Haploinsufficiency of human LIS1 (or PAFAHIB1) is responsible for the human neuronal migration defect lissencephaly. Recently, we found that LIS1 interacts with NUDEL, a homologue of the A. nidulans nuclear distribution mutant NudE. LIS1 and NUDEL together bind the cytoplasmic dynein heavy chain (CDHC) to regulate dynein motor function in non-neural cells. NUDEL is phosphorylated by Cdk5/p35, a complex essential for neuronal migration in mice, suggesting that the LIS1/NUDEL/CDHC complex is regulated by phosphorylation. Phosphorylated NUDEL binds to 14-3-3epsilon, a member of a large family of binding proteins that mediate subcellular localization or stability of phosphoproteins. These recent studies have provided a pathway through which LIS1 acts to regulate dynein motor function in non-neural cells. However, it is unknown if this pathway is critical for neuronal migration. It is tempting to speculate that the dysregulation of dynein motor function by the LIS1/NUDEL/CDHC complex is also responsible for the neuronal migration defects in mammals with reduced doses of LIS1, mice deficient for the Cdk5/p35 complex, and mice deficient for 14-3-3epsilon. We hypothesize that LIS1 forms a complex (LIS1/NUDEL/CDHC) that regulates dynein motor function during neuronal migration. The function of this complex is regulated by phosphorylation of NUDEL by Cdk5/p35 via binding and regulation of the cellular distribution and/or stability of NUDEL by 14-3-3epsilon. To address these hypotheses, we will utilize genetic and gene transfer approaches to modulate the levels of specific components of this pathway in mice or cells. The effects of these modifications on neuronal migration will be determined in vivo and in vitro using quantitative migration assays. To prove whether effects of these modifications on neuronal migration result from alterations in the LIS1/NUDEL/CDHC complex, we will employ cell biological and biochemical assays of cytoplasmic dynein function, including the cellular localization and biochemical interaction of members of this complex.
The specific aims of this application are: 1) determine whether LIS1 participates in neuronal migration by regulation of dynein motor function, 2) determine whether NUDEL participates with LIS1 to regulate dynein motor functions via Cdk5/p35 phosphorylation during neuronal migration; and 3) to determine if 14-3-3epsilon regulates the cellular distribution and/or stability of Cdk5-phosphorylated NUDEL during neuronal migration to regulate the activity of LIS1 and CDHC.
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