Author ORCID Identifier
Document Type
Dissertation
Date of Award
2024
Degree Name
Doctor of Philosophy (PhD)
Department
Basic Biomedical Science
First Advisor
Indra Chandrasekar
Abstract
The traditional functional roles of the actin associated motor protein nonmuscle myosin II (NM2) have been thoroughly studied. However, the nontraditional roles for NM2 in the context of membrane remodeling and protein trafficking events in cells have just recently been unraveled. Our work has found that genetic inactivation of NM2 protein isoforms MYH9 and MYH10 in the renal epithelium of adult mice leads to progressive kidney disease. Aberrant protein transport and localization in cells of the thick ascending limb (TAL) of the kidney nephron is accompanied by upregulation of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) pathway. The major objectives of my thesis work were to 1) confirm cargo protein transport defects in vitro using a novel immortalized TAL cell culture system 2) characterize a TALcell specific Myh9&10 cKO mouse model to conform the cell autonomous role for NM2 proteins in TAL cargo transport and 3) standardize and apply innovative electron and light microscopy methods to further characterize TAL cell structure and protein localization. Cell culture experiments show defects in protein trafficking upon genetic inactivation of Myh9&10 and TAL-specific Myh9&10 inactivation in adult mice leads to progressive kidney disease with TAL protein transport dysfunction and upregulation of ER stress and the UPR pathway. This prompted us to further investigate the ER in TAL cells. However, little information regarding in vivo TAL ER exists in the literature, and in vitro studies do not necessarily recapitulate in vivo ER characteristics. Using innovative electron and light microscopy methods, I have reconstructed and modeled numerous TAL organelles, including the ER, and reveal here in unprecedented detail their unique in vivo structural architecture. Because EM results do not provide information regarding protein identity, I have also established and refined experimental protocols for immunostaining and super-resolution imaging of kidney tissue sections. Structural analysis and protein localization studies show the highly organized nature of the basolateral region of TAL cells, where the ER, mitochondria, and basolateral plasma membrane are often in close proximity. NM2 protein resides in these areas and may be involved in a previously uncharacterized protein transport network whose function is highly dependent upon the complex and dynamic architecture of the basolateral plasma membrane.
Subject Categories
Biology | Cell Biology | Physiology
Keywords
Endoplasmic reticulum, Kidney, MYH9, Nonmuscle myosin II, Organelle architecture, Thick ascending limb
Number of Pages
231
Publisher
University of South Dakota
Recommended Citation
Busselman, Brook, "EXPLORING THE ROLE OF NONMUSCLE MYOSIN II IN RENAL THICK ASCENDING LIMB EPITHELIAL CELL PROTEIN TRAFFICKING AND ORGANELLE ARCHITECTURE" (2024). Dissertations and Theses. 243.
https://red.library.usd.edu/diss-thesis/243