Nonspecific Labeling Research Articles

Selective pH-Responsive Conjugation between a Pair of De Novo Discovered Peptides.

There is a demand for site-selective peptide/protein conjugation chemistry that is fully reversible in a stimulus-responsive manner. The contemporary methods for site-selective protein modification enable the preparation of homogeneous protein-small molecule conjugates, which are indispensable for drug delivery and chemical biology purposes, but such chemistries are usually irreversible. In contrast, the existing reversible protein labeling techniques are generally not site-selective. Here, we report an mRNA display-enabled de novo discovery of a pair of peptides which selectively react with each other to form a conjugate that is stable under neutral conditions (pH 7.5) but rapidly dissociates into the constituents at pH 10. A Cys thiol of peptide CP1 rapidly reacts (k1 = 340 M-1·s-1) with the isothiocyanate moiety in partner ITC6 to furnish a stable dithiocarbamate (t1/2 = 6 h at pH 7.5). We show that the pH-responsive nature of the reaction (conjugate's t1/2 = 5 min at pH 10) can be leveraged to utilize ITC6 (1) as a pull-down handle to selectively isolate CP1 from cell lysates and (2) as a temporary protecting group to protect CP1 from nonspecific Cys labeling reagents such as iodoacetamide. Altogether, the chemistry developed here complements the existing approaches and is applicable in a variety of chemical biology settings where selective reversible reactions are needed.

Enhancing substrate specificity of microbial transglutaminase for precise nanobody labeling

Streptomyces mobaraenesis transglutaminase (smTG) can be used for site-specific labeling of proteins with chemical groups. Here, we explored the use of modified smTG for the biosynthesis of nanobody-fluorophore conjugates (NFC). smTG catalyzes the conjugation of acyl donors containing glutamine with lysine-containing acceptors, which can lead to non-specific cross-linking. To achieve precise site-specific labeling, we employed molecular docking and virtual mutagenesis to redesign the enzyme's substrate specificity towards the peptide GGGGQR, a non-preferred acyl donor for smTG. Starting with a thermostable and highly active smTG variant (TGm2), we identified that single mutations G250H and Y278E significantly enhanced activity against GGGGQR, increasing it by 41 % and 1.13-fold, respectively. Notably, the Y278E mutation dramatically shifted the enzyme's substrate preference, with the activity ratio against GGGGQR versus the standard substrate CBZ-Gln-Gly rising from 0.05 to 0.93. In case studies, we used nanobodies 1C12 and 7D12 as labeling targets, catalyzing their conjugation with a synthetic fluorophore via smTG variants. Nanobodies fused with GGGGQR were successfully site-specifically labeled by TGm2-Y278E, in contrast to non-specific labeling observed with other variants. These results suggest that engineering smTG for site-specific labeling is a promising approach for the biosynthesis of antibody-drug conjugates.

Shining a light on fluorescent EV dyes: Evaluating efficacy, specificity and suitability by nano-flow cytometry.

Extracellular vesicles (EVs) are mediators of intercellular communication, recently recognised for their clinical applications. Accurate characterisation and quantification of EVs are critical for understanding of their function and clinical relevance. Many platforms utilise fluorescence for EV characterisation, frequently labelling surface proteins to identify EVs. The heterogeneity of EVs and the lack of a universal protein marker encourages the use of generic EV labelling methods, including membrane labelling. Using nano-flow cytometry, we evaluated six membrane dyes, including MemGlow and CellMask. Evaluation criteria included EV labelling efficacy, non-specific labelling of very low-density lipoproteins (VLDLs), brightness and dye aggregation. Significant variation was observed in dye performance, with certain dyes showing poor EV labelling efficacy or high affinity to VLDLs. Importantly, several promising candidates were identified for further investigation. Overall, this study highlights the importance of selecting appropriate membrane dyes for EV staining tailored to the aims of the study and the EV origin. MemGlow and CellMask proved favourable, allowing bright, sensitive staining of EV membranes with minimal aggregation. However, MemGlow showed an affinity to VLDLs, and CellMask requires additional sample handling for optimal labelling. These results contribute to deepening our understanding of EV membrane dyes, allowing for better dye selection and EV identification in future studies.

