Reversible peptide-protein interactions inside cells: enabling a new approach for achieving super-resolution imaging in live cells

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

Studies of electron temperature fluctuations in the core of Alcator C-Mod plasmas via correlation electron cyclotron emission

Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Nuclear Science and Engineering, 2013.Cataloged from PDF version of thesis.Includes bibliographical references (pages 55-56).Transport in tokamak plasmas is higher than predicted by neoclassical theory; this anomalous transport is believed to be attributed to turbulent fluctuations. New Correlation Electron Cyclotron Emission (CECE) experiments on Alcator C-Mod show lower levels of electron temperature fluctuations in the saturated ohmic confinement (SOC) regime than in the linear ohmic confinement (LOC) regime, however the lineaveraged density fluctuation data collected from ohmic plasmas previously showed the opposite trends. The apparent contradiction is explained by a change in the dominant turbulence modes in each confinement regime. Linear stability analysis shows that the LOC regime is dominated by trapped electron mode (TEM) turbulence and the SOC regime is on the border between the ion temperature gradient (ITG) and TEM turbulence modes being dominant. It is reasonable to believe that the TEM turbulence mode drives electron temperature fluctuations, which explains the higher electron temperature fluctuation levels seen in the LOC regime compared to the SOC regime.by Curran Y. M. Oi.S.B

Live‐cell super‐resolution imaging of actin using LifeAct‐14 with a PAINT‐based approach

We present direct‐LIVE‐PAINT, an easy‐to‐implement approach for the nanoscopic imaging of protein structures in live cells using labeled binding peptides. We demonstrate the feasibility of direct‐LIVE‐PAINT with an actin‐binding peptide fused to EGFP, the location of which can be accurately determined as it transiently binds to actin filaments. We show that direct‐LIVE‐PAINT can be used to image actin structures below the diffraction‐limit of light and have used it to observe the dynamic nature of actin in live cells. We envisage a similar approach could be applied to imaging other proteins within live mammalian cells

Imaging Proteins Sensitive to Direct Fusions Using Transient Peptide–Peptide Interactions

Fluorescence microscopy enables specific visualization of proteins in living cells and has played an important role in our understanding of the protein subcellular location and function. Some proteins, however, show altered localization or function when labeled using direct fusions to fluorescent proteins, making themdifficult to study in live cells. Additionally, the resolution of fluorescence microscopy is limited to ∼200 nm, which is 2 orders of magnitude larger than the size of most proteins. To circumvent these challenges, we previously developed LIVE-PAINT, a live-cell superresolution approach that takes advantage of short interacting peptides to transiently bind a fluorescent protein to the protein-ofinterest. Here, we successfully use LIVE-PAINT to image yeastmembrane proteins that do not tolerate the direct fusion of a fluorescent protein by using peptide tags as short as 5-residues. We also demonstrate that it is possible to resolve multiple proteins at the nanoscale concurrently using orthogonal peptide interaction pairs.KEYWORDS: membrane protein, protein−protein interaction, super-resolution microscopy, live-cell imaging, LIVE-PAINT, yeas

Correlation ECE diagnostic in Alcator C-Mod

Correlation ECE (CECE) is a diagnostic technique that allows measurement of small amplitude electron temperature, T[subscript e], fluctuations through standard cross-correlation analysis methods. In Alcator C-Mod, a new CECE diagnostic has been installed[Sung RSI 2012], and interesting phenomena have been observed in various plasma conditions. We find that local T[subscript e] fluctuations near the edge (ρ ~ 0:8) decrease across the linearto- saturated ohmic confinement transition, with fluctuations decreasing with increasing plasma density[Sung NF 2013], which occurs simultaneously with rotation reversals[Rice NF 2011]. T[subscript e] fluctuations are also reduced across core rotation reversals with an increase of plasma density in RF heated L-mode plasmas, which implies that the same physics related to the reduction of T[subscript e] fluctuations may be applied to both ohmic and RF heated L-mode plasmas. In I-mode plasmas, we observe the reduction of core T[subscript e] fluctuations, which indicates changes of turbulence occur not only in the pedestal region but also in the core across the L/I transition[White NF 2014]. The present CECE diagnostic system in C-Mod and these experimental results are described in this paper

