VIPER is a genetically encoded peptide tag for fluorescence and electron microscopy

Abstract

Many discoveries in cell biology rely on making specific proteins visible within their native cellular environment. There are various genetically encoded tags, such as fluorescent proteins, developed for fluorescence microscopy (FM). However, there are almost no genetically encoded tags that enable cellular proteins to be observed by both FM and electron microscopy (EM). Herein, we describe a technology for labeling proteins with diverse chemical reporters, including bright organic fluorophores for FM and electron-dense nanoparticles for EM. Our technology uses versatile interacting peptide (VIP) tags, a class of genetically encoded tag. We present VIPER, which consists of a coiled-coil heterodimer formed between the genetic tag, CoilE, and a probe-labeled peptide, CoilR. Using confocal FM, we demonstrate that VIPER can be used to highlight subcellular structures or to image receptor-mediated iron uptake. Additionally, we used VIPER to image the iron uptake machinery by correlative light and EM (CLEM). VIPER compared favorably with immunolabeling for imaging proteins by CLEM, and is an enabling technology for protein targets that cannot be immunolabeled. VIPER is a versatile peptide tag that can be used to label and track proteins with diverse chemical reporters observable by both FM and EM instrumentation.

A PopZ‐linked apical recruitment assay for studying protein–protein interactions in the bacterial cell envelope

“…A). Our first test pair in this case involved the heterodimeric coiled‐coil formed by CoilE and CoilR peptides (Moll et al , ; Doh et al , ). The bait was formed by fusing H3H4‐GFP with the control TM to CoilE (H3H4‐GFP–TM‐CoilE) so that the peptide would reside in the periplasm.…”

“…The H3H4‐GFP–tagged bait fusions were expressed from the medium copy plasmids pHCL149 ( C TM fusion), pHCL287 ( C TM‐CoilE fusion, CoilE in periplasm) and pHCL285 (fusion with FtsX of S. pneumoniae , Sp FtsX). CoilE and CoilR are designed peptides that interact with high affinity to form a heterodimeric coiled coil (Moll et al , ; Doh et al , ). The prey fusions were expressed from the genome‐integrated plasmids pHCL147 ( SS DsbA‐mScar), pHCL288 (CoilR‐mScar) and pHCL286 ( CC PcsB‐mScar).…”

Electron tomography of plant organelles and the outlook for correlative microscopic approaches

“…However, sensitivity and specificity are unpredictable because antibodies must bind to epitopes on the plastic surface (Kang, ). CLEM tools for localizing molecules in EM are rapidly developing, and some of them can be combined with ET to analyze the 3D organization of macromolecules in plant organelles (Clarke & Royle, ; Doh et al ., ).…”

Implementing VIPER for Imaging Cellular Proteins by Fluorescence Microscopy

“…One advantage of this approach is that the genetic tag is small-just 4 to 7 kDa. A second advantage is that reporter peptide labeling is typically restricted to the cell surface, which is useful for labeling and tracking transmembrane receptors (Yano et al, 2008) (i.e., in pulse-chase experiments (Doh et al, 2018; Lotze et al, 2018)). We named our coiled-coil tags Versatile Interacting Peptide (VIP) tags.…”

“…VIPER labeled sub-cellular structures in fixed cells and transmembrane receptors on live cells. Proteins could be imaged by FM or correlative light and EM (CLEM) (Doh et al, 2018).…”