Single-cell Fluorescence Microscopy Research Articles

In Situ Single-Cell Bacterial Imaging Provides Mechanistic Insight into the Photodynamic Action of Photosensitizer-Loaded Hydrogels.

Hydrogels, three-dimensional hydrophilic polymeric networks with high water retaining capacity, have gained prominence in wound management and drug delivery due to their tunability, softness, permeability, and biocompatibility. Electron-beam polymerized poly(ethylene glycol) diacrylate (PEGDA) hydrogels are particularly useful for phototherapies such as antimicrobial photodynamic therapy (aPDT) due to their excellent optical properties. This work takes advantage of the transparency of PEGDA hydrogels to investigate bacterial responses to aPDT at the single-cell level, in real-time and in situ. The photosensitizer methylene blue (MB) was loaded in PEGDA hydrogels by using two methods: reversible loading and irreversible immobilization within the 3D polymer network. MB release kinetics and singlet oxygen generation were studied, revealing the distinct behaviors of both hydrogels. Real-time imaging of Escherichia coli was conducted during aPDT in both hydrogel types, using the Min protein system to report changes in bacterial physiology. Min oscillation patterns provided mechanistic insights into bacterial photoinactivation, revealing a dependence on the hydrogel preparation method. This difference was attributed to the mobility of MB within the hydrogel, affecting its direct interaction with bacterial membranes. These findings shed light on the complex interplay between hydrogel properties and the bacterial response during aPDT, offering valuable insights for the development of antibacterial wound dressing materials. The study demonstrates the capability of real-time, single-cell fluorescence microscopy to unravel dynamic bacterial behaviors in the intricate environment of hydrogel surfaces during aPDT.

RICH2 decreases the mitochondrial number and affects mitochondrial localization in diffuse low-grade glioma-related epilepsy

Epilepsy, a common complication of diffuse low-grade gliomas (DLGGs; diffuse oligodendroglioma and astrocytoma collectively), severely compromises the quality of life of patients. DLGG epileptogenicity may primarily be generated by interactions between the tumor and the neocortex. Neuronal uptake of dysfunctional mitochondria from the extracellular environment can lead to abnormal neuronal discharge. Mitochondrial dysfunction is frequently observed in gliomas that can transmigrate across the plasma membranes. Here, we examined the role of the Rho GTPase-activating protein 44 (RICH2) in mitochondrial dynamics and DLGG-related epilepsy. We investigated the association between mitochondrial and RICH2 expression in human DLGG tissues using immunohistochemistry. We examined the association between RICH2 and epilepsy in nude mouse glioma models by electrophysiology. The effect of RICH2 on mitochondrial morphology and calcium motility were assessed by single cell fluorescence microscopy. Quantitative RT-PCR (qRT-PCR) and Western blot analysis were performed to characterize RICH2 induced expression changes in the genes related to mitochondrial dynamics, mitogenesis and mitochondrial function. We found that RICH2 expression was higher in oligodendroglioma than in astrocytoma and was correlated with better prognosis and higher epilepsy rate in patients. The expression of mitochondria may be associated with clinical DLGG-related epilepsy and reduced by RICH2 overexpression. And RICH2 could promote DLGG-related epilepsy in tumorigenic nude mice. RICH2 overexpression decreased calcium flow and the mitochondria released from glioma cells (SW1088 and U251) into the extracellular environment, potentially via downregulation of MFN-1/MFN-2 levels which suggests reduced mitochondrial fusion. In addition, we observed decreased mitochondrial trafficking into neurons (released from glioma cells and trafficked into neurons), which could explain the higher incidence of DLGG-related epilepsy due to reduced neuroprotection. Furthermore, RICH2 downregulated MAPK/ERK/HIF-1 pathway. In conclusion, these results suggest that RICH2 could promote epilepsy by (i) inhibiting mitochondrial fusion via MFN downregulation and Drp-1 upregulation; (ii) altering the MAPK/ERK/Hif-1 signaling axis. RICH2 may be a potential target in the treatment of DLGG-related epilepsy.

Sigma-1 Receptor Modulation by Ligands Coordinates Cancer Cell Energy Metabolism.

