Novel post-acquisition image processing to attenuate red blood cell autofluorescence for quantitative image analysis.

Quantitative analysis of microscopy images from samples stained with fluorescent probes necessitates a very low fluorescence background signal. In tissues prepared by immersion in a chemical fixative, followed by conventional processing for paraffin embedding, red blood cell autofluorescence across several imaging channels can be a nuisance. Although many protocols have been proposed to suppress red blood cell autofluorescence prior to microscopy imaging, in many instances they may not prove totally effective. Moreover, in environments such as core facilities where control over tissue processing and staining may not be feasible, methods to address autofluorescence via post-image acquisition processing may be of some advantage. To this end, we have developed an image analysis algorithm using a commercially based software platform to remove contaminating red blood cell autofluorescence during quantitative evaluation of the fluorescence signal from an immunostaining protocol. The method is based upon the low autofluorescence signal of red blood cells exhibited in the blue channel (used to detect DAPI nuclear signal of all cells), which can be subtracted from the total channel signal by increasing the threshold for DAPI signal in the nuclear detection settings during nuclear segmentation. With the contributing signal from the red blood cells eliminated, the specific immunostained signal for the antigen of interest could be determined. We believe that this simple algorithm performed on post-acquisition microscopy images will be of use for quantitative fluorescence analyses whenever red blood cell autofluorescence is present, especially in amounts where creating regions of interest for evaluation is not possible.

Src family kinase inhibitors blunt PACAP-induced PAC1 receptor endocytosis, phosphorylation of ERK, and the increase in cardiac neuron excitability.

Pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) activation of PAC1 receptors ( Adcyap1r1) significantly increases excitability of guinea pig cardiac neurons. This modulation of excitability is mediated in part by plasma membrane G protein-dependent activation of adenylyl cyclase and downstream signaling cascades. However, additional mechanisms responsible for the enhanced excitability are activated following internalization of the PAC1 receptor and endosomal signaling. Src family kinases play critical roles mediating endocytosis of many trophic factor and G protein-coupled receptors. The present study investigated whether Src family kinases also support the PACAP-induced PAC1 receptor internalization, phosphorylation of ERK, and enhanced neuronal excitability. Using human embryonic kidney cells stably expressing a green fluorescent protein-tagged PAC1 receptor, treatment with the Src family kinase inhibitor PP2 (10 µM) markedly reduced the PACAP-induced PAC1 receptor internalization, and in parallel, both PP2 and Src inhibitor 1 (Src-1, 2 µM) reduced ERK activation determined by Western blot analysis. In contrast, Src family kinase inhibitors did not eliminate a PACAP-induced rise in global calcium generated by inositol (1,4,5)-trisphosphate-induced release of calcium from endoplasmic reticulum stores. From confocal analysis of phosphorylated ERK immunostaining, PP2 treatment significantly attenuated PACAP activation of ERK in neurons within cardiac ganglia whole mount preparations. Intracellular recordings demonstrated that PP2 also significantly blunted a PACAP-induced increase in cardiac neuron excitability. These studies demonstrate Src-related kinase activity in PAC1 receptor internalization, activation of MEK/ERK signaling, and regulation of neuronal excitability. The present results provide further support for the importance of PAC1 receptor endosomal signaling as a key mechanism regulating cellular function.

Recruitment of endosomal signaling mediates the forskolin modulation of guinea pig cardiac neuron excitability.

Forskolin, a selective activator of adenylyl cyclase (AC), commonly is used to establish actions of G protein-coupled receptors (GPCRs) that are initiated primarily through activation of AC/cAMP signaling pathways. In the present study, forskolin was used to evaluate the potential role of AC/cAMP, which is a major signaling mechanism for the pituitary adenylate cyclase-activating polypeptide (PACAP)-selective PAC1 receptor, in the regulation of guinea pig cardiac neuronal excitability. Forskolin (5-10 µM) increases excitability in ~60% of the cardiac neurons. The forskolin-mediated increase in excitability was considered related to cAMP regulation of a cyclic nucleotide gated channel or via protein kinase A (PKA)/ERK signaling, mechanisms that have been linked to PAC1 receptor activation. However, unlike PACAP mechanisms, forskolin enhancement of excitability was not significantly reduced by treatment with cesium to block currents through hyperpolarization-activated nonselective cation channels (Ih) or by treatment with PD98059 to block MEK/ERK signaling. In contrast, treatment with the clathrin inhibitor Pitstop2 or the dynamin inhibitor dynasore eliminated the forskolin-induced increase in excitability; treatments with the inactive Pitstop analog or PP2 treatment to inhibit Src-mediated endocytosis mechanisms were ineffective. The PKA inhibitor KT5702 significantly suppressed the forskolin-induced change in excitability; further, KT5702 and Pitstop2 reduced the forskolin-stimulated MEK/ERK activation in cardiac neurons. Collectively, the present results suggest that forskolin activation of AC/cAMP/PKA signaling leads to the recruitment of clathrin/dynamin-dependent endosomal transduction cascades, including MEK/ERK signaling, and that endosomal signaling is the critical mechanism underlying the forskolin-induced increase in cardiac neuron excitability.

