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Most essential cellular functions are performed by proteins assembled into larger complexes. Fluorescence Polarization Microscopy (FPM) is a powerful technique that goes beyond traditional imaging methods by allowing researchers to measure not only the localization of proteins within cells, but also their orientation or alignment within complexes or cellular structures. FPM can be easily integrated into standard widefield microscopes with the addition of a polarization modulator. However, the extensive image processing and analysis required to interpret the data have limited its widespread adoption. To overcome these challenges and enhance accessibility, we introduce OOPS (Object-Oriented Polarization Software), a MATLAB package for object-based analysis of FPM data. By combining flexible image segmentation and novel object-based analyses with a high-throughput FPM processing pipeline, OOPS empowers researchers to simultaneously study molecular order and orientation in individual biological structures; conduct population assessments based on morphological features, intensity statistics, and FPM measurements; and create publication-quality visualizations, all within a user-friendly graphical interface. Here, we demonstrate the power and versatility of our approach by applying OOPS to punctate and filamentous structures.

Desmosomes are intercellular anchoring junctions that are crucial for maintaining the mechanical integrity of tissues routinely subjected to large forces, such as epithelia and cardiac muscle. Adhesion in desmosomes is mediated by a specialized class of transmembrane glycoproteins known as the desmosomal cadherins (DCs). Defects in desmosomal adhesion are associated with severe disease of the heart and skin and are frequently linked to mutations in the DCs. It is therefore important to understand the relationship between DC architecture and desmosome function. Accordingly, several groups have attempted to elucidate DC architecture—primarily using electron tomography (ET). However, ET studies of the desmosome are limited in spatial resolution by the inherent flexibility of the DCs, which has led to conflicting results. Consequently, the precise geometric arrangement of the DCs—and the underlying relationship between DC architecture and adhesive function—remain incompletely understood.Excitation-resolved fluorescence polarization microscopy (FPM) is a powerful technique which can be used to measure the order and orientation of fluorescently labeled biomolecules in their native cellular environments. In this dissertation, I leveraged the advantages of FPM by targeting flexible DC domains inaccessible to ET, providing several novel insights into DC architecture and dynamics. I showed that DC ectodomains are significantly more ordered than their intracellular counterparts, reflecting a drastic disparity in architecture between opposing sides of the plasma membrane. Notably, this may account for the unique ability of desmosomes to simultaneously maintain strong intercellular adhesion while also remaining plastic enough to facilitate rapid junctional remodeling in response to cellular cues. Indeed, the disparity between intracellular and extracellular architecture was consistent and broadly conserved across multiple DC isoforms, suggesting it is functionally relevant. Importantly, by statistically correlating FPM measurements with mathematical modeling informed by existing structural data, I was able to discriminate between competing models of DC architecture that could not be distinguished using ET. Finally, I found that DC organization and intercellular adhesive strength increase in a coordinated fashion during desmosome assembly. Taken together, my work has clarified the nanoscale architecture of the DCs, while also shedding light the poorly understood relationship between structure and function in the desmosome.

Most essential cellular functions are performed by proteins assembled into larger complexes. Fluorescence Polarization Microscopy (FPM) is a powerful technique that goes beyond traditional imaging methods by allowing researchers to measure not only the localization of proteins within cells, but also their orientation or alignment within complexes or cellular structures. FPM can be easily integrated into standard widefield microscopes with the addition of a polarization modulator. However, the extensive image processing and analysis required to interpret the data have limited its widespread adoption. To overcome these challenges and enhance accessibility, we introduce OOPS (Object-Oriented Polarization Software), a MATLAB-based analysis tool tailored for FPM data. This work highlights the distinctive features of our software, which empower researchers to efficiently manage large datasets; detect and analyze individual structures; conduct population assessments based on morphology, intensity, and polarization-specific parameters; and create publication-quality visualizations, all within a user-friendly graphical interface. Importantly, OOPS is adaptable to various sample types, labeling techniques, and imaging setups, facilitating in-depth analysis of diverse polarization-sensitive samples. Here, we demonstrate the power and versatility of our approach by applying OOPS to FPM images of both punctate and filamentous structures. OOPS is freely available under the GNU GPL 3.0 license and can be downloaded at https://github.com/Mattheyses-Lab/OOPS.

Early day before yesterday, with no previous intimation to me, my associate in the deanship of this School \" Passed thro' Glory's morning gate, And walked in Paradise.

TO William the Second the first of the dynasty brings heartiest congratulations and good wishes.

DEAR BRETHREN: I have been asked to address to you some words that may be considered a message from your Alma Mater as you gird yourselves for a fall campaign of aggressive evangelism. The author of the request cherishes a hope that any message adapted to be of service to you may prove to be of service to other ministerial brethren as well.