Multiresolution 3D Optical Mapping of Immune Cell Infiltrates in Mouse Asthmatic Lung.

Asthma is a chronic inflammatory airway disease driven by various infiltrating immune cell types into the lung. Optical microscopy has been used to study immune infiltrates in asthmatic lungs. Confocal laser scanning microscopy (CLSM) identifies the phenotypes and locations of individual immune cells in lung tissue sections by employing high-magnification objectives and multiplex immunofluorescence staining. In contrast, light-sheet fluorescence microscopy (LSFM) can visualize the macroscopic and mesoscopic architecture of whole-mount lung tissues in three dimensions (3D) by adopting an optical tissue-clearing method. Despite each microscopy method producing image data with unique resolution from a tissue sample, CLSM and LSFM have not been applied together because of different tissue-preparation procedures. Here, we introduce a new approach combining LSFM and CLSM into a sequential imaging pipeline. We built a new optical tissue clearing workflow in which the immersion clearing agent can be switched from an organic solvent to an aqueous sugar solution for sequential 3D LSFM and CLSM of mouse lungs. This sequential combination microscopy offered quantitative 3D spatial analyses of the distribution of immune infiltrates in the same mouse asthmatic lung tissue at the organ, tissue, and cell levels. These results show that our method facilitates multiresolution 3D fluorescence microscopy as a new imaging approach providing comprehensive spatial information for a better understanding of inflammatory lung diseases.

Three-dimensional spatial quantitative analysis of cardiac lymphatics in the mouse heart.

OBJECTIVE: Three-dimensional (3D) microscopy and image data analysis are necessary for studying the morphology of cardiac lymphatic vessels (LyVs) and their association with other cell types. We aimed to develop a methodology for 3D multiplexed lightsheet microscopy and highly sensitive and quantitative image analysis to identify pathological remodeling in the 3D morphology of LyVs in young adult mouse hearts with familial hypertrophic cardiomyopathy (HCM). METHODS: We developed a 3D lightsheet microscopy workflow providing a quick turn-around (as few as 5-6 days), multiplex fluorescence detection, and preservation of LyV structure and epitope markers. Hearts from non-transgenic and transgenic (TG) HCM mice were arrested in diastole, retrograde perfused, immunolabeled, optically cleared, and imaged. We built an image-processing pipeline to quantify LyV morphological parameters at the chamber and branch levels. RESULTS: Chamber-specific pathological alterations of LyVs were identified, and significant changes were seen in the right atrium (RA). TG hearts had a higher volume percent of ER-TR7+ fibroblasts and reticular fibers. In the RA, we found associations between ER-TR7+ volume percent and both LyV segment density and median diameter. CONCLUSIONS: This workflow and study enabled multi-scale analysis of pathological changes in cardiac LyVs of young adult mice, inviting ideas for research on LyVs in cardiac disease.

Nondestructive, multiplex three-dimensional mapping of immune infiltrates in core needle biopsy.

Enumeration of tumor-infiltrating lymphocytes (TILs) in H&E stained tissue sections has demonstrated limited value in predicting immune responses to cancer immunotherapy, likely reflecting the diversity of cell types and immune activation states among tumor infiltrates. Multiparametric flow cytometry enables robust phenotypic and functional analysis to distinguish suppression from activation, but tissue dissociation eliminates spatial context. Multiplex methods for immunohistochemistry (IHC) are emerging, but these interrogate only a single tissue section at a time. Here, we report transparent tissue tomography (T3) as a tool for three-dimensional (3D) imaging cytometry in the complex architecture of the tumor microenvironment, demonstrating multiplexed immunofluorescent analysis in core needle biopsies. Using T3 imaging, image processing and machine learning to map CD3+CD8+ cytotoxic T cells (CTLs) in whole core needle biopsies from Her2+ murine mammary tumors and human head and neck surgical specimens revealed marked inhomogeneity within single needle cores, confirmed by serial section IHC. Applying T3 imaging cytometry, we discovered a strong spatial correlation between CD3+CD8+ CTLs and microvasculature in the EGFR+ parenchyma, revealing significant differences among head and neck cancer patients. These results show that T3 offers simple and rapid access to three-dimensional and quantitative maps of the tumor microenvironment and immune infiltrate, offering a new diagnostic tool for personalized cancer immunotherapy.