Sigma-1 receptor (S1R) is an important endoplasmic reticulum chaperone with various functions in health and disease. The purpose of the current work was to elucidate the involvement of S1R in cancer energy metabolism under its basal, activated, and inactivated states. For this, two cancer cell lines that differentially express S1R were treated with S1R agonist, (+)-SKF10047, and antagonist, BD1047. The effects of the agonist and antagonist on cancer energy metabolism were studied using single-cell fluorescence microscopy analysis of real-time ion and metabolite fluxes. Our experiments revealed that S1R activation by agonist increases mitochondrial bioenergetics of cancer cells while decreasing their reliance on aerobic glycolysis. S1R antagonist did not have a major impact on mitochondrial bioenergetics of tested cell lines but increased aerobic glycolysis of S1R expressing cancer cell line. Our findings suggest that S1R plays an important role in cancer energy metabolism and that S1R ligands can serve as tools to modulate it.

Single Cell Analysis Reveals a New Microsporidia-Host Association in a Freshwater Lake.

Microsporidia are a large group of obligate intracellular eukaryotic parasites. Recent studies suggest that their diversity can be huge in freshwater lake ecosystems especially in the < 150-µm size fraction. However, little is known about their hosts and therefore their impact on the trophic food web functioning. In this study, single cell analysis and fluorescence microscopy were used to detect new host-parasite association within rotifer communities in lake Aydat (France). Our analysis showed the existence of a potential new species belonging to the Crispospora genus able of infecting the rotifer Kellicottia with a high prevalence (42.5%) suggesting that Microsporidia could have a great impact on the rotifer populations' regulation in lakes.

Transcription activation is enhanced by multivalent interactions independent of phase separation

Transcription factors (TFs) consist of a DNA-binding domain and an activation domain (AD) that are frequently considered to be independent and exchangeable modules. However, recent studies report that the physicochemical properties of the AD can control TF assembly at chromatin by driving phase separation into transcriptional condensates. Here, we dissected transcription activation by comparing different synthetic TFs at a reporter gene array with real-time single-cell fluorescence microscopy. In these experiments, binding site occupancy, residence time, and coactivator recruitment in relation to multivalent TF interactions were compared. While phase separation propensity and activation strength of the AD were linked, the actual formation of liquid-like TF droplets had a neutral or inhibitory effect on transcription activation. We conclude that multivalent AD-mediated interactions enhance the transcription activation capacity of a TF by increasing its residence time in the chromatin-bound state and facilitating the recruitment of coactivators independent of phase separation.

Effect of Thioflavin T on the Elongation Rate of Bacteria.

The growing field of bacterial electrophysiology examines the relationship between bacterial membrane potential and cell division, growth, sporulation, and biofilm formation. These experiments require Nernstian fluorescent dyes to monitor membrane potential. Our research uses single cell imaging to determine if a common fluorescent dye, Thioflavin T (ThT), affects the growth of bacteria. We use a combination of standard growth curve measurements and single cell imaging, both brightfield and fluorescence microscopy, to monitor the growth of Bacillus subtilis and Escherichia coli as a function of ThT concentration. Increased membrane potential (hyperpolarization) leads to increased intracellular accumulation of ThT: High fluorescence intensity is an indicator of hyperpolarization. Blue light is used to hyperpolarize a subpopulation of cells to monitor cellular elongation in response to increased cellular internalization of ThT. Single cell imaging shows that the elongation rates of B. subtilis and E. coli are decreased when these cells are incubated with ThT. At micromolar concentrations of ThT, this effect may be masked in standard growth curves, but is visible with single cell measurements on agarose pads. The increased cellular accumulation of ThT is a standard measure of hyperpolarization in bacterial electrophysiology. Growth curves, a bulk measurement, are typically used to determine suitable concentrations of ThT for use in experiments. Single cell measurements show that cells incubated with ThT have decreased elongation rates. This creates a potential experimental artifact that could lead to misinterpretation of data. Hyperpolarized cells internalize more ThT. This increased intracellular concentration of ThT, rather than the change in membrane potential, could lead to decreased growth. These experiments point toward the importance of single cell measurements to detect subtle changes in cell growth. We hope this research will be useful for other researchers in their choice of dye for the detection of membrane potential.

Metabolomics studies of cell-cell interactions using single cell mass spectrometry combined with fluorescence microscopy.

