Quantification Of Fluorescence Intensity Research Articles

Enteric Commensal Bacteria Induce Extracellular Signal-regulated Kinase Pathway Signaling via Formyl Peptide Receptor-dependent Redox Modulation of Dual Specific Phosphatase 3

The normal microbial occupants of the mammalian intestine are crucial for maintaining gut homeostasis, yet the mechanisms by which intestinal cells perceive and respond to the microbiota are largely unknown. Intestinal epithelial contact with commensal bacteria and/or their products has been shown to activate noninflammatory signaling pathways, such as extracellular signal-related kinase (ERK), thus influencing homeostatic processes. We previously demonstrated that commensal bacteria stimulate ERK pathway activity via interaction with formyl peptide receptors (FPRs). In the current study, we expand on these findings and show that commensal bacteria initiate ERK signaling through rapid FPR-dependent reactive oxygen species (ROS) generation and subsequent modulation of MAP kinase phosphatase redox status. ROS generation induced by the commensal bacteria Lactobacillus rhamnosus GG and the FPR peptide ligand, N-formyl-Met-Leu-Phe, was abolished in the presence of selective inhibitors for G protein-coupled signaling and FPR ligand interaction. In addition, pretreatment of cells with inhibitors of ROS generation attenuated commensal bacteria-induced ERK signaling, indicating that ROS generation is required for ERK pathway activation. Bacterial colonization also led to oxidative inactivation of the redox-sensitive and ERK-specific phosphatase, DUSP3/VHR, and consequent stimulation of ERK pathway signaling. Together, these data demonstrate that commensal bacteria and their products activate ROS signaling in an FPR-dependent manner and define a mechanism by which cellular ROS influences the ERK pathway through a redox-sensitive regulatory circuit.

A multicentre, double-masked, randomized, controlled trial assessing the effect of oral supplementation of omega-3 and omega-6 fatty acids on a conjunctival inflammatory marker in dry eye patients

To determine whether oral supplementation with omega-3 and omega-6 fatty acids can reduce conjunctival epithelium expression of the inflammatory marker human leucocyte antigen-DR (HLA-DR) in patients with dry eye syndrome (DES). This 3-month, double-masked, parallel-group, controlled study was conducted in nine centres, in France and Italy. Eligible adult patients with mild to moderate DES were randomized to receive a placebo containing medium-chain triglycerides or treatment supplement containing omega-3 and omega-6 fatty acids, vitamins and zinc. Treatment regimen was three capsules daily. Impression cytology (IC) was performed at baseline and at month 3 to assess the percentage of cells expressing HLA-DR and to evaluate fluorescence intensity, an alternate measure of HLA-DR. Dry eye symptoms and objective signs were also evaluated. Analyses were performed on the full analysis set (FAS) and per-protocol set (PPS). In total, 138 patients were randomized; 121 patients with available IC were included in the FAS, and of these, 106 patients had no major protocol deviations (PPS). In the PPS, there was a significant reduction in the percentage of HLA-DR-positive cells in the fatty acids group (p = 0.021). Expression of HLA-DR as measured by fluorescence intensity quantification was also significantly reduced in the fatty acids group [FAS (p = 0.041); PPS (p = 0.017)]. No significant difference was found for the signs and symptoms, but there was a tendency for improvement in patients receiving the fatty acids treatment. This study demonstrates that supplementation with omega-3 and omega-6 fatty acids can reduce expression of HLA-DR conjunctival inflammatory marker and may help improve DES symptoms.