Tumor-associated fibroblasts predominantly come from local and not circulating precursors.

Fibroblasts are common cell types in cancer stroma and lay down collagen required for survival and growth of cancer cells. Although some cancer therapy strategies target tumor fibroblasts, their origin remains controversial. Multiple publications suggest circulating mesenchymal precursors as a source of tumor-associated fibroblasts. However, we show by three independent approaches that tumor fibroblasts derive primarily from local, sessile precursors. First, transplantable tumors developing in a mouse expressing green fluorescent reporter protein (EGFP) under control of the type I collagen (Col-I) promoter (COL-EGFP) had green stroma, whereas we could not find COL-EGFP(+) cells in tumors developing in the parabiotic partner lacking the fluorescent reporter. Lack of incorporation of COL-EGFP(+) cells from the circulation into tumors was confirmed in parabiotic pairs of COL-EGFP mice and transgenic mice developing autochthonous intestinal adenomas. Second, transplantable tumors developing in chimeric mice reconstituted with bone marrow cells from COL-EGFP mice very rarely showed stromal fibroblasts expressing EGFP. Finally, cancer cells injected under full-thickness COL-EGFP skin grafts transplanted in nonreporter mice developed into tumors containing green stromal cells. Using multicolor in vivo confocal microscopy, we found that Col-I-expressing fibroblasts constituted approximately one-third of the stromal mass and formed a continuous sheet wrapping the tumor vessels. In summary, tumors form their fibroblastic stroma predominantly from precursors present in the local tumor microenvironment, whereas the contribution of bone marrow-derived circulating precursors is rare.

TRPC6 channel translocation into phagosomal membrane augments phagosomal function.

Defects in the innate immune system in the lung with attendant bacterial infections contribute to lung tissue damage, respiratory insufficiency, and ultimately death in the pathogenesis of cystic fibrosis (CF). Professional phagocytes, including alveolar macrophages (AMs), have specialized pathways that ensure efficient killing of pathogens in phagosomes. Phagosomal acidification facilitates the optimal functioning of degradative enzymes, ultimately contributing to bacterial killing. Generation of low organellar pH is primarily driven by the V-ATPases, proton pumps that use cytoplasmic ATP to load H(+) into the organelle. Critical to phagosomal acidification are various channels derived from the plasma membrane, including the anion channel cystic fibrosis transmembrane conductance regulator, which shunt the transmembrane potential generated by movement of protons. Here we show that the transient receptor potential canonical-6 (TRPC6) calcium-permeable channel in the AM also functions to shunt the transmembrane potential generated by proton pumping and is capable of restoring microbicidal function to compromised AMs in CF and enhancement of function in non-CF cells. TRPC6 channel activity is enhanced via translocation to the cell surface (and then ultimately to the phagosome during phagocytosis) in response to G-protein signaling activated by the small molecule (R)-roscovitine and its derivatives. These data show that enhancing vesicular insertion of the TRPC6 channel to the plasma membrane may represent a general mechanism for restoring phagosome activity in conditions, where it is lost or impaired.

Critical role of microbiota within cecal crypts on the regenerative capacity of the intestinal epithelium following surgical stress.

