DOI

https://doi.org/10.25772/PW2D-9Q93

Author ORCID Identifier

https://orcid.org/0000-0002-5358-8472

Defense Date

2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biomedical Engineering

First Advisor

Dr. Daniel E. Conway

Second Advisor

Dr. Jason Gleghorn

Third Advisor

Dr. Rebecca Heise

Fourth Advisor

Dr. Christopher Lemmon

Fifth Advisor

Dr. Gregory Walsh

Abstract

Recent advances in three-dimensional (3D) cell culture systems have provided key insights into the understanding of biochemical and physiological states of native tissue. A significant progress in the field of mechanobiology involves measuring cellular traction forces in a more native 3D environment. However, the effects of mechanical forces exerted across cellular junctions and the nuclear LINC complex, in an organized 3D system has not been investigated thus far. Epithelial cells spontaneously form acini (also known as cysts or spheroids) with a single, fluid-filled central lumen, when grown in 3D matrices. The size of the lumen is dependent on apical secretion of chloride ions, most notably by the CFTR channel, which has been suggested to establish pressure in the lumen due to water influx. In this work, we hypothesized that stretch induced via osmotic pressure is a major generator of increased cell-cell junction force in epithelial acini. Using FRET-force biosensors for E-cadherin we observed significant increases in the average tension per molecule for each protein in mature 3D acini as compared to 2D monolayers. Increases in CFTR activity resulted in increased E-cadherin forces, indicating that ionic gradients affect cellular tension. Direct measurements of pressure revealed that mature acini experience significant internal hydrostatic pressure (37 +/- 10.9 Pa). Changes in CFTR activity resulted in pressure and/or volume changes, both which affect E-cadherin tension. Increases in CFTR chloride secretion also induced YAP signaling and cellular proliferation. In order to recapitulate disruption of acinar homeostasis, we induced epithelial to mesenchymal transition (EMT). During the initial stages of EMT, there was a gradual decrease in E-cadherin force and lumen pressure that correlated with lumen infilling. Strikingly, increasing CFTR activity was sufficient to block EMT. Our results show that ion secretion, coupled with increased cellular tension, is an important regulator of morphogenesis and homeostasis in epithelial acini. Desmosomal cadherins are another important component of cell-cell junctions in many tissues. Loss or impairment of desmosomes presents with clinical phenotypes in the heart and skin as cardiac arrhythmias and skin blistering, respectively. Because heart and skin are tissues that are routinely subjected to large mechanical stresses, we hypothesized that desmosomes, similar to adherens junctions, would also experience significant tensile loading. To directly measure mechanical forces across desmosomes, we developed and validated a FRET-force biosensor. When expressed in human cardiomyocytes, the force sensor reported high tensile loading of DSG-2 during contraction. Additionally, when expressed in Madin-Darby canine kidney (MDCK-2) epithelial or epidermal (A431) monolayers, the sensor also reported tensile loading. Finally, we observed higher DSG-2 forces in 3D MDCK-2 acini when compared to 2D monolayers. Another critical structure of the cell is the linker of nucleoskeleton and cytoskeleton (LINC) complex that has recently been transpired as a potent mechano-sensor. Mutations in LINC complex components may be relevant to cancer, but how cell-level changes might translate into tissue-level malignancy is unclear. We hypothesized that the LINC complex was a necessary component for epithelial function. In this work, we show that inducible LINC complex disruption via (DN)-KASH1 in human mammary epithelial MCF-10A cells and canine kidney epithelial MDCK-2 cells mechanically destabilizes the acinus, suggesting that the LINC complex is necessary for acinar equilibrium. We observed that increased mechanical tension due to up regulation of Rho-kinase-dependent non-muscle myosin II activity coupled with asymmetric mitotic spindle orientation results in lumenal collapse. Additionally, cells expressing DN-KASH1 exhibited slower migration speeds in response to a scratch wound cell migration assay, consistent with prior reports in fibroblasts. Collectively, our findings provide a potential mechanistic explanation for how disruption of LINC complex may contribute to a loss of mechanical integrity in the epithelia, and how tensile forces across cell-cell junctions is an important regulator of epithelial organization and maintenance of homeostasis.

Rights

© Vani Narayanan

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

4-3-2020

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