Clustering and Activity Tuning of Kv1 Channels in Myelinated Hippocampal Axons

Precise localization of axonal ion channels is crucial for proper electrical and chemical functions of axons. In myelinated axons, Kv1 (Shaker) voltage-gated potassium (Kv) channels are clustered in the juxtaparanodal regions flanking the node of Ranvier. The clustering can be disrupted by deletion of various proteins in mice, including contactin-associated protein-like 2 (Caspr2) and transient axonal glycoprotein-1 (TAG-1), a glycosylphosphatidylinositol-anchored cell adhesion molecule. However, the mechanism and function of Kv1 juxtaparanodal clustering remain unclear. Here, using a new myelin coculture of hippocampal neurons and oligodendrocytes, we report that tyrosine phosphorylation plays a critical role in TAG-1-mediated clustering of axonal Kv1.2 channels. In the coculture, myelin specifically ensheathed axons but not dendrites of hippocampal neurons and clustered endogenous axonal Kv1.2 into internodes. The trans-homophilic interaction of TAG-1 was sufficient to position Kv1.2 clusters on axonal membranes in a neuron/HEK293 coculture. Mutating a tyrosine residue (Tyr⁴⁵⁸) in the Kv1.2 C terminus or blocking tyrosine phosphorylation disrupted myelin- and TAG-1-mediated clustering of axonal Kv1.2. Furthermore, Kv1.2 voltage dependence and activation threshold were reduced by TAG-1 coexpression. This effect was eliminated by the Tyr⁴⁵⁸ mutation or by cholesterol depletion. Taken together, our studies suggest that myelin regulates both trafficking and activity of Kv1 channels along hippocampal axons through TAG-1.

In vivo and in vitro surface display of heterologous proteins on Bacillus thuringiensis vegetative cells and spores

The Bacillus surface display system is a heat-stable platform for heterologous protein expression that preserves biological activity in an adverse environment. In the present study, a new cell surface display system that enables heterologous proteins to be displayed consecutively on the surface of Bacillus thuringiensis vegetative cells and spores in vivo and in vitro is reported. Using the N-terminal domain (Mbgn) of a peptidoglycan hydrolase (Mbg) as anchor, the green fluorescence protein (GFP) and a bacterial laccase (WlacD) can be targeted onto the surface of vegetative cells and spores in vivo in an active form. This was confirmed by GFP fluorescence intensity quantification, enzymatic activity measurement, immunofluorescence microscopy examination, and flow cytometry assay. Immobilization of externally added Mbgn-mediated fusion proteins in vitro to obtain actively displaying GFP and/or WlacD was subsequently achieved in B. thuringiensis, Bacillus subtilis and Bacillus cereus, demonstrating the versatility of the proposed system. Therefore, Mbgn-mediated in vivo and in vitro immobilization of foreign proteins represents an improved method of microbial display for extended biotechnological applications.

Sustained Glutamate Receptor Activation Down-regulates GABAB Receptors by Shifting the Balance from Recycling to Lysosomal Degradation

Metabotropic GABA(B) receptors are abundantly expressed at glutamatergic synapses where they control excitability of the synapse. Here, we tested the hypothesis that glutamatergic neurotransmission may regulate GABA(B) receptors. We found that application of glutamate to cultured cortical neurons led to rapid down-regulation of GABA(B) receptors via lysosomal degradation. This effect was mimicked by selective activation of AMPA receptors and further accelerated by coactivation of group I metabotropic glutamate receptors. Inhibition of NMDA receptors, blockade of L-type Ca(2+) channels, and removal of extracellular Ca(2+) prevented glutamate-induced down-regulation of GABA(B) receptors, indicating that Ca(2+) influx plays a critical role. We further established that glutamate-induced down-regulation depends on the internalization of GABA(B) receptors. Glutamate did not affect the rate of GABA(B) receptor endocytosis but led to reduced recycling of the receptors back to the plasma membrane. Blockade of lysosomal activity rescued receptor recycling, indicating that glutamate redirects GABA(B) receptors from the recycling to the degradation pathway. In conclusion, the data indicate that sustained activation of AMPA receptors down-regulates GABA(B) receptors by sorting endocytosed GABA(B) receptors preferentially to lysosomes for degradation on the expense of recycling. This mechanism may relieve glutamatergic synapses from GABA(B) receptor-mediated inhibition resulting in increased synaptic excitability.

Time Lapse in Vivo Visualization of Developmental Stabilization of Synaptic Receptors at Neuromuscular Junctions

The lifetime of nicotinic acetylcholine receptors (AChRs) in neuromuscular junctions (NMJs) is increased from <1 day to >1 week during early postnatal development. However, the exact timing of AChR stabilization is not known, and its correlation to the concurrent embryonic to adult AChR channel conversion, NMJ remodeling, and neuromuscular diseases is unclear. Using a novel time lapse in vivo imaging technology we show that replacement of the entire receptor population of an individual NMJ occurs end plate-specifically within hours. This makes it possible to follow directly in live animals changing stabilities of end plate receptors. In three different, genetically modified mouse models we demonstrate that the metabolic half-life values of synaptic AChRs increase from a few hours to several days after postnatal day 6. Developmental stabilization is independent of receptor subtype and apparently regulated by an intrinsic muscle-specific maturation program. Myosin Va, an F-actin-dependent motor protein, is also accumulated synaptically during postnatal development and thus could mediate the stabilization of end plate AChR.

