STRUCTURE AND FUNCTION OF CYTOPLASMIC DYNEIN

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Microtubule-based motility is critical to the life and growth of both individual cells and multicellular organisms. Malfunctions of the microtubule-based machines that provide for intracellular transport influence global aspects of cell behavior including chromosome segregation, cell division, ciliary and flagellar motility, endocytotic and secretory vesicle traffic, the organization of the cytoplasmic compartment, and the establishment of cell polarity. The interrelationships between these cellular behaviors and mechanisms of cellular differentiation and tissue morphogenesis are continually being recognized and are reflected in numerous medical problems including cancer, congenital chromosomal syndromes and birth defects. At a basic level, the function and regulation of microtubule-based machines is built into the associated motor enzymes that power them. This proposal seeks to understand the structure and function of the ubiquitous microtubule motor, cytoplasmic dynein. During the past grant period, we have conducted a biochemical, molecular, and genetic characterization of Drosophila cytoplasmic dynein heavy chain. In the next grant period, we propose to further study the structural and functional domains of the intrinsic components of the dynein motor complex, including the heavy, intermediate, and light chain polypeptides. Our cloning and characterization of the Dhc64C gene, as well as the isolation of recessive lethal mutations, has defined a functional dynein heavy chain (DHC) transcription unit. We will now modify this transcription unit to express altered DHC transgenes in vivo. The functional and biochemical properties of altered DHCs will be analyzed in vivo and in vitro to provide a molecular domain map of the dynein polypeptide. In addition, the DNA lesions of dynein mutations previously isolated in vivo by genetic screens will reveal new domains of functional significance within the DHC. The proposed analysis of the Drosophila 74 kD intermediate-, and 50-60 kD light chain genes in a developmental context will shed light on the role of these subunits in regulating the assembly and subcellular targeting of the dynein motor complex. We will clone the genes, analyze the distribution of their transcripts, and initiate a domain analysis of the gene products. The cytogenetic mapping of the genes and the genetic characterization of the loci will be pursued as necessary background for the future mutational analyses. Genetic analysis will be used to elucidate the full range of dynein function and to identify to novel genes and gene products that are required for dynein function. We will isolate and analyze extragenic enhancers and suppressors of the rough eye phenotype of the Glued and/or Dhc64C mutations. The proposed screens will capitalize on the ease of scoring defects in eye development and provide an excellent opportunity to investigate the participation of the dynein motor function in the coordinated nuclear movements, the regulation of cell division, and/or the signal transduction mechanisms that coincide with and/or direct the proper differentiation of eye.