Pilot--Assembly and Regulation of Dynein: Role of the Intermediate Chains

Morehouse College, Atlanta GA

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Abstract

The overall goal of my research is to determine the mechanism and regulation of dynein with particular[unreadable] emphasis on flagellar dyneins. Dynein is ubiquitous and essential family of microtubule motors that is[unreadable] involved in many vital biological processes such as left-right patterning during development, chromosome[unreadable] segregation, vesicular trafficking, and ciliary and flagellar motility. One of the major questions about dyneins[unreadable] that remains unanswered is: What is the mechanism of dynein isoform targeting and anchoring to cargo?[unreadable] This proposal focuses on the role of dynein intermediate chains in targeting dynein to cargo, taking[unreadable] advantage of the flagella inner arm dynein I1 in Chlamydomonas. The I1 dynein is targeted and anchored to[unreadable] a unique position on the A-microtubule. Additionally, chemical cross-linking studies revealed that the inner[unreadable] arm dynein I1 intermediate chains IC138 and IC140 each independently interact with a third 90 kDa protein. I postulate that the 90 kDa protein is either the third intermediate chain in I1 IC97,or more likely, a novel protein that mediated the docking of the I1 dynein complex to the microtubule. To test these ideas three aims are proposed: (1) Identify the 90 kDa protein. A novel enrichment procedure will be used to purify the cross-linked product and tandem mass spectrometry will be performed to identify the protein. Possible outcomes include the interacting protein is a previously unidentified protein, or the interacting protein is the third intermediate chain dynein in I1, IC97. (2) If the 90 kDa protein is a novel protein the gene will be cloned, mapped and antibodies generated. (3) If the mass spectrometry data reveal[unreadable] that the 90 kDa protein is IC97 then it will be important to map the interaction sites on IC138 and IC140.[unreadable] However, if the 90 kDa protein is a novel protein then the hypothesis that it is part of the docking mechanism[unreadable] for I1 will be tested. These data will define protein interactions within the axonemal inner arm dynein[unreadable] complex I1. The data will also reveal new principles for docking of dynein, leading to a better understanding[unreadable] of human diseases that result from defects in dynein assembly and regulation, such as primary cilia[unreadable] dyskinesia, defective left-right patterning during development, male infertility, and impaired female fertility.