Automated cell cluster analysis provides insight into multi-cell-type interactions between immune cells and their targets.

Understanding interactions between immune cells and their targets is an important step on the path to fully characterizing the immune system, and in doing so, learning how it combats disease. Many studies of these interactions have a narrow focus, often looking only at a binary result of whether or not a specific treatment was successful or only focusing on the interactions between two individual cells. Therefore, in an effort to more comprehensively study multicellular interactions among immune cells and their targets, we used in vitro longitudinal time-lapse imaging and developed an automated cell cluster analysis tool, or macro, to investigate the formation of cell clusters. In particular, we investigated the behavior of cancer-specific CD8+ and CD4+ T cells on how they interact around their targets: cancer cells and antigen-presenting cells. The macro that we established allowed us to examine these large-scale clustering behaviors taking place between those four cell types. Thus, we were able to distinguish directed immune cell clustering from random cell movement. Furthermore, this macro can be generalized to be applicable to systems consisting of any number of differently labeled species and can be used to track clustering behaviors and compare them to randomized simulations.

Role of c-Met/phosphatidylinositol 3-kinase (PI3k)/Akt signaling in hepatocyte growth factor (HGF)-mediated lamellipodia formation, reactive oxygen species (ROS) generation, and motility of lung endothelial cells.

Hepatocyte growth factor (HGF) mediated signaling promotes cell proliferation and migration in a variety of cell types and plays a key role in tumorigenesis. As cell migration is important to angiogenesis, we characterized HGF-mediated effects on the formation of lamellipodia, a pre-requisite for migration using human lung microvascular endothelial cells (HLMVECs). HGF, in a dose-dependent manner, induced c-Met phosphorylation (Tyr-1234/1235, Tyr-1349, Ser-985, Tyr-1003, and Tyr-1313), activation of PI3k (phospho-Yp85) and Akt (phospho-Thr-308 and phospho-Ser-473) and potentiated lamellipodia formation and HLMVEC migration. Inhibition of c-Met kinase by SU11274 significantly attenuated c-Met, PI3k, and Akt phosphorylation, suppressed lamellipodia formation and endothelial cell migration. LY294002, an inhibitor of PI3k, abolished HGF-induced PI3k (Tyr-458), and Akt (Thr-308 and Ser-473) phosphorylation and suppressed lamellipodia formation. Furthermore, HGF stimulated p47(phox)/Cortactin/Rac1 translocation to lamellipodia and ROS generation. Moreover, inhibition of c-Met/PI3k/Akt signaling axis and NADPH oxidase attenuated HGF- induced lamellipodia formation, ROS generation and cell migration. Ex vivo experiments with mouse aortic rings revealed a role for c-Met signaling in HGF-induced sprouting and lamellipodia formation. Taken together, these data provide evidence in support of a significant role for HGF-induced c-Met/PI3k/Akt signaling and NADPH oxidase activation in lamellipodia formation and motility of lung endothelial cells.

Anabaena cell ageing monitored with confocal fluorescence spectroscopy.

Cyanobacteria use a sophisticated system of pigments to collect light energy across the visible spectrum for photosynthesis. The pigments are assembled in structures called phycobilisomes, composed of phycoerythrocyanin, phycocyanin and allophycocyanin, which absorb energy and transfer it to chlorophyll in photosystem II reaction centres. All of the components of this system are fluorescent, allowing sensitive measurements of energy transfer using single cell confocal fluorescence microscopy. The native pigments can be interrogated without the use of reporters. Here, we use confocal fluorescence microscopy to monitor changes in the efficiency of energy transfer as single cells age, between the time they are born at cell division until they are ready to divide again. Alteration of fluorescence was demonstrated to change with the age of the cyanobacterial cell.

SLIT3-ROBO4 activation promotes vascular network formation in human engineered tissue and angiogenesis in vivo.