Quantification of Fluorescence Intensity of Labeled Human Mesenchymal Stem Cells and Cell Counting of Unlabeled Cells in Phase-Contrast Imaging: An Open-Source-Based Algorithm

Assessment of cell fate is indispensable to evaluate cell-based therapies in regenerative medicine. Therefore, a widely used technique is fluorescence labeling. A major problem still is the standardized, noninvasive, and reliable quantification of fluorescence intensity of adherent cell populations on single-cell level, since total fluorescence intensity must be correlated to the cell number. Consequently, the aim of the present study was to produce and validate an open-source-based algorithm, capable of measuring the total fluorescence intensity of cell populations and assessing the total cell number in phase-contrast images. To verify the algorithms' capacity to assess fluorescence intensity, human mesenchymal stem cells were transduced to stably express enhanced green fluorescent protein and results produced by the algorithm were compared to flow cytometry analysis. No significant differences could be observed at any time (p ≥ 0.443). For validation of the algorithm for cell counting in phase-contrast images, adherent human mesenchymal stem cells were manually counted and compared to results produced by the algorithm (correlation coefficient [CC] r = 0.975), nuclei staining (CC r = 0.997), and hemocytometer (CC r = 0.629). We conclude that applying the developed algorithm in routine practice allows robust, fast, and reproducible assessment of fluorescence intensity and cell numbers in simple large-scale microscopy. The method is easy to perform and open source based.

Therapeutic Potential of Proteasome Inhibition in Duchenne and Becker Muscular Dystrophies

Duchenne muscular dystrophy (DMD) and its milder allelic variant, Becker muscular dystrophy (BMD), result from mutations of the dystrophin gene and lead to progressive muscle deterioration. Enhanced activation of proteasomal degradation underlies critical steps in the pathogenesis of the DMD/BMD dystrophic process. Previously, we demonstrated that treatment with the proteasome inhibitor MG-132 rescues the cell membrane localization of dystrophin and the dystrophin glycoprotein complex in mdx mice, a natural genetic mouse model of DMD. The current work aims to thoroughly define the therapeutic potential in dystrophinopathies of Velcade, a drug that selectively blocks the ubiquitin-proteasome pathway. Velcade is particularly intriguing since it has been approved for the treatment of multiple myeloma. Therefore, its side effects in humans have been explored. Velcade effects were analyzed through two independent methodological approaches. First, we administered the drug systemically in mdx mice over a 2-week period. In this system, Velcade restores the membrane expression of dystrophin and dystrophin glycoprotein complex members and improves the dystrophic phenotype. In a second approach, we treated with the compound explants from muscle biopsies of DMD or BMD patients. We show that the inhibition of the proteasome pathway up-regulates dystrophin, alpha-sarcoglycan, and beta-dystroglycan protein levels in explants from BMD patients, whereas it increases the proteins of the dystrophin glycoprotein complex in DMD cases.

Activation of cAMP-dependent Signaling Induces Oxidative Modification of the Cardiac Na+-K+ Pump and Inhibits Its Activity

Cellular signaling can inhibit the membrane Na(+)-K(+) pump via protein kinase C (PKC)-dependent activation of NADPH oxidase and a downstream oxidative modification, glutathionylation, of the beta(1) subunit of the pump alpha/beta heterodimer. It is firmly established that cAMP-dependent signaling also regulates the pump, and we have now examined the hypothesis that such regulation can be mediated by glutathionylation. Exposure of rabbit cardiac myocytes to the adenylyl cyclase activator forskolin increased the co-immunoprecipitation of NADPH oxidase subunits p47(phox) and p22(phox), required for its activation, and increased superoxide-sensitive fluorescence. Forskolin also increased glutathionylation of the Na(+)-K(+) pump beta(1) subunit and decreased its co-immunoprecipitation with the alpha(1) subunit, findings similar to those already established for PKC-dependent signaling. The decrease in co-immunoprecipitation indicates a decrease in the alpha(1)/beta(1) subunit interaction known to be critical for pump function. In agreement with this, forskolin decreased ouabain-sensitive electrogenic Na(+)-K(+) pump current (arising from the 3:2 Na(+):K(+) exchange ratio) of voltage-clamped, internally perfused myocytes. The decrease was abolished by the inclusion of superoxide dismutase, the inhibitory peptide for the epsilon-isoform of PKC or inhibitory peptide for NADPH oxidase in patch pipette solutions that perfuse the intracellular compartment. Pump inhibition was also abolished by inhibitors of protein kinase A and phospholipase C. We conclude that cAMP- and PKC-dependent inhibition of the cardiac Na(+)-K(+) pump occurs via a shared downstream oxidative signaling pathway involving NADPH oxidase activation and glutathionylation of the pump beta(1) subunit.