Activation of MEK/ERK signaling contributes to the PACAP-induced increase in guinea pig cardiac neuron excitability.

Pituitary adenylate cyclase (PAC)-activating polypeptide (PACAP) peptides (Adcyap1) signaling at the selective PAC1 receptor (Adcyap1r1) participate in multiple homeostatic and stress-related responses, yet the cellular mechanisms underlying PACAP actions remain to be completely elucidated. PACAP/PAC1 receptor signaling increases excitability of neurons within the guinea pig cardiac ganglia, and as these neurons are readily accessible, this neuronal system is particularly amenable to study of PACAP modulation of ionic conductances. The present study investigated how PACAP activation of MEK/ERK signaling contributed to the peptide-induced increase in cardiac neuron excitability. Treatment with the MEK inhibitor PD 98059 blocked PACAP-stimulated phosphorylated ERK and, in parallel, suppressed the increase in cardiac neuron excitability. However, PD 98059 did not blunt the ability of PACAP to enhance two inward ionic currents, one flowing through hyperpolarization-activated nonselective cationic channels (Ih) and another flowing through low-voltage-activated calcium channels (IT), which support the peptide-induced increase in excitability. Thus a PACAP- and MEK/ERK-sensitive, voltage-dependent conductance(s), in addition to Ih and IT, modulates neuronal excitability. Despite prior work implicating PACAP downregulation of the KV4.2 potassium channel in modulation of excitability in other cells, treatment with the KV4.2 current blocker 4-aminopyridine did not replicate the PACAP-induced increase in excitability in cardiac neurons. However, cardiac neurons express the ERK target, the NaV1.7 sodium channel, and treatment with the selective NaV1.7 channel inhibitor PF-04856264 decreased the PACAP modulation of excitability. From these results, PACAP/PAC1 activation of MEK/ERK signaling may phosphorylate the NaV1.7 channel, enhancing sodium currents near the threshold, an action contributing to repetitive firing of the cardiac neurons exposed to PACAP.

Activation of MEK/ERK Signaling by PACAP in Guinea Pig Cardiac Neurons.

Pituitary adenylate cyclase-activating polypeptide (PACAP) signaling can increase guinea pig cardiac neuron excitability in part through extracellular signal-regulated kinase (ERK) activation. The present study examined the PACAP receptors and signaling cascades that stimulate guinea pig cardiac neuron ERK signaling using confocal microscopy to quantify PACAP-induced neuronal phosphorylated ERK (pERK) immunoreactivity. PACAP and maxadilan, but not vasoactive intestinal polypeptide (VIP), increased cardiac neuron pERK, implicating primary roles for PACAP-selective PAC1 receptor (Adcyap1r1) signaling rather than VPAC receptors (Vipr1 and Vipr2) in the generation of cardiac neuron pERK. The adenylyl cyclase (AC) activator forskolin, but not the protein kinase C (PKC) activator phorbol myristate acetate (PMA), increased pERK. Also, Bim1 did not blunt PACAP activation of pERK. Together, the results suggest PAC1 receptor signal transduction via Gs/adenylyl cyclase (AC)/cAMP rather than Gq/phospholipase C (PLC) generated neuronal pERK. Activator and inhibitor studies suggested that the PACAP-mediated pERK activation was PKA-dependent rather than an exchange protein directly activated by a cAMP (EPAC), PKA-independent mechanism. The PACAP-induced pERK was inhibited by the clathrin inhibitor Pitstop2 to block receptor internalization and endosomal signaling. We propose that the PACAP-mediated MEK/ERK activation in cardiac neurons involves both AC/cAMP/PKA signaling and PAC1 receptor internalization/activation of signaling endosomes.

Calcium influx, but not intracellular calcium release, supports PACAP-mediated ERK activation in HEK PAC1 receptor cells.