Successful implantation and long-term survival of engineered tissue grafts hinges on adequate vascularization of the implant. Endothelial cells are essential for patterning vascular structures, but they require supportive mural cells such as pericytes/mesenchymal stem cells (MSCs) to generate stable, functional blood vessels. While there is evidence that the angiogenic effect of MSCs is mediated via the secretion of paracrine signals, the identity of these signals is unknown. By utilizing two functionally distinct human MSC clones, we found that so-called "pericytic" MSCs secrete the pro-angiogenic vascular guidance molecule SLIT3, which guides vascular development by directing ROBO4-positive endothelial cells to form networks in engineered tissue. In contrast, "non-pericytic" MSCs exhibit reduced activation of the SLIT3/ROBO4 pathway and do not support vascular networks. Using live cell imaging of organizing 3D vascular networks, we show that siRNA knockdown of SLIT3 in MSCs leads to disorganized clustering of ECs. Knockdown of its receptor ROBO4 in ECs abolishes the generation of functional human blood vessels in an in vivo xenogenic implant. These data suggest that the SLIT3/ROBO4 pathway is required for MSC-guided vascularization in engineered tissues. Heterogeneity of SLIT3 expression may underlie the variable clinical success of MSCs for tissue repair applications.

Emerging Activity Patterns and Synaptogenesis in Dissociated Hippocampal Cultures.

Cultures of dissociated hippocampal neurons display a stereotypical development of network activity patterns within the first three weeks of maturation. During this process, network connections develop and the associated spiking patterns range from increasing levels of activity in the first two weeks to regular bursting activity in the third week of maturation. Characterization of network structure is important to examine the mechanisms underlying the emergent functional organization of neural circuits. To accomplish this, confocal microscopy techniques have been used and several automated synapse quantification algorithms based on (co)localization of synaptic structures have been proposed recently. However, these approaches suffer from the arbitrary nature of intensity thresholding and the lack of correction for random-chance colocalization. To address this problem, we developed and validated an automated synapse quantification algorithm that requires minimal operator intervention. Next, we applied our approach to quantify excitatory and inhibitory synaptogenesis using confocal images of dissociated hippocampal neuronal cultures captured at 5, 8, 14 and 20 days in vitro, the time period associated with the development of distinct neuronal activity patterns. As expected, we found that synaptic density increased with maturation, coinciding with increasing spiking activity in the network. Interestingly, the third week of the maturation exhibited a reduction in excitatory synaptic density suggestive of synaptic pruning that coincided with the emergence of regular bursting activity in the network.

Mechanisms of simultaneous linear and nonlinear computations at the mammalian cone photoreceptor synapse.

Neurons enhance their computational power by combining linear and nonlinear transformations in extended dendritic trees. Rich, spatially distributed processing is rarely associated with individual synapses, but the cone photoreceptor synapse may be an exception. Graded voltages temporally modulate vesicle fusion at a cone's ~20 ribbon active zones. Transmitter then flows into a common, glia-free volume where bipolar cell dendrites are organized by type in successive tiers. Using super-resolution microscopy and tracking vesicle fusion and postsynaptic responses at the quantal level in the thirteen-lined ground squirrel, Ictidomys tridecemlineatus, we show that certain bipolar cell types respond to individual fusion events in the vesicle stream while other types respond to degrees of locally coincident events, creating a gradient across tiers that are increasingly nonlinear. Nonlinearities emerge from a combination of factors specific to each bipolar cell type including diffusion distance, contact number, receptor affinity, and proximity to glutamate transporters. Complex computations related to feature detection begin within the first visual synapse.