Cell–cell interactions are critical for transmitting signals among cells and maintaining their normal functions from the single-cell level to tissues. In cancer studies, interactions between drug-resistant and drug-sensitive cells play an important role in the development of chemotherapy resistance of tumors. As metabolites directly reflect the cell status, metabolomics studies provide insight into cell–cell communication. Mass spectrometry (MS) is a powerful tool for metabolomics studies, and single cell MS (SCMS) analysis can provide unique information for understanding interactions among heterogeneous cells. In the current study, we utilized a direct co-culture system (with cell–cell contact) to study metabolomics of single cells affected by cell–cell interactions in their living status. A fluorescence microscope was utilized to distinguish these two types of cells for SCMS metabolomics studies using the Single-probe SCMS technique under ambient conditions. Our results show that through interactions with drug-resistant cells, drug-sensitive cancer cells acquired significantly increased drug resistance and exhibited drastically altered metabolites. Further investigation found that the increased drug resistance was associated with multiple metabolism regulations in drug-sensitive cells through co-culture such as the upregulation of sphingomyelins lipids and lactic acid and the downregulation of TCA cycle intermediates. The method allows for direct MS metabolomics studies of individual cells labeled with fluorescent proteins or dyes among heterogeneous populations.

Single-Cell RNA Sequencing Reveals Endothelial Cell Transcriptome Heterogeneity Under Homeostatic Laminar Flow.

Objective: Endothelial cells (ECs) that form the innermost layer of all vessels exhibit heterogeneous cell behaviors and responses to pro-angiogenic signals that are critical for vascular sprouting and angiogenesis. Once vessels form, remodeling and blood flow lead to EC quiescence, and homogeneity in cell behaviors and signaling responses. These changes are important for the function of mature vessels, but whether and at what level ECs regulate overall expression heterogeneity during this transition is poorly understood. Here, we profiled EC transcriptomic heterogeneity, and expression heterogeneity of selected proteins, under homeostatic laminar flow. Approach and Results: Single-cell RNA sequencing and fluorescence microscopy were used to characterize heterogeneity in RNA and protein gene expression levels of human ECs under homeostatic laminar flow compared to nonflow conditions. Analysis of transcriptome variance, Gini coefficient, and coefficient of variation showed that more genes increased RNA heterogeneity under laminar flow relative to genes whose expression became more homogeneous, although small subsets of cells did not follow this pattern. Analysis of a subset of genes for relative protein expression revealed little congruence between RNA and protein heterogeneity changes under flow. In contrast, the magnitude of expression level changes in RNA and protein was more coordinated among ECs in flow versus nonflow conditions. Conclusions: ECs exposed to homeostatic laminar flow showed overall increased heterogeneity in RNA expression levels, while expression heterogeneity of selected cognate proteins did not follow RNA heterogeneity changes closely. These findings suggest that EC homeostasis is imposed post-transcriptionally in response to laminar flow.

Single-Cell Imaging Reveals That Staphylococcus aureus Is Highly Competitive Against Pseudomonas aeruginosa on Surfaces.

Pseudomonas aeruginosa and Staphylococcus aureus frequently occur together in polymicrobial infections, and their interactions can complicate disease progression and treatment options. While interactions between P. aeruginosa and S. aureus have been extensively described using planktonic batch cultures, little is known about whether and how individual cells interact with each other on solid substrates. This is important because both species frequently colonize surfaces to form aggregates and biofilms in infections. Here, we performed single-cell time-lapse fluorescence microscopy, combined with automated image analysis, to describe interactions between P. aeruginosa PAO1 with three different S. aureus strains (Cowan I, 6850, JE2) during microcolony growth on agarose surfaces. While P. aeruginosa is usually considered the dominant species, we found that the competitive balance tips in favor of S. aureus on surfaces. We observed that all S. aureus strains accelerated the onset of microcolony growth in competition with P. aeruginosa and significantly compromised P. aeruginosa growth prior to physical contact. Upon direct contact, JE2 was the most competitive S. aureus strain, simply usurping P. aeruginosa microcolonies, while 6850 was the weakest competitor itself suppressed by P. aeruginosa. Moreover, P. aeruginosa reacted to the assault of S. aureus by showing increased directional growth and expedited expression of quorum sensing regulators controlling the synthesis of competitive traits. Altogether, our results reveal that quantitative single-cell live imaging has the potential to uncover microbial behaviors that cannot be predicted from batch culture studies, and thereby contribute to our understanding of interactions between pathogens that co-colonize host-associated surfaces during polymicrobial infections.

