Regulation of frizzled receptor activity at the plasma membrane : an interplay of the receptor, the trimeric G protein Go, and RGS protein and a scaffolding protein Kermit

G-protein-signaling pathways convey extracellular signals inside the cells and regulate distinct physiological responses. This type of signaling pathways consists of three major components: G-protein-coupled receptors (GPCRs), heterotrimeric G proteins (G-proteins) and downstream effectors. Upon ligand binding, GPCRs activate heterotrimeric G proteins to initiate the signaling cascade. Dysfunction of GPCR signaling correlates with numerous diseases such as diabetes, nervous and immune system deficiency, and cancer. As the signaling switcher, G-proteins (Gs, Gq/11, G12/13, and Gi/o) have been an appealing topic of research for decades. A heterotrimeric G-protein is composed of three subunits, the guanine nucleotide associated a-subunit, ß and y subunits. In general, the duration of signaling is determined by the lifetime of activated (GTP bound) Ga subunits. Identification of novel communication partners of Ga subunits appears to be an attractive way to understand the machinery of GPCR signaling. In our lab, we mainly focus on Gao, which is abundantly expressed in the nervous system. Here we present two novel interacting partners of Drosophila Gao: Dhit and Kermit, identified through yeast two-hybrid screening and genetic screening respectively. Dhit is characterized by a small size with a conserved RGS domain and an N-terminal cysteine rich motif. The RGS domain possesses the GAP (GTPase activating protein) activity towards G proteins. However, we found that Dhit exerts not only the GAP activity but also the GDI (guanine nucleotide dissociation inhibitor) activity towards Gao. The unexpected GDI activity is preserved in GAIP/RGS19 - a mammalian homologue of Dhit. Further experiments confirmed the GDI activity of Dhit and GAIP/RGS19 in Drosophila and mammalian cell models. Therefore, we propose that Dhit and its mammalian homologues modulate GPCR signaling by a double suppression of Ga subunits - suppression of their nucleotide exchange with GTP and acceleration of their hydrolysis of GTP. Kermit/GEPC was first identified as a binding partner of GAIP/RGS19 in a yeast two- hybrid screen. Instead of interacting with the Drosophila homologue of GAIP/RGS19 (Dhit), Kermit binds to Gao in vivo and in vitro. The functional consequence of Kermit/Gao interaction is the regulation of localization of Vang (one of the planar cell polarity core components) at the apical membrane. Overall, my work elaborated the action of Gao with its two interaction partners in Gao- mediated signaling pathway. Conceivably, the understanding of GPCR signaling including Gao and its regulators or effectors will ultimately shed light on future pharmaceutical research. - Les voies de signalisation médiées par les protéines G transmettent des signaux extracellulaires à l'intérieur des cellules pour réguler des réponses physiologiques distinctes. Cette voie de signalisation consiste en trois composants majeurs : les récepteurs couplés aux protéines G (GPCRs), les protéines G hétérotrimériques (G-proteins) et les effecteurs en aval. Suite à la liaison du ligand, les GPCRs activent les protéines G hétérotrimériques qui initient la cascade de signalisation. Des dysfonctions dans la signalisation médiée par les GPCRs sont corrélées avec de nombreuses maladies comme le diabète, des déficiences immunes et nerveuses, ainsi que le cancer. Puisque la voie de signalisation s'active et se désactive, les protéines G (Gs, Gq/11, G12/13 et Gi/o) ont été un sujet de recherche attrayant pendant des décennies. Une protéine G hétérotrimérique est composée de trois sous-unités, la sous-unité a associée au nucléotide guanine, ainsi que les sous-unités ß et y. En général, la durée du signal est déterminée par le temps de demi-vie des sous-unités Ga activées (Ga liées au GTP). Identifier de nouveaux partenaires de communication des sous-unités Ga se révèle être un moyen attractif de comprendre la machinerie de la signalisation par les GPCRs. Dans notre laboratoire nous nous sommes concentrés principalement sur Gao qui est exprimée de manière abondante dans le système nerveux. Nous présentons ici deux nouveaux partenaires qui interagissent avec Gao chez la drosophile: Dhit et Kermit, qui ont été identifiés respectivement par la méthode du yeast two-hybrid et par criblage génétique. Dhit est caractérisé par une petite taille, avec un domaine RGS conservé et un motif N- terminal riche en cystéines. Le domaine RGS contient une activité GAP (GTPase activating protein) pour les protéines G. Toutefois, nous avons découvert que Dhit exerce non seulement une activité GAP mais aussi une activité GDI (guanine nucleotide dissociation inhibitor) à l'égard de Gao. Cette activité GDI inattendue est préservée dans RGS19 - un homologue de Dhit chez les mammifères. Des expériences supplémentaires ont confirmé l'activité GDI de Dhit et de RGS19 chez Drosophila melanogaster et les modèles cellulaires mammifères. Par conséquent, nous proposons que Dhit et ses homologues mammifères modulent la signalisation GPCR par une double suppression des sous-unités Ga - suppression de leur nucléotide d'échange avec le GTP et une accélération dans leur hydrolyse du GTP. Kermit/GIPC a été premièrement identifié comme un partenaire de liaison de RGS19 dans le criblage par yeast two-hybrid. Au lieu d'interagir avec l'homologue chez la drosophile de RGS19 (Dhit), Kermit se lie à Gao in vivo et in vitro. La conséquence fonctionnelle de l'interaction Kermit/Gao est la régulation de la localisation de Vang, un des composants essentiel de la polarité planaire cellulaire, à la membrane apicale. Globalement, mon travail a démontré l'action de Gao avec ses deux partenaires d'interaction dans la voie de signalisation médiée par Gao. La compréhension de la signalisation par les GPCRs incluant Gao et ses régulateurs ou effecteurs aboutira à mettre en lumière de futurs axes dans la recherche pharmacologique.

