIS1 to be required for fast axonal transport, but only of larger vesicular cargo. This aspect of transport, we believe, may be particularly susceptible to disruption.
We are also exploring mechanisms for cytoplasmic dynein recruitment to kinetochores, cell cortical sites, and vesicular vs. pathogenic forms of cargo. We recently found a direct interaction between dynein and adenovirus through the major capsid protein hexon, which we have determined must be primed by transit of the virus through the endosomal pathway. Current studies involve the effects of host response mechanisms in regulating adenovirus transport and infectivity, and we are exploring the therapeutic and evolutionary significance of these results.
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).