Extracellular Labeling Research Articles

Fluorescence labeling strategies for cell surface expression of TRPV1.

Regulation of ion channel expression on the plasma membrane is a major determinant of neuronal excitability, and identifying the underlying mechanisms of this expression is critical to our understanding of neurons. Here, we present two orthogonal strategies to label extracellular sites of the ion channel TRPV1 that minimally perturb its function. We use the amber codon suppression technique to introduce a non-canonical amino acid (ncAA) with tetrazine click chemistry, compatible with a trans-cyclooctene coupled fluorescent dye. Additionally, by inserting the circularly permutated HaloTag (cpHaloTag) in an extracellular loop of TRPV1, we can incorporate a fluorescent dye of our choosing. Optimization of ncAA insertion sites was accomplished by screening residue positions between the S1 and S2 transmembrane domains with elevated missense variants in the human population. We identified T468 as a rapid labeling site (∼5 min) based on functional and biochemical assays in HEK293T/17 cells. Through adapting linker lengths and backbone placement of cpHaloTag on the extracellular side of TRPV1, we generated a fully functional channel construct, TRPV1exCellHalo, with intact wild-type gating properties. We used TRPV1exCellHalo in a single molecule experiment to track TRPV1 on the cell surface and validate studies that show decreased mobility of the channel upon activation. The application of these extracellular label TRPV1 (exCellTRPV1) constructs to track surface localization of the channel will shed significant light on the mechanisms regulating its expression and provide a general scheme to introduce similar modifications to other cell surface receptors.

Fluorescence labeling strategies for the study of ion channel and receptor cell surface expression: A comprehensive toolkit for extracellular labeling of TRPV1.

Regulation of ion channel expression on the plasma membrane is a major determinant of neuronal excitability, and identifying the underlying mechanisms of this expression is critical to our understanding of neurons. A critical aspect of measuring changes in ion channel expression is uniquely identifying ion channels located on the cell surface. To accomplish this goal we demonstrate two orthogonal strategies to label extracellular sites of the ion channel TRPV1 that minimally perturb the function of the channel: 1) We use the amber codon suppression technique to introduce a non-canonical amino acid (ncAA) with tetrazine click chemistry compatible with a trans-cyclooctene coupled fluorescent dye. 2) By inserting the circularly permutated HaloTag (cpHaloTag) in an extracellular loop of TRPV1, we incorporate a click-chemistry site for a chloroalkane-linked fluorescent dye of our choosing. Optimization of ncAA insertion sites was accomplished by screening residue positions between the S1 and S2 transmembrane domains with elevated missense variants in the human population, and we identified T468 as a rapid labeling site (~5 minutes) based on functional as well as biochemical assays in HEK293T/17 cells. After several rounds of adapting the linker lengths and backbone placement of cpHaloTag on the extracellular side of TRPV1, our efforts led to a channel construct that robustly expressed as a fully functional TRPV1exCellHalo fusion with intact wild-type gating properties. The TRPV1exCellHalo construct was used in a single molecule experiment to track TRPV1 on the cell surface and validate studies that show decreased mobility of the channel upon activation. The success of these extracellular label TRPV1 (exCellTRPV1) constructs as tools to track surface expression of the channel will shed significant light on the mechanisms regulating expression and provide a general scheme to introduce similar modifications to other cell surface receptors.

Abstract 86: Deep immuno-profiling of syngeneic tumor mouse models for preclinical studies