Reconditioning of the trabeculo-descemet's membrane with the 532-nm Nd : YAG (SLT) laser after deep sclerectomy.

The purpose of this study was to evaluate the intraocular pressure (IOP)-lowering effect of modified goniopuncture with the 532-nm Nd : YAG selective laser trabeculoplasty (SLT) laser on eyes after deep sclerectomy with collagen implant (DSCI). This was an interventional cased series. The effects of modified goniopuncture on eyes with insufficient IOP-lowering after DSCI were observed. Goniopuncture was performed using a Q-switched, frequency-doubled 532-nm Nd : YAG laser (SLT-goniopuncture, SLT-G). Outcome measures were amount of IOP-lowering and rapidity of decrease after laser intervention. In all, 10 eyes of 10 patients with a mean age of 71.0±7.7 (SD) years were treated with SLT-G. The mean time of SLT-G after DSCI procedure was 7.1±10.9 months. SLT-G decreased IOP from an average of 16.1±3.4 mm Hg to 14.2±2.8 mm Hg (after 15 min), 13.6±3.9 mm Hg (at 1 day), 12.5±4.1 mm Hg (at 1 month), and 12.6±2.5 (at 6 months) (P<0.0125). There were no complications related to the intervention. Patients in this series achieved an average 22.5% of IOP reduction after SLT-G. The use of the SLT laser appears to be an effective and safe alternative to the traditional Nd : YAG laser for goniopuncture in eyes after DSCI, with potential advantages related to non-perforation of trabeculo-descemet's membrane (TDM).

Ultrastructural changes of the internal limiting membrane removed during indocyanine green assisted peeling versus conventional surgery for an idiopathic epimacular membrane