A New Accurate and Reproducible Method to Assess Telomere Length by Flow-FISH.

Abstract 4719 BackgroundTelomeres have been related to many diseases including cancer, medullar insufficiency and to understand aging. To measure the average length of telomere repeats have been described at least three methods as Southern blot and quantitative fluorescence in situ hybridization using either digital fluorescence microscopy (Q-FISH) or flow cytometry (Flow-FISH). After two independents groups (Hultdin et al., 1998 and Rufer et al., 1999) have described different protocols using Flow-FISH to measure telomere lengths, the Southern blot and Q-FISH methods have been less used. However, when we were performed Hultdin et al., 1998 or Rufer et al., 1999 protocols were found some facts that could cause mistakes in the final results. Afterward, many steps of these protocols were explored to become the Flow-FISH results more accurate and reproducible. In our laboratory we developed a unlike protocol to assess the average length of telomere repeats by Flow-FISH taking advantage of the both Hultdin et al., 1998 and Rufer et al., 1999 techniques that was named Modified Hultdin and Rufer protocol. MethodsWe compare the telomere length repeats measure by three different methods of Flow-FISH with or lacking standard beads for calibrate fluorescence intensity, control cell line 1301 and compensation by DNA index of G0/G1 cells. ResultsWe demonstrated a linear relationship between the average length of telomere repeats by Hultdin et al., 1998 protocol and Modified Hultdin and Rufer protocol but showing that standard beads to quantify the fluorescence intensity was important to determine telomere length by Flow-FISH. However, the coefficient correlation (r) between the Rufer et al., 1999 protocol with Hultdin et al., 1998 and Modified Hultdin and Rufer protocols in our laboratory was poor, justifying of the internal control cell and compensation of the telomere length by DNA index of G0/G1 of unknown cells use. ConclusionsWe conclude that use of appropriate internal control cells with knowledge telomere length and DNA index is essential to improve accuracy and reproducibility of the telomere length measurement by Flow-FISH. Indeed the standard beads for fluorescence intensity quantification can allow intercenters comparison of the results. Disclosures:No relevant conflicts of interest to declare.

Confocal Nanoscanning, Bead Picking (CONA): PickoScreen Microscopes for Automated and Quantitative Screening of One-Bead One-Compound Libraries

Solid phase combinatorial chemistry provides fast and cost-effective access to large bead based libraries with compound numbers easily exceeding tens of thousands of compounds. Incubating one-bead one-compound library beads with fluorescently labeled target proteins and identifying and isolating the beads which contain a bound target protein, potentially represents one of the most powerful generic primary high throughput screening formats. On-bead screening (OBS) based on this detection principle can be carried out with limited automation. Often hit bead detection, i.e. recognizing beads with a fluorescently labeled protein bound to the compound on the bead, relies on eye-inspection under a wide-field microscope. Using low resolution detection techniques, the identification of hit beads and their ranking is limited by a low fluorescence signal intensity and varying levels of the library beads' autofluorescence. To exploit the full potential of an OBS process, reliable methods for both automated quantitative detection of hit beads and their subsequent isolation are needed. In a joint collaborative effort with Evotec Technologies (now Perkin-Elmer Cellular Technologies Germany GmbH), we have built two confocal bead scanner and picker platforms PS02 and a high-speed variant PS04 dedicated to automated high resolution OBS. The PS0X instruments combine fully automated confocal large area scanning of a bead monolayer at the bottom of standard MTP plates with semiautomated isolation of individual hit beads via hydraulic-driven picker capillaries. The quantification of fluorescence intensities with high spatial resolution in the equatorial plane of each bead allows for a reliable discrimination between entirely bright autofluorescent beads and real hit beads which exhibit an increased fluorescence signal at the outer few micrometers of the bead. The achieved screening speed of up to 200,000 bead assayed in less than 7 h and the picking time of approximately 1 bead/min allow exploitation of one-bead one-compound libraries with high sensitivity, accuracy, and speed.