Abstract The identification of the major cellular players involved in the progression of a type of cancer is a key step for the success of new immunotherapies for personalized medicine. Immune cells play critical functions in cancer, and mice with intact immune systems are vital to understanding tumor immunology. It is however a daunting challenge as complex relationships interplay between tumor cells and the immune system. Each component of innate immunity or adaptive immunity, such as T lymphocytes, macrophages or neutrophils, may be directed to a pro- or anti-tumor function. In order to cope with the complexity of the tumor microenvironment, it is necessary to use an experimental approach capable of characterizing the heterogeneity of the cell types present in the tumor, the evolution of their relative proportion and their fine-tuned functional specificity. This approach leads to the identification of new cellular biomarkers of different stages of tumor progression in order to identify new targets for therapeutic time-window of and improve cancer diagnosis To decipher the impact of immunotherapy treatments involved in the anti-tumor response, cellular phenotyping of leukocytes infiltrating a tumor but also those present in peripheral organs is necessary. Essentially based on extracellular labeling, this primary screen aims to quantify the different cell populations present in a syngenic tumor models on B6/N or BalbC genetical backgrounds. In order to increase our understanding in the precise mode of action of anti-PD1 treatment at the cellular level in sensitive and unsensitive models, we investigated immunophenotypes and responses to immune checkpoint inhibitor (ICI) of several hallmark syngeneic tumor models (MC38, CT26, B16F10, B16-OVA, RENCA, EMT6) in immunocompetent mouse models by flow and mass cytometry. We compared growth kinetics and profiled the immune cell composition of tumor microenvironment (TME), draining lymph node (dLN) and blood in order to establish immune-phenotypic cell signatures that correlates with treatment efficacy. Supervised, unsupervised and integrative data analysis as well as visualization tools are used to identify significant changes across different experimental settings. Our results indicate that each model possesses a unique tumor-immune infiltrate profile that can be modulated with immunotherapies. Overall, these studies provide an important resource of highly-characterized syngeneic tumor model and highlight the importance of tumor immune landscape variance across models that will drive selecting the most appropriate model to test novel immunotherapeutic agents and enhance our translation of knowledge from syngeneic models to human tumors. Citation Format: Anais Joachim, Emilie Maturin, Marielle Mello, Lilia Hadjem, Magali Grange, Olivier Deas, Ana Zarubica, Bernard Malissen, Hervé Luche, François Romagne, Stéphanie Blanchin. Deep immuno-profiling of syngeneic tumor mouse models for preclinical studies [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 86.

Abstract 2348: Mapping the interactome of matrisome targets using extracellular proximity labeling (ePL)

Abstract Classical methods to investigate protein-protein interactions (PPIs) are generally performed in non-living systems, yet in recent years new technologies utilizing proximity labeling (PL) have given researchers the tools to explore PPIs in living systems. PL has distinct advantages over traditional protein interactome studies, such as the ability to identify weak and transient interactions in vitro and in vivo. Most PL studies are performed on targets within or on the cell membrane. We describe a method to investigate PPIs within the extracellular compartment, using both BioID2 and TurboID, that we term extracellular PL (ePL). To demonstrate the utility of this modified technique, we investigate the interactome of the widely expressed matrisome protein Tissue inhibitors of metalloproteinases 2 (TIMP2). Tissue inhibitors of metalloproteinases (TIMPs) are a family of multi-functional proteins that were initially defined by their ability to inhibit the enzymatic activity of matrix metalloproteinases (MMPs), the major mediators of ECM breakdown and turnover. TIMP2 is a unique family member, with a broad expression profile that is expressed in both normal and diseased tissues, even in those with minimal metalloproteinase activity. Understanding the functional transformation of matrisome regulators, like TIMP2, during the evolution of tissue microenvironments associated with disease progression is essential to the development of ECM targeted therapeutics. This knowledge may also garner understanding of therapeutic resistance and the failure of conventional and next-generation cancer therapies. Using carboxyl- and amino-terminal fusion peptides of TIMP2 with BioID2 and TurboID, we describe the TIMP2 interactome in unique tissue compartments. We also illustrate how the TIMP2 interactome changes in the presence of different stimuli, in different cell lines, and with different reaction kinetics (BioID2 vs. TurboID); demonstrating the power of this technique and comparing our findings with classical PPI methods. We propose that the screening of matrisome targets in disease models using ePL will reveal new therapeutic targets for further comprehensive studies. Citation Format: David Peeney, Sadeechya Gurung, Josh Rich, Sasha Coates-Park, Yueqin Liu, William G. Stetler-Stevenson. Mapping the interactome of matrisome targets using extracellular proximity labeling (ePL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2348.

Abstract 4695: Expansion of the extracellular proximity labeling (ePL) technique to novel matrisome targets