In HEK cells expressing GFP-tagged PAC1Hop1 receptors, PACAP augments ERK phosphorylation through two parallel pathways: one through PACAP/PAC1 receptor internalization/endosome MEK/ERK signaling and the other through PLC/DAG/PKC activation. We examined whether elevation of intracellular calcium ([Ca(2+)]i) was required for either of the PACAP/PAC1 receptor-mediated ERK activation mechanisms. The PACAP (25 nM)-induced elevation of [Ca(2+)]i was greater with cells maintained in Ca(2+)-containing than in Ca(2+)-deficient solution, suggesting that both calcium release from internal stores and calcium influx contributed to the rise in [Ca(2+)]i. A thapsigargin-induced increase in [Ca(2+)]i also was greater with calcium in the external solution. OAG, the cell permeable analogue of DAG, increased [Ca(2+)]i, but only in Ca(2+)-containing solution. Decreasing external calcium or depleting internal calcium stores did not block PACAP-induced PAC1 receptor internalization. Omission of calcium from the external solution, but not thapsigargin pretreatment, significantly blunted PACAP-stimulated ERK phosphorylation. The PKC inhibitor BimI decreased PACAP-mediated ERK activation in both Ca(2+)-containing or Ca(2+)-deficient solutions. In contrast, following Pitstop 2 pretreatment to block endocytic mechanisms, PACAP activated ERK only when calcium was present in the external solution. We conclude that the endosome signaling pathway is largely calcium-independent whereas calcium influx appears necessary for the PLC/DAG/PKC component of PACAP-induced ERK activation.

PACAP-induced ERK activation in HEK cells expressing PAC1 receptors involves both receptor internalization and PKC signaling.

The pituitary adenylate cyclase-activating polypeptide (PACAP)-selective PAC1 receptor (Adcyap1r1) is a G protein-coupled receptor (GPCR) that activates adenylyl cyclase and PLC. Similar to many other GPCRs, our previous studies showed that the PAC1 receptor is internalized after ligand binding to form signaling endosomes, which recruit additional second messenger pathways. Using a human embryonic kidney (HEK 293) PAC1Hop1-EGFP receptor cell line, we have examined how different PAC1 receptor signaling mechanisms contribute to MEK/ERK activation. Unlike PAC1 receptor-stimulated adenylyl cyclase/cAMP production in the plasma membrane, PACAP-mediated ERK phosphorylation was partly dependent on receptor internalization, as determined by treatment with pharmacological inhibitors of endocytosis or temperature reduction, which also suppressed receptor internalization. Stimulation of cAMP generation by forskolin or exposure to the cell-permeable cAMP analogs 8-bromo-cAMP and dibutyryl cAMP had minimal effects on ERK phosphorylation in this system. The ability of reduced temperature (24°C) to consistently suppress ERK activation to a greater extent than the endocytosis inhibitors Pitstop 2 and dynasore indicated that other mechanisms, in addition to PAC1 internalization/endosome activation, were involved. Inhibition of PAC1 receptor-stimulated PLC/diacylglycerol/PKC signaling by bisindoylmaleimide I also attenuated ERK phosphorylation, and direct PKC activation with phorbol ester increased ERK phosphorylation in a temperature-dependent manner. Inhibition of PAC1 receptor endocytosis and PKC activation completely blocked PACAP-stimulated ERK activation. PACAP augmented phosphorylated ERK staining uniformly over the cytoplasm and nucleus, and PKC signaling facilitated nuclear phosphorylated ERK translocation. In sum, our results show that PACAP/PAC1 receptor endocytosis and PLC/diacylglycerol/PKC activation represent two complementary mechanisms contributing to PACAP-induced ERK activation.

Loss of PTB or negative regulation of Notch mRNA reveals distinct zones of Notch and actin protein accumulation in Drosophila embryo.

Polypyrimidine Tract Binding (PTB) protein is a regulator of mRNA processing and translation. Genetic screens and studies of wing and bristle development during the post-embryonic stages of Drosophila suggest that it is a negative regulator of the Notch pathway. How PTB regulates the Notch pathway is unknown. Our studies of Drosophila embryogenesis indicate that (1) the Notch mRNA is a potential target of PTB, (2) PTB and Notch functions in the dorso-lateral regions of the Drosophila embryo are linked to actin regulation but not their functions in the ventral region, and (3) the actin-related Notch activity in the dorso-lateral regions might require a Notch activity at or near the cell surface that is different from the nuclear Notch activity involved in cell fate specification in the ventral region. These data raise the possibility that the Drosophila embryo is divided into zones of different PTB and Notch activities based on whether or not they are linked to actin regulation. They also provide clues to the almost forgotten role of Notch in cell adhesion and reveal a role for the Notch pathway in cell fusions.

