Fluorescent Tracer Molecules Research Articles

Defibrillated Lignocellulose Recovery Guided by Plant Chemistry and Anatomy – A Pioneering Study with Lupinus angustifolius

AbstractThe strive toward sustainability increases the demand for bio‐based material production, forcing expansion of the biorefinery feedstock supply from forest wood to non‐woody materials such as agricultural residues. As a model organism for legume crops, the aptness of agricultural lupins as a lignocellulose feedstock is investigated. Principle chemical analysis combined with optotracing, in which the fluorescent tracer molecule Carbotrace 680 generates a visual map of the native tissues’ lignocellulose anatomy at sub‐cellular resolution, enables informed design of a mild recovery process. A streamlined conversion approach is then designed, yielding lignin‐containing microfibrillated cellulose. By monitoring defibrillation and delignification throughout the extraction process, the use of optotracing for non‐destructive fiber analytics at unprecedented details across all hierarchical structures of lignocellulosic materials is demonstrated. This crop valorization is a prime illustration of a holistic use of lupin biomass, with seeds serving as plant‐based food sources, and other parts as sources for lignocellulose‐based materials, thereby expanding both the biorefinery concept and feedstock supply.

Simple design for membrane-free microphysiological systems to model the blood-tissue barriers

Microphysiological systems (MPS) incorporate physiologically relevant microanatomy, mechanics, and cells to mimic tissue function. Reproducible and standardized in vitro models of tissue barriers, such as the blood-tissue interface (BTI), are critical for next-generation MPS applications in research and industry. Many models of the BTI are limited by the need for semipermeable membranes, use of homogenous cell populations, or 2D culture. These factors limit the relevant endothelial-epithelial contact and 3D transport, which would best mimic the BTI. Current models are also difficult to assemble, requiring precise alignment and layering of components. The work reported herein details the engineering of a BTI-on-a-chip (BTI Chip) that addresses current disadvantages by demonstrating a single layer, membrane-free design. Laminar flow profiles, photocurable hydrogel scaffolds, and human cell lines were used to construct a BTI Chip that juxtaposes an endothelium in direct contact with a 3D engineered tissue. A biomaterial composite, gelatin methacryloyl and 8-arm polyethylene glycol thiol, was used for in situ fabrication of a tissue structure within a Y-shaped microfluidic device. To produce the BTI, a laminar flow profile was achieved by flowing a photocurable precursor solution alongside phosphate buffered saline. Immediately after stopping flow, the scaffold underwent polymerization through a rapid exposure to UV light (<300 mJ/cm2). After scaffold formation, blood vessel endothelial cells were introduced and allowed to adhere directly to the 3D tissue scaffold, without barriers or phase guides. Fabrication of the BTI Chip was demonstrated in both an epithelial tissue model and blood-brain barrier (BBB) model. In the epithelial model, scaffolds were seeded with human dermal fibroblasts. For the BBB models, scaffolds were seeded with the immortalized glial cell line, SVGP12. The BTI Chip microanatomy was analyzed post facto by immunohistochemistry, showing the uniform production of a patent endothelium juxtaposed with a 3D engineered tissue. Fluorescent tracer molecules were used to characterize the permeability of the BTI Chip. The BTI Chips were challenged with an efflux pump inhibitor, cyclosporine A, to assess physiological function and endothelial cell activation. Operation of physiologically relevant BTI Chips and a novel means for high-throughput MPS generation was demonstrated, enabling future development for drug candidate screening and fundamental biological investigations.

285 (PB065) - Using Fluorescence Cross Correlation Spectroscopy to Monitor and Quantify Drug Target Occupancy in Cells, Tissues and Patient Biopsies