Fgf10 Regulates Transition Period of Cortical Stem Cell Differentiation to Radial Glia Controlling Generation of Neurons and Basal Progenitors

Radial glia (RG), the progenitors of cortical neurons and basal progenitors (BPs), differentiate from neuroepithelial cells (NCs) with stem cell properties. We show that the morphogen Fgf10 is transiently expressed by NCs coincident with the transition period of NC differentiation into RG. Targeted deletion of Fgf10 delays RG differentiation, whereas overexpression has opposing effects. Delayed RG differentiation in Fgf10 mutants occurs selectively in rostral cortex, paralleled by an extended period of symmetric NC divisions increasing progenitor number, coupled with delayed and initially diminished production of neurons and BPs. RG eventually differentiate in excess number and overproduce neurons and BPs rostrally resulting in tangential expansion of frontal areas and increased laminar thickness. Thus, transient Fgf10 expression regulates timely differentiation of RG, and through this function, determines both length of the early progenitor expansion phase and onset of neurogenesis and ultimately the number of progenitors and neurons fated to specific cortical areas.

Effect of Transient and Permanent Permeability Transition Pore Opening on NAD(P)H Localization in Intact Cells

To study the effect of mitochondrial permeability transition pore (PTP) opening on NAD(P)H localization, intact cells were exposed to the Ca(2+) ionophore A23187. PTP opening, mitochondrial membrane potential, mitochondrial volume, and NAD(P)H localization were assessed by time-lapse laser confocal microscopy using the calcein-cobalt technique, tetramethylrhodamine methyl ester, MitoTracker, and NAD(P)H autofluorescence, respectively. Concomitant with PTP opening, NAD(P)H fluorescence increased outside mitochondria. These events occurred in all cells and were prevented by cyclosporin A. Mitochondrial membrane potential was not systematically collapsed, whereas mitochondrial volume did not change, confirming that A23187 induced transient PTP opening in a subpopulation of cells and suggesting that mitochondrial swelling did not immediately occur after PTP opening in intact cells. NAD(P)H autofluorescence remained elevated after PTP opening, particularly after membrane potential had been collapsed by an uncoupler. Extraction of nucleotide for NAD(P)H quantification confirmed that PTP opening led to an increase in NAD(P)H content. Because the oxygen consumption rate decreased, whereas the lactate/pyruvate ratio increased after PTP opening in intact cells, we conclude that PTP opening inhibits respiration and dramatically affects the cytosolic redox potential in intact cells.

The Toca-1-N-WASP Complex Links Filopodial Formation to Endocytosis

The transducer of Cdc42-dependent actin assembly (Toca-1)-N-WASP complex was isolated as an essential cofactor for Cdc42-driven actin polymerization in vitro. Toca-1 consists of an N-terminal F-BAR domain, followed by a Cdc42 binding site (HR1 domain) and an SH3 domain, (the N-WASP interacting site). N-WASP is an activator of actin nucleation through the Arp2/3 complex. The aim of the present study was to investigate the cellular function of the Toca-1-N-WASP complex. We report that Toca-1 induces filopodia and neurites as does N-WASP in N1E115 neuroblastoma cells. Toca-1 requires the F-BAR domain, Cdc42 binding site, and SH3 domain to induce filopodia. Toca-1 and N-WASP both require each other to induce filopodia. The expression of Toca-1 and N-WASP affects the distribution, size, and number of Rab5 positive membranes. Toca-1 interacts directly with N-WASP in filopodia and Rab5 membrane as seen by Forster resonance energy transfer. Thus the Toca-1-N-WASP complex localizes to and induces the formation of filopodia and endocytic vesicles. Last, three inhibitors of endocytosis, Dynamin-K44A, Eps15Delta95/295, and clathrin heavy chain RNA interference, block Toca-1-induced filopodial formation. Taken together, these data suggest that the Toca-1-N-WASP complex can link filopodial formation to endocytosis.