Permeabilization of Drosophila embryos for introduction of small molecules.

Pharmacological manipulations in the Drosophila embryo have been hindered by the impermeability of the eggshell. The ultimate barrier to delivery of small molecule solutes to the embryo is the waxy layer that lies beneath the external chorion layers and encases the underlying vitelline membrane of the eggshell. Conventional protocols call for heptane or octane to permeablize the dechorionated eggshell however, these solvents are toxic and can result in low viability. Furthermore, heptane and octane require transition of the embryo between aqueous and organic phase solvents making it challenging to avoid desiccation. Here we describe an embryo permeabilization solvent (EPS) composed of d-limonene and plant-derived surfactants that is water miscible and highly effective in rendering the dechorionated eggshell permeable. EPS permeabilization enables embryo uptake of several different dyes of various molecular mass up to 995Da. We find that the embryo undergoes an age-dependent decrease in the ability to be permeabilized in the first six to eight hours after egg laying. This apparent developmental change in the vitelline membrane contributes to the heterogeneity in permeabilization seen even among closely staged embryos. However, using fluorescent properties of Rhodamine B dye and various conditions of EPS treatment we demonstrate the ability to obtain optimally permeabilized viable embryos. We also demonstrate the ability to assess teratogenic activity of several compounds applied to embryos in vitro, using both early and late developmental endpoints. Application of the method to transgenic strains carrying GFP-reporter genes results in a robust readout of pharmacological alteration of embryogenesis. The straightforward and rapid nature of the manipulations needed to prepare batches of permeabilized embryos has the potential of establishing the Drosophila embryo as an alternative model in toxicology and for small molecule screening in a high-throughput format.

Methylmercury disruption of embryonic neural development in Drosophila.

Methylmercury (MeHg) is a potent environmental neurotoxin that preferentially targets the developing embryonic nervous system. While a number of cytotoxic mechanisms of MeHg have been characterized in differentiated cells its mode of action in the developing nervous system in vivo is less clear. Studies in primate and rodent models demonstrate aberrant cell migration and disorganized patterning of cortical layers in the brain following MeHg exposure. However, defining the molecular and cellular pathways targeted by MeHg will require more genetically accessible animal models. In this study, we instigate a method of in vitro MeHg exposure using Drosophila embryos. We demonstrate dose-dependent inhibition of embryonic development with MeHg revealed by a failure of embryos to hatch to the larval stage. In addition, we document definitive phenotypes in neural development showing abnormalities in neuronal and glial cell patterning consistent with disrupted migration. We observe pronounced defects in neurite outgrowth in both central and peripheral neurons. Ectopic expression of the Nrf2 transcription factor in embryos, a core factor in the antioxidant response element (ARE) pathway, enhances embryonic development and hatching in the presence of MeHg, illustrating the power of this model for investigation of candidate MeHg tolerance genes. Our data establish a utility for the Drosophila embryo model as a platform for elucidating MeHg sensitive pathways in neural development.

Direct localization of the 51 and 24 kDa subunits of mitochondrial complex I by three-dimensional difference imaging.

Complex I is the largest complex in the respiratory chain, and the least understood. We have determined the 3D structure of complex I from Yarrowia lipolytica lacking the flavoprotein part of the N-module, which consists of the 51 kDa (NUBM) and the 24 kDa (NUHM) subunits. The reconstruction was determined by 3D electron microscopy of single particles. A comparison to our earlier reconstruction of the complete Y. lipolytica complex I clearly assigns the two flavoprotein subunits to an outer lobe of the peripheral arm of complex I. Localizing the two subunits allowed us to fit the X-ray structure of the hydrophilic fragment of complex I from Thermus thermophilus. The fit that is most consistent with previous immuno-electron microscopic data predicts that the ubiquinone reducing catalytic center resides in the second peripheral lobe, while the 75 kDa subunit is placed near the previously seen connection between the peripheral arm and the membrane arm protrusions.

The anterior cardiac plexus: an intrinsic neurosecretory site within the stomatogastric nervous system of the crab Cancer productus.