Fluorescence Cross Correlation Spectroscopy (FCCS) is a singly molecule sensitive method to analyze molecular interactions. The approach has been employed in the past by Intana to set up high throughput screens, based on affinity or enzymatic activities. Similarly, we have investigated the formation and assembly of large protein complexes and studied complex targets as channels, transporters and even GPCRs. Among many other advantages, FCCS allows measurements in cellular lysates, bypassing protein purification and retaining the physiological environment. Consequently, assay development is straight forward and allows tackling also demanding targets. With this study we aimed to quantify the absolut drug target occupancy in cell in order to correlate the ratio of inhibited target molecules to the physiological, i.e. therapeutic effect. In order to measure unlabeled drugs and targets without purification of either binding partner for subsequent analysis Intana has set up an FCCS based assay format making use of fluorescent antibodies to label targets without interfering with drug target interactions and fluorescent tracer to quantify the unoccupied fraction of targets. For this samples are processed to cellular lysates using proprietary protocols and co-incubated with 2 different antibodies, carrying 2 different fluorescent labels and binding independently on 2 epitopes on the target. By this the total concentration of targets in the lysate can be precisely quantified. In a second step a fluorescent tracer molecule, which binds into the same pocket as the drug of interest is added to the lysate to quantify all unoccupied targets. In addition, a fluorescent antibody directed against the target is added for increase sensitivity. Knowing the exact concentration of targets in the sample and the concentration of targets that is bound to the tracer yields a precise quantification of target occupancy in the sample. Our approach was tested in cells, tissues, xenograft tumors and clinical patient samples. Data can help to guide cellular and animal experiments and to correlate or stratify patient responses to drug treatments. Conflict of interest: Ownership: Intana Bioscince GmbH, siTOOLs Biotech GmbH, Alpine Antiviral GmbH.

Cover Feature: Structural Properties Dictating Selective Optotracer Detection of Staphylococcus aureus (ChemBioChem 11/2022)

The cover feature illustrates how the chemical design of optotracers can be used to enable selective detection of the Gram-positive cocci Staphylococcus aureus in monocultures and when present in cultures mixed with the Gram-negative rod Salmonella Enteritidis. This is the first study to show that the chemical structure and photophysics of the optotracer molecules, which are conformation-sensitive fluorescent tracer molecules detecting peptide- and carbohydrate-based biopolymers, can be used as key tunable characteristics for the development of optotracers for selective detection of bacterial species. More information can be found in the Full Paper by A. Richter-Dahlfors et al.

Structural Properties Dictating Selective Optotracer Detection of Staphylococcus aureus.

Optotracers are conformation‐sensitive fluorescent tracer molecules that detect peptide‐ and carbohydrate‐based biopolymers. Their binding to bacterial cell walls allows selective detection and visualisation of Staphylococcus aureus (S. aureus). Here, we investigated the structural properties providing optimal detection of S. aureus. We quantified spectral shifts and fluorescence intensity in mixes of bacteria and optotracers, using automatic peak analysis, cross‐correlation, and area‐under‐curve analysis. We found that the length of the conjugated backbone and the number of charged groups, but not their distribution, are important factors for selective detection of S. aureus. The photophysical properties of optotracers were greatly improved by incorporating a donor‐acceptor‐donor (D‐A‐D)‐type motif in the conjugated backbone. With significantly reduced background and binding‐induced on‐switch of fluorescence, these optotracers enabled real‐time recordings of S. aureus growth. Collectively, this demonstrates that chemical structure and photophysics are key tunable characteristics in the development of optotracers for selective detection of bacterial species.

Optotracing for selective fluorescence-based detection, visualization and quantification of live S. aureus in real-time

Methods for bacterial detection are needed to advance the infection research and diagnostics. Based on conformation-sensitive fluorescent tracer molecules, optotracing was recently established for dynamic detection and visualization of structural amyloids and polysaccharides in the biofilm matrix of gram-negative bacteria. Here, we extend the use of optotracing for detection of gram-positive bacteria, focussing on the clinically relevant opportunistic human pathogen Staphylococcus aureus. We identify a donor-acceptor-donor-type optotracer, whose binding-induced fluorescence enables real-time detection, quantification, and visualization of S. aureus in monoculture and when mixed with gram-negative Salmonella Enteritidis. An algorithm-based automated high-throughput screen of 1920 S. aureus transposon mutants recognized the cell envelope as the binding target, which was corroborated by super-resolution microscopy of bacterial cells and spectroscopic analysis of purified cell wall components. The binding event was essentially governed by hydrophobic interactions, which permitted custom-designed tuning of the binding selectivity towards S. aureus versus Enterococcus faecalis by appropriate selection of buffer conditions. Collectively this work demonstrates optotracing as an enabling technology relevant for any field of basic and applied research, where visualization and detection of S. aureus is needed.

