Real-time Secretion Research Articles

Sub-second release of Neurotransmitters from CD4+ T lymphocytes

Abstract The neuroimmune system is complex and is regulated by small chemical messengers released from resident immune and neuronal cells. Catecholamines like dopamine and norepinephrine are important chemical messengers responsible for initiating and propagating messages between neurons and immune cells; however, the mechanism and dynamics of this signaling are relatively unknown. The presence of the intracellular machinery to synthesize and interact with catecholamines in T lymphocytes has been known for over a decade; however, direct measurement of catecholamine secretion in real time from immune cells has not been possible. Here, we have discovered that Naïve CD4+ T cells are capable of releasing catecholamines on a sub-second time scale. We have used a revolutionary technique, often used in the neuroscience field, called fast-scan cyclic voltammetry (FSCV) to measure sub-second fluctuations of catecholamines from isolated CD4+ T cells. This work provides direct evidence that T cells are capable of releasing catecholamines; potentially as a feedback communicator to resident sympathetic neurons in host immune organs. These results also prove that immune cells can signal on the same time scale as neurons. This work will hopefully open the door to enable more sophisticated studies at the neuron-immune synapse in the future and the understanding of neuro-immune based diseases.

Continuous pH monitoring using a sensor for the early detection of anastomotic leaks.

Anastomotic leaks (AL) and staple line leaks are a serious post-operative complication that can develop following bariatric surgery. The delay in the onset of symptoms following a leak usually results in reactive diagnostics and treatment, leading to increased patient morbidity and mortality, and a clinical and economic burden on both the patient and the hospital. Despite support in literature for pH as a biomarker for early detection of AL, the current methods of pH detection require significant clinician involvement and resources. Presented here is a polyaniline (PANI)-based pH sensor that can be connected inline to surgical drains to continuously monitor peritoneal secretion in real time for homeostatic changes in pH. During this study, the baseline peritoneal fluid pH was measured in two pigs using the PANI sensor and verified using a benchtop pH probe. The PANI sensor was then utilized to continuously monitor the changes in the pH of peritoneal effluent, as a gastric leak was simulated. The inline sensors were able to detect the resulting local changes in drainage pH within 10 min of leak induction. The successful implementation of this sensor in clinical practice can both enable high efficiency continuous monitoring of patient status and drastically decrease the time required to detect AL, thus potentially decreasing the clinical and economic burden incurred by gastric leaks.

Subsecond release of catecholamines from CD4+ T lymphocytes

Abstract The neuroimmune system is complex and is regulated by small chemical messengers released from resident immune and neuronal cells. Catecholamines like dopamine and norepinephrine are important chemical messengers responsible for initiating and propagating messages between neurons and immune cells; however, the mechanism and dynamics of this signaling is relatively unknown. The presence of the intracellular machinery to synthesize and interact with catecholamines in T lymphocytes has been known for over a decade; however, direct measurement of catecholamine secretion in real time from immune cells has not been possible. Here, we have discovered that CD4+ T cells are capable of releasing catecholamines on a subsecond time scale. We have used a revolutionary technique, often used in the neuroscience field, called fast-scan cyclic voltammetry (FSCV) to measure subsecond fluctuations of catecholamines from isolated CD4+ T cells. This work provides direct evidence that T cells are capable of releasing catecholamines; potentially as a feedback communicator to resident sympathetic neurons in host immune organs. These results also prove that immune cells can signal on the same time scale as neurons . This work will hopefully open the door to enable more sophisticated studies at the neuron-immune synapse in the future. National Institute of Health R01AI151552

Preparation of Whole-mount Mouse Islets on Vascular Extracellular Matrix for Live Islet Cell Microscopy.