Photolysis of caged sphingosine-1-phosphate induces barrier enhancement and intracellular activation of lung endothelial cell signaling pathways.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that mediates cellular functions by ligation via G protein-coupled S1P receptors. In addition to its extracellular action, S1P also has intracellular effects; however, the signaling pathways modulated by intracellular S1P remain poorly defined. We have previously demonstrated a novel pathway of intracellular S1P generation in human lung endothelial cells (ECs). In the present study, we examined the role of intracellular S1P generated by photolysis of caged S1P on EC barrier regulation and signal transduction. Intracellular S1P released from caged S1P caused mobilization of intracellular calcium, induced activation of MAPKs, redistributed cortactin, vascular endothelial cadherin, and ß-catenin to cell periphery, and tightened endothelial barrier in human pulmonary artery ECs. Treatment of cells with pertussis toxin (PTx) had no effect on caged S1P-mediated effects on Ca(2+) mobilization, reorganization of cytoskeleton, cell adherens junction proteins, and barrier enhancement; however, extracellular S1P effects were significantly attenuated by PTx. Additionally, intracellular S1P also activated small GTPase Rac1 and its effector Ras GTPase-activating-like protein IQGAP1, suggesting involvement of these proteins in the S1P-mediated changes in cell-to-cell adhesion contacts. Downregulation of sphingosine kinase 1 (SphK1), but not SphK2, with siRNA or inhibition of SphK activity with an inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (CII) attenuated exogenously administrated S1P-induced EC permeability. Furthermore, S1P1 receptor inhibitor SB649164 abolished exogenous S1P-induced transendothelial resistance changes but had no effect on intracellular S1P generated by photolysis of caged S1P. These results provide evidence that intracellular S1P modulates signal transduction in lung ECs via signaling pathway(s) independent of S1P receptors.

Quantal release of free radicals during exocytosis of phagosomes.

Secretion of lysosomes and related organelles is important for immune system function. High-resolution membrane capacitance techniques were used to track changes in membrane area in single phagocytes during opsonized polystyrene bead uptake and release. Secretagogue stimulation of cells preloaded with beads resulted in immediate vesicle discharge, visualized as step increases in capacitance. The size of the increases were consistent with phagosome size. This hypothesis was confirmed by direct observation of dye release from bead-containing phagosomes after secretagogue stimulation. Capacitance recordings of exocytosis were correlated with quantal free radical release, as determined by amperometry. Thus, phagosomes undergo regulated secretion in macrophages, one function of which may be to deliver sequestered free radicals to the extracellular space.

Subcellular localization of the J-protein Sis1 regulates the heat shock response.

Cells exposed to heat shock induce a conserved gene expression program, the heat shock response (HSR), encoding protein homeostasis (proteostasis) factors. Heat shock also triggers proteostasis factors to form subcellular quality control bodies, but the relationship between these spatial structures and the HSR is unclear. Here we show that localization of the J-protein Sis1, a cofactor for the chaperone Hsp70, controls HSR activation in yeast. Under nonstress conditions, Sis1 is concentrated in the nucleoplasm, where it promotes Hsp70 binding to the transcription factor Hsf1, repressing the HSR. Upon heat shock, Sis1 forms an interconnected network with other proteostasis factors that spans the nucleolus and the surface of the endoplasmic reticulum. We propose that localization of Sis1 to this network directs Hsp70 activity away from Hsf1 in the nucleoplasm, leaving Hsf1 free to induce the HSR. In this manner, Sis1 couples HSR activation to the spatial organization of the proteostasis network.

Neuronal BIN1 Regulates Presynaptic Neurotransmitter Release and Memory Consolidation.

BIN1, a member of the BAR adaptor protein family, is a significant late-onset Alzheimer disease risk factor. Here, we investigate BIN1 function in the brain using conditional knockout (cKO) models. Loss of neuronal Bin1 expression results in the select impairment of spatial learning and memory. Examination of hippocampal CA1 excitatory synapses reveals a deficit in presynaptic release probability and slower depletion of neurotransmitters during repetitive stimulation, suggesting altered vesicle dynamics in Bin1 cKO mice. Super-resolution and immunoelectron microscopy localizes BIN1 to presynaptic sites in excitatory synapses. Bin1 cKO significantly reduces synapse density and alters presynaptic active zone protein cluster formation. Finally, 3D electron microscopy reconstruction analysis uncovers a significant increase in docked and reserve pools of synaptic vesicles at hippocampal synapses in Bin1 cKO mice. Our results demonstrate a non-redundant role for BIN1 in presynaptic regulation, thus providing significant insights into the fundamental function of BIN1 in synaptic physiology relevant to Alzheimer disease.

