RESUMEN
Twenty-five chimera compounds of Pitstop® 1 and 2 were synthesised and screened for their ability to block the clathrin terminal domain-amphiphysin protein-protein interaction (NTD-PPI using an ELISA) and clathrin mediated endocytosis (CME) in cells. Library 1 was based on Pitstop 2, but no notable clathrin PPI or in-cell activity was observed. With the Pitstop 1, 16 analogues were produced with 1,8-naphthalic imide core as a foundation. Analogues with methylene spaced linkers and simple amides showed a modest to good range of PPI inhibition (7.6 to 42.5 mM, naphthyl 39 and 4-nitrophenyl 40 respectively) activity. These data reveal the importance of the naphthalene sulfonate moiety, with no des-SO3 analogue displaying PPI inhibition. This was consistent with the observed analogue docked poses within the clathrin terminal domain Site 1 binding pocket. Further modifications targeted the naphthalene imide moiety, with the installation of 5-Br (45a), 5-OH (45c) and 5-propyl ether (45d) moieties. Among them, the OH 45c and propyl ether 45d retained PPI inhibition, with propyl ether 45d being the most active with a PPI inhibition IC50 = 7.3 mM. This is 2x more potent than Pitstop® 2 and 3x more potent than Pitstop 1.
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Exocytosis and endocytosis are essential physiological processes and are of prime importance for brain function. Neurotransmission depends on the Ca2+-triggered exocytosis of synaptic vesicles (SVs). In neurons, exocytosis is spatiotemporally coupled to the retrieval of an equal amount of membrane and SV proteins by compensatory endocytosis. How exocytosis and endocytosis are balanced to maintain presynaptic membrane homeostasis and, thereby, sustain brain function is essentially unknown. We combine mouse genetics with optical imaging to show that the SV calcium sensor Synaptotagmin 1 couples exocytic SV fusion to the endocytic retrieval of SV membranes by promoting the local activity-dependent formation of the signaling lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) at presynaptic sites. Interference with these mechanisms impairs PI(4,5)P2-triggered SV membrane retrieval but not exocytic SV fusion. Our findings demonstrate that the coupling of SV exocytosis and endocytosis involves local Synaptotagmin 1-induced lipid signaling to maintain presynaptic membrane homeostasis in central nervous system neurons.
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Vesículas Sinápticas , Sinaptotagmina I , Animales , Ratones , Endocitosis/fisiología , Exocitosis/fisiología , Lípidos , Transmisión Sináptica , Vesículas Sinápticas/metabolismo , Sinaptotagmina I/genética , Sinaptotagmina I/metabolismoRESUMEN
Phosphatidylinositol 3-kinase type 2α (PI3KC2α) and related class II PI3K isoforms are of increasing biomedical interest because of their crucial roles in endocytic membrane dynamics, cell division and signaling, angiogenesis, and platelet morphology and function. Herein we report the development and characterization of PhosphatidylInositol Three-kinase Class twO INhibitors (PITCOINs), potent and highly selective small-molecule inhibitors of PI3KC2α catalytic activity. PITCOIN compounds exhibit strong selectivity toward PI3KC2α due to their unique mode of interaction with the ATP-binding site of the enzyme. We demonstrate that acute inhibition of PI3KC2α-mediated synthesis of phosphatidylinositol 3-phosphates by PITCOINs impairs endocytic membrane dynamics and membrane remodeling during platelet-dependent thrombus formation. PITCOINs are potent and selective cell-permeable inhibitors of PI3KC2α function with potential biomedical applications ranging from thrombosis to diabetes and cancer.
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Fosfatidilinositol 3-Quinasa , Fosfatidilinositol 3-Quinasas , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositoles , Fosfatos de Fosfatidilinositol/metabolismoRESUMEN
Cytokinesis mediates the final separation of a mother cell into two daughter cells. Septins are recruited to the cleavage furrow at an early stage. During cytokinetic progression the septin cytoskeleton is constantly rearranged, ultimately leading to a concentration of septins within the intercellular bridge (ICB), and to the formation of two rings adjacent to the midbody that aid ESCRT-dependent abscission. The molecular mechanisms underlying this behavior are poorly understood. Based on observations that septins can associate with actin, microtubules and associated motors, we review here established roles of septins in mammalian cytokinesis, and discuss, how septins may support cytokinetic progression by exerting their functions at particular sites. Finally, we discuss how this might be assisted by phosphoinositide-metabolizing enzymes.