Cecal crypts represent a unique niche that are normally occupied by the commensal microbiota. Due to their density and close proximity to stem cells, microbiota within cecal crypts may modulate epithelial regeneration. Here we demonstrate that surgical stress, a process that invariably involves a short period of starvation, antibiotic exposure, and tissue injury, results in cecal crypt evacuation of their microbiota. Crypts devoid of their microbiota display pathophysiological features characterized by abnormal stem cell activation as judged by leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) staining, expansion of the proliferative zone toward the tips of the crypts, and an increase in apoptosis. In addition, crypts devoid of their microbiota display loss of their regenerative capacity as assessed by their ability to form organoids ex vivo. When a four-member human pathogen community isolated from the stool of a critically ill patient is introduced into the cecum of mice with empty crypts, crypts become occupied by the pathogens and further disruption of crypt homeostasis is observed. Fecal microbiota transplantation restores the cecal crypts' microbiota, normalizes homeostasis within crypts, and reestablishes crypt regenerative capacity. Taken together, these findings define an emerging role for the microbiota within cecal crypts to maintain epithelial cell homeostasis in a manner that may enhance recovery in response to the physiological stress imposed by the process of surgery. NEW & NOTEWORTHY: This study provides novel insight into the process by which surgical injury places the intestinal epithelium at risk for colonization by pathogenic microbes and impairment of its regenerative capacity via loss of its microbiota. We show that fecal transplant restores crypt homeostasis in association with repopulation of the microbiota within cecal crypts.

CFTR regulates phagosome acidification in macrophages and alters bactericidal activity.

Acidification of phagosomes has been proposed to have a key role in the microbicidal function of phagocytes. Here, we show that in alveolar macrophages the cystic fibrosis transmembrane conductance regulator Cl- channel (CFTR) participates in phagosomal pH control and has bacterial killing capacity. Alveolar macrophages from Cftr-/- mice retained the ability to phagocytose and generate an oxidative burst, but exhibited defective killing of internalized bacteria. Lysosomes from CFTR-null macrophages failed to acidify, although they retained normal fusogenic capacity with nascent phagosomes. We hypothesize that CFTR contributes to lysosomal acidification and that in its absence phagolysosomes acidify poorly, thus providing an environment conducive to bacterial replication.

Three-dimensional spatial quantitative analysis of cardiac lymphatics in the mouse heart.

Objective: 3D microscopy and image data analysis are necessary for studying the morphology of cardiac lymphatic vessels (LyVs) and association with other cell types. We aimed to develop a methodology for 3D multiplexed lightsheet microscopy and highly sensitive and quantitative image analysis to identify pathological remodeling in the 3D morphology of LyVs in young adult mouse hearts with familial hypertrophic cardiomyopathy (HCM). Methods: We developed a 3D lightsheet microscopy workflow providing a quick turn-around (as few as 5-6 days), multiplex fluorescence detection, and preservation of LyV structure and epitope markers. Hearts from non-transgenic (NTG) and transgenic (TG) HCM mice were arrested in diastole, retrograde perfused, immunolabeled, optically cleared, and imaged. We built an image processing pipeline to quantify LyV morphological parameters at the chamber and branch levels. Results: Chamber-specific pathological alterations of LyVs were identified, but most significantly in the right atrium (RA). TG hearts had a higher volume fraction of ER-TR7 + fibroblasts and reticular fibers. In the RA, we found associations between ER-TR7 + volume fraction and both LyV segment density and median diameter. Conclusions: This workflow and study enabled multi-scale analysis of pathological changes in cardiac LyVs of young adult mice, inviting ideas for research on LyVs in cardiac disease.

Hypoxia sensing requires H2S-dependent persulfidation of olfactory receptor 78.

Oxygen (O2) sensing by the carotid body is critical for maintaining cardiorespiratory homeostasis during hypoxia. Hydrogen sulfide (H2S) signaling is implicated in carotid body activation by low O2. Here, we show that persulfidation of olfactory receptor 78 (Olfr78) by H2S is an integral component of carotid body activation by hypoxia. Hypoxia and H2S increased persulfidation in carotid body glomus cells and persulfidated cysteine240 in Olfr78 protein in heterologous system. Olfr78 mutants manifest impaired carotid body sensory nerve, glomus cell, and breathing responses to H2S and hypoxia. Glomus cells are positive for GOlf, adenylate cyclase 3 (Adcy3) and cyclic nucleotide-gated channel alpha 2 (Cnga2), key molecules of odorant receptor signaling. Adcy3 or Cnga2 mutants exhibited impaired carotid body and glomus cell responses to H2S and breathing responses to hypoxia. These results suggest that H2S through redox modification of Olfr78 participates in carotid body activation by hypoxia to regulate breathing.