Aberrant accrual of BIN1 near Alzheimer's disease amyloid deposits in transgenic models.

Bridging integrator 1 (BIN1) is the most significant late-onset Alzheimer's disease (AD) susceptibility locus identified via genome-wide association studies. BIN1 is an adaptor protein that regulates membrane dynamics in the context of endocytosis and membrane remodeling. An increase in BIN1 expression and changes in the relative levels of alternatively spliced BIN1 isoforms have been reported in the brains of patients with AD. BIN1 can bind to Tau, and an increase in BIN1 expression correlates with Tau pathology. In contrast, the loss of BIN1 expression in cultured cells elevates Aß production and Tau propagation by insfluencing endocytosis and recycling. Here, we show that BIN1 accumulates adjacent to amyloid deposits in vivo. We found an increase in insoluble BIN1 and a striking accrual of BIN1 within and near amyloid deposits in the brains of multiple transgenic models of AD. The peri-deposit aberrant BIN1 localization was conspicuously different from the accumulation of APP and BACE1 within dystrophic neurites. Although BIN1 is highly expressed in mature oligodendrocytes, BIN1 association with amyloid deposits occurred in the absence of the accretion of other oligodendrocyte or myelin proteins. Finally, super-resolution microscopy and immunogold electron microscopy analyses highlight the presence of BIN1 in proximity to amyloid fibrils at the edges of amyloid deposits. These results reveal the aberrant accumulation of BIN1 is a feature associated with AD amyloid pathology. Our findings suggest a potential role for BIN1 in extracellular Aß deposition in vivo that is distinct from its well-characterized function as an adaptor protein in endocytosis and membrane remodeling.

Functional analysis of c-Met/hepatocyte growth factor pathway in malignant pleural mesothelioma.

c-Met receptor tyrosine kinase (RTK) has not been extensively studied in malignant pleural mesothelioma (MPM). In this study, c-Met was overexpressed and activated in most of the mesothelioma cell lines tested. Expression in MPM tissues by immunohistochemistry was increased (82%) in MPM in general compared with normal. c-Met was internalized with its ligand hepatocyte growth factor (HGF) in H28 MPM cells, with robust expression of c-Met. Serum circulating HGF was twice as high in mesothelioma patients as in healthy controls. There was a differential growth response and activation of AKT and extracellular signal-regulated kinase 1/2 in response to HGF for the various cell lines. Dose-dependent inhibition (IC50 < 2.5 micromol/L) of cell growth in mesothelioma cell lines, but not in H2052, H2452, and nonmalignant MeT-5A (IC50 > 10 micromol/L), was observed with the small-molecule c-Met inhibitor SU11274. Furthermore, migration of H28 cells was blocked with both SU11274 and c-Met small interfering RNA. Abrogation of HGF-induced c-Met and downstream signaling was seen in mesothelioma cells. Of the 43 MPM tissues and 7 cell lines, we have identified mutations within the semaphorin domain (N375S, M431V, and N454I), the juxtamembrane domain (T1010I and G1085X), and an alternative spliced product with deletion of the exon 10 of c-Met in some of the samples. Interestingly, we observed that the cell lines H513 and H2596 harboring the T1010I mutation exhibited the most dramatic reduction of cell growth with SU11274 when compared with wild-type H28 and nonmalignant MeT-5A cells. Ultimately, c-Met would be an important target for therapy against MPM.

Light Activation of Calcein Inhibits Vesicle Release of Catecholamines.