Abstract The extracellular matrix (ECM) is a complex and dynamic meshwork of extracellular macromolecules that plays a key role in cancer progression and metastasis. The matrisome encompasses the genes encoding ECM and ECM-associated secretory proteins, and elucidating the protein-protein interactions (PPIs) of the matrisome is a promising area of study in the quest for a better understanding of cancer pathology and therapeutics. Proximity labeling, a powerful technique for investigating protein interactomes based on enzyme-fusion constructs and affinity chromatography, has considerable potential in detailing the protein-protein interactions of the matrisome. We have recently had success in developing a proximity labeling approach based on fusion constructs containing the biotinylating enzymes BioID2 and TurboID. We apply this approach to gain new insights into the interactome of the matrisome protein tissue inhibitor of metalloproteinase 2 (TIMP2), a protein found to have antitumorigenic potential. At present, we seek to expand this approach to other ECM protein targets, including tissue inhibitor of metalloproteinase 3 (TIMP3) and thrombospondin-1 (THBS-1). TIMP3 has been shown to possess anti-tumor properties, with loss of TIMP3 function associated with poor prognosis in multiple cancers. THBS-1 is a large (129 kDa) ECM protein that is relatively promiscuous with diverse functions in the tumor microenvironment. Expression of THBS-1 is frequently lost during oncogenesis, and such loss is associated with poor prognosis in some cancers. We describe how ePL can be expanded to these matrisome targets in different cellular systems (adherent vs. suspension) and show that ePL assays can be performed with stable and transient transgene expression. We show that ePL is a suitable method for studying PPIs in novel matrisome targets and expect to apply the technique to further targets in the near future. Citation Format: Joshua Rich, Sadeechya Gurung, Sukhbir Kaur, David Roberts, William Stetler-Stevenson, David Peeney. Expansion of the extracellular proximity labeling (ePL) technique to novel matrisome targets. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4695.

Human Forebrain Organoid-Derived Extracellular Vesicle Labeling with Iron Oxides for In Vitro Magnetic Resonance Imaging.

The significant roles of extracellular vesicles (EVs) as intracellular mediators, disease biomarkers, and therapeutic agents, make them a scientific hotspot. In particular, EVs secreted by human stem cells show significance in treating neurological disorders, such as Alzheimer’s disease and ischemic stroke. However, the clinical applications of EVs are limited due to their poor targeting capabilities and low therapeutic efficacies after intravenous administration. Superparamagnetic iron oxide (SPIO) nanoparticles are biocompatible and have been shown to improve the targeting ability of EVs. In particular, ultrasmall SPIO (USPIO, <50 nm) are more suitable for labeling nanoscale EVs due to their small size. In this study, induced forebrain neural progenitor cortical organoids (iNPCo) were differentiated from human induced pluripotent stem cells (iPSCs), and the iNPCo expressed FOXG1, Nkx2.1, α-catenin, as well as β-tubulin III. EVs were isolated from iNPCo media, then loaded with USPIOs by sonication. Size and concentration of EV particles were measured by nanoparticle tracking analysis, and no significant changes were observed in size distribution before and after sonication, but the concentration decreased after labeling. miR-21 and miR-133b decreased after sonication. Magnetic resonance imaging (MRI) demonstrated contrast visualized for the USPIO labeled EVs embedded in agarose gel phantoms. Upon calculation, USPIO labeled EVs exhibited considerably shorter relaxation times, quantified as T2 and T2* values, reducing the signal intensity and generating higher MRI contrast compared to unlabeled EVs and gel only. Our study demonstrated that USPIO labeling was a feasible approach for in vitro tracking of brain organoid-derived EVs, which paves the way for further in vivo examination.

Amyloid Pathology in the Central Auditory Pathway of 5XFAD Mice Appears First in Auditory Cortex.

Effective treatment of Alzheimer's disease (AD) will hinge on early detection. This has led to the search for early biomarkers that use non-invasive testing. One possible early biomarker is auditory temporal processing deficits, which reflect central auditory pathway dysfunction and precede cognitive and memory declines in AD. Gap detection is a measure of auditory temporal processing, is impaired in human AD, and is also impaired in the 5XFAD mouse model of AD. Gap detection deficits appear as early as postnatal day 60 in 5XFAD mice, months before cognitive deficits or cell death, supporting gap detection as an early biomarker. However, it remains unclear how gap detection deficits relate to the progression of amyloid pathology in the auditory system. To determine the progression of amyloid pathology throughout the central auditory system and across age in 5XFAD mice. We quantified intracellular and extracellular antibody labelling of Aβ42 in 6 regions of the central auditory system from p14 to p150. Pathology appeared first in primary auditory cortex (A1) as intracellular accumulation of Aβ42 in layer 5 pyramidal neurons by age p21. Extracellular plaques appeared later, by age p90, in A1, medial geniculate body, and inferior colliculus. Auditory brainstem structures showed minimal amyloid pathology. We also observed pathology in the caudal pontine reticular nucleus, a brainstem structure that is outside of the central auditory pathway but which is involved in the acoustic startle reflex. These results suggest that Aβ42 accumulation, but not plaques, may impair gap detection.