A versatile approach to quality control of protein-based receptor layers by reversible, nonspecific staining for multiplex SPRi immunosensing

SPRi protein arrays for rapid, label-free immunodetection offer an attractive approach for high throughput biosensing and analysis of molecular interactions. However, their routine use for quantitative analysis requires adequate reproducibility and homogeneity of receptor assemblies manufactured ex-situ using microdispensing techniques. There is also an emerging need to develop versatile and non-destructive methods for the determination of protein surface density for quality control of the immobilization process. Here, we report on a simple, one-step method of surface proteins visualization by their reversible, non-specific staining with anionic dyes: Ponceau S (PS), Amido Black (AB) or Coomassie Brilliant Blue G (CBB-G). Using rabbit IgG antibody and transferrin as model receptors, we demonstrate the possibility of protein surface density determination in a broad range up to ∼ 3 ng∙mm−2 (for a 2D monolayer) and up to at least 12.5 ng∙mm−2 (for a 3D hydrogel-coated substrate). The ranges of dynamic response for the developed methods cover the typical surface densities required for biosensing and studies of the binding kinetics. It was demonstrated that each of the investigated dyes manifests advantages in specified applications. While CBB-G shows the highest sensitivity of SPRi response, AB requires the gentlest labeling conditions, and PS shows the highest association rate and ease of washing out, even from protein layers of high density. The feasibility of the developed method for the quality control of SPRi microarray was confirmed. The advantages of the new, dye-based, non-specific labeling method over immunolabeling in terms of simplicity of implementation and versatility have also been highlighted.

N-Acyl-N-Alkyl Sulfonamide Probes for Ligand-Directed Covalent Labeling of GPCRs: The Adenosine A2B Receptor as Case Study.

Small molecular tool compounds play an essential role in the study of G protein-coupled receptors (GPCRs). However, tool compounds most often occupy the orthosteric binding site, hampering the study of GPCRs upon ligand binding. To overcome this problem, ligand-directed labeling techniques have been developed that leave a reporter group covalently bound to the GPCR, while allowing subsequent orthosteric ligands to bind. In this work, we applied such a labeling strategy to the adenosine A2B receptor (A2BAR). We have synthetically implemented the recently reported N-acyl-N-alkyl sulfonamide (NASA) warhead into a previously developed ligand and show that the binding of the A2BAR is not restricted by NASA incorporation. Furthermore, we have investigated ligand-directed labeling of the A2BAR using SDS-PAGE, flow cytometric, and mass spectrometry techniques. We have found one of the synthesized probes to specifically label the A2BAR, although detection was hindered by nonspecific protein labeling most likely due to the intrinsic reactivity of the NASA warhead. Altogether, this work aids the future development of ligand-directed probes for the detection of GPCRs.

Classification of single extracellular vesicles in a double nanohole optical tweezer for cancer detection

A major challenge in cancer prognostics is finding early biomarkers that can accurately identify cancer. Circulating tumor cells are rare and circulating tumor DNA can not provide information about the originating cell. Extracellular vesicles (EVs) contain cell specific information, are abundant in fluids, and have unique properties between cancerous and non-cancerous. Fluorescence measurements have limitations from intrinsic fluorescent background signals, photobleaching, non-specific labelling, and EV structural modifications. Here, we demonstrate a label-free approach to classification of 3 different EVs, derived from non-malignant, non-invasive cancerous, and invasive cancerous cell lines. Using double nanohole optical tweezers, the scattering from single trapped EVs is measured, and using a 1D convolutional neural network, we are able to classify the time series optical signal into its respective EV class with greater than 90% accuracy.

Next-Generation Metabolic Glycosylation Reporters Enable Detection of Protein O-GlcNAcylation in Living Cells without S-Glyco Modification.