Calcein, a fluorescent fluid phase marker, has been used to track and visualize cellular processes such as synaptic vesicle fusion. It is also the fluorophore for live cells in the commonly used Live/Dead viability assay. In pilot studies designed to determine fusion pore open size and vesicle movement in secretory cells, imaging analysis revealed that calcein reduced the number of vesicles released from the cells when stimulated with nicotine. Using amperometry to detect individual vesicle release events, we show that when calcein is present in the media, the number of vesicles that fuse with the cellular membrane is reduced when cells are stimulated with either nicotine or high K+. Experimentally, amperometric electrodes are not undergoing fouling in the presence of calcein. We hypothesized that calcein, when activated by light, releases reactive oxygen species that cause a reduction in secreted vesicles. We show that when calcein is protected from light during experimentation, little to no reduction of vesicle secretion occurred. Therefore, photoactivated calcein can cause deleterious results for measurements of cellular processes, likely to be the result of release of reactive oxygen species.

Improved deconvolution of very weak confocal signals.

Deconvolution is typically used to sharpen fluorescence images, but when the signal-to-noise ratio is low, the primary benefit is reduced noise and a smoother appearance of the fluorescent structures. 3D time-lapse (4D) confocal image sets can be improved by deconvolution. However, when the confocal signals are very weak, the popular Huygens deconvolution software erases fluorescent structures that are clearly visible in the raw data. We find that this problem can be avoided by prefiltering the optical sections with a Gaussian blur. Analysis of real and simulated data indicates that the Gaussian blur prefilter preserves meaningful signals while enabling removal of background noise. This approach is very simple, and it allows Huygens to be used with 4D imaging conditions that minimize photodamage.

High resolution optical imaging of infarction in intact organs.

We describe a method to visualize green fluorescent protein (GFP)-labeled cells in intact organs through combined confocal and reflected laser light imaging. This method allows us a three-dimensional (3-D) view of specific cell types in situ. Imaging of tissues from transgenic mice in which the endothelial cells are labeled with GFP under the control of endothelial-specific tyrosine receptor kinase 2 (TIE2) shows the spatial distribution of the GFP-labeled endothelial cells in intact organs. We have used this method to examine the tissue necrosis in the intact heart and kidney resulting from myocardial and renal infarction. In myocardial infarction produced by surgically occluding the left anterior descending coronary artery, the border of the infarct was highly cellular and showed a disrupted endothelial network and scar tissue appearing as a dense layer of reflection. The induced renal infarction produced by ligating the renal artery in the pedicle showed a clear infarct border in the affected kidney. The 3-D reconstruction of specific cell types in the context of the surrounding tissues should be useful for studying the overall organization and the relationship between different structures in the intact organ in normal and disease states.

A mouse model for studying intrahepatic islet transplantation.

Intrahepatic human islet transplantation has raised hopes for a cure for diabetes mellitus, especially in patients with type 1 diabetes; however, the need for a substantial amount of islets and, in many instances, repeated transplantations demonstrates underlying problems with this procedure, such as failure of angiogenesis and immunologic rejection. Studies using rodent models may be helpful in improving the success of islet transplantation. However, most of the studies using rodents for islet transplantation have been under the kidney capsule rather than the liver. Using islets from transgenic mice expressing green fluorescent protein under the control of mouse insulin I promoter, the authors have developed a method with which to visualize histologic and pathologic changes in intraportally transplanted islets and surrounding hepatic tissue using reflected light confocal imaging. Initial events 24 hr after islet transplantation in the liver include beta-cell loss and hepatic ischemic injuries.

Mitogen-activated protein kinases and retinal ischemia.