In situ cell-type-specific cell-surface proteomic profiling in mice

Cell-surface proteins (CSPs) mediate intercellular communication throughout the lives of multicellular organisms. However, there are no generalizable methods for quantitative CSP profiling in specific cell types in vertebrate tissues. Here, we present in situ cell-surface proteome extraction by extracellular labeling (iPEEL), a proximity labeling method in mice that enables spatiotemporally precise labeling of cell-surface proteomes in a cell-type-specific environment in native tissues for discovery proteomics. Applying iPEEL to developing and mature cerebellar Purkinje cells revealed differential enrichment in CSPs with post-translational protein processing and synaptic functions in the developing and mature cell-surface proteomes, respectively. A proteome-instructed invivo loss-of-function screen identified a critical, multifaceted role for Armh4 in Purkinje cell dendrite morphogenesis. Armh4 overexpression also disrupts dendrite morphogenesis; this effect requires its conserved cytoplasmic domain and is augmented by disrupting its endocytosis. Our results highlight the utility of CSP profiling in native mammalian tissues for identifying regulators of cell-surface signaling.

Alleviation of cognitive deficits via upregulation of chondroitin sulfate biosynthesis by lignan sesamin in a mouse model of neuroinflammation

Lignans are plant-derived compounds that act as partial estrogen agonists. Chondroitin sulfate proteoglycans (CSPGs) represent one of the major components of the extracellular matrix. Here we aimed to understand the role of sesamin (SES), a major lignan compound, in the biosynthesis and degradation of CSPGs in the mouse hippocampus because CSPGs play a key role in the regulation of cognitive functions through the promotion of adult neurogenesis. The expression of the pro-inflammatory cytokine interleukin-1β was decreased by SES administration in the hippocampus of lipopolysaccharide (LPS)-treated mice, a model of neuroinflammation-induced cognitive deficits. The expression of genes related to biosynthesis and degradation of CSPGs in the hippocampus of LPS-treated mice was both increased and decreased by SES administration. Further, the diffuse extracellular matrix labeling of CSPGs by Wisteria floribunda agglutinin (WFA) in the hippocampus of LPS-treated mice was increased by SES administration. The densities of neural stem cells, late transit-amplifying cells, and newborn-granule cells in the hippocampus of LPS-treated mice were also increased by SES administration. Moreover, SES-induced alterations in gene expression, WFA labeling, and adult neurogenesis in LPS-treated mice were more evident in the dorsal hippocampus (center of cognition) than in the ventral hippocampus (center of emotion). Neither LPS nor SES administration affected locomotor activity, anxiety-like behavior, and depression-related behavior. However, impairments in contextual memory and sensorimotor gating in LPS-treated mice were recovered by SES administration. Our results show that SES can promote adult hippocampal neurogenesis through the upregulation of CSPGs, which may alleviate cognitive deficits induced by neuroinflammation.

Abstract 998: Extracellular proximity labeling (ePL) as a tool to identify protein-protein interactions in the tumor microenvironment

Abstract The extracellular matrix (ECM) is a dynamic niche that is extensively reshaped in the development of the tumor microenvironment (TME). Our current understanding of ECM function and dynamics is largely informed by identification of protein-protein interactions (PPIs) using co-immunoprecipitation (co-IP) techniques that may miss transient and weak/unstable interactions. Recent advances in proximity labeling techniques have greatly expanded the interactome networks of numerous intracellular proteins, however these tools have yet to be extended to study PPIs in the ECM. We have recently optimized a systematic approach to identify PPIs in the ECM using fusion constructs of the biotinylating enzymes, BioID2 and TurboID, with the widely expressed matrix regulator TIMP2. BioID2 and TurboID offer differing reaction kinetics that may provide complimentary information on extracellular PPIs (ePPIs). Matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs) are crucial regulators of ECM structure and composition. TIMPs are widely expressed multifunctional proteins that serve to promote ECM homeostasis that is often perturbed in many cancers and chronic disorders. Although biochemical data suggests that TIMPs are promiscuous proteins, the TIMP interactome is poorly defined. We have optimized a protocol for the identification of ePPIs for the TIMP family of proteins. Fusion constructs equipped with a promiscuous biotin ligase (BioID2/TurboID) fused to the N- or C-terminal of full length TIMP2 were packaged into retroviral vectors for cellular delivery. Cells were exposed to the extracellular proximity labelling (ePL) fusion proteins and processed in our optimized analysis pipeline. ePPIs were identified via streptavidin pulldown and proteomic techniques. We present our optimized ePL pipeline and show that this technique is an effective tool for the identification of novel ePPIs for multiple extracellular targets. Citation Format: Sadeechya Gurung. Extracellular proximity labeling (ePL) as a tool to identify protein-protein interactions in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 998.

