Date of Defense

4-19-2019

Date of Graduation

4-2019

Department

Biological Sciences

First Advisor

Pamela Hoppe

Second Advisor

Kathryn Docherty

Abstract

The UNC-82 kinase of C. elegans is necessary for proper sarcomere and body wall muscle development, but its mechanism of action is not known. UNC-82 specifically localizes to the M-line and coordinates the organization of the myosin thick filaments in the sarcomeres. UNC-82 is a ~1600 amino acid protein with an N-terminal kinase catalytic domain and a large C-terminal region of unknown function. To elucidate the function of the C-terminal region, we created Caenorhabditis elegans organisms that express a mutant UNC-82 in which the kinase domain cannot be activated. Site-directed mutagenesis of a DNA clone encoding the unc-82 kinase domain produced a mutant version in which the threonine that is phosphorylated in the active kinase was replaced with alanine, which cannot be phosphorylated. We injected this mutant clone with the remaining unc-82 gene into C. elegans gonads and characterized the distribution of the mutant protein in worm progeny expressing the transgene. Defects in the localization pattern of the mutant UNC-82 protein were observed – including discontinuous striation patterns of UNC-82 signal at the M-line, and abnormal round accumulations throughout the myocytes. The disruption in protein patterning is different from that seen with a previously studied catalytically-impaired UNC-82 protein, which localized normally in the presence of wild-type UNC-82 protein and formed abnormal accumulations only when it was the only UNC-82 protein in the cell. An additional difference from the previously studied unc-82 mutants was an increased death rate of F1 generation worms expressing the transgene. A published study in mouse found that expression of the UNC-82 mammalian ortholog SNARK containing the analogous activation loop mutation caused muscle cell death, suggesting that the stimulation of the apoptotic pathway is an additional effect of this mutant. Therefore, our evidence suggests that blocking activation loop phosphorylation of UNC-82 has severe consequences for both UNC-82 protein localization and cell viability that are unlike those seen in other unc-82 mutants. Due to the homology between unc-82 and AMPK-related kinases in vertebrates, we speculate that further research with this transgene will uncover significant findings that could provide further insight into vertebrate (including human) muscle physiology and development.

Access Setting

Honors Thesis-Restricted

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