LIVE-PAINT Supplementary Videos

We present LIVE-PAINT, a new approach to super-resolution fluorescent imaging inside live cells. In LIVE-PAINT only a short peptide sequence is fused to the protein being studied, unlike conventional super-resolution methods, which rely on directly fusing the biomolecule of interest to a large fluorescent protein, organic fluorophore, or oligonucleotide. LIVE-PAINT works by observing the blinking of localized fluorescence as this peptide is reversibly bound by a protein that is fused to a fluorescent protein. We have demonstrated the effectiveness of LIVE-PAINT by imaging a number of different proteins inside live S. cerevisiae. Not only is LIVE-PAINT widely applicable, easily implemented, and the modifications minimally perturbing, but we also anticipate it will extended data acquisition times compared to those previously possible with methods that involve direct fusion to a fluorescent protein.Oi, Curran; Horrocks, Mathew; Regan, Lynne. (2020). LIVE-PAINT Supplementary Videos, [dataset]. University of Edinburgh. Quantitative Biology, Biochemistry and Biotechnology. https://doi.org/10.7488/ds/2801

LIVE-PAINT Supplementary Videos: Compressed versions

Compressed versions of the files of Oi, Curran; Horrocks, Mathew; Regan, Lynne. (2020). LIVE-PAINT Supplementary Videos, [dataset]. University of Edinburgh. Quantitative Biology, Biochemistry and Biotechnology. https://doi.org/10.7488/ds/2801. ## File listing ## * "SupplementaryMovie1_-_mKO_blinking_compressed.mp4" LIVE-PAINT blinking behavior shown in S. cerevisiae using the fluorescent protein mKO and the reversible interaction pair SYNZIP17-SYNZIP18. Scale bar is 1 micron. * "SupplementaryMovie2_-_mOrange_blinking_compressed.mp4" LIVE-PAINT blinking behavior shown in S. cerevisiae using the fluorescent protein mOrange and the reversible interaction pair SYNZIP17-SYNZIP18. Scale bar is 1 micron. * "SupplementaryMovie3_-_cofilin_tracking_compressed.mp4" Video tracking cofilin in S. cerevisiae, where cofilin is C-terminally tagged with SYNZIP18 and labeled with separately expressed SYNZIP17-mNeonGreen. Scale bar is 5 microns. * "SupplementaryMovie4_-_cofilin_tracking_compressed.mp4" Tracking the diffusion of a single cofilin "spot" in S. cerevisiae, where cofilin is C-terminally tagged with SYNZIP18 and labeled with separately expressed SYNZIP17-mNeonGreen. Scale bar is 1 micron.Oi, Curran; Horrocks, Mathew; Regan, Lynne. (2020). LIVE-PAINT Supplementary Videos: Compressed versions, [moving image]. University of Edinburgh. Quantitative Biology, Biochemistry and Biotechnology. https://doi.org/10.7488/ds/2839

LIVE-PAINT Supporting Datasets

We present LIVE-PAINT, a new approach to super-resolution fluorescent imaging inside live cells. In LIVE-PAINT only a short peptide sequence is fused to the protein being studied, unlike conventional super-resolution methods, which rely on directly fusing the biomolecule of interest to a large fluorescent protein, organic fluorophore, or oligonucleotide. LIVE-PAINT works by observing the blinking of localized fluorescence as this peptide is reversibly bound by a protein that is fused to a fluorescent protein. We have demonstrated the effectiveness of LIVE-PAINT by imaging a number of different proteins inside live S. cerevisiae. Not only is LIVE-PAINT widely applicable, easily implemented, and the modifications minimally perturbing, but we also anticipate it will extend data acquisition times compared to those previously possible with methods that involve direct fusion to a fluorescent protein.Oi, Curran; Horrocks, Mathew; Gidden, Zoe; Regan, Lynne. (2020). LIVE-PAINT Supporting Datasets, [dataset]. University of Edinburgh. Quantitative Biology, Biochemistry and Biotechnology. https://doi.org/10.7488/ds/2859