Microfluidic Prototyping by Xurography to Engineer Fully‐lumenized Microvessels In Vitro

Vascular permeability (VP) is a measure of the barrier function (BF) of an intact endothelial cell (EC) monolayer. This parameter is especially important in physiology, as deregulation of permeability leads to leaky blood vessels with a transvascular imbalance of chemical gradients, fluidic pressure, and in some instances excessive trafficking of blood‐borne cells into the surrounding tissue. Regulation of VP is a highly coordinated process. However, monitoring VP in vivo can be challenging as it typically requires invasive and labor intensive intravital microscopy. Here we present an in vitro alternative by developing a microfluidic model of a blood vessel analog that permits characterization of endothelial BF under the influence of different extracellular matrix (ECM) and perivascular cellular constituents. Our model relies on the manufacturing process known as xurography, an inexpensive and rapid print‐and‐peel prototyping method for assembling multiple layers of polymers. This method has been recently applied to bypass the need for high‐end fabrication facilities that are not readily available at all research institutions. However, to date most experimental platforms manufactured with xurography have focused on biochemical studies with few cell culture‐based applications. Briefly, after using a cutting plotter to form a channel (.8 mm x 8 mm) on a PDMS layer (~ 400 um thick), the device is constructed through a series of bindings to other PDMS layers (3 x ~ 250 um) and biopsy punching that form two regions. The inlet and outlet ports for cell seeding (2mm and 4mm) are located at the extremes of the model. An area for ECM hydrogels is allocated in the middle, with a PDMS rod placed in the center (Fig 1). After the construction, collagen type I at 6mg/ml is inserted into the central region. Once polymerized the PDMS rod is pulled to leave a lumen, permitting the addition of ECs into the formed ductal structure. In order to assess BF, the vessels were stained and imaged with confocal microscopy for the intercellular adherent junction molecule, VE‐cadherin as well as cytoskeletal proteins. To quantify VP, a fluorescent tracer molecule was infused in the vessel, and this parameter was determined by measuring changes of intensity as the dye permeated from the vessel to the surrounding hydrogel. Importantly, our results indicated the vessel analog demonstrated VP on the same order of magnitude as similar studies with ECs growing in static conditions (Fig 2). Notably, the microdevice allows the addition of cells that can support EC function. These findings will be fundamental as we explore the crosstalk between cells that serve in regulating blood vessel BF. Moreover, the inexpensive fabrication process can be adapted seamlessly by research institutions across the nation to probe further into vascular biology.Support or Funding InformationFunding was provided by the National Heart, Lung, and Blood Institute (R01HL141941) and The Mark Foundation for Cancer Research ASPIRE Award. MC‐M acknowledges funding from a NIH Diversity Supplement (R01HL141941).Microfluidic model design and Confocal Imaging ‐ A) Assembly of microfluidic vessel analogue through binding of layers after fabrication. B) Staining for cytoskeletal protein, cell nuclei, and VE‐cadherin.Figure 1Apparent Permeability: Quantification of Vascular Permeability. Images of fluorescent dye permeating through the endothelial cell wall (time point ‐ 0 to 5 min.)Figure 2

Development of hydrogels based on oxidized cellulose sulfates and carboxymethyl chitosan

Cellulose or chitosan represent highly abundant biopolymers possessing excellent biocompatibility that is required in tissue engineering. Both, cellulose and chitosan can be used to form hydrogels that can replace soft human tissues like cartilage. Hence, we developed here an approach to oxidize cellulose after sulfation, which was then crosslinked with carboxymethyl chitosan (CMCh). Sulfation was performed either by direct or acetosulfation reaching different sulfation degrees of DSSulf = 0.8–2.0. Subsequently oxidation of cellulose sulfate (CS) was performed with sodium periodate, which yielded aldehyde contents of DSAld = 0.1–0.3. Since oxidation requires the presence of vicinal hydroxyl groups in the anhydroglucose unit (AGU) of CS, higher sulfation degree as obtained by direct sulfation including hydroxyl groups at C2 and C3-position yielded lower aldehyde contents. On the contrary, regioselective sulfation at C6-position by acetosulfation was more suitable to achieve higher oxidation degrees of CS. Consequently, hydrogel formation obtained by chemical crosslinking of oxidized cellulose sulfate (oCS) with CMCh was fast within seconds when oxidation degree was high, but sulfation degree low. Moreover gel formation lasted almost 24 h when sulfation degree was high. It could also be shown that hydrogels based on oCS with a DSAld of 0.28 or higher were stable for 25 days when incubated in phosphate-buffered saline (PBS) or Dulbeccos modified Eagle medium (DMEM). Studies with pH dependent fluorescent tracer molecules could show that the intrinsic pH value in hydrogels was slightly acidic (sim pH 6.4) when they were incubated in PBS at pH 7.4. Mass transfer and homogeneity of the gel network was studied by NMR finding that diffusion of water molecules was not hindered inside the hydrogels.