Pancreatic islet β cells preferentially secrete insulin toward the plasma membrane, making contact with the capillary extracellular matrix (ECM). Isolated islets separated from the exocrine acinar cells are the best system for cell biology studies of primary β cells, whereas isolated islets lose their capillary network during ex vivo culture. Providing the appropriate extracellular signaling by attaching islets to vascular ECM-coated surfaces can restore the polarized insulin secretion toward the ECM. The guided secretion toward ECM-coated glass coverslips provides a good model for recording insulin secretion in real time to study its regulation. Additionally, β cells attached to the ECM-coated coverslips are suitable for confocal live imaging of subcellular components including adhesion molecules, cytoskeleton, and ion channels. This procedure is also compatible for total internal reflection fluorescence (TIRF) microscopy, which provides optimal signal-to-noise ratio and high spatial precision of structures close to the plasma membrane. In this article, we describe the optimized protocol for vascular ECM-coating of glass coverslips and the process of attachment of isolated mouse islets on the coverslip. This preparation is compatible with any high-resolution microscopy of live primary β cells. Key features • Optimized coating procedure to attach isolated islets, compatible for both confocal and TIRF microscopy. • The ECM-coated glass coverslip functions as the artificial capillary surface to guide secretion toward the coated surface for optimal imaging of secretion events. • Shows the process of islets attachment to the ECM-coated surface in a 6-day ex vivo culture.

Lab-on-a-Chip System for Developing and Fluorescence Imaging a Three-Dimensional Model of Pancreatic Islets Under Flow Conditions

Introduction Diabetes mellitus is a group of metabolic diseases characterized by elevated blood sugar resulting from a defect in the production of insulin by pancreatic β-cells or tissue insensitivity for this peptide hormone [1]. The most common form of the disease is diabetes mellitus type 2 (insulin-dependent), which is diagnosed in about 80% of all patients [2]. In patients with this type of diabetes, both the efficiency and the secretion of insulin are disturbed [3]. For this reason, it is very important to thoroughly understand the kinetics of insulin release in response to various conditions, which can help develop effective therapy. The most important cells for this disease are insulin secreting β-cells and their antagonist - glucagon-secreting α-cells [4]. In our research, we focused on imitate the pancreatic islet structure, which can be a universal model for testing the impact of various environmental factors on disease development. The idea of using a Lab-on-a-chip system for three-dimensional islet cells culture came from the possibility of mimicking in vivo conditions. This can give a wider and more accurate view of the pancreatic islets function than the cell culture in static conditions. Lab-on-a-chip system In our research we present a Lab-on-a-chip system in which a pancreatic “pseudoislet” model will be developed. This system is composed of biocompatible, non-toxic and transparent materials, such as: poly(dimethylsiloxane) (PDMS) and thin glass. Thanks to this, it is possible to conduct cell culture and observe the results in various types of microscopes. The geometry of Lab-on-a-chip system consist of two elliptical cell culture chambers, one for the test sample and second for the reference sample. In in each of the chambers there are 15 round microtraps. Each of the microtraps is made of 6 micropillars. This solution forces the dense packing of cells and their aggregations. The geometry of the developed system is consistent with the culture wells on a standard multi-well plate, which allows measurements in a multi-well plate reader. Methods All experiments were performed using two commercially purchased pancreatic islets cell lines: β-cells (INS-1E) and α-cells (α-TC1-6). Appropriate α-cells and β-cells ratios were selected to reflect the morphology and composition of the pancreatic islet found in the body. The cells were introduced through the inlet to the Lab-on-a-chip system and placed for 24h in an incubator to form spherical aggregates (“pseudoislets”). In the next stage immunostaining protocol using primary and secondary antibody solutions was developed. Due to the use this method and confocal microscope it was possible to confirm the correct distribution of cells in the obtained aggregate. To confirm cells viability previously prepared solution of Calcein AM and Propidium Iodide were introduced through the microsystem inlet with a flow rate 5µL/min for 10 min. After 10 min incubation (37oC, 5% CO2) the viability of the cells was determined using fluorescence microscope. Proliferation of the ells was measured using spectrofluorimetric multi-well plate reader after incubation with AlamarBlue solution. The “pseudoislets” were then stimulated with various glucose concentrations (imitation of health and disease state). After this, the standard ELISA test procedure was adapted to the microfluidic conditions to examine the insulin secretion profile from the obtained “pseudoislets”. Results and Conclusions It was confirmed that 15 spherical α and β-cell aggregates with diameters of 160-180 µm were obtained in one culture chamber using the method described earlier. As was expected the participation of β-cells in the core and α-cells on the periphery of the aggregate was confirmed by the use of immunostaining method. Moreover, the compatibility of the designed microsystem with the multi-well plate reader allowed to monitor cell proliferation and viability in situ during the culture. A high viability after 24 and 48 hours of culture in the microchip was confirmed and was 97% and 95% respectively. The cells maintained their morphology, function and high level of proliferation for up to 48 hours of culture. Moreover, in the developed model health and disease conditions were simulated and the level of insulin secreted from this model was determined using the ELISA test. At this stage, a research model corresponding to the model in vivo conditions was obtained. The next step will be to develop a method for analyzing insulin secretion in real time (such as Surface Plasmon Resonance). This study presents basic research and in the future, this model could be utilized to simulate diabetes, testing new drugs and therapy in diabetes mellitus treatment.