Protein O-GlcNAcylation is a ubiquitous posttranslational modification of cytosolic and nuclear proteins involved in numerous fundamental regulation processes. Investigation of O-GlcNAcylation by metabolic glycoengineering (MGE) has been carried out for two decades with peracetylated N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine derivatives modified with varying reporter groups. Recently, it has been shown that these derivatives can result in non-specific protein labeling termed S-glyco modification. Here, we report norbornene-modified GlcNAc derivatives with a protected phosphate at the anomeric position and their application in MGE. These derivatives overcome two limitations of previously used O-GlcNAc reporters. They do not lead to detectable S-glyco modification, and they efficiently react in the inverse-electron-demand Diels-Alder (IEDDA) reaction, which can be carried out even within living cells. Using a derivative with an S-acetyl-2-thioethyl-protected phosphate, we demonstrate the protein-specific detection of O-GlcNAcylation of several proteins and the protein-specific imaging of O-GlcNAcylation inside living cells by Förster resonance energy transfer (FRET) visualized by confocal fluorescence lifetime imaging microscopy (FLIM).

Specific nanoprobe design for MRI: Targeting laminin in the blood-brain barrier to follow alteration due to neuroinflammation.

Chronic neuroinflammation is characterized by increased blood-brain barrier (BBB) permeability, leading to molecular changes in the central nervous system that can be explored with biomarkers of active neuroinflammatory processes. Magnetic resonance imaging (MRI) has contributed to detecting lesions and permeability of the BBB. Ultra-small superparamagnetic particles of iron oxide (USPIO) are used as contrast agents to improve MRI observations. Therefore, we validate the interaction of peptide-88 with laminin, vectorized on USPIO, to explore BBB molecular alterations occurring during neuroinflammation as a potential tool for use in MRI. The specific labeling of NPS-P88 was verified in endothelial cells (hCMEC/D3) and astrocytes (T98G) under inflammation induced by interleukin 1β (IL-1β) for 3 and 24 hours. IL-1β for 3 hours in hCMEC/D3 cells increased their co-localization with NPS-P88, compared with controls. At 24 hours, no significant differences were observed between groups. In T98G cells, NPS-P88 showed similar nonspecific labeling among treatments. These results indicate that NPS-P88 has a higher affinity towards brain endothelial cells than astrocytes under inflammation. This affinity decreases over time with reduced laminin expression. In vivo results suggest that following a 30-minute post-injection, there is an increased presence of NPS-P88 in the blood and brain, diminishing over time. Lastly, EAE animals displayed a significant accumulation of NPS-P88 in MRI, primarily in the cortex, attributed to inflammation and disruption of the BBB. Altogether, these results revealed NPS-P88 as a biomarker to evaluate changes in the BBB due to neuroinflammation by MRI in biological models targeting laminin.

Tracing the origin of alveolar stem cells in lung repair and regeneration

Alveolar type 2 (AT2) cells are stem cells of the alveolar epithelia. Previous genetic lineage tracing studies reported multiple cellular origins for AT2 cells after injury. However, conventional lineage tracing based on Cre-loxP has the limitation of non-specific labeling. Here, we introduced a dual recombinase-mediated intersectional genetic lineage tracing approach, enabling precise investigation of AT2 cellular origins during lung homeostasis, injury, and repair. We found AT1 cells, being terminally differentiated, did not contribute to AT2 cells after lung injury and repair. Distinctive yet simultaneous labeling of club cells, bronchioalveolar stem cells (BASCs), and existing AT2 cells revealed the exact contribution of each to AT2 cells post-injury. Mechanistically, Notch signaling inhibition promotes BASCs but impairs club cells' ability to generate AT2 cells during lung repair. This intersectional genetic lineage tracing strategy with enhanced precision allowed us to elucidate the physiological role of various epithelial cell types in alveolar regeneration following injury.

Standard Radio-Iodine Labeling Protocols Impaired the Functional Integrity of Mesenchymal Stem/Stromal Cell Exosomes.

Mesenchymal stem/stromal cells (MSCs) are an extensively studied cell type in clinical trials due to their easy availability, substantial ex vivo proliferative capacity, and therapeutic efficacy in numerous pre-clinical animal models of disease. The prevailing understanding suggests that their therapeutic impact is mediated by the secretion of exosomes. Notably, MSC exosomes present several advantages over MSCs as therapeutic agents, due to their non-living nature and smaller size. However, despite their promising therapeutic potential, the clinical translation of MSC exosomes is hindered by an incomplete understanding of their biodistribution after administration. A primary obstacle to this lies in the lack of robust labels that are highly sensitive, capable of directly and easily tagging exosomes with minimal non-specific labeling artifacts, and sensitive traceability with minimal background noise. One potential candidate to address this issue is radioactive iodine. Protocols for iodinating exosomes and tracking radioactive iodine in live imaging are well-established, and their application in determining the biodistribution of exosomes has been reported. Nevertheless, the effects of iodination on the structural or functional activities of exosomes have never been thoroughly examined. In this study, we investigate these effects and report that these iodination methods abrogate CD73 enzymatic activity on MSC exosomes. Consequently, the biodistribution of iodinated exosomes may reflect the biodistribution of denatured exosomes rather than functionally intact ones.