Assessment of Histiotrophic Nutrition Using Fluorescent Probes.

Histiotrophic nutrition is a process whereby the rodent visceral yolk sac (VYS) internalizes exogenous macromolecules, degrades them, and sends the degradation products to the embryo. Quantification and visualization of histiotrophic nutrition can be accomplished using fluorescent tracer molecules such as fluorescein isothiocyanate-conjugated albumin (FITC-albumin). The methods are simple and can provide complimentary functional and structural information in studies of the effects of embryotoxicants on visceral yolk sac function.

Molecular Probe Diffusion in Thin Polymer Films: Evidence for a Layer with Enhanced Mobility Far above the Glass Temperature.

We studied experimentally the influence of interfaces on the dynamics in thin polymer films at temperatures far above the glass temperature (Tg + 80 °C). Polyisoprene (PI) was employed as a model system. We examined glass substrate supported films with thicknesses (d) spanning the range from 10 μm to 10 nm that correspond to d/Rg from 400 to 1, where Rg is the polymer radius of gyration. We employed fluorescence correlation spectroscopy (FCS) to monitor the translational diffusion of small fluorescent tracer molecules, dispersed at nanomolar concentrations in the PI matrix. In thick films, a single diffusion process correlated to the bulk segmental dynamics of the matrix polymer was present. However, when the film thickness was smaller than the normal dimension of the FCS observation volume, a second, faster diffusion process appeared, reflecting enhanced segmental dynamics near the free surface. Our results provide direct experimental evidence for the existence of a layer with enhanced mobility near the free surface of supported PI films at temperatures as high as 80 °C above the bulk Tg.

RepA-WH1, the agent of an amyloid proteinopathy in bacteria, builds oligomeric pores through lipid vesicles.

RepA-WH1 is a disease-unrelated protein that recapitulates in bacteria key aspects of human amyloid proteinopathies: i) It undergoes ligand-promoted amyloidogenesis in vitro; ii) its aggregates are able to seed/template amyloidosis on soluble protein molecules; iii) its conformation is modulated by Hsp70 chaperones in vivo, generating transmissible amyloid strains; and iv) causes proliferative senescence. Membrane disruption by amyloidogenic oligomers has been found for most proteins causing human neurodegenerative diseases. Here we report that, as for PrP prion and α-synuclein, acidic phospholipids also promote RepA-WH1 amyloidogenesis in vitro. RepA-WH1 molecules bind to liposomes, where the protein assembles oligomeric membrane pores. Fluorescent tracer molecules entrapped in the lumen of the vesicles leak through these pores and RepA-WH1 can then form large aggregates on the surface of the vesicles without inducing their lysis. These findings prove that it is feasible to generate in vitro a synthetic proteinopathy with a minimal set of cytomimetic components and support the view that cell membranes are primary targets in protein amyloidoses.

HIV-Tat Protein Forms Phosphoinositide-dependent Membrane Pores Implicated in Unconventional Protein Secretion

HIV-Tat has been demonstrated to be secreted from cells in a phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-dependent manner. Here we show that HIV-Tat forms membrane-inserted oligomers, a process that is accompanied by changes in secondary structure with a strong increase in antiparallel β sheet content. Intriguingly, oligomerization of HIV-Tat on membrane surfaces leads to the formation of membrane pores, as demonstrated by physical membrane passage of small fluorescent tracer molecules. Although membrane binding of HIV-Tat did not strictly depend on PI(4,5)P2 but, rather, was mediated by a range of acidic membrane lipids, a functional interaction between PI(4,5)P2 and HIV-Tat was critically required for efficient membrane pore formation by HIV-Tat oligomers. These properties are strikingly similar to what has been reported previously for fibroblast growth factor 2 (FGF2), providing strong evidence of a common core mechanism of unconventional secretion shared by HIV-Tat and fibroblast growth factor 2.