Quantifying single-cell secretion in real time using resonant hyperspectral imaging

Cell communication is primarily regulated by secreted proteins, whose inhomogeneous secretion often indicates physiological disorder. Parallel monitoring of innate protein-secretion kinetics from individual cells is thus crucial to unravel systemic malfunctions. Here, we report a label-free, high-throughput method for parallel, in vitro, and real-time analysis of specific single-cell signaling using hyperspectral photonic crystal resonant technology. Heterogeneity in physiological thrombopoietin expression from individual HepG2 liver cells in response to platelet desialylation was quantified demonstrating how mapping real-time protein secretion can provide a simple, yet powerful approach for studying complex physiological systems regulating protein production at single-cell resolution.

Challenges in Developing Protein Secretion Assays at a Single-Cell Level.

In addition to direct physical interactions between/among cells, the secretion of humoral factors from living cells is a critical process for cell-cell communications. A well-known extracellular signaling event is mediated by immune cell cytokines/chemokines. Because cell-cell communication is crucial in immune cell sociology, protein secretion assays first attracted a broad range of immunology interests. Now that we have entered an era of systems biology, cell-cell interactions mediated by secreted molecules should be revisited to understand the dynamics and homeostasis of the cell society as a whole. Of more importance, recent advances in detection and microfluidics technologies enable us to monitor protein secretion in real time rather than as a snapshot from the past, which gives us an opportunity to more deeply understand the logic of mammalian cell sociology. This chapter reviews the recent progress in and future direction of protein secretion assays, particularly from a mammalian cell sociology viewpoint.

Tracking Proteins Secreted by Bacteria: What's in the Toolbox?

Bacteria have acquired multiple systems to expose proteins on their surface, release them in the extracellular environment or even inject them into a neighboring cell. Protein secretion has a high adaptive value and secreted proteins are implicated in many functions, which are often essential for bacterial fitness. Several secreted proteins or secretion machineries have been extensively studied as potential drug targets. It is therefore important to identify the secretion substrates, to understand how they are specifically recognized by the secretion machineries, and how transport through these machineries occurs. The purpose of this review is to provide an overview of the biochemical, genetic and imaging tools that have been developed to evaluate protein secretion in a qualitative or quantitative manner. After a brief overview of the different tools available, we will illustrate their advantages and limitations through a discussion of some of the current open questions related to protein secretion. We will start with the question of the identification of secreted proteins, which for many bacteria remains a critical initial step toward a better understanding of their interactions with the environment. We will then illustrate our toolbox by reporting how these tools have been applied to better understand how substrates are recognized by their cognate machinery, and how secretion proceeds. Finally, we will highlight recent approaches that aim at investigating secretion in real time, and in complex environments such as a tissue or an organism.

Plasmonic nanohole array biosensor for label-free and real-time analysis of live cell secretion.

