DEVELOPMENT OF NOVEL METHODS FOR IMAGING MEMBRANE PROTEIN STOICHIOMETRY AT THE SINGLE MOLECULE LEVEL AND FOR FABRICATION OF A SINGLE MOLECULE PROTEIN-DNA IMAGING DEVICE

Single molecule fluorescence imaging techniques have revolutionized the way we study biology by offering methods to examine the behavior and arrangement of individual molecules and the molecular mechanisms underlying biological processes. These techniques permit the investigation of transient states and critical heterogeneities undetectable by ensemble measurements and genome-wide assays. In this dissertation, I detail a series of projects, performed as a member of the laboratory of Warren Zipfel, in which new single molecule methods were created and applied to address important biological questions. A technique designed to study protein-DNA interactions at the single molecule level in a high-throughput fashion is called “DNA curtains.” The nanopatterned microfluidic devices necessary for these experiments have previously been fabricated using electron-beam lithography. We developed a simplified, cost effective, and more accessible method of fabricating these devices. Due to their modular DNA binding domain, transcription activator-like effectors (TALEs) have potential to be used in the study of gene function and gene editing with medical and agricultural applications. Understanding the target search mechanism of TALEs is important to developing more efficient and accurate ways to design and deliver TALE proteins. In my first project, we investigated TALEs using “DNA curtains” in an effort to elucidate the details of this search mechanism. Many single molecule techniques require the sample to be observed in vitro in order to isolate the biomolecule of interest. As a result, physiological behavior may not be preserved. In my second project, we developed a method named Single Protein Recovery After Dilution (SPReAD) that addresses this limitation by enabling protein stoichiometry and function to be studied in vivo. My final project is an investigation of the functional composition of metabotropic glutamate receptors (mGluRs). Glutamate acts as both a neurotransmitter and neuromodulator in the central nervous system. These neuromodulatory effects are mediated by mGluRs and their improper function has been linked to schizophrenia and Fragile X Syndrome. Understanding the stoichiometry of mGluR complexes is necessary to the development of pharmacological compounds which modulate their signaling. We investigated the interaction between Group I mGluRs at the single molecule level in vivo utilizing our SPReAD technique