Date of Award

6-2018

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Dr. Pamela Hoppe

Second Advisor

Dr. Wendy Beane

Third Advisor

Dr. Donald Kane

Fourth Advisor

Dr. Blair Szymczyna

Keywords

Myosin, AMPK, thick filament assembly, kinase, sarcomere organization

Abstract

The mechanism of assembly of sarcomeric myosins into elongating thick filaments is not well understood. The results of this study suggest a novel mechanistic model in which the AMPK-related kinase UNC-82, which is orthologous to mammalian ARK5/NUAK1 and SNARK/NUAK2, acts as an assemblase on sarcomeric myosin molecules, mediating their addition into the elongating thick filaments of C. elegans body-wall muscle. The classical genetic crosses performed in this study produced over 53 different worm strains that contain thick-filament-affecting mutations and/or transgenes in single-, double- or triple-mutant combinations. The phenotypes of these strain were documented and analyzed using viability assays, brood analysis, thrashing assays, immunocytochemistry and polarized light microscopy. This in-depth analysis revealed a vital stoichiometric relationship of UNC-82 kinase with the three homologous myosin-related proteins that are the major structural components of the thick filament: myosin A, the minor filament-initiating myosin; myosin B, the major myosin required for locomotion in adult worms; and paramyosin, the headless myosin that forms the core of the long thick filament structure. The importance of UNC-82 protein dosage in proper filament formation suggests a structural role for UNC-82, which is consistent with the large number of amino acids (more than 1200) that lie outside the kinase catalytic domain. Experiments with a mutation that impairs only the catalytic activity of the kinase domain suggest that UNC-82 enzymatic activity is specifically required for addition of paramyosin and myosin A, but not myosin B, to elongating filaments. Using chimeric myosin transgenes that are combinations of sequences from both myosin A and myosin B, we have mapped the myosin-A-specific requirement for UNC-82 to a 531-amino-acid region of the coiled-coil rod. The results of this study expand the understanding of the mechanism of thick filament elongation whereby UNC-82 acts to modulate the interactions between myosins A and B with paramyosin in elongating thick filaments. This new knowledge provides important evidence to support further investigation of the UNC-82/NUAK/ARK5/SNARK protein kinase as a potential drug target in managing cardiac and skeletal related myopathies.

Access Setting

Dissertation-Open Access

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