PURPOSE: Mitogen-activated protein kinases (MAPKs), consisting of three major enzymes-extracellular signal-regulated kinase (ERK), p38, and c-jun N-terminal kinase (JNK)-couple cell-surface receptors to critical regulatory targets and gene transcription. We hypothesized that MAPKs are differentially expressed and have distinct functions after retinal ischemia. METHODS: Rats were subjected to retinal ischemia by elevation of intraocular pressure. Changes in MAPK expression were examined by Western blot of whole retinal homogenates and by immunohistochemistry of retinal cryosections. Phosphorylated (activated) ERK, p38, and JNK proteins were localized by fluorescent double labeling. The functional significance of activated MAPKs was assessed using pharmacological antagonists. Specific MAPK blockade was documented by kinase assay and immunohistochemistry for phosphorylated target proteins. The outcome after ischemia was examined with electroretinography (ERG), by measuring retinal cell layer thickness in paraffin-embedded sections, and by TUNEL staining on retinal cryosections. Data were analyzed using ANOVA and post hoc t-test, with P < 0.05 considered significant. RESULTS: Expression of phosphorylated JNK and p38 increased significantly after ischemia and followed a specific time course, beginning at 1 hour, and persisting up to 1 week later. JNK and p38 were expressed in the nuclei of ganglion and amacrine cells, the outer plexiform layer, the nerve fiber layer, and the axonal terminals of bipolar cells. Phosphorylated ERK was expressed in Müller cells, peaking at 1 to 6 hours after ischemia. Blocking activation of p38 or ERK significantly improved recovery of the ERG b-wave after ischemia, dramatically decreased thinning of the inner nuclear layers, and decreased the percentage of TUNEL-positive cells. CONCLUSIONS: The MAPKs each demonstrate a specific cellular distribution after ischemia, and ERK and p38 are linked to apoptosis. Blockade of p38 or ERK provides significant protection from ischemic damage, suggesting a novel therapeutic role for MAPK inhibition in neuroprotection.

Longitudinal confocal microscopy imaging of solid tumor destruction following adoptive T cell transfer.

A fluorescence-based, high-resolution imaging approach was used to visualize longitudinally the cellular events unfolding during T cell-mediated tumor destruction. The dynamic interplay of T cells, cancer cells, cancer antigen loss variants, and stromal cells-all color-coded in vivo-was analyzed in established, solid tumors that had developed behind windows implanted on the backs of mice. Events could be followed repeatedly within precisely the same tumor region-before, during and after adoptive T cell therapy-thereby enabling for the first time a longitudinal in vivo evaluation of protracted events, an analysis not possible with terminal imaging of surgically exposed tumors. T cell infiltration, stromal interactions, and vessel destruction, as well as the functional consequences thereof, including the elimination of cancer cells and cancer cell variants were studied. Minimal perivascular T cell infiltrates initiated vascular destruction inside the tumor mass eventually leading to macroscopic central tumor necrosis. Prolonged engagement of T cells with tumor antigen-crosspresenting stromal cells correlated with high IFNγ cytokine release and bystander elimination of antigen-negative cancer cells. The high-resolution, longitudinal, in vivo imaging approach described here will help to further a better mechanistic understanding of tumor eradication by T cells and other anti-cancer therapies.

Time-course imaging of therapeutic functional tumor vascular normalization by antiangiogenic agents.

We describe here new technology that enables noninvasive imaging of therapeutic functional normalization of tumor blood vessels by antiangiogenic agents. Noninvasive variable-magnification in vivo-fluorescence imaging as well as fluorescence tomography was used to visualize functional vessel normalization. Changes in the same vessel before and after drug treatment were imaged with high resolution in real time. Differences in vascular responses to the mTOR inhibitor rapamycin and to an anti-VEGF antibody were functionally imaged. Tumor vessel normalization was shown by significantly reduced leakiness and subsequent improved tumor delivery of Paclitaxel-BODPY as well as by normalized morphology. The tumor vascular pool agent, AngioSense(750), was retained only in tumors after either anti-VEGF antibody or rapamycin treatment, as visualized by noninvasive fluorescence tomography. The antiangiogenic therapy normalized vessels, which significantly enhanced the antitumor efficacy of paclitaxel because of increased drug penetration throughout the tumor. The optical imaging technology described here is thus a powerful, noninvasive, time-course imaging tool of functional tumor vessel normalization and its therapeutic consequences.