Polyglutamine protein aggregates are dynamic.

Protein aggregation and the formation of inclusion bodies are hallmarks of the cytopathology of neurodegenerative diseases, including Huntington's disease, Amyotropic lateral sclerosis, Parkinson's disease and Alzheimer's disease. The cellular toxicity associated with protein aggregates has been suggested to result from the sequestration of essential proteins that are involved in key cellular events, such as transcription, maintenance of cell shape and motility, protein folding and protein degradation. Here, we use fluorescence imaging of living cells to show that polyglutamine protein aggregates are dynamic structures in which glutamine-rich proteins are tightly associated, but which exhibit distinct biophysical interactions. In contrast, the interaction between wild-type, but not mutant, Hsp70 exhibits rapid kinetics of association and dissociation similar to interactions between Hsp70 and thermally unfolded substrates. These studies provide new insights into the composite organization and formation of protein aggregates and show that molecular chaperones are not sequestered into aggregates, but are instead transiently associated.

CLC-3 channels modulate excitatory synaptic transmission in hippocampal neurons.

It is well established that ligand-gated chloride flux across the plasma membrane modulates neuronal excitability. We find that a voltage-dependent Cl(-) conductance increases neuronal excitability in immature rodents as well, enhancing the time course of NMDA receptor-mediated miniature excitatory postsynaptic potentials (mEPSPs). This Cl(-) conductance is activated by CaMKII, is electrophysiologically identical to the CaMKII-activated CLC-3 conductance in nonneuronal cells, and is absent in clc-3(-/-) mice. Systematically decreasing [Cl(-)](i) to mimic postnatal [Cl(-)](i) regulation progressively decreases the amplitude and decay time constant of spontaneous mEPSPs. This Cl(-)-dependent change in synaptic strength is absent in clc-3(-/-) mice. Using surface biotinylation, immunohistochemistry, electron microscopy, and coimmunoprecipitation studies, we find that CLC-3 channels are localized on the plasma membrane, at postsynaptic sites, and in association with NMDA receptors. This is the first demonstration that a voltage-dependent chloride conductance modulates neuronal excitability. By increasing postsynaptic potentials in a Cl(-) dependent fashion, CLC-3 channels regulate neuronal excitability postsynaptically in immature neurons.

Disease-causing mutations in the cystic fibrosis transmembrane conductance regulator determine the functional responses of alveolar macrophages.

Alveolar macrophages (AMs) play a major role in host defense against microbial infections in the lung. To perform this function, these cells must ingest and destroy pathogens, generally in phagosomes, as well as secrete a number of products that signal other immune cells to respond. Recently, we demonstrated that murine alveolar macrophages employ the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel as a determinant in lysosomal acidification (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933-944). Lysosomes and phagosomes in murine cftr(-/-) AMs failed to acidify, and the cells were deficient in bacterial killing compared with wild type controls. Cystic fibrosis is caused by mutations in CFTR and is characterized by chronic lung infections. The information about relationships between the CFTR genotype and the disease phenotype is scarce both on the organismal and cellular level. The most common disease-causing mutation, DeltaF508, is found in 70% of patients with cystic fibrosis. The mutant protein fails to fold properly and is targeted for proteosomal degradation. G551D, the second most common mutation, causes loss of function of the protein at the plasma membrane. In this study, we have investigated the impact of CFTR DeltaF508 and G551D on a set of core intracellular functions, including organellar acidification, granule secretion, and microbicidal activity in the AM. Utilizing primary AMs from wild type, cftr(-/-), as well as mutant mice, we show a tight correlation between CFTR genotype and levels of lysosomal acidification, bacterial killing, and agonist-induced secretory responses, all of which would be expected to contribute to a significant impact on microbial clearance in the lung.

Multiplex Three-Dimensional Mapping of Macromolecular Drug Distribution in the Tumor Microenvironment.

