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.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-03232005-094829 |
| Date | 25 March 2005 |
| Creators | Soukoulis, Victor |
| Contributors | John Penn, David Bader, Guoqiang Gu, Raymond DuBois, Vivien Casagrande |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-03232005-094829/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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