MOLECULAR GENETICS OF CYTOPLASMIC DYNEIN

Univ Of Massachusetts Med Sch Worcester, Worcester MA

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Abstract

Cytoplasmic dynein is a microtubule motor protein thought to be responsible for many aspects of intracellular motility. This laboratory described cytoplasmic dynein as multisubunit complex responsible for retrograde, or minus end-directed force production. In the first project period of this grant we have cloned and characterized all of its subunits. We have also cloned and characterized subunits of the multisubunit dynactin complex, which has been implicated in cytoplasmic dynein function, though its mechanism of action is unknown. We will use the recombinant wild-type and mutant probes and methods developed during the preceding project period to address the major outstanding issues regarding cytoplasmic dynein function: its full range of functions, how it is targeted to specific organelles, how its activity and distribution are regulated, and how it produces force. We will define the motor domain of the heavy chain using truncation and point mutant forms of the HC expressed in Hi-5 insect cells and COS-7 mammalian cells. We will define the tertiary structural organization of the molecule by defining subunit interaction and by ultrastructural means. We will determine the function of the putative nucleotidase domain of the cytoplasmic dynein light intermediate chains to determine their role in cytoplasmic dynein regulation. We will explore the function and interaction between cytoplasmic dynein and dynactin in cells overexpressing the p5O subunit of dynactin and other wild-type and mutant subunits in transiently and stably transfected mammalian cells. We will define the stage of mitosis at which cytoplasmic dynein acts and the range of organelle types under cytoplasmic dynein control. We will determine whether dynactin mediates the interaction of cytoplasmic dynein with organelles and kinetochores, or whether it regulates cytoplasmic dynein activity. Finally, we will identify polypeptides responsible for specificity in the subcellular targeting of cytoplasmic dynein. Understanding the range of functions and the mechanism of action of cytoplasmic dynein are of considerable importance to issues of human health. For example, cytoplasmic dynein appears to be responsible for retrograde axonal transport, which is required for growth factor activity, and, therefore, neuronal viability and may be important in the etiology of neurodegenerative diseases. Recent evidence suggests an involvement in brain developmental defects. Cytoplasmic dynein may also have a role in some aspects of mitosis, and, as such, may represent a potential target for the chemotherapeutic treatment of neoplastic diseases.