AFM and Fluorescence Microscopy of Single Cells with Simultaneous Mechanical Stimulation via Electrically Stretchable Substrates.

We have developed a novel experimental set-up that simultaneously, (i) applies static and dynamic deformations to adherent cells in culture, (ii) allows the visualization of cells under fluorescence microscopy, and (iii) allows atomic force microscopy nanoindentation measurements of the mechanical properties of the cells. The cell stretcher device relies on a dielectric elastomer film that can be electro-actuated and acts as the cell culture substrate. The shape and position of the electrodes actuating the film can be controlled by design in order to obtain specific deformations across the cell culture chamber. By using optical markers we characterized the strain fields under different electrode configurations and applied potentials. The combined setup, which includes the cell stretcher device, an atomic force microscope, and an inverted optical microscope, can assess in situ and with sub-micron spatial resolution single cell topography and elasticity, as well as ion fluxes, during the application of static deformations. Proof of performance on fibroblasts shows a reproducible increase in the average cell elastic modulus as a response to applied uniaxial stretch of just 4%. Additionally, high resolution topography and elasticity maps on a single fibroblast can be acquired while the cell is deformed, providing evidence of long-term instrumental stability. This study provides a proof-of-concept of a novel platform that allows in situ and real time investigation of single cell mechano-transduction phenomena with sub-cellular spatial resolution.

Two Metabolic Fuels, Glucose and Lactate, Differentially Modulate Exocytotic Glutamate Release from Cultured Astrocytes.

Astrocytes have a prominent role in metabolic homeostasis of the brain and can signal to adjacent neurons by releasing glutamate via a process of regulated exocytosis. Astrocytes synthesize glutamate de novo owing to the pyruvate entry to the citric/tricarboxylic acid cycle via pyruvate carboxylase, an astrocyte specific enzyme. Pyruvate can be sourced from two metabolic fuels, glucose and lactate. Thus, we investigated the role of these energy/carbon sources in exocytotic glutamate release from astrocytes. Purified astrocyte cultures were acutely incubated (1h) in glucose and/or lactate-containing media. Astrocytes were mechanically stimulated, a procedure known to increase intracellular Ca2+ levels and cause exocytotic glutamate release, the dynamics of which were monitored using single cell fluorescence microscopy. Our data indicate that glucose, either taken-up from the extracellular space or mobilized from the intracellular glycogen storage, sustained glutamate release, while the availability of lactate significantly reduced the release of glutamate from astrocytes. Based on further pharmacological manipulation during imaging along with tandem mass spectrometry (proteomics) analysis, lactate alone, but not in the hybrid fuel, caused metabolic changes consistent with an increased synthesis of fatty acids. Proteomics analysis further unveiled complex changes in protein profiles, which were condition-dependent and generally included changes in levels of cytoskeletal proteins, proteins of secretory organelle/vesicle traffic and recycling at the plasma membrane in aglycemic, lactate or hybrid-fueled astrocytes. These findings support the notion that the availability of energy sources and metabolic milieu play a significant role in gliotransmission.

Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform.

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience comorbidity related to chronic inflammation despite antiretroviral therapy. However, these inflammatory signaling has not been fully characterized. The role of early entry events on the activation of cellular signaling events and downstream gene expression has not been captured at the single-cell level. Here the authors describe a method that applies principles of live-cell fluorescence microscopy to an automated single-cell platform that cultures and images cells over user-customized time courses, allowing for high-throughput analysis of dynamic cellular processes. This assay can track single-cell live fluorescence microscopy of early events that immediately follow HIV-1 infection, notably the influx of calcium that accompanies exposure to the virus and the development of productive infection using a fluorescent reporter virus. MT-4 cells are loaded with a calcium-sensitive dye and cultured in isolated pens on a nanofluidic device. The cultured cells are infected with an HIV-1 reporter virus (HIV-1 NLCI). A fluorescence microscope positioned above the nanofluidic device measures calcium influx over an 8-min time course following acute HIV-1 exposure. HIV-1 productive infection is measured in those same cells over a 4-day interval. Imaging data from these time courses are analyzed to define virus-host receptor interactions and signaling pathway dynamics. The authors present an integrated, scalable alternative to traditional imaging methods using a novel optofluidic platform capable of single-cell sorting, culturing, imaging, and software automation. This assay can measure the kinetics of events under various conditions, including cell type, agonist, or antagonist effect, while measuring an array of parameters. This is the first established method for nanofluidic high-throughput longitudinal single-cell culture and imaging: This technique can be broadly adapted to study cellular signaling kinetics and dynamic molecular interactions.

