Characteristics of genetic tags for correlative light and electron microscopy.

Fluorescence microscopy is indispensable in live cell studies of fluorescently-labeled proteins, but has limited resolution and context. Electron microscopy offers high-resolution imaging of cellular ultrastructure, including membranes, organelles, and other nanoscale features. However, identifying proteins by EM remains a substantial challenge. There is potential to combine the strengths of both FM and EM through correlative light and EM (CLEM), and bridging the two modalities enables new discoveries and biological insights. CLEM enables cellular proteins to be observed dynamically, across size scales, and in relationship to sub-cellular structures. A central limitation to using CLEM is the scarcity of methods for labeling proteins with CLEM reporters. This review will describe the characteristics of genetic tags for CLEM that are available today, including fixation-resistant fluorescent proteins, 3,3'-diaminobenzidine (DAB)-based tags, metal-chelating tags, DNA origami tags, and VIP tags.

Orthogonal Versatile Interacting Peptide Tags for Imaging Cellular Proteins.

Genetic tags are transformative tools for investigating the function, localization, and interactions of cellular proteins. Most studies today are reliant on selective labeling of more than one protein to obtain comprehensive information on a protein's behavior in situ. Some proteins can be analyzed by fusion to a protein tag, such as green fluorescent protein, HaloTag, or SNAP-Tag. Other proteins benefit from labeling via small peptide tags, such as the recently reported versatile interacting peptide (VIP) tags. VIP tags enable observations of protein localization and trafficking with bright fluorophores or nanoparticles. Here, we expand the VIP toolkit by presenting two new tags: TinyVIPER and PunyVIPER. These two tags were designed for use with MiniVIPER for labeling up to three distinct proteins at once in cells. Labeling is mediated by the formation of a high-affinity, biocompatible heterodimeric coiled coil. Each tag was validated by fluorescence microscopy, including observation of transferrin receptor 1 trafficking in live cells. We verified that labeling via each tag is highly specific for one- or two-color imaging. Last, the self-sorting tags were used for simultaneous labeling of three protein targets (i.e., TOMM20, histone 2B, and actin) in fixed cells, highlighting their utility for multicolor microscopy. MiniVIPER, TinyVIPER, and PunyVIPER are small and robust peptide tags for selective labeling of cellular proteins.

Investigating ß-Lactam Drug Targets in Mycobacterium tuberculosis Using Chemical Probes.

Tuberculosis (TB), caused by the bacterial pathogen Mycobacterium tuberculosis (Mtb), infects 10 million people a year. An estimated 25% of humans harbor latent TB infections, an asymptomatic form of the disease. In both active and latent infections, Mtb relies on cell wall peptidoglycan for viability. In the current work, we synthesized fluorescent analogues of ß-lactam antibiotics to study two classes of enzymes that maintain Mtb's peptidoglycan: penicillin-binding proteins (PBPs) and l,d-transpeptidases (LDTs). This set of activity-based probes included analogues of three classes of ß-lactams: a monobactam (aztreonam-Cy5), a cephalosporin (cephalexin-Cy5), and a carbapenem (meropenem-Cy5). We used these probes to profile enzyme activity in protein gel-resolved lysates of Mtb. All three out-performed the commercial reagent Bocillin-FL, a penam. Meropenem-Cy5 was used to identify ß-lactam targets by mass spectrometry, including PBPs, LDTs, and the ß-lactamase BlaC. New probes were also used to compare PBP and LDT activity in two metabolic states: dormancy and active replication. We provide the first direct evidence that Mtb dynamically regulates the enzymes responsible for maintaining peptidoglycan in dormancy. Lastly, we profiled drug susceptibility in lysates and found that meropenem inhibits PBPs, LDTs, and BlaC.

MiniVIPER Is a Peptide Tag for Imaging and Translocating Proteins in Cells.