Molecular dynamics study of rhodamine 6G diffusion atn-decane–water interfaces

Two-dimensional diffusion of a rhodamine 6G fluorescent tracer molecule at the n-decane-water interface was studied with all-atom molecular dynamics simulations. In agreement with experimental data, we find increased mobility of the tracer at the n-decane-water interfaces in comparison to its mobility in bulk water. Orientational ordering of water and n-decane molecules near the interface is observed, and may change the interfacial viscosity as suggested to explain the experimental data. However, the restricted rotational motion of the rhodamine molecule at the interface suggests that the Saffman-Delbrück model may be a more appropriate approximation of rhodamine diffusion at n-decane-water interfaces, and, without any decrease in interfacial viscosity, suggests faster diffusion consistent with both experimental and simulation values.

Single-molecule diffusion in freely suspended smectic films.

We present a study of the molecular diffusion in freely suspended smectic-A liquid crystal films with thicknesses ranging from 20 down to only two molecular layers. The molecular mobility is directly probed by determining the trajectories of single, fluorescent tracer molecules. We demonstrate, using several different smectic compounds, that a monotonic increase of the diffusion coefficient with decreasing film thickness is a general phenomenon. In two-layer films, the diffusion is enhanced by a factor of 3 to 5 compared to the corresponding bulk smectic phase. Molecular dynamics simulations of freely suspended smectic films are presented which support the experimental results.

Simultaneous Measurement of Kidney Function by Dynamic Contrast Enhanced MRI and FITC-Sinistrin Clearance in Rats at 3 Tesla: Initial Results

Glomerular filtration rate (GFR) is an essential parameter of kidney function which can be measured by dynamic contrast enhanced magnetic resonance imaging (MRI-GFR) and transcutaneous approaches based on fluorescent tracer molecules (optical-GFR). In an initial study comparing both techniques in separate measurements on the same animal, the correlation of the obtained GFR was poor. The goal of this study was to investigate if a simultaneous measurement was feasible and if thereby, the discrepancies in MRI-GFR and optical-GFR could be reduced. For the experiments healthy and unilateral nephrectomised (UNX) Sprague Dawley (SD) rats were used. The miniaturized fluorescent sensor was fixed on the depilated back of an anesthetized rat. A bolus of 5 mg/100 g b.w. of FITC-sinistrin was intravenously injected. For dynamic contrast enhanced perfusion imaging (DCE-MRI) a 3D time-resolved angiography with stochastic trajectories (TWIST) sequence was used. By means of a one compartment model the excretion half-life (t1/2) of FITC-sinistrin was calculated and converted into GFR. GFR from DCE-MRI was calculated by fitting pixel-wise a two compartment renal filtration model. Mean cortical GFR and GFR by FITC-sinistrin were compared by Bland-Altman plots and pair-wise t-test. Results show that a simultaneous GFR measurement using both techniques is feasible. Mean optical-GFR was 4.34±2.22 ml/min (healthy SD rats) and 2.34±0.90 ml/min (UNX rats) whereas MRI-GFR was 2.10±0.64 ml/min (SD rats) and 1.17±0.38 ml/min (UNX rats). Differences between healthy and UNX rats were significant (p<0.05) and almost equal percentage difference (46.1% and 44.3%) in mean GFR were assessed with both techniques. Overall mean optical-GFR values were approximately twice as high compared to MRI-GFR values. However, compared to a previous study, our results showed a higher agreement. In conclusion, the possibility to use the transcutaneous method in MRI may have a huge impact in improving and validating MRI methods for GFR assessment in animal models.

Optical Investigation of the Intergrowth Structure and Accessibility of Brønsted Acid Sites in Etched SSZ-13 Zeolite Crystals by Confocal Fluorescence Microscopy

Template decomposition followed by confocal fluorescence microscopy reveals a tetragonal-pyramidal intergrowth of subunits in micrometer-sized nearly cubic SSZ-13 zeolite crystals. In order to accentuate intergrowth boundaries and defect-rich areas within the individual large zeolite crystals, a treatment with an etching NaOH solution is applied. The defective areas are visualized by monitoring the spatial distribution of fluorescent tracer molecules within the individual SSZ-13 crystals by confocal fluorescence microscopy. These fluorescent tracer molecules are formed at the inner and outer crystal surfaces by utilizing the catalytic activity of the zeolite in the oligomerization reaction of styrene derivatives. This approach reveals various types of etching patterns that are an indication for the defectiveness of the studied crystals. We can show that specially one type of crystals, denoted as core-shell type, is highly accessible to the styrene molecules after etching. Despite the large crystal dimensions, the whole core-shell type SSZ-13 crystal is utilized for catalytic reaction. Furthermore, the confocal fluorescence microscopy measurements indicate a nonuniform distribution of the catalytically important Brønsted acid sites underlining the importance of space-resolved measurements.