Purpose:To evaluate the histological features of cellular retinal fragments on the internal limiting membrane (ILM) removed during epiretinal membrane peeling surgery with and without the aid of ICG diluted in 5% glucose Methods:ILM specimens removed from 88 eyes undergoing vitrectomy and membrane peeling surgery for idiopathic epiretinal membrane between 1995 and 2003 were reviewed retrospectively. Surgery was performed in all cases by the same surgeon using the same technique. ICG was diluted with 5% glucose. Histological analysis focused on the presence and characteristics of retinal structures on the retinal surface of the ILM. Statistical analysis compared the results between group I (conventional surgery without ICG) and group II (ICG-assisted peeling) Results:Seventy-one eyes underwent EMM surgery without the aid of ICG (group I) and seventeen underwent EMM ICG-assisted surgery assisted using ICG (group II). The amount of Muller cell debris on the retinal surface of the ILM was more significant in the group I (no ICG) than in the group II (ICG) (40.8 versus 11.8; p = 0.024). Large fragments of Muller cells were more frequently observed in the group I (no ICG) than in the group II (ICG) (63.4% versus 23.5%; p= 0.003).The presence of larger retinal elements such as neural axons and vessels were observed attached to retinal face of the ILM in 5 (7%) cases of the no-ICG group. No such retinal elements were detected in any of the histological ILM specimens of the ICG-assisted group Conclusions:The use of ICG diluted with 5% glucose in the aid of ILM removal during epiretinal membrane surgery was associated with less retinal debris attached to retinal face of the ILM compared to surgery in which ICG was not used. Our findings contradict previous reports in the literature, in which use of ICG diluted with balanced salt solution (BSS) was associated with more retinal fragments attached to the retinal face of the ILM. According to our results, we hypothesize that diluting ICG with 5% glucose may decrease the adhesion of the ILM to the underlying retinal layers such that less retinal debris is removed with peeling of the ILM.

Quantitative proteomics identifies the membrane-associated peroxidase GPx8 as a cellular substrate of the hepatitis C virus NS3-4A protease.

The hepatitis C virus (HCV) NS3-4A protease is not only an essential component of the viral replication complex and a prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. It cleaves and thereby inactivates two crucial adaptor proteins in viral RNA sensing and innate immunity, mitochondrial antiviral signaling protein (MAVS) and TRIF, a phosphatase involved in growth factor signaling, T-cell protein tyrosine phosphatase (TC-PTP), and the E3 ubiquitin ligase component UV-damaged DNA-binding protein 1 (DDB1). Here we explored quantitative proteomics to identify novel cellular substrates of the NS3-4A protease. Cell lines inducibly expressing the NS3-4A protease were analyzed by stable isotopic labeling using amino acids in cell culture (SILAC) coupled with protein separation and mass spectrometry. This approach identified the membrane-associated peroxidase GPx8 as a bona fide cellular substrate of the HCV NS3-4A protease. Cleavage by NS3-4A occurs at Cys 11, removing the cytosolic tip of GPx8, and was observed in different experimental systems as well as in liver biopsies from patients with chronic HCV. Overexpression and RNA silencing studies revealed that GPx8 is involved in viral particle production but not in HCV entry or RNA replication. Conclusion: We provide proof-of-concept for the use of quantitative proteomics to identify cellular substrates of a viral protease and describe GPx8 as a novel proviral host factor targeted by the HCV NS3-4A protease. (Hepatology 2014;59:423-433).

Membrane lipid composition affects plant heat sensing and modulates Ca ( 2+) -dependent heat shock response.

Understanding how plants sense and respond to heat stress is central to improve crop tolerance and productivity. Recent findings in Physcomitrella patensdemonstrated that the controlled passage of calcium ions across the plasma membrane regulates the heat shock response (HSR). To investigate the effect of membrane lipid composition on the plant HSR, we acclimated P. patens to a slightly elevated yet physiological growth temperature and analysed the signature of calcium influx under a mild heat shock. Compared to tissues grown at 22°C, tissues grown at 32°C had significantly higher overall membrane lipid saturation level and, when submitted to a short heat shock at 35°C, displayed a noticeably reduced calcium influx and a consequent reduced heat shock gene expression. These results show that temperature differences, rather than the absolute temperature, determine the extent of the plant HSR and indicate that membrane lipid composition regulates the calcium-dependent heat-signaling pathway.