Abstract 2588: Bioengineering of site-specific antibody labeling from basic research to spatial omics platforms

Abstract Over the last few years, spatial multi-omics platforms have emerged as a powerful tool for characterizing cancer biology in multiple dimensions. Notably, these systems typically rely on multiplex immunolabeling. Conventionally, multiplexing is accomplished through carefully selecting primary antibodies from different animal species, and in combination with their corresponding polyclonal secondary antibodies. However, given the practical complication related to the species availability of primary antibodies, the multiplexing labeling is still under constraint due to the necessity for matched secondary species labels and the availability. For decades, non-specific labeling of primary antibodies through amine- or thiol-reactive chemistries was widely performed. Evolving research and commercial products have sought to overcome this antibody-based multiplexing limitations by developing enzymatic reaction-directed chemical reaction or antibody targeting proteins that could contain affinity to the Fc or Fab regions of antibodies. However, these methods mentioned above have substantive limitations and side-effects on affinity, conjugation number, buffer incompatibilities, storage incompatibilities, insufficient conjugation, and uncontrolled stoichiometric labeling. Herein, we introduce an efficient, sustainable, reproducible site-specific antibody covalent labeling strategy through engineering anti-IgG secondary nanobodies. These infer the stability, high-affinity of species specific and easy-to-produce IgG binders with the permanence of controlled activation for proximity-driven intermolecular covalent conjugation, with a promise for efficient multiplexity. This labeling technology addresses antibody labeling challenges through currently available approaches, and would substantially enhance multiplexity in a wide variety of research and clinical settings. Citation Format: Kai Tao, Matthew J. Rames, MingChong Dai, Gulsu Sener, Samuel Olson, Srivathsan Ranganathan, Sila Ozdemir, Adam Oken, Michael Brasino, Randy Armstrong, Yu-Jui Chiu, Ece Eksi, Bruce Branchaud, Sadik Esener. Bioengineering of site-specific antibody labeling from basic research to spatial omics platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2588.

Isoform-Selective Fluorescent Labeling of 14-3-3σ by Acrylamide-Containing Fusicoccins.

The 14-3-3 family of proteins is central to the regulation of signaling pathways driven by serine/threonine kinases. In humans, 14-3-3 consists of seven highly conserved isoforms, yet the function of each isoform remains to be fully elucidated. Synthetic agents capable of isoform-specific fluorescent labeling of 14-3-3 would provide a useful tool for studying in depth the biological roles of isoforms. In this study, the 14-3-3σ isoform was evaluated, which possesses a unique Cys38, and a natural product-based fluorescent labeling agent was designed by introducing an acrylamide group and a fluorescent dye to fusicoccin (FC). In vitro evaluation demonstrated that 12-hydroxy 1 and 2 exhibit 14-3-3σ selective labeling activity over 14-3-3ζ in the presence of a mode-3 phospholigand. Furthermore, 2 was shown to label 14-3-3σ in cell lysate in the presence of a C-terminal mode-3 phosphopeptide derived from ERα, with no apparent nonspecific labeling. These results indicate that 2 is capable of selective fluorescent detection of 14-3-3σ upon binding to mode-3 phospholigand under biologically relevant conditions.

Tissue Clearing To Localize Microplastics via Three-Dimensional Imaging of Whole Organisms.

