Date of Defense

5-1-2015

Date of Graduation

7-2015

Department

Biological Sciences

First Advisor

Pamela Hoppe

Second Advisor

Cindy Linn

Third Advisor

NaTasha Schiller

Abstract

In the striated muscle cells of the organism Caenorhabditis elegans, both UNC-82 and UNC-89 proteins are located at the M-line and are required for proper assembly of thick filament proteins. I suggest that UNC-89 is the enzyme that phosphorylates UNC-82, activating it to organize paramyosin and myosin into filaments. To test this, I created novel double mutant strains with different mutations in the UNC-89 and UNC-82 kinases. Alleles include unc-82(e1323), which has a premature stop codon in the kinase domain that likely results in a protein null, and unc-82(e1220), which is a missense allele making a presumed kinase-dead product. I crossed these lines with unc-89(su75) worms, which express only UNC-89 isoforms that lack the kinase domain, and whose muscle cells contain birefringent paramyosin aggregates (Guy Benian, personal communication). Polarized light microscopy was used to visualize birefringent organized thick filament proteins in muscle. UV light was used to track localization of a full-length UNC-82::GFP fusion construct as well as a full-length myosin A::GFP transgene. My data demonstrate that UNC-82::GFP localizes to the abnormal paramyosin aggregates. Antibody staining also was utilized to observe the location of paramyosin and myosin. My data is consistent with a linear phosphorylation pathway acting on UNC-89, UNC-82 and paramyosin. I also found that double mutants null UNC-89 and null UNC-82 could not create a viable line, suggesting that both proteins play a vital role in proper myosin organization. I recommend further studies of thick filament organization with the creation of a kinase-dead UNC-89 allele.

Access Setting

Honors Thesis-Restricted

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