Understanding how mechanical forces direct tissue organization during development as well as in homeostasis and disease, are among the big unsolved questions in biology and regenerative medicine [1–15]. Fluorescence microscopy has proven to be particularly useful in unraveling these mysteries [16]. To enhance the usefulness of fluorescence microscopy, fluorescence probes that respond to mechanical strain or tension are of particular interest [17–20]. In this thesis we focus on issues related to the design of, and the interpretation of data produced by fluorescent reporters of mechanical strain or tension.
Specific aim 1 (chapter 3)
Explore the possibility of using mechanical force to disturb the fluorescence of a GFP molecule. We describe computer simulations of the GFP molecule where a computer model of the GFP molecule was subjected to various amounts of mechanical tension. We found that depending on the amount of tension we applied, the GFP molecule changed its conformation in fairly specific ways that resulted in tension-dependent stable structures. The end-to-end distances of the protein termini in these states were found to be in good agreement with experiments performed with an atomic force microscope [21].
To determine if these different structures have different fluorescence properties we engineered
mutations of two popular varieties of GFP: EGFP and EYFP. The mutations were designed to mimic the structures of the molecule as seen in the computer model when it was subjected to mechanical tension. We found that the fluorescence properties of the mutants changed differently depending on the variety of GFP that we started with. The detailed information that we were able to derive from the simulations and genetically engineered mutants may be useful in the design of FRET sensors that use GFP-based molecules for the fluorescent donor and acceptor as well as in the design of novel tension or strain sensors that use only single GFP molecules.
Specific aim 2 (chapter 4)
Examine how FRET measurements are made and interpreted when using the fluorescence lifetime approach with a pulsed laser and photon counting detectors where the key measurement parameter is the average arrival time of the photons. We theoretically analyze a particular case consisting of a semi-stiff polymer with donors placed randomly along the polymer with an acceptor placed at its end. We show how to interpret measurements of the average arrival time of the photons in terms of the length of the polymer, and then show how the model may be extended to more general cases. This interpretation of the average arrival time of the photons allows length measurements of polymers having lengths significantly longer than polymers labeled with single donor and acceptor fluorescent molecules. This model may be applied to a number of systems of biological interest including double stranded DNA as well as certain long and relatively stiff protein molecules such as the extracellular matrix protein fibronectin.
Specific aim 3 (chapter 5)
Apply the semi-stiff polymer FRET model to the extracellular matrix protein fibronectin which has been labeled so that the FRET signal changes in response to changes in the molecule's conformation [17,22]. Fibronectin's four free cysteines are labeled with FRET acceptors and the extensive free lysines are randomly labeled with FRET donors. We show that the FRET measurements of fibronectin monomers interpreted according to the model presented in chapter 4 result in contour lengths that are in good agreement with contour lengths of fibronectin obtained by imaging molecules adsorbed to
surfaces with electron microscopy and then tracing their length [23,24].
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