Macromolecular cancer drugs such as therapeutic antibodies and nanoparticles are well known to display slow extravasation and incomplete penetration into tumors, potentially protecting cancer cells from therapeutic effects. Conventional assays to track macromolecular drug delivery are poorly matched to the heterogeneous tumor microenvironment, but recent progress on optical tissue clearing and three-dimensional (3D) tumor imaging offers a path to quantitative assays with cellular resolution. Here, we apply transparent tissue tomography (T3) as a tool to track perfusion and delivery in the tumor and to evaluate target binding and vascular permeability. Using T3, we mapped anti-programmed cell death protein-ligand 1 (PD-L1) antibody distribution in whole mouse tumors. By measuring 3D penetration distances of the antibody drug out from the blood vessel boundaries into the tumor parenchyma, we determined spatial pharmacokinetics of anti-PD-L1 antibody drugs in mouse tumors. With multiplex imaging of tumor components, we determined the distinct distribution of anti-PD-L1 antibody drug in the tumor microenvironment with different PD-L1 expression patterns. T3 imaging revealed CD31+ capillaries are more permeable to anti-PD-L1 antibody transport compared with the blood vessels composed of endothelium supported by vascular fibroblasts and smooth muscle cells. T3 analysis also confirmed that isotype IgG antibody penetrates more deeply into tumor parenchyma than anti-Her2 or anti-EGFR antibody, which were restrained by binding to their respective antigens on tumor cells. Thus, T3 offers simple and rapid access to 3D, quantitative maps of macromolecular drug distribution in the tumor microenvironment, offering a new tool for development of macromolecular cancer therapeutics.

O-GlcNAcylation Enhances Double-Strand Break Repair, Promotes Cancer Cell Proliferation, and Prevents Therapy-Induced Senescence in Irradiated Tumors.

The metabolic reprogramming associated with characteristic increases in glucose and glutamine metabolism in advanced cancer is often ascribed to answering a higher demand for metabolic intermediates required for rapid tumor cell growth. Instead, recent discoveries have pointed to an alternative role for glucose and glutamine metabolites as cofactors for chromatin modifiers and other protein posttranslational modification enzymes in cancer cells. Beyond epigenetic mechanisms regulating gene expression, many chromatin modifiers also modulate DNA repair, raising the question whether cancer metabolic reprogramming may mediate resistance to genotoxic therapy and genomic instability. Our prior work had implicated N-acetyl-glucosamine (GlcNAc) formation by the hexosamine biosynthetic pathway (HBP) and resulting protein O-GlcNAcylation as a common means by which increased glucose and glutamine metabolism can drive double-strand break (DSB) repair and resistance to therapy-induced senescence in cancer cells. We have examined the effects of modulating O-GlcNAcylation on the DNA damage response (DDR) in MCF7 human mammary carcinoma in vitro and in xenograft tumors. Proteomic profiling revealed deregulated DDR pathways in cells with altered O-GlcNAcylation. Promoting protein O-GlcNAc modification by targeting O-GlcNAcase or simply treating animals with GlcNAc protected tumor xenografts against radiation. In turn, suppressing protein O-GlcNAcylation by blocking O-GlcNAc transferase activity led to delayed DSB repair, reduced cell proliferation, and increased cell senescence in vivo. Taken together, these findings confirm critical connections between cancer metabolic reprogramming, DDR, and senescence and provide a rationale to evaluate agents targeting O-GlcNAcylation in patients as a means to restore tumor sensitivity to radiotherapy. IMPLICATIONS: The finding that the HBP, via its impact on protein O-GlcNAcylation, is a key determinant of the DDR in cancer provides a mechanistic link between metabolic reprogramming, genomic instability, and therapeutic response and suggests novel therapeutic approaches for tumor radiosensitization.

Dynamin regulates focal exocytosis in phagocytosing macrophages.