Lipid diffusion in planar membranes investigated by fluorescence correlation spectroscopy

Investigation of lipid lateral mobility in biological membranes and their artificial models provides information on membrane dynamics and structure; methods based on optical microscopy are very convenient for such investigations. We focus on fluorescence correlation spectroscopy (FCS), explain its principles and review its state of the art versions such as 2-focus, Z-scan or scanning FCS, which overcome most artefacts of standard FCS (especially those resulting from the need for an external calibration) making it a reliable and versatile method. FCS is also compared to single particle tracking and fluorescence photobleaching recovery and the applicability and the limitations of the methods are briefly reviewed. We discuss several key questions of lateral mobility investigation in planar lipid membranes, namely the influence which membrane and aqueous phase composition (ionic strength and sugar content), choice of a fluorescent tracer molecule, frictional coupling between the two membrane leaflets and between membrane and solid support (in the case of supported membranes) or presence of membrane inhomogeneities has on the lateral mobility of lipids. The recent FCS studies addressing those questions are reviewed and possible explanations of eventual discrepancies are mentioned.

Sonoporation by Ultrasound-Activated Microbubble Contrast Agents: Effect of Acoustic Exposure Parameters on Cell Membrane Permeability and Cell Viability

This work investigates the effect of ultrasound exposure parameters on the sonoporation of KHT-C cells in suspension by perflutren microbubbles. Variations in insonating acoustic pressure (0.05 to 3.5 MPa), pulse frequency (0.5 to 5.0 MHz), pulse repetition frequency (10 to 3000 Hz), pulse duration (4 to 32 micros) and insonation time (0.1 to 900 s) were studied. The number of cells permeabilised to a fluorescent tracer molecule (70 kDa FITC-dextran) and the number of viable cells were measured using flow cytometry. The effect of exposure on the microbubble population was measured using a Coulter counter. Cell viability and membrane permeability were found to depend strongly on the acoustic exposure conditions. Cell permeability increased and viability decreased with increasing peak negative pressure, pulse repetition frequency, pulse duration and insonation time and with decreasing pulse centre frequency. The highest therapeutic ratio (defined as the ratio of permeabilised to nonviable cells) achieved was 8.8 with 32 +/- 4% permeabilization and 96 +/- 1% viability at 570 kPa peak negative pressure, 8 micros pulse duration, 3 kHz pulse repetition frequency, 500 kHz centre frequency and 12 s insonation time with microbubbles at 3.3% volume concentration. These settings correspond to an acoustic energy density (E(SPPA)) of 3.12 J/cm(2). Cell permeability and viability did not correlate with bubble disruption. The results indicate that ultrasound exposure parameters can be optimized for therapeutic sonoporation and that bubble disruption is a necessary but insufficient indicator of ultrasound-induced permeabilization.

Molecular tracer diffusion in thin azobenzene polymer layers

Translational diffusion of fluorescent tracer molecules in azobenzene polymer layers is studied at different temperatures and under illumination using the method of fluorescence recovery after photobleaching. Diffusion is clearly observed in the dark above the glass transition temperature, while homogeneous illumination at 488nm and 100mW∕cm2 does not cause any detectable diffusion of the dye molecules within azobenzene layers. This implies that the viscosity of azobenzene layers remains nearly unchanged under illumination with visible light in the absence of internal or external forces.

Pannexin 1 in erythrocytes: Function without a gap

ATP is a widely used extracellular signaling molecule. The mechanism of ATP release from cells is presently unresolved and may be either vesicular or channel-mediated. Erythrocytes release ATP in response to low oxygen or to shear stress. In the absence of vesicles, the release has to be through channels. Erythrocytes do not form gap junctions. Yet, here we show with immunohistochemical and electrophysiological data that erythrocytes express the gap junction protein pannexin 1. This protein, in addition to forming gap junction channels in paired oocytes, can also form a mechanosensitive and ATP-permeable channel in the nonjunctional plasma membrane. Consistent with a role of pannexin 1 as an ATP release channel, ATP release by erythrocytes was attenuated by the gap junction blocker carbenoxolone. Furthermore, under conditions of ATP release, erythrocytes took up fluorescent tracer molecules permeant to gap junction channels.