Interpretation of the Wingless Gradient Requires Signaling-Induced Self-Inhibition

In a classical view of development, a cell can acquire positional information by reading the local concentration of a morphogen independently of its neighbors. Accordingly, in Drosophila, the morphogen Wingless produced in the wing's prospective distal region activates target genes in a dose-dependent fashion to organize the proximodistal pattern. Here, we show that, in parallel, Wingless triggers two nonautonomous inhibitory programs that play an important role in the establishment of positional information. Cells flanking the source of Wingless produce a negative signal (encoded by notum) that inhibits Wingless signaling in nearby cells. Additionally, in response to Wingless, all prospective wing cells produce an unidentified signal that dampens target gene expression in surrounding cells. Thus, cells influence each other's response to Wingless through at least two modes of lateral inhibition. Without lateral inhibition, some cells acquire ectopic fates. Lateral inhibition may be a general mechanism behind the interpretation of morphogen gradients.

98 HETEROCHROMATIN REPROGRAMMING IN MOUSE EARLY DEVELOPMENT

Heterochromatin is essential for epigenetic gene silencing and nuclear chromatin architecture. Early mouse development is accompanied by dynamic epigenetic changes and heterochromatin restructuring in the cell nuclei of cleavage stage embryos. We have previously shown that disruption of heterochromatin markers such as DNA methylation following somatic nuclear transfer causes developmental failure (Beaujean N et al. 2004 Biol. Reprod. 71, 185–193). The aim of the present study was to investigate the transitions and maturation of heterochromatin during normal development to improve nuclear reprogramming technology and understand developmental abnormalities caused by epigenetic alterations in somatic nuclear transfer and assisted reproductive procedures. Mouse pre- and post-implantation embryos and ES cells from B6CBAF1 mice were used in experiments employing the following methodologies. Embryos and ES cells were stained by immunofluorescence to detect heterochromatin proteins and epigenetic markers. Images were captured using laser confocal microscopy. Relative quantification of fluorescent intensity was performed using Zeiss LSM-meta and WCIF-ImageJ software. mRNA of Histone methyltransferase (HMTase), suv39h, suv420h, ehmt, eset, and demethyltransferase (DHMTase), jmjd1a, jmjd2a, jmjd2c were quantified by real-time PCR. siRNA and specific inhibitors were used to study the function of suv39h, ehmt2, jmjd2c genes that may be responsible for heterochromatin reprogramming. Our results demonstrated that H3K9me3, H3K9me2, H4K20me2, H4K20me3, HP1α and HP1β are reprogrammed during early development. The gene expression results showed the dynamic and temporal gene expression of HMTases and DHMTase during development. Inhibition of Ehmt2 and jmjd2c caused preimplantation developmental arrest. Furthermore, we found chromatin modification differences in the heterochromatin of ES cells, ICM and epiblast. We conclude that heterochromatin reprogramming might be essential for development because it may contribute to chromatin architecture, thus influencing gene expression. HMTases and DHTMases could be implicated in the mechanism of heterochromatin reprogramming. It is possible that Ehmt2 and jmjd2c play an important role in preimplantation development by modifying chromatin globally and at the local gene level. This work was supported by a The Royal Thai Studentship. We acknowledge the contributions of technical assistance from T. O’Connor, B. Wongtawan and P. Travers.

Ubiquitination and Endocytosis of Cell Adhesion Molecule DM-GRASP Regulate Its Cell Surface Presence and Affect Its Role for Axon Navigation

DM-GRASP, cell adhesion molecule of the immunoglobulin superfamily, has been shown to promote growth and navigation of axons. We here demonstrate that clustering of DM-GRASP in the plasma membrane induces its rapid internalization via dynamin- and clathrin-dependent endocytosis, which is controlled by phosphatidylinositol 3-kinase and mitogen-activated protein kinase ERK. The clustering of DM-GRASP activates ERK; the intensity and duration of ERK activation by DM-GRASP do not depend on rapid clathrin-mediated internalization of DM-GRASP. Moreover, the preference of retinal ganglion cell axons for DM-GRASP-coated micro-lanes requires clathrin-mediated endocytosis for the appropriate axonal turning reactions at substrate borders. Because the intracellular domain of DM-GRASP does not contain motifs for direct interactions with the endocytosis machinery, we performed a yeast two-hybrid screen to identify intracellular proteins mediating the uptake of DM-GRASP and isolated ubiquitin. Immunoprecipitation of DM-GRASP coexpressed with ubiquitin revealed that one or two ubiquitin(s) are attached to the intracellular domain of cell surface-resident DM-GRASP. Furthermore, elevated ubiquitination levels result in a decrease of cell surface-resident DM-GRASP as well as in the amount of total DM-GRASP. The endocytosis rate is not affected, but the delivery to multivesicular bodies is increased, indicating that DM-GRASP ubiquitination enhances its sorting into the degradation pathway. Together, our data show that ubiquitination and endocytosis of DM-GRASP in concert regulate its cell surface concentration, which is crucial for its function in axon navigation.

