Date of Award

Spring 5-1-2025

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Committee Advisor

Clarissa Henry

Second Committee Member

Samuel T Hess

Third Committee Member

Melody Neely

Additional Committee Members

Greg Cox

Ben King

Abstract

Healthy muscle fibers move bone by contracting and transducing force to the extracellular matrix in the tendon region, which connects to bone, producing movement. In the extracellular matrix, laminin proteins bind to integrin and dystroglycan proteins, which span the muscle cell membrane and bind to the actin cytoskeleton inside the muscle cell. This connection facilitates cellular communication and is required for muscle attachment. But in muscular dystrophy and dystroglycanopathies, progressive diseases that affect both muscle and neurological health, muscle fibers detach from the extracellular matrix and fail to function properly. We found that laminin deposition in the extracellular matrix and muscle health improved after oxidized nicotinamide adenine dinucleotide (NAD+) treatment of dystroglycan mutant and integrin mutant zebrafish muscular dystrophy models. But in one dystroglycanopathy mutant, lacking dystroglycan glycosylation, NAD+ did not improve muscle health. Glycosylation is known to influence protein localization and clustering. Therefore, we hypothesized that mechanistically, NAD+ alters the nanoscale localization of dystroglycan and integrin and that their nanoscale localization is crucial for muscle attachment. To measure subcellular localization of these laminin receptors in whole, intact zebrafish muscle, which had not been done before, we tested the feasibility of fluorescence photoactivation localization microscopy (FPALM). Results were consistent with confocal data. We used FPALM to measure dystroglycan organization in a zebrafish model of integrin α7 muscular dystrophy. Preliminary FPALM data indicated smaller dystroglycan cluster areas in integrin α7 mutants compared to non-mutants. To further investigate membrane protein organization and neuromuscular health, we generated a tetraspanin CD151 mutant zebrafish. CD151 creates membrane microdomains and binds to integrins but has been ignored in muscle research. Preliminary data showed that CD151 mutants have altered neuromuscular structure and minor muscle defects, and that overexpression of CD151 increased aberrant muscle fiber boundary crossings in putative dystroglycan mutant.

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