Nuclear pushing and pulling in three-dimensional matrices

James Matthew Cowan

doi:10.17918/00011170

Cells migrating through complex three-dimensional (3D) environments must solve the problem of how to move the large and relatively stiff nucleus through confining spaces. A range of mechanisms have evolved as solutions, varying by cell type. However, it is unclear to what extent cells can access mechanisms used by other cell types, or what factors may trigger use of a particular mechanism. Gliomas, a pathological cell type, often acquire the aberrant and highly lethal facility for robust and adaptable migration. In 3D, their mesenchymal phenotype, requirement for non-muscle myosin II (NMII) contractility, and potential use of a NMII-mediated contractile mechanism to pull their nucleus forward raised the question of whether glioma cells are able to migrate using a similar nuclear-pulling mechanism as dermal fibroblasts. In this thesis I examine how glioma stem-like cells (GSCs) translocate their nucleus in dense 3D extracellular matrix and compare their movement to the well characterized fibroblast nuclear-pulling mechanism. In chapter 1, I discuss our current understanding of various molecular, mechanical and environmental factors that govern cell migration in 3D, with a particular focus on how cells move the nucleus in confined space. In chapter 2, I detail methodologies that enable the side-by-side comparison of GSCs with dermal fibroblasts, including GSC culturing technique, an assay to monitor and thereby avoid cell differentiation, and a procedure for embedding cells in 3D matrix for live-cell microscopic imaging. In chapter 3, I test whether GSCs use a nuclear-pulling mechanism similar to dermal fibroblasts and find that in 3D collagen matrices GSCs use rear-contractility to push the nucleus forward. I additionally find that activation of mechanosensitive ion channels increases cell contractility in both GSCs and fibroblasts without inducing a migratory mode switch, and whereas fibroblasts gain an increased ability to remodel matrix, GSCs do not exhibit similar increased capability. This suggests in GSCs a relatively lower exertion of traction forces on the matrix, and points to integrin-based adhesiveness as a possible factor that enables activation of a nuclear pulling mechanism. Finally, in chapter 4, I further consider the questions raised by the findings in chapter 3, note gaps and areas for future investigation, and contextualize the work of this thesis in a broader context.

Nuclear pushing and pulling in three-dimensional matrices

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Nuclear pushing and pulling in three-dimensional matrices

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