Date of Award

Spring 4-17-2025

Embargo Period

4-24-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Regenerative Medicine and Cell Biology

College

College of Graduate Studies

First Advisor

Amy Engevik

Second Advisor

Jessica Hartman

Third Advisor

Melinda Engevik

Fourth Advisor

Robin Muise-Helmericks

Fifth Advisor

Katherine Chetta

Abstract

Background: Myosin Vb (Myo5b) is an essential molecular motor responsible for delivering diverse cargo to the apical membrane of enterocytes. Mutations in Myo5b cause congenital enteropathies, but its broader role in maintaining the integrity of the intestinal brush border under normal physiological conditions and in disease remain unclear. We hypothesize that Myo5b participates in the assembly and maintenance of the intestinal brush border by transporting members of the intermicrovillar adhesion complex (IMAC) and the xenobiotic efflux pump P-glycoprotein (P-gp) to the apical membrane of enterocytes. We further hypothesize that dysregulation of the myosin chaperone protein, UNC45A, and downregulation of Myo5b play critical roles in Colorectal Adenocarcinoma (COAD). Methods: We used multiple murine models, a swine model, and intestinal organoids that lack functional Myo5b to examine IMAC and P-gp trafficking. Human intestinal tissue was used to determine the localization of P-gp and Myo5b in the setting of intestinal diseases. Functional assays to measure P-gp efflux activity were performed in human organoids and colorectal cancer (CRC) cells. To investigate Myo5b and UNC45A dysregulation in COAD, we used multiple human databases and validated findings in human organoids and CRC cells. Results: In Myo5b deficient models, IMAC components and P-gp were mislocalized from the apical membrane to the subapical domain. Similarly, immunofluorescent staining in human inflammatory bowel disease tissue showed decreased levels of Myo5b and P-gp. The efflux activity of P-gp in CRC cells and organoids was significantly reduced by inhibition of Myosin V and its inhibition decreased cell viability when treated with the chemotherapeutic drug doxorubicin. In COAD, we found that Myo5b mRNA and protein levels were reduced compared to nontumor tissue and identified its promoter region to be hypermethylated in human tissue and in CRC cells. Evaluating UNC45A using the same methods revealed reduced UNC45A protein levels in COAD. We identified upregulated UNC45A targeting miRNAs in COAD tissue and in CRC cell lines. Conclusions: These findings not only define new apical trafficking cargoes for Myo5b but demonstrates its role in maintaining the physical and biochemical barrier of the intestine. Targeting Myo5b mediated pathways could provide new therapeutic strategies for intestinal disorders and cancer.

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