Abstract 3836: Mapping the interactome of the endogenous metalloproteinase inhibitor TIMP2 using extracellular proximity labeling (ePL)

Abstract Tissue inhibitors of metalloproteinases (TIMPs) are a family of endogenous proteins that were initially defined by their ability to inhibit the enzymatic activity of matrix metalloproteinases (MMPs), the major mediators of ECM breakdown and turnover. TIMPs are important regulators of ECM structure, function and homeostasis. Since their original discovery, additional MMP-independent functions have been attributed to TIMP family members leading to their designation as multifunctional proteins with discrete functional domains. TIMP2 is a unique family member, with a broad expression profile that is expressed in both normal and diseased tissues, even in those with minimal metalloproteinase activity. Understanding the functional transformation of matrisome regulators, like TIMP-2, during the dynamic evolution of tissue microenvironments associated with disease progression is essential to the development of ECM targeted therapeutics. This knowledge may also garner understanding of therapeutic resistance and the failure of conventional and next-generation cancer therapies. Interactome studies are predominantly performed in non-living systems, and very rarely performed in complex culture systems such as 3D spheroids/co-cultures. To investigate the TIMP-2 interactome in complex biological systems we recently developed carboxyl- and amino-terminal fusion genes of TIMP-2 with the promiscuous biotinylating peptides BioID2 and TurboID. Packaged for retroviral delivery, this system gives us great flexibility in assessing the TIMP-2 interactome in simple versus complex 3D co-culture systems. We show, for the first time, that conditions can be optimized to support proximity labeling reactions in the extracellular milieu, representing an important technical resource for extracellular matrix biologists. Using this method, we identify novel interacting partners for TIMP-2 that provide insight into the tissue-specific actions of this protein in both healthy and diseased tissues. Citation Format: David Peeney, Sadeechya Gurung, Yueqin Liu, Carolyn Lazaroff, Christopher Richie, William G. Stetler-Stevenson. Mapping the interactome of the endogenous metalloproteinase inhibitor TIMP2 using extracellular proximity labeling (ePL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3836.

Imaging flow cytometry challenges the usefulness of classically used extracellular vesicle labeling dyes and qualifies the novel dye Exoria for the labeling of mesenchymal stromal cell–extracellular vesicle preparations

Background aimsExtracellular vesicles (EVs) are involved in mediating intercellular communication processes. An important goal within the EV field is the study of the biodistribution of EVs and the identification of their target cells. Considering that EV uptake is assumed to be important for EVs in mediating intercellular communication processes, labeling with fluorescent dyes has emerged as a broadly distributed strategy for the identification of EV target cells and tissues. However, the accuracy and specificity of commonly utilized labeling dyes have not been sufficiently analyzed. MethodsBy combining recent advances in imaging flow cytometry for the phenotypic analysis of single EVs and aiming to identify target cells for EVs within therapeutically relevant mesenchymal stromal cell (MSC)-EV preparations, the authors explored the EV labeling efficacy of various fluorescent dyes, specifically carboxyfluorescein diacetate succinimidyl ester, calcein AM, PKH67, BODIPY TR ceramide (Thermo Fisher Scientific, Darmstadt, Germany) and a novel lipid dye called Exoria (Exopharm Limited, Melbourne, Australia). ResultsThe authors’ analyses qualified Exoria as the only dye that specifically labeled EVs within the MSC-EV preparations. Furthermore, the authors demonstrated that Exoria labeling did not interfere with the immunomodulatory properties of the MSC-EV preparations as tested in a multi-donor mixed lymphocyte reaction assay. Within this assay, labeled EVs were differentially taken up by different immune cell types. ConclusionsOverall, the results qualify Exoria as an appropriate dye for the labeling of EVs derived from the authors’ MSC-EV preparations. This study also demonstrates the need for the development of next-generation EV characterization tools that are able to localize and confirm the specificity of EV labeling.

