Molecular Genetic Analysis of the Role of M-Line Proteins in Thick Filament Assembly and Attachment in C. Elegans

Date of Award


Degree Name

Doctor of Philosophy


Biological Sciences

First Advisor

Dr. Pamela Hoppe

Second Advisor

Dr. Todd Barkman

Third Advisor

Dr. Jian Yao

Fourth Advisor

Dr. Jennifer Schisa


C.elegens, muscle development, UNC-82 kinase, UNC-89/obscurin, thick filament assembly, M-line proteins


The M-line, a component of the sarcomere unit of striated muscle tissue, plays a crucial role in muscle contraction and therefore in locomotion in nematodes and other organisms. Mutations affecting M-line proteins have phenotypes that are consistent with the M-line functioning as the site of attachment for the myosin-containing thick filaments. In C. elegans the central portion of the thick filament, which attaches to the M-line, is formed by myosin A. The mechanism of thick filament attachment to the M-line in vertebrates and in C. elegans has not been fully defined. Mutations in several different M-line proteins, key among them UNC-82 kinase, UNC-98/ZnF, UNC-96, and UNC-89/obscurin, disrupt thick filament structure, suggesting that they play a role in guiding assembly of the thick filament itself. The objective of this study was to determine the role of different M-line proteins in building myosin filaments or mediating the attachment of the filaments to the M-line structure in striated muscle. The molecular components that link myosin A to the large structural M-line component UNC- 89/obscurin were investigated using in vivo assays testing the ability of UNC-89/obscurin to associate with myosin A within the contractile apparatus or in ectopic accumulations of filamentous myosin A in different mutant backgrounds. The study involved performing classical genetic crosses and immunocytochemistry.

The results showed that UNC-98/ZnF, but not UNC-82 kinase or UNC-96, is required for association of UNC-89/obscurin with ectopic myosin structures. However, UNC-89/obscurin localization to the M-line in the contractile apparatus can occur independently of UNC-98/ZnF and the UNC-98/ZnF-binding site at the C-terminus of myosin A. The study also resolved that UNC-89/obscurin organization and localization during embryonic elongation cannot occur normally in the absence of myosin A. Experiments using chimeric transgenes revealed that UNC-82 kinase interacts with the N-terminal region of the myosin A rod while UNC-98/ZnF interacts with the C-terminal end of the rod.

The present study also aimed to identify a kinase involved in the activation of unc-82- kinase, an important enzyme in the development of the muscle in C. elegans whose activation system is still unknown. For that, some known kinases of this worm were selected following different reasoning and then tested as potential activators. Some kinases were selected based on the level of expression of these genes in nematode muscle cells and other ones, based on the bibliographic review of enzymes that activate mammalian kinases orthologous to unc-82-kinase. Morphological inspection of muscle tissue in mutants of the candidate kinases found none that presented atypical morphology in the muscle tissue, and therefore it was not possible to suggest a link between them and the activation of unc-82. AMPK-related kinases are activated by phosphorylation of threonine in the activation loop by an upstream kinase. The effect of blocking phosphorylation of the UNC-82 activation loop by making a T to A mutation using either CRISPR or extrachromosomal array technology produced the unexpected result of lethality. The observation that unphosphorylated UNC-82 causes lethality suggests that UNC-82 may always be phosphorylated, perhaps by multiple different kinases, or auto-phosphorylation or kinome reprogramming.

Access Setting

Dissertation-Abstract Only

Restricted to Campus until


This document is currently not available here.