Microscopy allows researchers to interrogate proteins within a cellular context. To deliver protein-specific contrast, we developed a new class of genetically encoded peptide tags called versatile interacting peptide (VIP) tags. VIP tags deliver a reporter to a target protein via the formation of a heterodimer between the peptide tag and an exogenously added probe peptide. We report herein a new VIP tag named MiniVIPER, which is comprised of a MiniE-MiniR heterodimer. We first demonstrated the selectivity of MiniVIPER by labeling three cellular targets: transferrin receptor 1 (TfR1), histone protein H2B, and the mitochondrial protein TOMM20. We showed that either MiniE or MiniR could serve as the genetically encoded tag. Next, we demonstrated MiniVIPER's versatility by generating five spectrally distinct probe peptides to label tagged TfR1 on live cells. Lastly, we demonstrated two new applications for VIP tags. First, we used MiniVIPER in combination with another VIP tag, VIPER, to selectively label two different proteins in a single cell (e.g., TfR1 with H2B or TOMM20). Second, we used MiniVIPER to translocate a fluorescent protein to the nucleus through in situ dimerization of mCherry with H2B-mEmerald. In summary, MiniVIPER is a new peptide tag that enables multitarget imaging and artificial dimerization of proteins in cells.

Fluorescent probes for investigating peptidoglycan biosynthesis in mycobacteria.

Tuberculosis killed 1.5 million people in 2018. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the most deadly infectious bacteria in the world. A strength of mycobacterial pathogens - their formidable cell wall - could also be one of their greatest molecular vulnerabilities. As in other bacteria, peptidoglycan (PG) maintenance and integrity is essential to mycobacterial survival. But Mtb PG is unique, and a better understanding of its biosynthetic machinery could lead to new drugs or more effective treatment regimens. Such investigations are being accelerated by the application of fluorescent probes, including those based on vancomycin, ß-lactams, PG stem mimics, d-amino acids, and reactive glycans. This review will describe how fluorescent probes are being used to uncover new information on the regulation and drug susceptibility of two classes of enzymes that fortify the Mtb PG: the penicillin-binding proteins and the L,D-transpeptidases.

Generation of CoilR Probe Peptides for VIPER-labeling of Cellular Proteins.

Versatile Interacting Peptide (VIP) tags are a new class of genetically-encoded tag designed for imaging cellular proteins by fluorescence and electron microscopy. In 2018, we reported the VIPER tag ( Doh et al., 2018 ), which contains two elements: a genetically-encoded peptide tag (i.e., CoilE) and a probe peptide (i.e., CoilR). These two peptides deliver contrast to a protein of interest by forming a specific, high-affinity heterodimer. The probe peptide was designed with a single cysteine residue for site-specific modification via thiol-maleimide chemistry. This feature can be used to attach a variety of biophysical reporters to the peptide, including bright fluorophores for fluorescence microscopy or electron-dense nanoparticles for electron microscopy. In this Bio-Protocol, we describe our methods for expressing and purifying recombinant CoilR. Additionally, we describe protocols for making fluorescent or biotinylated probe peptides for labeling CoilE-tagged cellular proteins. This protocol is complemented by two other Bio-Protocols outlining the use of VIPER ( Doh et al., 2019a and 2019b).

Imaging VIPER-labeled Cellular Proteins by Correlative Light and Electron Microscopy.

Advances in fluorescence microscopy (FM), electron microscopy (EM), and correlative light and EM (CLEM) offer unprecedented opportunities for studying diverse proteins and nanostructures involved in fundamental cell biology. It is now possible to visualize and quantify the spatial organization of cellular proteins and other macromolecules by FM, EM, and CLEM. However, tagging and tracking cellular proteins across size scales is restricted by the scarcity of methods for attaching appropriate reporter chemistries to target proteins. Namely, there are few genetic tags compatible with EM. To overcome these issues we developed Versatile Interacting Peptide (VIP) tags, genetically-encoded peptide tags that can be used to image proteins by fluorescence and EM. VIPER, a VIP tag, can be used to label cellular proteins with bright, photo-stable fluorophores for FM or electron-dense nanoparticles for EM. In this Bio-Protocol, we provide an instructional guide for implementing VIPER for imaging a cell-surface receptor by CLEM. This protocol is complemented by two other Bio-Protocols outlining the use of VIPER ( Doh et al., 2019a and 2019b).