Morphology and receptive field organization of a temporal processing region in Apteronotus albifrons

The timing system of weakly electric fishes is vital for many behavioral processes, but the system has been relatively unexplored in Apteronotus albifrons. This paper describes the receptive fields of phase-locked neurons in the midbrain of A. albifrons, in combination with neuroanatomy and electron microscopy (EM) to delineate a phase-locked area in this fish, the magnocellular mesencephalic nucleus (MMN). The MMN was isolated electrophysiologically through the detection of phase-locked field potentials of high amplitude. Single-cell recordings were made with a sharp electrode while a phase-locked modulated stimulus was provided to the fish. Receptive field centers of phase-locked neurons in MMN were consistent with tuberous electroreceptor density maps from previous studies, but no receptive field centers were found in the posterior 50% of the body. Intracellular and extracellular labeling of MMN revealed three cell populations: giant cells with large somata (19-24µm) and their axonal arborizations which span across the entire extent of MMN, axon terminals from spherical cells of the electrosensory lateral line lobe (ELL), and small cell somata (3-7µm) along with their projections which extend outside the nucleus. EM revealed multiple gap junction and chemical synapses within MMN. Our results indicate that MMN is a dedicated temporal processing center in A. albifrons.

Application of a Novel In Situ Activity Assay to Detect Lysyl Oxidase Like‐2 (LOXL2)

IntroductionLysyl oxidase like‐2 (LOXL2) is an enzyme of the lysyl oxidase (LOX) super family, which are amine oxidases that initialize collagen and elastin cross‐linking to form a stable and organized matrix. Augmented LOXL2 expression is highly associated with diverse disease processes with a known element of matrix accumulation including fibrosis, pulmonary hypertension, and cancer metastasis. To date, lack of sensitive and specific assays that can detect LOXL2 catalytic function in situ in the extracellular matrix (ECM) of cells and tissue has limited the ability to fully elucidate the role of LOXL2 in pathobiological mechanisms. Existing assays are limited in dynamic range and are unfeasible to interrogate matrix‐associated LOXL2 as they require LOXL2 to be in solution. We developed a novel in situ activity assay that overcomes these limitations. Our previous proof‐of‐concept experiments showed that the assay is able to detect overexpression of LOXL2 or loss of enzymatic function in cell culture. Here, we applied the assay in biologically relevant specimens.Materials and MethodsIn our assay, we utilize the strong reaction between hydrazide and aldehyde to label the LOX catalyzed allysine (−CHO) residues with biotin hydrazide (BHZ). This allows for a robust method of visualizing all ECM protein sidechains that have been catalyzed by LOXs via biotin‐streptavidin interaction. This assay has been incorporated in a few of our studies on LOXL2. First, we tested the expression and activity level of various LOXL2 mutant proteins overexpressed in A7r5 cells via adenoviral delivery. Second, we used this assay on A7r5 cells to study the modulation of LOXL2 activity when processed by protease Factor Xa (FXa). Lastly, we tested expression of LOXL2 in aortas from wild type and LOXL2+/− mice, which were cut into 5mm rings and transected open. In all experiments, samples were incubated with 50–150nM BHZ for 24 hours to allow for extracellular matrix deposition and labeling of LOXL2 catalysis. Biotinylation of allysines and expression of LOXL2 were then visualized by confocal immunofluorescence (IF) microscopy.ResultsAs seen in Figure 1, cells overexpressing LOXL2‐wt showed significantly increased biotinylation, suggesting an increase in LOXL2 activity as expected. Fibrous structures can also be seen for longer BHZ incubation as new matrix form. Whereas cells overexpressing LOXL2DM showed no difference in biotinylation compared to control, as LOXL2DM cannot catalyze oxidative deamination to produce allysines. Additional conditions have also been tested to study the functions of different domains of LOXL2. Activity assay on cells exposed to FXa processing showed that processing disrupted LOXL2 catalysis. Lastly, we were able to clearly visualize significant difference in LOXL2 expression in Young and Old WT vs. LOXL2 +/− aorta, confirming that the assay is feasible not only in cellular experiments but also on tissue samples.ConclusionsWe were able to visualize LOXL2 reaction in the ECM by IF microscopy through the biotinylation by BHZ. This study demonstrates different applications of a novel in situ activity assay for LOXL2 in both cell culture and tissue samples, which can be impactful for future research that studies LOXL2 as a therapeutic target.Support or Funding InformationJHU ACCM StAAR grant and MedImmuneIF images of A7r5 cells overexpressing wild type LOXL2 and enzymatically inactive LOXL2DM. Cells were incubated with biotin hydrazide for 24 hours.Figure 1IF confocal Zstack maximum projection images of aortas from Young (&lt;16 weeks) and Old (&gt;52 weeks) WT and LOXL2+/− mice.Figure 2

Proximal Tubule Epithelial Cell Reabsorption of the Drug Delivery Protein Elastin‐like Polypeptide