Simultaneous Recording of Multiple Signaling Events by Frequency- and Spectrally-Tuned Multiplexing of Fluorescent Probes

Fluorescent probes that change their spectral properties upon binding to specific ligands, e.g., second messengers, or changes in the membrane potential (Vm) are invaluable tools to study cellular signal transduction. Here, we introduce a novel technique that facilitates simultaneous recording of multiple fluorescent probes: frequency- and spectrally-tuned multiplexing (FASTM). FASTM uses phase-sensitive signal detection, which renders various combinations of probes for Vm and ions accessible for multiplexing. Using stopped-flow fluorimetry, we show that FASTM allows for simultaneous recording of rapid changes in Ca2+, pH, Na+, and Vm with high sensitivity, millisecond time resolution, and minimal cross-talk. Moreover, FASTM is compatible with opto-chemical tools (e.g., caged compounds) used for manipulating signal transduction with light. Finally, FASTM is suited for multiplexing of genetically-encoded FRET biosensors and synthetic probes using single-cell fluorescence microscopy. Altogether, FASTM opens new experimental avenues for interrogating cellular signal transduction processes with high temporal resolution.

A single point mutation converts a proton-pumping rhodopsin into a red-shifted, turn-on fluorescent sensor for chloride.

The visualization of chloride in living cells with fluorescent sensors is linked to our ability to design hosts that can overcome the energetic penalty of desolvation to bind chloride in water. Fluorescent proteins can be used as biological supramolecular hosts to address this fundamental challenge. Here, we showcase the power of protein engineering to convert the fluorescent proton-pumping rhodopsin GR from Gloeobacter violaceus into GR1, a red-shifted, turn-on fluorescent sensor for chloride in detergent micelles and in live Escherichia coli. This non-natural function was unlocked by mutating D121, which serves as the counterion to the protonated retinylidene Schiff base chromophore. Substitution from aspartate to valine at this position (D121V) creates a binding site for chloride. The binding of chloride tunes the pKa of the chromophore towards the protonated, fluorescent state to generate a pH-dependent response. Moreover, ion pumping assays combined with bulk fluorescence and single-cell fluorescence microscopy experiments with E. coli, expressing a GR1 fusion with a cyan fluorescent protein, show that GR1 does not pump ions nor sense membrane potential but instead provides a reversible, ratiometric readout of changes in extracellular chloride at the membrane. This discovery sets the stage to use natural and laboratory-guided evolution to build a family of rhodopsin-based fluorescent chloride sensors with improved properties for cellular applications and learn how proteins can evolve and adapt to bind anions in water.

Cathelicidin Peptides Restrict Bacterial Growth via Membrane Perturbation and Induction of Reactive Oxygen Species.

All metazoans produce antimicrobial peptides (AMPs) that have both broad antimicrobial and immunomodulatory activity. Cathelicidins are AMPs that preferentially kill Gram-negative bacteria in vitro, purportedly by assembling into higher-order structures that perforate the membrane. We utilized high-resolution, single-cell fluorescence microscopy to examine their mechanism of action in real time. Engineered cathelicidins rapidly bound to Gram-negative and Gram-positive cells and penetrated the cytoplasmic membrane. Rapid failure of the peptidoglycan superstructure in regions of active turnover caused leakage of cytoplasmic contents and the formation of membrane-bound blebs. A mutation anticipated to destabilize interactions between cathelicidin subunits had no effect on bactericidal activity, suggesting that cathelicidins have activities beyond perforating the membrane. Nanomolar concentrations of cathelicidins, although not bactericidal, reduced the growth rate of Gram-negative and Gram-positive bacteria. The cells exhibited expression changes in multiple essential processes, including protein synthesis, peptidoglycan biosynthesis, respiration, and the detoxification of reactive oxygen species (ROS). Time-lapse imaging revealed that ROS accumulation preceded bleb formation, and treatments that reduced cellular ROS levels overcame these bactericidal effects. We propose that that the primary effect of cathelicidins is to induce the production of ROS that damage bacterial molecules, leading to slowed growth or cell death. Given their low circulating levels in vivo, AMPs may serve to slow bacterial population expansion so that cellular immunity systems can respond to and battle the infection.IMPORTANCE Antimicrobial peptides (AMPs) are an important part of the mammalian innate immune system in the battle against microbial infection. How AMPs function to control bacteria is not clear, as nearly all activity studies use nonphysiological levels of AMPs. We monitored peptide action in live bacterial cells over short time frames with single-cell resolution and found that the primary effect of cathelicidin peptides is to increase the production of oxidative molecules that cause cellular damage in Gram-positive and Gram-negative bacteria.