Implementing VIPER for Imaging Cellular Proteins by Fluorescence Microscopy.

Genetically-encoded tags are useful tools for multicolor and multi-scale cellular imaging. Versatile Interacting Peptide (VIP) tags, such as VIPER, are new genetically-encoded tags that can be used in various imaging applications. VIP tags consist of a coiled-coil heterodimer, with one peptide serving as the genetic tag and the other ("probe peptide") delivering a reporter compatible with imaging. Heterodimer formation is rapid and specific, allowing proteins to be selectively labeled for live-cell and fixed-cell imaging. In this Bio-Protocol, we include a detailed guide for implementing the VIPER technology for imaging receptors on live cells and intracellular targets in fixed cells. This protocol is complemented by two other Bio-Protocols outlining the use of VIPER (Doh et al., 2019a and 2019b).

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

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.

Versatile Interacting Peptide (VIP) Tags for Labeling Proteins with Bright Chemical Reporters.

Fluorescence microscopy is an essential tool for the biosciences, enabling the direct observation of proteins in their cellular environment. New methods that facilitate attachment of photostable synthetic fluorophores with genetic specificity are needed to advance the frontiers of biological imaging. Here, we describe a new set of small, selective, genetically encoded tags for proteins based on a heterodimeric coiled-coil interaction between two peptides: CoilY and CoilZ. Proteins expressed as a fusion to CoilZ were selectively labeled with the complementary CoilY fluorescent probe peptide. Fluorophore-labeled target proteins were readily detected in cell lysates with high specificity and sensitivity. We found that these versatile interacting peptide (VIP) tags allowed rapid and specific delivery of bright organic dyes or quantum dots to proteins displayed on living cells. Additionally, we validated that either CoilY or CoilZ could serve as the VIP tag, which enabled us to observe two distinct cell-surface protein targets with this one heterodimeric pair.

Small-Molecule Probes Reveal Esterases with Persistent Activity in Dormant and Reactivating Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) is the deadliest bacterial pathogen in the world. An estimated one-third of humans harbor Mtb in a dormant state. These asymptomatic, latent infections impede tuberculosis eradication due to the long-term potential for reactivation. Dormant Mtb has reduced enzymatic activity, but hydrolases that remain active facilitate pathogen survival. We targeted Mtb esterases, a diverse set of enzymes in the serine hydrolase family, and studied their activities using both activity-based probes (ABPs) and fluorogenic esterase substrates. These small-molecule probes revealed functional esterases in active, dormant, and reactivating cultures. Using ABPs, we identified five esterases that remained active in dormant Mtb, including LipM (Rv2284), LipN (Rv2970c), CaeA (Rv2224c), Rv0183, and Rv1683. Three of these, CaeA, Rv0183, and Rv1683, were catalytically active in all three culture conditions. Fluorogenic probes additionally revealed LipH (Rv1399c), Culp1 (Rv1984c), and Rv3036c esterase activity in dormant and active cultures. Esterases with persistent activity are potential diagnostic biomarkers or therapeutic targets for Mtb-infected individuals with latent or active tuberculosis.

Profiling Esterases in Mycobacterium tuberculosis Using Far-Red Fluorogenic Substrates.

Enzyme-activated, fluorogenic probes are powerful tools for studying bacterial pathogens, including Mycobacterium tuberculosis (Mtb). In prior work, we reported two 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO)-derived acetoxymethyl ether probes for esterase and lipase detection. Here, we report four-carbon (C4) and eight-carbon (C8) acyloxymethyl ether derivatives, which are longer-chain fluorogenic substrates. These new probes demonstrate greater stability and lipase reactivity than the two-carbon (C2) acetoxymethyl ether-masked substrates. We used these new C4 and C8 probes to profile esterases and lipases from Mtb. The C8-masked probes revealed a new esterase band in gel-resolved Mtb lysates that was not present in lysates from nonpathogenic M. bovis (bacillus Calmette-Guérin), a close genetic relative. We identified this Mtb-specific enzyme as the secreted esterase Culp1 (Rv1984c). Our C4- and C8-masked probes also produced distinct Mtb banding patterns in lysates from Mtb-infected macrophages, demonstrating the potential of these probes for detecting Mtb esterases that are active during infections.