Elastin like polypeptide (ELP) is a protein composed of a pentapeptide repeat domain found in human tropoelastin. ELP is used for drug delivery by altering the ELP coding sequence to attach drugs, therapeutic peptides, or therapeutic proteins. One of the focuses of our group has been development of ELP – delivered therapeutics for treatment of kidney disease. Thus, understanding how ELP is filtered, reabsorbed, and distributed within the kidney is of key importance. In vivo studies in our lab showed that ELPs accumulated preferentially in the kidney after systemic administration. Renal deposition and intra‐renal distribution was dependent on ELP size, with smaller ELPs accumulating exclusively in the renal cortex and larger ELPs having a more mixed cortical and medullary distribution. All ELPs were present at high levels in tubular epithelial cells. However, whether the tubular distribution is the result of ELP reabsorption after filtration and whether the amount or rate of reabsorption is dependent on ELP size is unknown.To address this issue, uptake and intracellular distribution of fluorescein‐5‐malemide labeled ELPs of varying sizes were determined in primary human proximal tubule epithelial cells (HRPteC). HRPteC were seeded in a 6 well plate (4x104/well) and incubated with fluorescein labeled ELP (10μM) for various times (24hr, 6hr, 4hr, 2hr and 1hr). HRPteC were collected, and mean cellular fluorescence intensity was measured using flow cytometry. Percentage of ELP internalization was determined by quenching the extracellular fluorescein label with trypan blue (0.13% v/v), then repeating the flow cytometric analysis to measure only intracellular fluorescence. To obtain a qualitative representation of ELP interaction with kidney cells, HRPteC were seeded in an 8‐chamber glass slide (2x103 cells/chamber) and incubated with fluorescein labeled ELP (10μM) for various times as above. Following fixation and staining with 4′,6‐diamidino‐2‐phenylindole (DAPI), cells were imaged by laser scanning confocal microscopy. Our data demonstrate that a longer incubation time led to a greater accumulation of ELP in the proximal tubule epithelial cells. Analysis of internalization rate revealed that all ELPs entered the cells slowly over the course of a 24‐hour time frame. This finding was supported by confocal microscopy. Between 1 and 12 hours, localization of ELP occurred at the cellular membrane of the proximal tubule cells, but at time points between 12 and 24 hours, ELP was visible in punctate cytoplasmic structures likely to be endocytic vesicles. A smaller ELP‐63 (~25 kDa) accumulated in cells at higher levels than did a larger ELP‐159 (~62 kDa) after 24 hours of incubation (390.7 ±5.0 versus 651.6 ±42.7 RFU; p&lt;0.05). These data suggest that there may be a common mechanism for tubular epithelial cell uptake of ELPs of various sizes, but that smaller ELPs may be internalized to a greater extent than larger ELPs. The results of these and future studies to determine the exact renal handling of ELPs of various sizes will be crucial to the design of ELPs for delivery of therapeutics to specific renal compartments or cell types.

Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates.

Employing self-labelling protein tags for the attachment of fluorescent dyes has become a routine and powerful technique in optical microscopy to visualize and track fused proteins. However, membrane permeability of the dyes and the associated background signals can interfere with the analysis of extracellular labelling sites. Here we describe a novel approach to improve extracellular labelling by functionalizing the SNAP-tag substrate benzyl guanine (“BG”) with a charged sulfonate (“SBG”). This chemical manipulation can be applied to any SNAP-tag substrate, improves solubility, reduces non-specific staining and renders the bioconjugation handle impermeable while leaving its cargo untouched. We report SBG-conjugated fluorophores across the visible spectrum, which cleanly label SNAP-fused proteins in the plasma membrane of living cells. We demonstrate the utility of SBG-conjugated fluorophores to interrogate class A, B and C G protein-coupled receptors (GPCRs) using a range of imaging approaches including nanoscopic superresolution imaging, analysis of GPCR trafficking from intra- and extracellular pools, in vivo labelling in mouse brain and analysis of receptor stoichiometry using single molecule pull down.

Bioorthogonal labeling with tetrazine-dyes for super-resolution microscopy

Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye. We discovered that photoinduced electron transfer (PET) from the excited dye to tetrazine is the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. Upon reaction with dienophiles quenching interactions are reduced resulting in a considerable increase in fluorescence intensity. Efficient and specific labeling of all tetrazine-dyes investigated permits super-resolution microscopy with high signal-to-noise ratio even at the single-molecule level. The different cell permeability of tetrazine-dyes can be used advantageously for specific intra- and extracellular labeling of proteins and highly sensitive fluorescence imaging experiments in fixed and living cells.