Cell secretion dynamics plays a central role in physiological and disease processes. Due to its various temporal profiles, it is essential to implement a precise detection scheme for continuous monitoring of secretion in real time. The current fluorescent and colorimetric approaches hinder such applications due to their multiple time-consuming steps, molecular labeling, and especially the 'snapshot' endpoint readouts. Here, we develop a nanoplasmonic biosensor for real-time monitoring of live cell cytokine secretion in a label-free configuration. Our nanoplasmonic biosensor is composed of gold nanohole arrays supporting extraordinary optical transmission (EOT), which enables sensitive and high-throughput analysis of biomolecules. The nanobiosensor is integrated with an adjustable microfluidic cell module for the analysis of live cells under well-controlled culture conditions. We achieved an outstanding sensitivity for the detection of vascular endothelial growth factor (VEGF) directly in complex cell media. Significantly, the secretion dynamics from live cancer cells were monitored and quantified for 10 hours while preserving good cell viability. This novel approach of probing cytokine secretion activity is compatible with conventional inverted microscopes found in a common biology laboratory. With its simple optical set-up and label-free detection configuration, we anticipate our nanoplasmonic biosensor to be a powerful tool as a lab-on-chip device to analyze cellular activities for fundamental cell research and biotechnologies.

A reusable electrochemical proximity assay for highly selective, real-time protein quantitation in biological matrices.

Rapid and specific quantitation of a variety of proteins over a wide concentration range is highly desirable for biosensing at the point-of-care, in clinical laboratories, and in research settings. Our recently developed electrochemical proximity assay (ECPA) is a target-flexible, DNA-directed, direct-readout protein quantitation method with detection limits in the low femtomolar range, making it particularly amenable to point-of-care detection. However, consistent quantitation in more complex matrices is required at the point-of-care, and improvements in measurement speed are needed for clinical and research settings. Here, we address these concerns with a reusable ECPA, where a gentle regeneration of the surface DNA monolayer (used to capture the proximity complex) is achieved enzymatically through a novel combination of molecular biology and electrochemistry. Strategically placed uracils in the DNA sequence trigger selective cleavage of the backbone, releasing the assembled proximity complex. This allows repeated protein quantitation by square-wave voltammetry (SWV)—as quickly as 3 min between runs. The process can be repeated up to 19 times on a single electrode without loss of assay sensitivity, and currents are shown to be highly repeatable with similar calibrations using seven different electrodes. The utility of reusable ECPA is demonstrated through two important applications in complex matrices: (1) direct, quantitative monitoring of hormone secretion in real time from as few as five murine pancreatic islets and (2) standard addition experiments in unspiked serum for direct quantitation of insulin at clinically relevant levels. Results from both applications distinguish ECPA as an exceptional tool in protein quantitation.

Real-time imaging of type III secretion: Salmonella SipA injection into host cells

Many pathogenic and symbiotic Gram-negative bacteria employ type III secretion systems to inject "effector" proteins into eukaryotic host cells. These effectors manipulate signaling pathways to initiate symbiosis or disease. By using time-lapse microscopy, we have imaged delivery of the Salmonella type III effector protein SipA/SspA into animal cells in real time. SipA delivery mostly began 10-90 sec after docking and proceeded for 100-600 sec until the bacterial SipA pool (6 +/- 3 x 10(3) molecules) was exhausted. Similar observations were made for the effector protein SopE. This visualization of type III secretion in real time explains the efficiency of host cell manipulation by means of this virulence system.

Monitoring secretion in real time: capacitance, amperometry and fluorescence compared

Techniques for measuring exocytosis, endocytosis and vesicle cycling in living cells in real time have resulted in a rapid expansion in the knowledge of these processes in neurons and other secretory cells. Several experimental approaches, developed during the past decade, have played key roles in this expansion. In this review we focus on three techniques: electrophysiological methods for monitoring membrane capacitance, electrochemical methods for detecting released secretory contents and optical methods for imaging membranes of endosomes and recycled vesicles that are stained with fluorescent dyes. Each technique has contributed unique and complementary information about the vesicle cycle, advancing our knowledge of the kinetics of membrane fusion and retrieval, the identity of the secretory contents and the spatial patterns and directional pathways involved in secretory membrane recycling. Naturally, each technique has inherent limitations; some of these shortcomings have recently been resolved by using more than one method simultaneously.