Understanding the biological impacts of plastic pollution requires an effective methodology to detect unlabeled microplastics in environmental samples. Detecting unlabeled microplastics in an organism generally requires a digestion protocol, which results in the loss of spatial information on the distribution of microplastic within the organism and could lead to the disappearance of the smaller plastics. Fluorescence microscopy allows visualization of ingested microplastics but many labeling strategies are nonspecific and label biomass, thus limiting our ability to distinguish internalized plastics. While prelabeled plastics can be used to avoid nonspecific labeling, this approach precludes the detection of environmental microplastics in organisms. Also, using prelabeled microplastics can affect the viability of the organism and impact plastic uptake. Thus, a method was developed that employs nonspecific labeling with a tissue-clearing technique. Briefly, unlabeled microplastics are stained with a fluorescent dye after ingestion by the organism. The tissue-clearing technique then removes tissue-bound dye while rendering the structurally intact organism transparent. The internalized plastics remain stained and can be visualized in the cleared tissue with fluorescence microscopy. The technique is demonstrated using polystyrene beads in living aquatic organismsTigriopus californicusandDaphnia magnaand by spiking a model vertebrate (Cephalochordata) with different microplastics.

Solvent accessibility of a GPCR transmembrane domain probed by in-membrane chemical modification (IMCM).

G protein-coupled receptors (GPCRs) transmit signals from drugs across cell membranes, leading to associated physiological effects. To study the structural basis of the transmembrane signalling, in-membrane chemical modification (IMCM) has previously been introduced for 19 F-labelling of GPCRs expressed in Spodoptera frugiperda (Sf9) insect cells. Here, IMCM is used with the A2A adenosine receptor (A2A AR) expressed in Pichia pastoris; 19 F-NMR revealed nearly complete solvent protection of the A2A AR transmembrane domain in the membrane and in 2,2-didecylpropane-1,3-bis-β-D-maltopyranoside (LMNG)/cholesteryl hemisuccinate (CHS) micelles, and extensive solvent accessibility for A2A AR in n-dodecyl β-D-maltoside (DDM)/CHS micelles. No Cys residue dominated non-specific labelling with 2,2,2-trifluoroethanethiol. These observations yield an improved protocol for IMCM 19 F-labelling of GPCRs and new insights into variable solvent accessibility for function-related characterization of GPCRs.

Bright-field to fluorescence microscopy image translation for cell nuclei health quantification.

Microscopy is a widely used method in biological research to observe the morphology and structure of cells. Amongst the plethora of microscopy techniques, fluorescent labeling with dyes or antibodies is the most popular method for revealing specific cellular organelles. However, fluorescent labeling also introduces new challenges to cellular observation, as it increases the workload, and the process may result in nonspecific labeling. Recent advances in deep visual learning have shown that there are systematic relationships between fluorescent and bright-field images, thus facilitating image translation between the two. In this article, we propose the cross-attention conditional generative adversarial network (XAcGAN) model. It employs state-of-the-art GANs (GANs) to solve the image translation task. The model uses supervised learning and combines attention-based networks to explore spatial information during translation. In addition, we demonstrate the successful application of XAcGAN to infer the health state of translated nuclei from bright-field microscopy images. The results show that our approach achieves excellent performance both in terms of image translation and nuclei state inference.

Comparison of Different Competitive Proteome Profiling Approaches in Target Identification of Covalent Inhibitors.

Competitive proteome profiling is a powerful approach for the identification of targets of small molecules. This approach usually employs an inhibitor-derived probe or a cysteine-reactive probe such as an IA-alkyne in a comparison between inhibitor-treated and untreated samples, thus enabling distinction between genuine targets and nonspecific labeling. We have developed an active probe derived from an EGFR inhibitor, afatinib, and a cysteine reactive probe, an alkyne-containing α,β-unsaturated amide, to compare their characterization of cellular targets. In both approaches, myosin heavy chain 9 (MYH9) was identified as an off-target. Subsequent functional validation experiments suggested that MYH9 might be involved in the function of afatinib.

Probing Nascent RNA with Metabolic Incorporation of Modified Nucleosides.

