UBC Theses and Dissertations
Interpretation of fluorescence microscopy experiments on cell surface receptor dynamics with stochastic and deterministic mathematical models Herrera Reyes, Alejandra Donaji
Fluorescence microscopy has provided cellular biologists with quantifiable data, that can be paired with mathematical models to discover the mechanics of the imaged processes. We developed mathematical models to analyze data from two fluorescence techniques: direct Stochastic Optical Reconstruction Microscopy (dSTORM) and fluorescence recovery after photobleaching (FRAP). dSTORM is a super-resolution technique that uses photo-switchable fluorophores to achieve nanometer resolution images, allowing us to visualize the organization of proteins at nano-scales. However, dSTORM images can suffer from recording a single photo-switchable fluorophore multiple times, possibly creating artificial features. This is specially relevant in the analysis of membrane B-cell receptors clustering, where spatial clustering might relate to immune activation. I developed a protocol to estimate the number of unique fluorophores present in the experiment by coupling their temporal (with a Markov-chain model) and spatial (with a Gaussian mixture model) dynamics within a maximum likelihood framework. Previous studies have used the temporal information, but they have not coupled it with the spatial information (both localization and localization estimation error). I tested my protocol on simulated data, well-characterized DNA origami data and B-cell receptor data with positive results. My model is general enough to apply to other biological systems besides B-cell data and will enhance a microscopy technique that is widely used in biological applications. FRAP can be used to quantify the mobility of membrane proteins. We used it on live Drosophila organisms to study the outside-in pathway in cell adhesion to the extracellular matrix (ECM). We developed an ODE model to describe the recycling of the membrane protein, integrin, in charge of the adhesions. We found that both integrin and ECM ligands stabilize outside-in signalling and that relevant chemical treatments do not balance mutant integrin activation but stabilize the adhesions in control organisms. We also analyzed inside-out activation with a similar ODE model and by labeling the cytosolic protein talin. We found that talin is sensitive to increases and decreases in applied force. Disruptions of the intracellular force negatively affected adhesion stability. Increasing the force resulted in a faster assembly of new adhesions, whereas decreased forces increased the talin turnover.
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