Super-resolution imaging of proteins in live cells using reversibly interacting peptide pairs
Creators: name: Gidden, Zoe
Publisher: name: The University of Edinburgh
Year: 2024
Subjects: Super-resolution imaging
proteins in live cells
reversibly interacting peptide pairs
live cell super-resolution imaging
Photo-activated localisation microscopy (PALM)
LIVE-PAINT
live-cell super-resolution microscopy

description: Super-resolution techniques have revolutionised our ability to observe cellular structures with significantly higher resolution than traditional microscopy. Despite the number of super-resolution microscopy techniques available, live cell super-resolution imaging remains challenging. For example, while Photo-activated localisation microscopy (PALM) can be used in vivo, it necessitates the direct fusion of a fluorophore to the protein of interest. This approach can be problematic because a direct fusion to a fluorescent protein can disrupt the normal function and localisation of the protein being studied. Moreover, once the fluorescent protein is photobleached, no more data can be collected from that molecule. In this thesis, I describe the development and use of LIVE-PAINT, a novel live-cell super-resolution microscopy technique. In LIVE-PAINT, a peptide-protein or peptidepeptide pair, one fused to the protein of interest and the other to a fluorescent protein, reversibly interact. When the peptide pair bind, a blink is observed, and the precise location can be determined. In a few minutes, enough binding events occur to generate an image of the protein of interest with a resolution of around 20 nanometres. Initially, this work optimises and applies LIVE-PAINT for diffraction-limited and super-resolution imaging of proteins within live budding yeast cells. I then demonstrate that the small peptide tag used to label the protein of interest makes LIVE-PAINT a valuable tool for imaging proteins that are sensitive to direct fusions to fluorescent proteins. In addition, I validate that LIVE-PAINT enables replenishment of signal throughout imaging. This is because the imaging peptide, the peptide-labelled fluorescent protein, is expressed separately from the target protein, creating a pool of imaging peptides within the cell that can replenish those that are photobleached during imaging. I utilise this property of LIVE-PAINT to track moving proteins over long periods of time. Subsequently, I describe how I adapted the LIVE-PAINT system to apply this technique to the more complex environment of live mammalian cells. I show that LIVE-PAINT successfully yields diffraction-limited and super-resolution images of proteins located in various organelles. This is the first time that interacting peptide pairs have been used to facilitate point accumulation for imaging in nanoscale topography (PAINT) based super-resolution imaging in live mammalian cells. These results are obtained through both transient transfections of labelled proteins and stably integrated versions. Through this work I generate several new cell lines which can be shared with other researchers allowing them to use this technique to gain new insights into the proteins they study. Furthermore, this thesis explores improvements to the LIVE-PAINT method. I demonstrate that peptides as small as 5 residues can be used for LIVE-PAINT imaging. This will broaden the applicability of LIVE-PAINT to a wider range of proteins that cannot tolerate modifications. To harness the increased brightness of synthetic fluorescent dyes compared to fluorescent proteins, I developed mammalian cell lines expressing a HaloTag fused to a LIVE-PAINT peptide. I show that the exogenous addition of the binding partner to HaloTag, HaloLigand, labelled with a synthetic dye, to these cells, enables LIVE-PAINT imaging with synthetic dyes. Lastly, I validate that LIVE-PAINT can be multiplexed by using orthogonal peptide-protein pairs to image two proteins concurrently in live cells. In summary, this thesis presents the development and optimisation of LIVE-PAINT, an innovative peptide-based super-resolution imaging technique tailored for live cell imaging. While this work explores select applications of LIVE-PAINT, it is anticipated that this novel technique will have a broad spectrum of applications.