Sequentially aerated membrane biofilm reactors for autotrophic nitrogen removal: microbial community composition and dynamics

Membrane-aerated biofilm reactors performing autotrophic nitrogen removal can be successfully applied to treat concentrated nitrogen streams. However, their process performance is seriously hampered by the growth of nitrite oxidizing bacteria (NOB). In this work we document how sequential aeration can bring the rapid and long-term suppression of NOB and the onset of the activity of anaerobic ammonium oxidizing bacteria (AnAOB). Real-time quantitative polymerase chain reaction analyses confirmed that such shift in performance was mirrored by a change in population densities, with a very drastic reduction of the NOB Nitrospira and Nitrobacter and a 10-fold increase in AnAOB numbers. The study of biofilm sections with relevant 16S rRNA fluorescent probes revealed strongly stratified biofilm structures fostering aerobic ammonium oxidizing bacteria (AOB) in biofilm areas close to the membrane surface (rich in oxygen) and AnAOB in regions neighbouring the liquid phase. Both communities were separated by a transition region potentially populated by denitrifying heterotrophic bacteria. AOB and AnAOB bacterial groups were more abundant and diverse than NOB, and dominated by the r-strategists Nitrosomonas europaea and Ca. Brocadia anammoxidans, respectively. Taken together, the present work presents tools to better engineer, monitor and control the microbial communities that support robust, sustainable and efficient nitrogen removal

Seed priming and sulfur effects on soybean cell membrane stability and yield in saline soil

The objective of this work was to determine the effects of seed priming and sulfur application on cell membrane characteristics, seedling emergence, chlorophyll content and grain yield of soybean (Glycine max) in saline soil. A complete-block design in 4x3 factorial arrangement with three replicates was used to test four types of seed priming (water, auxin, gibberellin and non-priming) and three levels of sulfate availability (0, 70 and 140 kg ha-1 K2SO4). The soil had a silty loam texture with an electrical conductivity of 3.61 ds m-1, a pH of 8.2 and a saturation percentage of about 46%. Seed priming had significant effects on mean emergence rate (MER), emergence percentage, relative water content (RWC) of leaves, relative chlorophyll content, time of maturity, shoot length and grain yield. The highest values for these variables were observed in the priming treatments, except for the time of maturity. Sulfur application had significant effects on MER, shoot length, RWC, membrane injury index and grain yield. Priming treatments provide greater emergence rates and grain yields and interact sinergicaly with sulfur rates.

Oligogalacturonide defense signals in plants: large fragments interact with the plasma membrane in vitro.

Oligogalacturonides are plant cell wall-derived regulatory molecules which stimulate defense gene expression during pathogenesis. In vitro, these compounds enhance the phosphorylation of an approximately 34-kDa protein (pp34) in purified plasma membranes from potato and tomato leaves. We now show that polygalacturonate-enhanced phosphorylation of pp34 occurs in plasma membranes purified from tomato roots, hypocotyls, and stems and from undifferentiated potato cells. Furthermore, a similar phosphorylation is detected in leaf plasma membranes from soybean, a plant distantly related to tomato. Purified oligogalacturonides 13 to at least 26 residues long stimulate pp34 thiophosphorylation in vitro. This stimulation pattern differs from the induction of many known defense responses in vivo, where a narrower range of smaller fragments, between approximately 10 and 15 residues long, are active. On the basis of these differences we suggest that observed effects of applied exogenous oligogalacturonides on defense responses may not necessarily reflect the situation during pathogenesis. The cell wall could act as a barrier to many exogenous oligo- and polygalacturonides as well as other large regulatory ligands.

GLUT8 subcellular localization and absence of translocation to the plasma membrane in PC12 cells and hippocampal neurons.

