Dyneins are motor proteins that move cellular cargos along microtubules toward their minus ends. There are two sub-classes of dynein. Cytoplasmic dynein is a single isoform that is involved in various essential cellular functions, including retrograde vesicle transport in the cytoplasm and in nerve axons, organellar movement, orientation of the mitotic spindle, disassembly of centromeric checkpoint proteins, and nuclear migration. Axonemal dynein occurs as approximately 12 isoforms, encoded by distinct genes, that are responsible for the beating movements of cilia and sperm flagella. In all dyneins the motor unit consists of six AAA protomers disposed in tandem on a single """"""""heavy chain"""""""" polypeptide of unusually high molecular mass (>500 kDa). These AAA protomers form a hexameric ring from which the microtubule-binding domain protrudes on a short stalk. Binding and hydrolysis of ATP at certain of the AAA protomers is believed to cause cyclic changes in the angle of the protruding stalk and so translocate the dynein along the microtubule to which it is attached. Sequence analysis suggests that the structure of the AAA protomers in dynein resembles that of other members of the AAA ATPase family, with their closest relationship being to AAA protomers of the recently discovered protein, midasin. The long-term goal of this project is to understand the relationship between structure and function in dynein isoforms.
The specific aims are to use techniques of cell and molecular biology: 1) to express individual structural domains of dynein in bacteria or in eukaryote cell cultures in order to obtain relatively low molecular weight forms of these proteins that retain subsets of their functional properties; 2) to use site-directed mutagenesis of the dynein motor to characterize the cooperative ATP-dependent structural changes in the AAA protomers and microtubule-binding stalk that are believed to underlie the ability of dynein to translocate along a microtubule; 3) to use antibodies to define an experimental map of the motor surface and of the changes that occur during the ATPase cycle; 4) to relate the structural and enzymatic properties of the AAA protomers in dynein to those of the AAA protomers in midasin. Defects in dynein underlie many human health problems, including male infertility, bronchiactosis, lissencephaly, primary cilia diskinesia, and defective retrograde transport in nerve axons that can result in amyotrophic lateral sclerosis (ALS).
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