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MG53 DEFICIENCY DELAYS WOUND HEALING
by
Matthew Orange
B.S., Gettysburg College - 2005
Thesis Advisor: Jianjie Ma, Ph.D.
Graduate Program in Physiology and Integrative Biology
School of Public Health
2nd Floor Conference Room 258
Piscataway
Tuesday, July 31, 2012
10:00 a.m.
Abstract
Plasma membrane repair is more than a passive process. Experimental observations in various cell types show that membrane interruptions too large to be thermodynamically closed are repaired by a protein mediated mechanism. Mitsugumin 53 (MG53) is a protein of the membrane repair machinery in striated muscle fibers. In skeletal muscle, it protects against damaging forces and deficiency causes myopathy. In cardiac muscle, MG53 confers a protective effect against ischemia-reperfusion injury. While the cardiac and skeletal muscle phenotype of MG53 deficient mice (mg53-/-) is characterized, little is known about healing of non-muscle tissues in this mouse. We investigated the function of MG53 in keratinocytes and its contribution to skin wound healing. The presence of extracellular recombinant human MG53 (rhMG53) protected primary human keratinocytes from mechanical damage. Expression of GFP-MG53 in cultured human keratinocytes confirmed that the protein responded to chemically or mechanically induced membrane damage in the same fashion as that previously observed in muscle fibers. While Western blot of protein from mouse skin showed MG53 expression, it was not expressed in human or mouse keratinocytes. Immunohistochemistry revealed that expression of MG53 in mouse skin was specific to the panniculus carnosus, a subdermal striated muscle layer that has limited distribution throughout human skin. Excisional wounding revealed delayed healing of mg53-/- mice when compared to wild-type. Our secondary objective was to investigate complementary function of MG53 and dysferlin, another sarcolemma repair protein that fuses MG53 delivered intracellular vesicles to the membrane at injury sites. The double knockout mouse (mg53-/-dysf-/-) displayed additive myopathy and repair defect, indicating the functions of MG53 and dysferlin are more complicated than that of two points along a linear pathway to repair. We hypothesized that the proteins repaired membrane compartments other than sarcolemma, specifically the transverse tubule (t-tubule) membrane. MG53 deficient muscle fibers were unable to survive osmotic stress designed to belabor the t-tubule membrane. Our data shows that MG53 is more than a sarcolemma repair protein, as its presence affects the ability of skin to heal and of t-tubules to deal with osmotic stress.
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