GLUT8 is a high-affinity glucose transporter present mostly in testes and a subset of brain neurons. At the cellular level, it is found in a poorly defined intracellular compartment in which it is retained by an N-terminal dileucine motif. Here we assessed GLUT8 colocalization with markers for different cellular compartments and searched for signals, which could trigger its cell surface expression. We showed that when expressed in PC12 cells, GLUT8 was located in a perinuclear compartment in which it showed partial colocalization with markers for the endoplasmic reticulum but not with markers for the trans-Golgi network, early endosomes, lysosomes, and synaptic-like vesicles. To evaluate its presence at the plasma membrane, we generated a recombinant adenovirus for the expression of GLUT8 containing an extracellular myc epitope. Cell surface expression was evaluated by immunofluorescence microscopy of transduced PC12 cells or primary hippocampal neurons exposed to different stimuli. Those included substances inducing depolarization, activation of protein kinase A and C, activation or inhibition of tyrosine kinase-linked signaling pathways, glucose deprivation, AMP-activated protein kinase stimulation, and osmotic shock. None of these stimuli-induced GLUT8 cell surface translocation. Furthermore, when GLUT8myc was cotransduced with a dominant-negative form of dynamin or GLUT8myc-expressing PC-12 cells or neurons were incubated with an anti-myc antibody, no evidence for constitutive recycling of the transporter through the cell surface could be obtained. Thus, in cells normally expressing it, GLUT8 was associated with a specific intracellular compartment in which it may play an as-yet-uncharacterized role.

Dissection of hormone-responsive plasma membrane to nucleus signaling of Bravis Radix : its role in vascular differentiation and root meristem growth

AbstractPlants continuously grow during their complete life span and understanding the mechanisms that qualitatively regulate their traits remains a challenging topic in biology. The hormone auxin has been identified as a crucial molecule for shaping plant growth, as it has a role in most developmental processes. In the root, the directional, so-called polar transport of auxin generates a peak of concentration that specifies and maintains the stem cell niche and a subsequent gradient of decreasing concentration that also regulates cell proliferation and differentiation. For these reasons, auxin is considered the main morphogen of the root, as it is fundamental for its organization and maintenance. Recently, in Arabidopsis thaliana, a natural variation screen allowed the discovery of BREVIS RADIX (BRX) gene as a limiting factor for auxin responsive gene expression and thus for root growth.In this study, we discovered that BRX is a direct target of auxin that positively feeds back on auxin signaling, as a transcriptional co-regulator, through interaction with the Auxin Response Factor (ARF) MONOPTEROS (MP), modulating the auxin gene response magnitude during the transition between division and differentiation in the root meristem. Moreover, we provide evidence that BRX is activated at the plasma membrane level as an associated protein before moving into the nucleus to modulate cellular growth.To investigate the discrepancy between the auxin concentration and the expression pattern of its downstream targets, we combined experimental and computational approaches. Expression profiles deviating from the auxin gradient could only be modeled after intersection of auxin activity with the observed differential endocytosis pattern and with positive auto- regulatory feedback through plasma- membrane-to-nucleus transfer of BRX. Because BRX is required for expression of certain auxin response factor targets, our data suggest a cell-type-specific endocytosis-dependent input into transcriptional auxin perception. This input sustains expression of a subset of auxin-responsive genes across the root meristem's division and transition zones and is essential for meristem growth. Thus, the endocytosis pattern provides specific positional information to modulate auxin response. RésuméLes plantes croissent continuellement tout au long de leur cycle de vie. Comprendre et expliquer les mécanismes impliqués dans ce phénomène reste à l'heure actuelle, un défi. L'hormone auxine a été identifiée comme une molécule essentielle à la régulation de la croissance des plantes, car impliquée dans la plupart des processus développementaux. Dans la racine, le transport polaire de l'auxine, par la génération d'un pic de concentration, spécifie et maintient la niche de cellules souches, et par la génération d'un gradient de concentration, contrôle la prolifération et la différentiation cellulaire. Puisque l'auxine est essentielle pour l'organisation et la maintenance du système racinaire, il est considéré comme son principal morphogène. Récemment, dans la plante modèle, Arabidopsis thalinana, un criblage des variations génétique a permis d'identifier le gène Brevis radix (BRX) comme facteur limitant l'expression des gènes de réponse à l'auxine et par là même, la croissance de la racine.Dans ce travail, nous avons découvert que BRX est une cible direct de l'auxine qui rétroactive positivement le signalement de l'hormone, agissant ainsi comme un régulateur transcriptionnel à travers l'interaction avec la protéine Monopteros (MP) de la famille des facteurs de réponse à l'auxine (Auxin Responsive Factor, ARF), et modulant ainsi la magnitude de la réponse des gènes reliés à l'auxine durant la division et la différentiation cellulaire dans le méristème de la racine. De plus, nous fournissons des preuves que BRX est activées au niveau de la membrane plasmique, tel une protéine associée se déplaçant à l'intérieur du noyau et modulant la croissance cellulaire.Pour mener à bien l'investigation des divergences entre la concentration de l'auxine et les schémas d'expression de ses propres gènes cibles, nous avons combiné les approches expérimentales et computationnelles. Les profiles d'expressions déviant du gradient d'auxine pourraient seulement être modéliser après intersection de l'activité de l'auxine avec les schémas différentiels d'endocytose observés et les boucles de rétroaction positives et autorégulatrices par le transfert de BRX de la membrane plasmique au noyau. Puisque BRX est requis pour l'expression de certains gènes cibles des facteurs de réponse à l'auxine, nos données suggèrent une contribution dépendante d'une endocytose spécifique au type de cellule dans la perception transcriptionnelle à l'auxine Cette contribution soutient l'expression d'un sous-set de gène de réponse à l'auxine dans la division du méristème racinaire et la zone de transition, et par conséquent, est essentielle pour la croissance méristematique. Ainsi, le schéma d'endocytose fournit des informations positionnelles spécifiques à la modulation de la réponse à l'auxine.