Potential of Fibroblast Cell Therapy for Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin-blistering disorder caused by mutations in the COL7A1 gene that lead to reduced type-VII collagen and defective anchoring fibrils at the dermal-epidermal junction (DEJ). Presently there are no effective treatments for this disorder. Recent mouse studies have shown that intradermal injections of normal human fibroblasts can generate new human type-VII collagen and anchoring fibrils at the DEJ. To assess potential clinical benefits in humans, we gave single intradermal injections of allogeneic fibroblasts to five subjects with RDEB. We noted increased type-VII collagen at the DEJ at 2 weeks and at 3 months following injection and increased anchoring fibrils, although none of these had normal morphology. No adverse effects, clinical or immunopathologic, were noted. We believe the major effect of allogeneic fibroblasts is to increase the recipients' own COL7A1 mRNA levels with greater deposition of mutant type-VII collagen at the DEJ and formation of additional rudimentary anchoring fibrils. Nevertheless, this mutant protein may be partially functional and capable of increasing adhesion at the DEJ. This is the first study demonstrating that intradermal injections of allogeneic fibroblasts have therapeutic potential in human subjects with RDEB.

A planar interdigitated ring electrode array via dielectrophoresis for uniform patterning of cells

Uniform patterning of cells is highly desirable for most cellular studies involving cell–cell interactions but is often difficult in an in vitro environment. This paper presents the development of a collagen-coated planar interdigitated ring electrode (PIRE) array utilizing positive dielectrophoresis to pattern cells uniformly. Key features of the PIRE design include: (1) maximizing length along the edges where the localized maximum in the electric field exists; (2) making the inner gap slightly smaller than the outer gap in causing the electric field strength near the center of a PIRE being generally stronger than that near the outer edge of the same PIRE. Results of human hepatocellular carcinoma cells, HepG2, adhered on a 6 × 6 PIRE array show that cells patterned within minutes with good uniformity (48 ± 6 cells per PIRE). Cell viability test revealed healthy patterned cells after 24 h that were still confined to the collagen-coated PIREs. Furthermore, quantification of fluorescence intensity of living cells shows an acceptable reproducibility of cell viability among PIREs (mean normalized intensity per PIRE was 1 ± 0.138). The results suggest that the PIRE array would benefit applications that desire uniform cellular patterning, and improve both response and reproducibility of cell-based biosensors.

Annexin A6-induced Inhibition of Cytoplasmic Phospholipase A2 Is Linked to Caveolin-1 Export from the Golgi

The molecular mechanisms regulating the exit of caveolin from the Golgi complex are not fully understood. Cholesterol and sphingolipid availability affects Golgi vesiculation events and involves the activity of cytoplasmic phospholipase A(2) (cPLA(2)). We recently demonstrated that high expression levels of annexin A6 (AnxA6) perturb the intracellular distribution of cellular cholesterol, thereby inhibiting caveolin export from the Golgi complex. In the present study we show that in Chinese hamster ovary cells overexpressing AnxA6, sequestration of cholesterol in late endosomes, leading to reduced amounts of cholesterol in the Golgi, inhibits cPLA(2) activity and its association with the Golgi complex. This correlates with the blockage of caveolin export from the Golgi in cells treated with methyl arachidonyl fluorophosphonate, a Ca(2+)-dependent cPLA(2) inhibitor. AnxA6-mediated down-regulation of cPLA(2) activity was overcome upon the addition of exogenous cholesterol or transfection with small interfering RNA targeting AnxA6. These findings indicate that AnxA6 interferes with caveolin transport through the inhibition of cPLA(2).