The Vallee lab is interested in a variety of biological phenomena involving motor proteins, with a major emphasis on cytoplasmic dynein. We described it originally 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 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 and in normal development. Lissencephaly (smooth brain) arises from mutations in the dynein regulator, LIS1. Investigation of the role of LIS1 in developing rat brain involves transfection of neural progenitor cells by in utero electroporation. shRNAs are used to interfere with expression of LIS1, cytoplasmic dynein, and a variety of additional dynein regulators, including NudE, NudEL, BicD2, and CENP-F. Fluorescent fusion protein markers are co-expressed to monitor cellular and subcellular behavior to understand the specific roles of these factors in the mechanism of neuronal migration, morphogenesis, and proliferation. These studies have led to a model for how LIS1 mutations cause lissencephaly (Tsai et al., 2005; Tsai et al., 2007). They have also led to models for how the forces generated by cytoplasmic dynein in developing neurons contribute to neural progenitor cell migration and division.
A recent focus of the lab has been on the earliest stages of neurogenesis in the CNS, addressing the unusual behavior of the radial glial progenitor (RGP) cells. These highly elongated cells span the developing neocortex from the ventricles to the brain surface, and serve as guides for migrating neurons. In addition, RGP cells multiply to give rise to most neurons and glial cells in the developing brain, and also to adult neural stem cells. The RGP cells exhibit a remarkable form of cell cycle-dependent nuclear oscillation. Nuclei divide at the surface of the ventricle, ascend basally during G1, undergo S-phase, and then return to the ventricle during G2 to divide again. The physiological purpose and underlying mechanism for this highly conserved behavior have remained largely unexplored until recently. The lab has found that, in rat brain, kinesin-3 is responsible for basal nuclear migration, and cytoplasmic dynein for apical migration (Tsai et al., 2010). Recent work has revealed that nuclear pores in RGP cells recruit cytoplasmic dynein using two G2-specific mechanisms (Hu et al., 2013). How this mechanism is triggered in early G2, and how mitosis is delayed until nuclei contact the ventricular surface, remain the focus of additional studies.
The lab has also investigated the molecular mechanisms by which LIS1 and other dynein regulators control dynein function. A number of factors regulate dynein targeting to subcellular sites. We have found LIS1, aided by NudE, to bind to the dynein motor domain during its powerstroke, and to stabilize the interaction of dynein with microtubules during this phase of the crossbridge cycle (McKenney et al., 2010) (with lab of S. Gross, UC Irvine). The result is a substantial increase in total forces generated by groups of dynein molecules, e. g., those associated with nuclei in the developing brain. Current work is focused on understanding the specific effects of these proteins on the dynein motor domain. We are also dissecting the dynein motor domain (with A. Gennerich, Albert Einstein College of Medicine) to understand the complex intramolecular, as well as intermolecular mechanisms involved in dynein motor regulation.
As a complement to these studies, we continue to investigate mechanisms of dynein regulation in a variety of cellular processes, including vesicular transport in axons and nonneuronal cells, growth cone and lamellipodial formation and function, and mitosis. We are also investigating mechanisms used by a non-physiological form of dynein cargo, adenovirus, to hijack dynein and other motor proteins for use in transport to the nucleus. We have used biochemical methods to identify motor proteins and their specific subunits recruited to adenovirus, and the capsid components which serve as receptors (Bremner et al., 2009). We recently found that PKA activation during adenovirus infection results in phosphorylation of the dynein LIC1 subunit. The result is displacement of dynein from physiological organelles for recruitment by the virus. We are using high temporal and spatial resolution particle tracking analysis (Yi et al., 2011) to monitor the behavior of fluorescently tagged virus in infected cells. These studies are aimed at determining the role of microtubule plus and minus end-directed motors in the overall infection process. Our data suggest that motor proteins recruited to the virus could facilitate infection, or, alternatively act in defense of the cell. These studies have suggested the existence of novel host defense mechanisms, an understanding of which may be of general importance in controlling host-pathogen competition.
References
Bremner, K. H., Scherer, J., Yi, J., Vershinin, M., Gross, S. P., and Vallee, R. B. (2009). Adenovirus transport via direct interaction of cytoplasmic dynein with the viral capsid hexon subunit. Cell Host Microbe 6:523-535.
*Hu, D.J., *Baffet A.D., *Nayak T., Akhmanova A., Doye V., Vallee R.B. (2013) Dynein Recruitment to Nuclear Pores Activates Apical Nuclear Migration and Mitotic Entry in Brain Progenitor Cells. Cell, in press
*McKenney, R. J., *Vershinin, M., +Vallee R. B., and +Gross, S. P. (2010) LIS1 and NudE induce a persistent dynein force-producing state. Cell 141:304-314
*Tsai, J.-T., *Lian, W.-N., Kemal, S., Kriegstein, A., and Vallee, R. B. (2010) Kinesin 3 and Cytoplasmic Dynein Mediate Interkinetic Nuclear Migration in Neural Stem Cells. Nature Neurosci. 13:1463-1471.
Tsai, J.W., Bremner, K.H. , and Vallee, R.B. 2007. Dual subcellular roles for LIS1 and dynein in radial neuronal migration in live brain tissue. Nature Neurosci. 10:970-9.
Tsai, J. W.; Chen, Y.; Kriegstein, A. R.; Vallee, R. B. (2005) LIS1 RNA interference blocks neural stem cell division, morphogenesis, and motility at multiple stages. J. Cell Biol. 170:935-45.
Yi, J, Ori-McKenney, K. M., McKenney, R. J., Vershinin, M., Gross, S. P., and Vallee, R. B. (2011). High resolution imaging reveals indirect coordination of opposite motors and LIS1 role in high-load axonal transport. J. Cell Biol. 195:193-201.