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 protein subcellular location and function. Some proteins, however, show altered localization and/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 two 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. FOR TABLE OF CONTENTS ONLY O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/547312v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@ce1d9borg.highwire.dtl.DTLVardef@1f9b5c8org.highwire.dtl.DTLVardef@10d92ccorg.highwire.dtl.DTLVardef@20ad5d_HPS_FORMAT_FIGEXP M_FIG C_FIG