Phagocytosis in macrophages is thought to involve insertion of cytoplasmic vesicles at sites of membrane expansion before particle ingestion ("focal" exocytosis). Capacitance (Cm) measurements of cell surface area were biphasic, with an initial rise indicative of exocytosis followed by a fall upon phagocytosis. Unlike other types of regulated exocytosis, the Cm rise was insensitive to intracellular Ca2+, but was inhibited by guanosine 5'-O-(2-thio)diphosphate. Particle uptake, but not Cm rise, was affected by phosphatidylinositol 3-kinase inhibitors. Inhibition of actin polymerization eliminated the Cm rise, suggesting possible coordination between actin polymerization and focal exocytosis. Introduction of anti-pan-dynamin IgG blocked Cm changes, suggesting that dynamin controls focal exocytosis and thereby phagocytosis. Similarly, recombinant glutathione S-transferase*amphiphysin-SH3 domain, but not a mutated form that cannot bind to dynamin, inhibited both focal exocytosis and phagocytosis. Immunochemical analysis of endogenous dynamin distribution in macrophages revealed a substantial particulate pool, some of which localized to a presumptive endosomal compartment. Expression of enhanced green fluorescent protein*dynamin-2 showed a motile dynamin pool, a fraction of which migrated toward and within the phagosomal cup. These results suggest that dynamin is involved in the production and/or movement of vesicles from an intracellular organelle to the cell surface to support membrane expansion around the engulfed particle.

Multiplex three-dimensional optical mapping of tumor immune microenvironment.

Recent developments in optical tissue clearing and microscopic imaging have advanced three-dimensional (3D) visualization of intact tissues and organs at high resolution. However, to expand applications to oncology, critical limitations of current methods must be addressed. Here we describe transparent tissue tomography (T3) as a tool for rapid, three-dimensional, multiplexed immunofluorescent tumor imaging. Cutting tumors into sub-millimeter macrosections enables simple and rapid immunofluorescence staining, optical clearing, and confocal microscope imaging. Registering and fusing macrosection images yields high resolution 3D maps of multiple tumor microenvironment components and biomarkers throughout a tumor. The 3D maps can be quantitatively evaluated by automated image analysis. As an application of T3, 3D mapping and analysis revealed a heterogeneous distribution of programmed death-ligand 1 (PD-L1) in Her2 transgenic mouse mammary tumors, with high expression limited to tumor cells at the periphery and to CD31+ vascular endothelium in the core. Also, strong spatial correlation between CD45+ immune cell distribution and PD-L1 expression was revealed by T3 analysis of the whole tumors. Our results demonstrate that a tomographic approach offers simple and rapid access to high-resolution three-dimensional maps of the tumor immune microenvironment, offering a new tool to examine tumor heterogeneity.

Mitochondrial mechanisms of neuronal rescue by F-68, a hydrophilic Pluronic block co-polymer, following acute substrate deprivation.

Global brain ischemia can lead to widespread neuronal death and poor neurologic outcomes in patients. Despite detailed understanding of the cellular and molecular mechanisms mediating neuronal death following focal and global brain hypoxia-ischemia, treatments to reduce ischemia-induced brain injury remain elusive. One pathway central to neuronal death following global brain ischemia is mitochondrial dysfunction, one consequence of which is the cascade of intracellular events leading to mitochondrial outer membrane permeabilization. A novel approach to rescuing injured neurons from death involves targeting cellular membranes using a class of synthetic molecules called Pluronics. Pluronics are triblock copolymers of hydrophilic poly[ethylene oxide] (PEO) and hydrophobic poly[propylene oxide] (PPO). Evidence is accumulating to suggest that hydrophilic Pluronics rescue injured neurons from death following substrate deprivation by preventing mitochondrial dysfunction. Here, we will review current understanding of the nature of interaction of Pluronic molecules with biological membranes and the efficacy of F-68, an 80% hydrophilic Pluronic, in rescuing neurons from injury. We will review data indicating that F-68 reduces mitochondrial dysfunction and mitochondria-dependent death pathways in a model of neuronal injury in vitro, and present new evidence that F-68 acts directly on mitochondria to inhibit mitochondrial outer membrane permeabilization. Finally, we will present results of a pilot, proof-of-principle study suggesting that F-68 is effective in reducing hippocampal injury induced by transient global ischemia in vivo. By targeting mitochondrial dysfunction, F-68 and other Pluronic molecules constitute an exciting new approach to rescuing neurons from acute injury.

