Date of Award

Spring 2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biophysics and Biochemistry

First Advisor

Regan, Lynne

Abstract

Cells are the basic unit of life and, within cells, thousands of unique proteins work in concert to perform a vast array of tasks. Visualizing and tracking proteins inside live cells is therefore critical to understanding the behavior of these proteins in vivo. The invention of fluorescence microscopy has enabled proteins to be tagged and tracked using fluorescent molecules. More recently, the development of super-resolution microscopy has enabled very high resolution images of proteins in cells to be collected, both in vitro and in vivo.Currently, one major challenge in super-resolution microscopy is the fact that many proteins are not amenable to tagging and imaging using existing methods. For example, many proteins mislocalize or misfunction when fused to another protein as large as a fluorescent protein. Similarly, proteins with short half-lives are difficult to image, because they are degraded before a fused fluorescent protein has time to mature and become fluorescent. In this dissertation I present a new super-resolution imaging method called Live cell Imaging using reVersible intEractions - Point Accumulation In Nanoscale Topography (LIVE-PAINT). In this technique, reversible peptide-protein interaction pairs are used to transiently associate a fluorescent protein with a protein of interest. To implement LIVE-PAINT, I fused one half of a peptide-protein interaction pair to a protein I want to image at its genomic locus, thus labeling all copies of the protein in the cell with a peptide tag. Then, I separately fused the other half of the peptide-protein interaction pair to a fluorescent protein and integrated the construct into the genome, under control of the galactose inducible promoter. When both constructs are expressed concurrently, binding events between the protein of interest and fluorescent protein are mediated by the peptide-protein interaction pair. I have demonstrated that LIVE-PAINT can be performed using coiled coil interaction pairs and peptide-tetratricopeptide interaction pairs with a range of binding affinities between approximately 1 and 300 nM. I have also shown that LIVE-PAINT can be performed using many different color fluorescent proteins, demonstrating the flexibility of the method. LIVE-PAINT has many strengths which make it a useful new super-resolution tool. One example of this is given by proteins which do not tolerate direct fusions to fluorescent protein. I have tagged several putative plasma membrane proteins which localize to the vacuole when directly fused to fluorescent proteins and shown they localize to the plasma membrane as expected when tagged using peptide-protein interaction pairs and imaged with LIVE-PAINT. This putative localization to the plasma membrane is also confirmed by immunostaining data in one case. I have also demonstrated that LIVE-PAINT enables signal replenishment. In my work, the peptide-protein interactions used to tag the protein of interest are reversible and I restrict the illumination volume during imaging. This means that after a fluorescent protein unbinds from a protein of interest, another one can diffuse in from a part of the cell outside the illumination volume and bind in its place. Because the fluorescent protein is expressed separately from the protein of interest, much larger constructs can be reversibly associated to a protein of interest without increasing the size of the fusion to the protein of interest. To show this, I expressed a tandem array of three identical fluorescent proteins and demonstrated that this construct could be used for LIVE-PAINT imaging without any noticeable effect on the proper localization or function of the protein of interest. An additional benefit of the fact that the fluorescent protein is expressed separately from the protein of interest is that the expression level of the fluorescent protein is therefore not directly tied to the expression level of the protein of interest. This property of LIVE-PAINT makes it a good tool for imaging very low and very high abundance proteins, which suffer from too little or too much fluorescent signal in traditional fluorescence microscopy approaches. Thus, I have shown that LIVE-PAINT is a useful new super-resolution imaging technique and there are a number of applications for which it is uniquely well suited. LIVE-PAINT is particularly useful for studying proteins which are not amenable to direct fusion to fluorescent proteins, proteins which are short-lived, and proteins which are expressed at a very low or very high level.

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