Synthesis of a far-red fluorophore and its use as an esterase probe in living cells.

We report the synthesis of a new far-red fluorophore, 1,3-dichloro-7-hydroxy-2H-spiro[acridine-9,1'-cyclohexane]-2',5'-diene-2,4'-dione (DSACO), which was modified to make two esterase probes: DSACO-2-AME and DSACO-7-AME. Both probes act as "turn-on" substrates for esterases and lipases. DSACO-2-AME exhibited efficient esterase-activated fluorescence inside living cells and is a stable, far-red alternative for the widely-used fluorescein diacetate.

Far-red fluorogenic probes for esterase and lipase detection.

Fluorogenic enzyme probes go from a dark to a bright state following hydrolysis and can provide a sensitive, real-time readout of enzyme activity. They are useful for examining enzymatic activity in bacteria, including the human pathogen Mycobacterium tuberculosis. Herein, we describe two fluorogenic esterase probes derived from the far-red fluorophore 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). These probes offer enhanced optical properties compared to existing esterase probes because the hydrolysis product, DDAO, excites above 600 nm while retaining a good quantum yield (ϕ=0.40). We validated both probes with a panel of commercially available enzymes alongside known resorufin- and fluorescein-derived esterase substrates. Furthermore, we used these probes to reveal esterase activity in protein gel-resolved mycobacterial lysates. These probes represent new tools for esterase detection and characterization and should find use in a variety of applications.

An expanded set of fluorogenic sulfatase activity probes.

Fluorogenic probes that are activated by an enzymatic transformation are ideally suited for profiling enzyme activities in biological systems. Here, we describe two fluorogenic enzyme probes, 3-O-methylfluorescein-sulfate and resorufin-sulfate, that can be used to detect sulfatases in mycobacterial lysates. Both probes were validated with a set of commercial sulfatases and used to reveal species-specific sulfatase banding patterns in a gel-resolved assay of mycobacterial lysates. The fluorogenic probes described here are suitable for various assays and provide a starting point for creating new sulfatase probes with improved selectivity for mycobacterial sulfatases.

Sulfatase-activated fluorophores for rapid discrimination of mycobacterial species and strains.

Most current diagnostic tests for tuberculosis do not reveal the species or strain of pathogen causing pulmonary infection, which can lead to inappropriate treatment regimens and the spread of disease. Here, we report an assay for mycobacterial strain assignment based on genetically conserved mycobacterial sulfatases. We developed a sulfatase-activated probe, 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)-sulfate, that detects enzyme activity in native protein gels, allowing the rapid detection of sulfatases in mycobacterial lysates. This assay revealed that mycobacterial strains have distinct sulfatase fingerprints that can be used to judge both the species and lineage. Our results demonstrate the potential of enzyme-activated probes for rapid pathogen discrimination for infectious diseases.

Chemical strategies for tagging and imaging the proteome.

Proteomic visualization serves as a complement to proteomic identification. In recent years, chemical biologists have made rapid progress developing new methods to tag and image defined sets of proteins. These researchers have requisitioned cellular machinery to place small, reactive analogues into biomolecules. The analogue has been labeled subsequently using a selective ligation reaction. Many groups have demonstrated the efficacy of the copper-catalyzed or strain-promoted azide-alkyne ligation; both enable rapid and precise labeling in complex biological mixtures. This review provides an overview of the methods which have been optimized to tag and fluorophore-label biomolecules for imaging subsets of the proteome in bacterial and mammalian cells. With the approaches described herein, it should be possible to image cells as they undergo changes over time.