biostudies-literatureGidden ZYale UniversityUK Dementia Research InstituteWellcome TrustNational Institute of General Medical SciencesBiotechnology and Biological Sciences Research CouncilNIGMS NIH HHS10633-10641https://www.ebi.ac.uk/biostudies/studies/S-EPMC10683072biostudies-literatureUnknown23(22)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 them difficult 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 super-resolution approach that takes advantage of short interacting peptides to transiently bind a fluorescent protein to the protein-of-interest. Here, we successfully use LIVE-PAINT to image yeast membrane 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.Nano lettersImaging Proteins Sensitive to Direct Fusions Using Transient Peptide-Peptide Interactions.PMC10683072BB/M010996/1R01 GM118528226437/Z/22/ZNIH R01 GM118528Konieczna ZGidden Zde Moliner FVendrell MJohnston EJBhaskar HRosser SJOi CMochrie SGJRegan LHorrocks MHMendive-Tapia LfalseImaging 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 them difficult 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 super-resolution approach that takes advantage of short interacting peptides to transiently bind a fluorescent protein to the protein-of-interest. Here, we successfully use LIVE-PAINT to image yeast membrane 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.2023-01-01T00:00:00Z2023 Nov2025-04-22T08:10:42.265Z2025-02-19T04:44:16.938ZS-EPMC106830723791677010.1021/acs.nanolett.3c03780