Date of Award

Fall 11-2014

Embargo Period

7-19-2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Mechanical Engineering

Advisor(s)

Philip LeDuc

Abstract

Cell-substrate interactions influence various cellular processes such as morphology, motility, proliferation and differentiation. Actin dynamics within cells have been shown to be influenced by substrate stiffness, as NIH 3T3 fibroblasts grown on stiffer substrates tend to exhibit more prominent actin stress fiber formation. Circular dorsal ruffles (CDRs) are transient actin-rich ring-like structures within cells, induced by various growth factors, such as the platelet-derived growth factor (PDGF). CDRs grow and shrink in size after cells are stimulated with PDGF, eventually disappearing ten of minutes after stimulation. As substrate stiffness affect actin structures and cell motility, and CDRs are actin structures which have been previously linked to cell motility and macropinocytosis, the role of substrate stiffness on the properties of CDRs in NIH 3T3 fibroblasts and how they proceed to affect cell behavior is investigated. Cells were seeded on Poly-dimethylsiloxane (PDMS) substrates of various stiffnesses and stimulated with PDGF to induce CDR formation. It was found that an increase in substrate stiffness increases the lifetime of CDRs, but did not affect their size. A mathematical model of the signaling pathways involved in CDR formation is developed to provide insight into this lifetime and size dependence, and is linked to substrate stiffness via Rac-Rho antagonism. CDR formation did not affect the motility of cells seeded on 10 kPa stiff substrates, but is shown to increase localized lamellipodia formation in the cell via the diffusion of actin from the CDRs to the lamellipodia. To further probe the influence of cell-substrate interactions on cell behavior and actin dynamics, a two dimensional system which introduces a dynamically changing, reversible and localized substrate stiffness environment is constructed. Cells are seeded on top of thin PDMS nano-membranes, and are capable of feeling through the thin layer, experiencing the stiffness of the polyacrylamide substrates below the nano-membrane. The membranes are carefully re-transplanted on top of other polyacrylamide substrates with differing stiffnesses. This reversible dynamic stiffness system is a novel approach which would help in the investigation of the influence of reversible dynamic stiffness environments on cell morphology, motility, proliferation and differentiation in various cells types.

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