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Type of Document Dissertation Author Towns, William L URN etd-12082006-160054 Title Impact of Transfected Cytoplasmic Dynein Intermediate Chain on Dynein-Based Transport Degree Doctor of Philosophy Department Biological Sciences Advisory Committee
Advisor Name Title Kevin Vaughan Committee Chair Holly Goodson Committee Member Joe OTousa Committee Member Ted Hinchcliffe Committee Member Keywords
- Dynein
- transport
- Intermediate chain
- transfection
Date of Defense 2006-12-07 Availability restricted Abstract IMPACT OF TRANSFECTED CYTOPLASMIC DYNEIN INTERMEDIATE CHAIN ON DYNEIN-BASED TRANSPORTAbstract
by
William L. Towns
Cytoplasmic dynein is a microtubule motor protein responsible for the transport of multiple cargos. In previous work, we found that overexpression of dynein IC tagged with GFP (IC-GFP) disrupted dynein cargo transport in vivo. This phenotype was found to be weakened or strengthened when IC-GFP mutants mimicking the phospho (S84D IC-GFP) or de-phospho (S84A IC-GFP) IC states were overexpressed. To determine by what mechanism IC-GFP expression causes this phenotype, we performed a series of biochemical assays on IC-GFP or S84D IC-GFP-transfected cell lysates. IC-GFP expression did not disrupt dynein complex assembly. IC-GFP did not incorporate into native dynein or dimerize with dynein IC, but it recruited a limited amount of the dynein light chains LC8 and Tctex-1. S84D IC-GFP expression did not alter any of these behaviors, suggesting that these characteristics are not phosphorylation dependent. These results suggest that IC-GFP disrupts cargo transport without directly interfering with native dynein or its subunits. To determine the minimum sufficient region of IC necessary to generate the cargo disruption phenotype, we performed live-cell analysis on cells expressing the p150Glued-binding region of IC (1-100 IC-GFP). 1-100 IC-GFP expression caused dynein cargo transport disruption similar to IC-GFP, and this behavior was weakened or strengthened when serine 84 was mutated to aspartic acid or alanine respectively. 1-100 IC-GFP expression did not disrupt dynein complex assembly and did not interact with native dynein IC, LC8, or Tctex-1. None of these behaviors were altered by serine 84 mutation. These results suggest that the p150Glued-binding region of IC is sufficient to cause cargo disruption. To determine if IC-GFP is capable of competitive binding to dynein cargo, we performed binding assays with purified phagosomes. Recombinant IC-GFP and S84A IC-GFP were capable of binding to phagosomes, while S84D IC-GFP exhibited reduced organelle binding. Two-dimensional gel analysis of IC-GFP and S84D IC-GFP-transfected cell lysate revealed that IC-GFP and not S84D IC-GFP can be phosphorylated by the cell, These findings suggest that IC-GFP disrupts dynein transport by competitive binding to p150Glued, and this binding is regulated by serine 84 phosphorylation.
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