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Dissertation
Intracellular pressure and myosin II activity control epithelial tissue integrity and 3D mesenchymal cell migration
Doctor of Philosophy (Ph.D.), Drexel University
Dec 2021
DOI:
https://doi.org/10.17918/00000896
Abstract
Intracellular pressure can drive changes in cell morphology and behavior. Intracellular pressure is a function of non-muscle myosin II (NMII), which causes increased pressure due to the squeezing of the cytoplasm by actomyosin filaments. Our lab has previously shown that precise compartmentalization of intracellular pressure by a nuclear piston drives 3D migration in dermal fibroblasts through crosslinked matrices. However, there are still many unknowns about the generation and role of intracellular pressure in cell migration. In this thesis, I explore how myosin II and intracellular pressure-based changes drive epithelial cell function and 3D mesenchymal migration. First, I investigated the role of intracellular pressure in epithelial MDCK cells which we found to have significantly higher cytoplasmic pressure compared to dermal fibroblasts. We tested the hypothesis that changes in pressure occur concurrently as cells undergo epithelial-mesenchymal transition (EMT) and that high pressure maintains epithelial phenotypes. We find that triggering EMT in MDCK cells using hepatocyte growth factor (HGF) significantly lowers intracellular pressure, which corresponds with lamellipodia formation and increased cellular velocity. We find that inhibition of NMII causes similar decrease in pressure and increase in lamellipodia and velocity, which suggests that these changes in epithelial cell motility that occur through decreased pressure are NMII-dependent. Conversely, increasing intracellular pressure through treatment with hypo-osmotic media abrogates changes caused by HGF treatment, suggesting that changes in pressure are necessary for MDCK cells to become more motile in nature. In addition, we find that low pressure causes increased paracellular flow of ions between adjacent cells, suggesting that high pressure maintains epithelial barrier function. Altogether, these data suggest that myosin II regulation of intracellular pressure in epithelial cells is necessary for maintaining epithelial phenotypes, such as barrier function and low motility and that pressure decreases as cells undergo EMT and become more mesenchymal and motile. In the second portion of my thesis, I investigated the role of the cytolinker protein plectin in driving nuclear translocation during 3D migration by connecting NMII-generated force to the nucleus. Previous work from our lab showed that vimentin intermediate filaments and actomyosin filaments work in concert to drive 3D nuclear translocation in dermal fibroblasts migrating through crosslinked matrices. However, we did not know how these networks interacted or how exactly NMII force was linked to the nucleus. Through my work, I found that actin and vimentin are crosslinked through plectin in a myosin II-dependent manner in 2D fibroblasts. These plectin-vimentin interactions are mechanosensitive and respond to focal adhesion engagement, NMII contractility, and substrate stiffness. This mechanosensitive 2D plectin complex slows down 2D migration, but is critical for nuclear translocation during 3D migration in both crosslinked and non-crosslinked matrices, the former of which triggers nuclear piston-based migration. Specifically, plectin plays a role in compartmentalization of pressure and loss of plectin causes pressure to be equalized within 3D fibroblasts. Lastly, we found that plectin organized vimentin filaments perinuclearly and polarized focal adhesions and NMII in 3D fibroblasts to drive nuclear piston-based migration. Altogether, these data suggest that plectin is critical for the nuclear piston machinery as it organizes the cytoskeleton and aids in compartmentalization of pressure to drive 3D migration.
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Details
- Title
- Intracellular pressure and myosin II activity control epithelial tissue integrity and 3D mesenchymal cell migration
- Creators
- Pragati Chengappa Marks
- Contributors
- Ryan J. Petrie (Advisor)Elias Spiliotis (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- x, 142 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Biology; College of Arts and Sciences; Drexel University
- Other Identifier
- 991015757596604721