Genetic Analysis of the Regulation of Cytoplasmic Dynein

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Cytoplasmic dynein is a multisubunit complex that functions as a microtubule-associated motor required for nuclear movement and positioning, assembly of the spindle, chromosome segregation, organization of Golgi, ER to Golgi trafficking, and retrograde transport of organelles in axons. An additional multisubunit complex known as dynactin is required for dynein function and interaction with various cargoes. The dynein heavy chain is one of the largest proteins in the cell (4000 residues) with the C-terminal two-thirds forming the motor domain and the N-terminal region forming an extended tail that functions in cargo interaction. Cytoplasmic dynein function is essential in metazoans (e.g., mouse and Drosophila); however, in filamentous fungi, dynein is not essential for viability and this has greatly simplified genetic analysis of this enormous complex. Using the filamentous fungus Neurospora crassa (for which the complete genome sequence was recently published), 1000 mutants defective for cytoplasmic dynein or dynactin function have been isolated, including 300 independent dynein heavy chain mutants. Sequence analysis of cloned dynein/dynactin genes as well as the determination of the entire N. crassa genome sequence has revealed that this complex is highly conserved in N. crassa relative to metazoans supporting the use of N. crass as a model system for the analysis of dynein structure, function and regulation. In the work to be done under this award, the vast array of N. crassa dynein mutants will be used to explore the regulation of dynein motor activity. Previous work has shown that N. crassa cytoplasmic dynein motor ATPase activity is inactivated by phosphorylation in mutants defective in dynactin function (i.e., cargo interaction) or function of the LIS1 complex. This inactivation occurs not by phosphorylation of the dynein heavy chain, but by phosphorylation of small proteins (8- and 20-kDa) associated with the heavy chain. To determine the identity of these proteins, dynein heavy chain will be tagged, affinity purified and the associated proteins will be resolved by 2D gels. The heavy chain- associated polypeptides will be excised from the 2D gels and their identity determined by using mass spectrometry analysis in conjunction with the complete N. crassa genome sequence. The effects of null mutations in the genes encoding these dynein-associated subunits will be determined by assaying dynein ATPase activity. Additional work will focus on defining the effects of specific heavy chain mutations. A minimum of 100 independent dynein heavy chain mutations will be identified for those mutants that produce full-length polypeptide. These mutants will be examined for defects in dynein ATPase activity, microtubule binding and association with other dynein subunits. The information from this analysis will complement the work described above and will provide insight into the mechanism by which the dynein motor translocates along microtubules. Cytoplasmic dynein is the most complicated motor operating in the cytoplasm, and N. crassa represents the only model organism in which it is possible to rapidly isolate hundreds of mutants defective in dynein function. Completion of this work will be of great benefit in understanding the regulation of the dynein motor activity, and it will provide the first large-scale genetic dissection of the dynein heavy chain. Undergraduate students of various minority groups have played important roles in current and past research activities, and additional students will be encouraged to participate in these research efforts.