Single-Cell Analyses of Human Eosinophils at High Resolution to Understand Compartmentalization and Vesicular Trafficking of Interferon-Gamma

Human eosinophils release numerous cytokines that are pre-synthesized and stored within their cytoplasmic-specific (secretory) granules. For example, high levels of interferon-gamma (IFN-γ) are constitutively expressed in these cells, but the intracellular compartments involved in the transport and release of this cytokine remain to be established. In this work, we used a single-cell approach to investigate the subcellular localization of IFN-γ in human eosinophils stimulated or not with tumor necrosis factor alpha (TNF-α) or CC-chemokine ligand 11 CCL11 (eotaxin-1), inflammatory mediators that induce eosinophil activation and secretion. A pre-embedding immunonanogold transmission electron microscopy (TEM) technique that combines optimal epitope preservation and access to membrane microdomains was applied to detect precise localization of IFN-γ in combination with computational quantitative analyses. In parallel, degranulation processes and formation of eosinophil sombrero vesicles (EoSVs), large transport carriers involved in the transport of granule-derived cytokines, were investigated. Quantitative TEM revealed that both CCL11 and TNF-α-activated eosinophils significantly increased the total number of EoSVs compared to the unstimulated group, indicating that this vesicular system is actively formed in response to cell activation. Ultrastructural immunolabeling identified a robust pool of IFN-γ on secretory granules in both unstimulated and stimulated cells. Moreover, EoSVs carrying IFN-γ were seen around or/and in contact with secretory granules and also distributed in the cytoplasm. Labeling was clearly associated with EoSV membranes. The total number of IFN-γ-positive EoSVs was significantly higher in stimulated compared to unstimulated cells, and these labeled vesicles had a differential distribution in the cytoplasm of activated cells, being significantly higher in the cell periphery compared with the inner cell, thus revealing intracellular IFN-γ mobilization for release. IFN-γ extracellular labeling was found at the cell surface, including on extracellular vesicles. Our results provide direct evidence that human eosinophils compartmentalize IFN-γ within secretory granules and identify, for the first time, a vesicular trafficking of IFN-γ associated with large transport carriers. This is important to understand how IFN-γ is trafficked and secreted during inflammatory responses.

Super-Resolution Imaging of the Extracellular Space in Living Brain Tissue

The extracellular space (ECS) of the brain has an extremely complex spatial organization, which has defied conventional light microscopy. Consequently, despite a marked interest in the physiological roles ofbrain ECS, its structure and dynamics remain largelyinaccessible for experimenters. We combined 3D-STED microscopy and fluorescent labeling of theextracellular fluid to develop super-resolution shadow imaging (SUSHI) of brain ECS in living organotypic brain slices. SUSHI enables quantitative analysis of ECS structure and reveals dynamics on multiple scales in response to a variety of physiological stimuli. Because SUSHI produces sharp negative images of all cellular structures, it also enables unbiased imaging of unlabeled brain cells with respect to their anatomical context. Moreover, the extracellular labeling strategy greatly alleviates problems of photobleaching and phototoxicity associated with traditional imaging approaches. As a straightforward variant of STED microscopy, SUSHI provides unprecedented access to the structure and dynamics of live brain ECS and neuropil.

Surface electrodes record and label brain neurons in insects.

We used suction electrodes to reliably record the activity of identified ascending auditory interneurons from the anterior surface of the brain in crickets. Electrodes were gently attached to the sheath covering the projection area of the ascending interneurons and the ringlike auditory neuropil in the protocerebrum. The specificity and selectivity of the recordings were determined by the precise electrode location, which could easily be changed without causing damage to the tissue. Different nonauditory fibers were recorded at other spots of the brain surface; stable recordings lasted for several hours. The same electrodes were used to deliver fluorescent tracers into the nervous system by means of electrophoresis. This allowed us to retrograde label the recorded auditory neurons and to reveal their cell body and dendritic structure in the first thoracic ganglion. By adjusting the amount of dye injected, we specifically stained the ringlike auditory neuropil in the brain, demonstrating the clusters of cell bodies contributing to it. Our data provide a proof that surface electrodes are a versatile tool to analyze neural processing in small brains of invertebrates.NEW & NOTEWORTHY We show that surface suction electrodes can be used to monitor the activity of auditory neurons in the cricket brain. They also allow delivering electrophoretically a fluorescent tracer to label the structure of the recorded neurons and the local neuropil to which the electrode was attached. This new extracellular recording and labeling technique is a versatile and useful method to explore neural processing in invertebrate sensory and motor systems.