Low-dimensional Dynamics of Two Coupled Biological Oscillators.

The circadian clock and the cell cycle are two biological oscillatory processes that coexist within individual cells. These two oscillators were found to interact, which can lead to their synchronization. Here, we develop a method to identify a low-dimensional stochastic model of the coupled system directly from time-lapse imaging in single cells. In particular, we infer the coupling and non-linear dynamics of the two oscillators from thousands of mouse and human single-cell fluorescence microscopy traces. This coupling predicts multiple phase-locked states showing different degrees of robustness against molecular fluctuations inherent to cellular-scale biological oscillators. For the 1:1 state, the predicted phase-shifts upon period perturbations were validated experimentally. Moreover, the phase-locked states are temperature-independent and evolutionarily conserved from mouse to human, hinting at a common underlying dynamical mechanism. Finally, we detect a signature of the coupled dynamics in a physiological context, explaining why tissues with different proliferation states exhibited shifted circadian clock phases.

Glycogen Synthase Kinase 3 Beta Controls Presenilin-1-Mediated Endoplasmic Reticulum Ca2+ Leak Directed to Mitochondria in Pancreatic Islets and beta-Cells

Background/AimsIn pancreatic β-cells, the intracellular Ca2+ homeostasis is an essential regulator of the cells’ major functions. The endoplasmic reticulum (ER) as interactive intracellular Ca2+ store balances cellular Ca2+. In this study basal ER Ca2+ homeostasis was evaluated in order to reveal potential β-cell-specificity of ER Ca2+ handling and its consequences for mitochondrial Ca2+, ATP and respiration.MethodsThe two pancreatic cell lines INS-1 and MIN-6, freshly isolated pancreatic islets, and the two non-pancreatic cell lines HeLA and EA.hy926 were used. Cytosolic, ER and mitochondrial Ca2+ and ATP measurements were performed using single cell fluorescence microscopy and respective (genetically-encoded) sensors/dyes. Mitochondrial respiration was monitored by respirometry. GSK3β activity was measured with ELISA.ResultsAn atypical ER Ca2+ leak was observed exclusively in pancreatic islets and β-cells. This continuous ER Ca2+ efflux is directed to mitochondria and increases basal respiration and organellar ATP levels, is established by GSK3β-mediated phosphorylation of presenilin-1, and is prevented by either knockdown of presenilin-1 or an inhibition/knockdown of GSK3β. Expression of a presenlin-1 mutant that mimics GSK3β-mediated phosphorylation established a β-cell-like ER Ca2+ leak in HeLa and EA.hy926 cells. The ER Ca2+ loss in β-cells was compensated at steady state by Ca2+ entry that is linked to the activity of TRPC3.ConclusionPancreatic β-cells establish a cell-specific ER Ca2+ leak that is under the control of GSK3β and directed to mitochondria, thus, reflecting a cell-specific intracellular Ca2+ handling for basal mitochondrial activity.

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy.

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Bimodal Expression of the Salmonella Typhimurium spv Operon.

The well-studied spv operon of Salmonellatyphimurium is important for causing full virulence in mice and both the regulation and function of the Spv proteins have been characterized extensively over the past several decades. Using quantitative single-cell fluorescence microscopy, we demonstrate the spv regulon to display a bimodal expression pattern that originates in the bimodal expression of the SpvR activator. The spv expression pattern is influenced by growth conditions and the specific Styphimurium strain used, but does not require Salmonella-specific virulence regulators. By monitoring real-time promoter kinetics, we reveal that SpvA has the ability to impart negative feedback on spvABCD expression without affecting spvR expression. Together, our data suggest that the SpvA protein counteracts the positive feedback loop imposed by SpvR, and could thus be responsible for dampening spvABCD expression and coordinating virulence protein production in time. The results presented here yield new insights in the intriguing regulation of the spv operon and adds this operon to the growing list of virulence factors exhibiting marked expression heterogeneity in Styphimurium.