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Title page for ETD etd-03232005-094829


Type of Document Dissertation
Author Soukoulis, Victor
Author's Email Address victor.soukoulis@vanderbilt.edu
URN etd-03232005-094829
Title Characterization of CytLEK1 as a Novel Regulator of the LIS1 Pathway
Degree PhD
Department Cell and Developmental Biology
Advisory Committee
Advisor Name Title
Raymond DuBois Committee Chair
David Bader Committee Member
Guoqiang Gu Committee Member
John Penn Committee Member
Vivien Casagrande Committee Member
Keywords
  • Nde1
  • LIS1
  • NudE
  • cytoskeleton
  • microtubule
Date of Defense 2005-03-11
Availability unrestricted
Abstract
LIS1 and NudE(L) are partner proteins in a conserved pathway regulating the function of dynein and microtubules. Members of the LIS1 pathway play a critical role in fundamental cellular processes, such as differentiation, proliferation, and migration. Therefore, determining the precise nature of their actions will lead to significant insight into the complex process of embryogenesis. In this document, I present data revealing that cytLEK1, a large protein containing a spectrin repeat and multiple leucine zippers, is a novel component of this pathway through its direct interaction with NudE, as determined by a yeast two-hybrid screen. This is the first time that the cytLEK1 protein has been linked to a molecular pathway. I identified the exact binding domains in each molecule, and co-immunoprecipitation and colocalization studies confirmed the specificity of the interaction between cytLEK1 and NudE. Confocal deconvolution analysis revealed that cytLEK1 exhibits colocalization with endogenous NudE and with the known NudE binding partners, LIS1 and dynein. By localizing the NudE-binding domain of cytLEK1 to a small domain within the molecule, I was able to disrupt cytLEK1 function using a dominant negative approach, in addition to LEK1 knockdown, and thus examine the role of the cytLEK1-NudE interaction in cells. Consistent with a defect in the LIS1 pathway, disruption of cytLEK1 function resulted in alteration of cellular morphology and microtubule organization. Additionally, cells exhibited a severe inability to repolymerize their microtubule networks after nocodazole challenge. I also present data here examining, for the first time, the expression and localization of cytLEK1 in various cells and tissues. These experiments provide further evidence suggestive of the action of cytLEK1 as a regulator of the LIS1 pathway. Taken together, my studies reveal that cytLEK1 is essential for cellular functions regulated by the LIS1 pathway and may play a critical role during murine development.
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