Molecular Mechanisms of Neuronal Migrations

University Of Virginia, Charlottesville VA

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Abstract

DESCRIPTION (provided by applicant): Lissencephaly is a debilitating genetic disorder which affects the ability of progenitor neurons to progress properly through the cell cycle and migrate to their target locations in the brain cortex. As a result the cortex is malformed with lack of typical gyrations, leading to severe retardation, epileptic seizures and other symptoms. The affected gene, LIS1, is a component of a regulatory pathway that modulates the function of the molecular motor dynein and its associated 1.2 MDa complex dynactin. These huge molecular complexes mediate the attachment of cell cortex, nucleus, kinetochores, select organelles and other cargo to microtubules at the (+) ends. The proteins that make up these pathways are stringently conserved from fungi to man, consistent with their vital biological roles. Our strategic objective is to elucidate the structure-function relationships in these proteins and to explain their normal physiological roles. Aside from the LIS1 protein, our research targets several functionally related proteins including Nde1, Ndel1, NudC and components of the dynactin complex. We are uniquely equipped to address these problems using both biophysical methods and functional assays. We will use synergistic and complementary combinations of crystallography, NMR, EPR and other spectroscopic methods to explore the structural complexity of these proteins and their dynamics. Moreover, we will elucidate how these proteins are involved in the critical phase of mitotic cell division, i.e. in the attachment of the chromosome kinetochores to the microtubules of the mitotic spindle. These investigations may provide detailed explanation why neuronal cells fail to progress through the cell cycle to generate mature, functional cells. They may also shed lights on phenomena closely related to cancer, such as cell proliferation and migration. To dissect the functionality of Nud proteins in mitosis we will use a unique in vitro assay based on reconstituted mitotic spindle and chromosomes from Xenopus oocyte extracts. To complement these functional studies, we will use cutting-edge mass spectrometry analysis to identify the protein-protein interactions networks in the brain involving the Nud family of proteins. A comparison of results based on the Xenopus assays with the pull-down assays using mouse brain extracts will provide us with a unique database for Nud-protein interactions and invaluable insight into their function.