Inositol (1,4,5)-trisphosphate dynamics and intracellular calcium oscillations in pancreatic beta-cells.

Glucose-stimulated insulin secretion is associated with transients of intracellular calcium concentration ([Ca2+]i) in the pancreatic beta-cell. We tested the hypothesis that inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] [Ca2+]i release is incorporated in glucose-induced [Ca2+]i oscillations in mouse islets and MIN6 cells. We found that depletion of intracellular Ca2+ stores with thapsigargin increased the oscillation frequency by twofold and inhibited the slow recovery phase of [Ca2+]i oscillations. We employed a pleckstrin homology domain-containing fluorescent biosensor, phospholipase C partial differential pleckstrin homology domain-enhanced green fluorescent protein, to visualize Ins(1,4,5)P3 dynamics in insulin-secreting MIN6 cells and mouse islets in real time using a video-rate confocal system. In both types of cells, stimulation with carbamoylcholine (CCh) and depolarization with KCl results in an increase in Ins(1,4,5)P3 accumulation in the cytoplasm. When stimulated with glucose, the Ins(1,4,5)P3 concentration in the cytoplasm oscillates in parallel with oscillations of [Ca2+]i. Maximal accumulation of Ins(1,4,5)P3 in these oscillations coincides with the peak of [Ca2+]i and tracks changes in frequencies induced by the voltage-gated K+ channel blockade. We show that Ins(1,4,5)P3 release in insulin-secreting cells can be stimulated by depolarization-induced Ca2+ flux. We conclude that Ins(1,4,5)P3 concentration oscillates in parallel with [Ca2+]i in response to glucose stimulation, but it is not the driving force for [Ca2+]i oscillations.

Visualizing calcium signaling in cells by digitized wide-field and confocal fluorescent microscopy.

Calcium (Ca2+) is a fundamentally important component of cellular signal transduction. Dynamic changes in the concentration of Ca2+ ([Ca2+]) in the cytoplasm and within organelles are tightly controlled and regulate a diverse array of biological activities, including fertilization, cell division, gene expression, cellular metabolism, protein biosynthesis, secretion, muscle contraction, intercellular communication, and cell death. Measurement of intracellular [Ca2+] is essential to understanding the role of Ca2+ and for defining the underlying regulatory mechanisms in any cellular process. A broad range of synthetic and biosynthetic fluorescent Ca2+ sensors are available that enable the visualization and quantification of subcellular spatio-temporal [Ca2+] gradients. This chapter describes the application of wide-field digitized video fluorescence microfluorometry and confocal microscopy to quantitatively image Ca2+ in cells with high temporal and spatial resolution.

Flow cytometric analysis of phosphorylated histone H2AX following exposure to ionizing radiation in human microvascular endothelial cells.

We applied a flow cytometric method to quantify IR-induced histone H2AX phosphorylation at serine 139 (gammaH2AX) and compared those values to those obtained using a standard microscopy based foci counting method. After PFA fixation, methanol permeabilization was suitable for both FITC- or Alexa647-gammaH2AX. In contrast, Alexa647-gammaH2AX was not suitable for ethanol permeabilization. Antibody concentrations at 1-2 microg/ml yielded the highest gammaH2AX positive percentage for both antibodies. Without DAPI staining, gammaH2AX formation can be measured as a relative fold increase. Values determined by bivariant flow cytometric analysis and those obtained using microscopic foci formation exhibited a good quantitative correlation. Values obtained by both methods could vary according to the gating or threshold setting used. gammaH2AX positive cells increased as a function of radiation dose (2-16 Gy) followed by a dose-dependent decay. The free radical scavenger N-acetyl-L-cysteine (NAC), if administered at a concentration of 4 mM 30 min before IR, was effective in reducing IR-induced gammaH2AX formation in all phases of the cell cycle. We have developed a simplified and quantitative flow cytometry based method to measure IR-induced gammaH2AX in cells and demonstrated strong correlation to values obtained by a standard automated digital microscopic foci analysis along with NIH ImageJ custom macro software.