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 emphasis on flagellar dyneins. Dynein is ubiquitous and essential family of microtubule motors that is involved in many vital biological processes such as left-right patterning during development, chromosome segregation, vesicular trafficking, and ciliary and flagellar motility. One of the major questions about dyneins that remains unanswered is: What is the mechanism of dynein isoform targeting and anchoring to cargo? This proposal focuses on the role of dynein intermediate chains in targeting dynein to cargo, taking advantage of the flagella inner arm dynein I1 in Chlamydomonas. The I1 dynein is targeted and anchored to a unique position on the A-microtubule. Additionally, chemical cross-linking studies revealed that the inner 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 that the 90 kDa protein is IC97 then it will be important to map the interaction sites on IC138 and IC140. However, if the 90 kDa protein is a novel protein then the hypothesis that it is part of the docking mechanism for I1 will be tested. These data will define protein interactions within the axonemal inner arm dynein complex I1. The data will also reveal new principles for docking of dynein, leading to a better understanding of human diseases that result from defects in dynein assembly and regulation, such as primary cilia dyskinesia, defective left-right patterning during development, male infertility, and impaired female fertility.