Membrane insertion and antibody recognition of a glycosylphosphatidylinositol-anchored protein: an optical study.

The kinetics of binding of a glycolipid-anchored protein (the promastigote surface protease, PSP) to planar lecithin bilayers is studied by an integrated optics technique, in which the bilayer membrane is supported on an optical wave guide and the phase velocities of guided light modes in the wave guide are measured. From these velocities, the optical parameters of the membrane and PSP layers deposited on the waveguide are determined, yielding in particular the mass of PSP bound to the membrane, which is followed in real time. From a comparison of the binding rates of PSP and PSP from which the lipid moiety has been removed, it is shown that the lipid moiety plays a key role in anchoring the protein to the membrane. Specific and nonspecific binding of antibodies to membrane-anchored PSP is also investigated. As little as a fifth of a monolayer of PSP is sufficient to suppress the appreciable nonspecific binding of antibodies to the membrane.

Structure of the glycosyl-phosphatidylinositol membrane anchor of the Leishmania major promastigote surface protease.

In common with many other plasma membrane glycoproteins of eukaryotic origin, the promastigote surface protease (PSP) of the protozoan parasite Leishmania contains a glycosyl-phosphatidylinositol (GPI) membrane anchor. The GPI anchor of Leishmania major PSP was purified following proteolysis of the PSP and analyzed by two-dimensional 1H-1H NMR, compositional and methylation linkage analyses, chemical and enzymatic modifications, and amino acid sequencing. From these results, the structure of the GPI-containing peptide was found to be Asp-Gly-Gly-Asn-ethanolamine-PO4-6Man alpha 1-6Man alpha 1-4GlcN alpha 1-6myo-inositol-1-PO4-(1-alkyl-2-acyl-glycerol). The glycan structure is identical to the conserved glycan core regions of the GPI anchor of Trypanosoma brucei variant surface glycoprotein and rat brain Thy-1 antigen, supporting the notion that this portion of GPIs are highly conserved. The phosphatidylinositol moiety of the PSP anchor is unusual, containing a fully saturated, unbranched 1-O-alkyl chain (mainly C24:0) and a mixture of fully saturated unbranched 2-O-acyl chains (C12:0, C14:0, C16:0, and C18:0). This lipid composition differs significantly from those of the GPIs of T. brucei variant surface glycoprotein and mammalian erythrocyte acetylcholinesterase but is similar to that of a family of glycosylated phosphoinositides found uniquely in Leishmania.