The discovery of previously unknown functional roles of RNA in biological systems has led to increased interest in revealing novel RNA molecules as therapeutic targets and the development of tools to better understand the role of RNA in cells. RNA metabolic labeling broadens the scope of studying RNA by incorporating of unnatural nucleobases and nucleosides with bioorthogonal handles that can be utilized for chemical modification of newly synthesized cellular RNA. Such labeling of RNA provides access to applications including measurement of the rates of synthesis and decay of RNA, cellular imaging for RNA localization, and selective enrichment of nascent RNA from the total RNA pool. Several unnatural nucleosides and nucleobases have been shown to be incorporated into RNA by endogenous RNA synthesis machinery of the cells. RNA metabolic labeling can also be performed in a cell-specific manner, where only cells expressing an essential enzyme incorporate the unnatural nucleobase into their RNA. Although several discoveries have been enabled by the current RNA metabolic labeling methods, some key challenges still exist: (i) toxicity of unnatural analogues, (ii) lack of RNA-compatible conjugation chemistries, and (iii) background incorporation of modified analogues in cell-specific RNA metabolic labeling. In this Account, we showcase work done in our laboratory to overcome these challenges faced by RNA metabolic labeling.To begin, we discuss the cellular pathways that have been utilized to perform RNA metabolic labeling and study the interaction between nucleosides and nucleoside kinases. Then we discuss the use of vinyl nucleosides for metabolic labeling and demonstrate the low toxicity of 5-vinyluridine (5-VUrd) compared to other widely used nucleosides. Next, we discuss cell-specific RNA metabolic labeling with unnatural nucleobases, which requires the expression of a specific phosphoribosyl transferase (PRT) enzyme for incorporation of the nucleobase into RNA. In the course of this work, we discovered the enzyme uridine monophosphate synthase (UMPS), which is responsible for nonspecific labeling with modified uracil nucleobases. We were able to overcome this background labeling by discovering a mutant uracil PRT (UPRT) that demonstrates highly specific RNA metabolic labeling with 5-vinyluracil (5-VU). Furthermore, we discuss the optimization of inverse-electron-demand Diels-Alder (IEDDA) reactions for performing chemical modification of vinyl nucleosides to achieve covalent conjugation of RNA without transcript degradation. Finally, we highlight our latest endeavor: the development of mutually orthogonal chemical reactions for selective labeling of 5-VUrd and 2-vinyladenosine (2-VAdo), which allows for potential use of multiple vinyl nucleosides for simultaneous investigation of multiple cellular processes involving RNA. We hope that our methods and discoveries encourage scientists studying biological systems to include RNA metabolic labeling in their toolkit for studying RNA and its role in biological systems.

Development of a novel flow cytometry method for detecting pneumococcal-specific B cells.

Antigen-specific B cell identification by flow cytometry is crucial for investigating their immunophenotype, subset distribution, and kinetics post-infection or immunization. Methods using biotinylated polysaccharide antigens have been described, but there is still room for improvement regarding sensitivity and applicability. The aim of this study was the development and validation of a multimer bead-based method for detecting pneumococcal polysaccharide serotypes (PS)-specific B cells following pneumococcal immunization. PS was chemically biotinylated and mounted on anti-biotin beads, and labeled with phycoerythrin (PE)-conjugated anti-biotin antibody to form a PS-multimer used for cell staining. Labeled beads were washed to remove excess fluorochrome and diminish non-specific labeling and background noise. Optimal ratios of PS-bead conjugate to PE and PS-multimer to cells were determined with titration assays. Comparison between the PS-multimer and a PS-PE monomer revealed enhanced detection of PS-specific cells and considerable signal amplification, attributed to the multimeric form of the detection probe and increased availability of antigen epitopes. To validate the specificity of the method, a competition assay using unbound PS was performed. Following pre-incubation with increasing PS concentrations, detection of PS-specific B cells with the PS-multimer was inhibited in a stepwise manner. Pre-incubation with excess PS completely blocked the fluorescent signal. This novel bead-based flow cytometry approach is a sensitive method demonstrating high specificity. It generated enhanced signals, provided clear-cut results, and was easily applicable, not requiring B cell pre-enrichment. It could be modified to adapt other antigens of interest, especially polysaccharides and proteins that could be used to probe antigen-specific B cell responses. The study of such responses may elucidate the underlying mechanisms involved in the establishment of long-term protection, provide evidence-based rationale for improving currently available vaccines and vaccination strategies, and pave the way for future vaccine development.

