Development of Novel Bioengineering Tools for Investigating Cell Mechanotransduction in vitro

This dissertation presents research conducted on the design and development of three bioengineering tools for studying cell mechanotransduction on a live imaging platform, and for measuring response to stimuli in real time. These three tools are categorized in the following two areas: 1) dynamically tunable hydrogel substrate to probe cell behavior, and 2) fluorescent imaging markers to measure cell response to external stimuli./nCells live in a complex environment filled with neighboring cells, biochemical signals, and the extracellular matrix (ECM). The ECM is not to be overlooked as simply a structure for cells to live and thrive on. It is a rich treasure trove of information, not only because it harbors a diverse array of chemokines and signaling molecules, but also for its own properties. The ECM provides mechanical cues to direct a variety of cellular behaviors. The study of mechanotransduction is, therefore, aimed at elucidating the biochemical and physical processes through which these mechanical cues are able to bring about certain cell responses./nAlthough the mechanotransduction of cells has been studied to some extend on a population level, we are only beginning to analyze these pathways on a single cell basis. Cell populations can be fairly heterogeneous, and study at the population level often misses the complexity and nuances of cellular response. Furthermore, treatments are often applied to elicit cell behavior hours, if not days, before a response is measured in a population of fixed cells. Cells, on the other hand, can respond to local events on the order of seconds. Without methodologies of study that correspond to this time scale, a lot of details are missed. As such, more imaging tools need to be developed to both probe and measure cell response to mechanical cues on a single cell level in real time./nIn this dissertation, we aim to develop and characterize three bionegineering tools for real time live probing and response measurement on the time scale of seconds. NISO-crosslinked hydrogels allow for temporal, spatial, and magnitude control of stiffness change in the cell’s own substrate. This system is easily incorporated into live imaging platforms, taking advantage of the commonly available 405nm laser line on many fluorescence microscopes to elicit the change in the gel. With the use of confocal microscopy, precise spatial control over area of change is possible. While NISO provides a method of externally controlling the mechanical properties of the extracellular environment, the fLOV2-based fluorescent markers and nesprin tension sensor developed in the subsequent chapters provide further means to measure the response to such external stimuli. fLOV2 is a small plant-derived fluorescent protein, less than half the size of popular GFP-family proteins. Its small size and photophysical properties give it certain distinct advantages. Paired with mRuby2 as a FRET acceptor, the fLOV2/mRuby2 coupling rivals current FRET pairs in many aspects and its reduced size present opportunities for FRET sensors in which steric hindrance is of concern. The nesprin tension sensor aims to advance current understanding of mechanotransduction via physical pathways by directing the study into the nucleus. Together, this set of tools is a powerful kit for furthering understanding of the mechanisms of cell mechanotransduction.

Advisor: Li, Song
Marriott, Gerard