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Single Molecule Microscopy of Protein-Containing Phospholipid Vesicles – Blake Ford

Blake FordFor my summer research I work with graduate student, Noah Robinson, in Dr. Kyoung’s lab. The project is focused on developing a novel method to examine weak, short-lived, protein-protein interactions. These interactions are compelling because a broad variety of interactions involved in signaling and biochemical pathways in cells fall into this transient category. Transient interactions are notoriously difficult to study due to their inherent short lived nature, and our work should allow for new, exciting discoveries.

BF1To study these complex interactions, we will trap molecules in vesicle “nano-chambers” in order to monitor them using a single molecule fluorescence microscope constructed in-house.

 

This new nano-reaction chamber approach is motivated by the need to overcome limits of conventional methodologies to study transient protein-protein interaction. Observing many molecules, as done in current methodologies, results in an average of signals and these transient interactions we are interested in are not detected. By engineering a system where multiple single molecules are trapped and probed in a small volume using fusion methods, it will be possible to examine the functional mechanisms of specific protein-protein interactions in macromolecular complexes of signaling pathways. This work will lead to identify a new class of anti-disease targets.

My primary role is that of data analyst. I convert raw real-time videos into information about individual nano-chambers. I then examine this data searching for convincing signals and evidences of fusion events in the effort of showing optimized conditions for the working system. I have also developed a protocol on how to use the MATLAB program for each step of the imaging and data analysis process. Additionally, I have created sample holders, which consist of micron-sized inlet and outlet holes that vesicles will be immobilized in. Finally, I have worked to find, read, and relay primary literature in an effort to bring new ideas into the project.