Identification of macrophages in normal and injured mouse tissues using reporter lines and antibodies

Reliable tools for macrophage identification in mouse tissues are critical for studies investigating inflammatory and reparative responses. Transgenic reporter mice and anti-macrophage antibodies have been used as “specific pan-macrophage” markers in many studies; however, organ-specific patterns of expression and non-specific labeling of other cell types, such as fibroblasts, may limit their usefulness. Our study provides a systematic comparison of macrophage labeling patterns in normal and injured mouse tissues, using the CX3CR1 and CSF1R macrophage reporter lines and anti-macrophage antibodies. Moreover, we tested the specificity of macrophage antibodies using the fibroblast-specific PDGFRmathrm{alpha } reporter line. Mouse macrophages exhibit organ-specific differences in expression of macrophage markers. Hepatic macrophages are labeled for CSF1R, Mac2 and F4/80, but lack CX3CR1 expression, whereas in the lung, the CSF1R+/Mac2+/Mac3+ macrophage population is not labeled with F4/80. In the splenic red pulp, subpopulations of CSF1R+/F4/80+/Mac3+cells were labeled with Mac2, CX3CR1 and lysozyme M. In the kidney, Mac2, Mac3 and lysozyme M labeled a fraction of the CSF1R+ and CX3CR1+ macrophages, but also stained tubular epithelial cells. In normal hearts, the majority of CSF1R+ and CX3CR1+ cells were not detected with anti-macrophage antibodies. Myocardial infarction was associated with marked expansion of the CSF1R+ and CX3CR1+ populations that peaked during the proliferative phase of cardiac repair, and also expressed Mac2, Mac3 and lysozyme M. In normal mouse tissues, a small fraction of cells labeled with anti-macrophage antibodies were identified as PDGFRmathrm{alpha }+ fibroblasts, using a reporter system. The population of PDGFRmathrm{alpha }+ cells expressing macrophage markers expanded following injury, likely reflecting emergence of cellular phenotypes with both fibroblast and macrophage characteristics. In conclusion, mouse macrophages exhibit remarkable heterogeneity. Selection of the most appropriate markers for identification of macrophages in mouse tissues is dependent on the organ and the pathologic condition studied.

Cy3-RgIA-5727 Labels and Inhibits α9-Containing nAChRs of Cochlear Hair Cells.

Efferent cholinergic neurons inhibit sensory hair cells of the vertebrate inner ear through the combined action of calcium-permeable α9α10-containing nicotinic acetylcholine receptors (nAChRs) and associated calcium-dependent potassium channels. The venom of cone snails is a rich repository of bioactive peptides, many with channel blocking activities. The conopeptide analog, RgIA-5474, is a specific and potent antagonist of α9α10-containing nAChRs. We added an alkyl functional group to the N-terminus of the RgIA-5474, to enable click chemistry addition of the fluorescent cyanine dye, Cy3. The resulting peptide, Cy3-RgIA-5727, potently blocked mouse α9α10 nAChRs expressed in Xenopus oocytes (IC50 23 pM), with 290-fold less activity on α7 nAChRs and 40,000-fold less activity on all other tested nAChR subtypes. The tight binding of Cy3-RgIA-5727 provided robust visualization of hair cell nAChRs juxtaposed to cholinergic efferent terminals in excised, unfixed cochlear tissue from mice. Presumptive postsynaptic sites on outer hair cells (OHCs) were labeled, but absent from inner hair cells (IHCs) and from OHCs in cochlear tissue from α9-null mice and in cochlear tissue pre-incubated with non-Cy3-conjugated RgIA-5474. In cochlear tissue from younger (postnatal day 10) mice, Cy3-RgIA-5727 also labeled IHCs, corresponding to transient efferent innervation at that age. Cy3 puncta in Kölliker’s organ remained in the α9-null tissue. Pre-exposure with non-Cy3-conjugated RgIA-5474 or bovine serum albumin reduced this non-specific labeling to variable extents in different preparations. Cy3-RgIA-5727 and RgIA-5474 blocked the native hair cell nAChRs, within the constraints of application to the excised cochlear tissue. Cy3-RgIA-5727 or RgIA-5474 block of efferent synaptic currents in young IHCs was not relieved after 50 min washing, so effectively irreversible.