The stomatogastric nervous system (STNS) of decapod crustaceans is modulated by both locally released and circulating substances. In some species, including chelate lobsters and freshwater crayfish, the release zones for hormones are located both intrinsically to and at some distance from the STNS. In other crustaceans, including Brachyuran crabs, the existence of extrinsic sites is well documented. Little, however, is known about the presence of intrinsic neuroendocrine structures in these animals. Putative intrinsic sites have been identified within the STNS of several crab species, though ultrastructural confirmation that these structures are in fact neuroendocrine in nature remains lacking. Using a combination of anatomical techniques, we demonstrate the existence of a pair of neurosecretory sites within the STNS of the crab Cancer productus. These structures, which we have named the anterior cardiac plexi (ACPs), are located on the anterior cardiac nerves (acns), which overlie the cardiac sac region of the foregut. Each ACP starts several hundred micro m from the origin of the acn and extends distally for up to several mm. Transmission electron microscopy done on these structures shows that nerve terminals are present in the peripheral portion of each acn, just below a well defined epineurium. These terminals contain dense-core and, occasionally, electron-lucent vesicles. In many terminals, morphological correlates of hormone secretion are evident. Immunocytochemistry shows that the ACPs are immunopositive for FLRFamide-related peptide. All FLRFamide labeling in the ACPs originates from four axons, which descend to these sites through the superior oesophageal and stomatogastric nerves. Moreover, these FLRFamide-immunopositive axons are the sole source of innervation to the ACPs. Collectively, our results suggest that the STNS of C. productus is not only a potential target site for circulating hormones, but also serves as a neuroendocrine release center itself.

Immunocytochemical evidence for nitric oxide- and carbon monoxide-producing neurons in the stomatogastric nervous system of the crayfish Cherax quadricarinatus.

Nitric oxide (NO) and carbon monoxide (CO) have been shown to serve neuromodulatory roles in both vertebrates and invertebrates. Here, we use antibodies to their respective biosynthetic enzymes, nitric oxide synthase (NOS) and heme oxygenase 2 (HO-2), to map the distribution of putative gas-producing neurons in the stomatogastric nervous system (STNS) of the crayfish Cherax quadricarinatus. In this species, NOS immunolabeling is found in the neuropil of the stomatogastric ganglion (STG). This staining originates from two immunopositive axons that project to the STG through the superior oesophageal and stomatogastric nerves, presumably from cell bodies located in the commissural ganglia (CoGs). HO-2 immunoreactivity is present in small diameter fibers and varicosities in the periphery of nerves located in the anterior portion of the STNS. This labeling originates from approximately 12 somata in each CoG. Transmission electron microscopy done on the nerves of the anterior STNS shows they contain a neuroendocrine plexus. Collectively, our results indicate that NO- and CO-producing neurons are likely to exist in the crayfish STNS. Moreover, these gases appear to be produced by distinct subsets of the neurons present there. The localization of NO to the STG neuropil suggests that it serves as a locally released modulator or is involved in the local release of other substances within this ganglion. The presence of CO in the neurohemal plexus of the anterior STNS suggests that it serves as a circulating hormone or is involved in the control of neuroendocrine release from this plexus.

Activity and distribution of paxillin, focal adhesion kinase, and cadherin indicate cooperative roles during zebrafish morphogenesis.

We investigated the focal adhesion proteins paxillin and Fak, and the cell-cell adhesion protein cadherin in developing zebrafish (Danio rerio) embryos. Cadherins are expressed in presomitic mesoderm where they delineate cells. The initiation of somite formation coincides with an increase in the phosphorylation of Fak, and the accumulation of Fak, phosphorylated Fak, paxillin, and fibronectin at nascent somite boundaries. In the notochord, cadherins are expressed on cells during intercalation, and phosphorylated Fak accumulates in circumferential rings where the notochord cells contact laminin in the perichordal sheath. Subsequently, changes in the orientations of collagen fibers in the sheath suggest that Fak-mediated adhesion allows longitudinal expansion of the notochord, but not lateral expansion, resulting in notochord elongation. Novel observations showed that focal adhesion kinase and paxillin concentrate at sites of cell-cell adhesion in the epithelial enveloping layer and may associate with actin cytoskeleton at epithelial junctions containing cadherins. Fak is phosphorylated at these epithelial junctions but is not phosphorylated on Tyr397, implicating a noncanonical mechanism of regulation. These data suggest that Fak and paxillin may function in the integration of cadherin-based and integrin-based cell adhesion during the morphogenesis of the early zebrafish embryo.