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PURPOSE: For surgical treatment of inguinal hernia, large-pore, lightweight mesh has been shown to offer advantages over small-pore, heavyweight options in terms of chronic post-operative inguinal pain, but without the disadvantage of having to deal with an increased recurrence rate. Limited data are available for the mesh plug repair technique. Therefore, the primary aim of this study is to compare large-pore, lightweight mesh versus small-pore, heavyweight mesh for mesh plug repair with regard to chronic pain and recurrences in elective primary unilateral hernias. In addition, we report our experience in repairing recurrent hernias. METHODS: Using a modified version of the questionnaire from the Danish Hernia Registry, two groups were surveyed: elective primary unilateral hernias and recurrent unilateral hernias. In both groups small-pore, heavyweight mesh (HWM) and lightweight, large-pore mesh (LWM) were compared with respect to chronic pain and recurrences. Propensity score matching (PS) was carried out on a pool of 1,782 patients. Effect sizes were assessed by using Cohen's d and Cramer's V. RESULTS: If the questionnaire item 'lump/swelling' is considered as a surrogate for recurrence (clinically verified in our study), the results in primary hernias after HWM show a 6.0% recurrence rate and 7.3% after LWM (p = 0.487) with a mean follow up of 31,3 months in HWM and 29,2 months in LWM respectively. The questionnaire item 'pain impacting on work/leisure activities' was answered with Yes in 11.5% following HWM and in 10.5% following LWM (p = 0.665). After the evaluation of the overall surgical results, we did not find differences. Comparing primary and recurrent hernia repair we found below small effect size differences with respect to the items of the questionnaire. CONCLUSIONS: The use of LWM in repairing elective unilateral primary hernias by the mesh plug technique does not result in less chronic pain and more recurrences when compared with HWM. Recurrent hernias repaired by the mesh plug technique may have same outcomes like in primary hernia repair when considering the magnitude of effect sizes.
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Hernia Inguinal/cirugía , Herniorrafia/métodos , Puntaje de Propensión , Mallas Quirúrgicas , Adulto , Anciano , Dolor Crónico/prevención & control , Procedimientos Quirúrgicos Electivos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Dolor Postoperatorio/prevención & control , Recurrencia , Estudios Retrospectivos , Encuestas y CuestionariosRESUMEN
BACKGROUND: Development of platelet precursor cells, megakaryocytes (MKs), implies an increase in their size; formation of the elaborate demarcation membrane system (DMS); and extension of branched cytoplasmic structures, proplatelets, that will release platelets. The membrane source(s) for MK expansion and proplatelet formation have remained elusive. OBJECTIVE: We hypothesized that traffic of membranes regulated by phosphatidylinositol 3-monophosphate (PI3P) contributes to MK maturation and proplatelet formation. RESULTS: In immature MKs, PI3P produced by the lipid kinase Vps34 is confined to perinuclear early endosomes (EE), while in mature MKs PI3P shifts to late endosomes and lysosomes (LE/Lys). PI3P partially colocalized with the plasma membrane marker phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) and with LE/Lys in mature MKs, suggests that PI3P-containing LE/Lys membranes contribute to MK expansion and proplatelet formation. Consistently, we found that sequestration of PI3P, specific pharmacological inhibition of Vps34-mediated PI3P production, or depletion of PI3P by PI3-phosphatase (MTM1)-mediated hydrolysis potently blocked proplatelet formation. Moreover, Vps34 inhibition led to the intracellular accumulation of enlarged LE/Lys, and decreased expression of surface LE/Lys markers. Inhibiting Vps34 at earlier MK stages caused aberrant DMS development. Finally, inhibition of LE/Lys membrane fusion by a dominant negative mutant of the small GTPase Rab7 or pharmacological inhibition of PI3P conversion into PI(3,5)P2 led to enlarged LE/Lys, reduced surface levels of LE/Lys markers, and decreased proplatelet formation. CONCLUSION: Our results suggest that PI3P-positive LE/Lys contribute to the membrane growth and proplatelet formation in MKs by their translocation to the cell periphery and fusion with the plasma membrane.
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Megacariocitos , Fosfatos de Fosfatidilinositol , Plaquetas , Endosomas , LisosomasRESUMEN
Compartmentalization of membrane transport and signaling processes is of pivotal importance to eukaryotic cell function. While plasma membrane compartmentalization and dynamics are well known to depend on the scaffolding function of septin GTPases, the roles of septins at intracellular membranes have remained largely elusive. Here, we show that the structural and functional integrity of the Golgi depends on its association with a septin 1 (SEPT1)-based scaffold, which promotes local microtubule nucleation and positioning of the Golgi. SEPT1 function depends on the Golgi matrix protein GM130 (also known as GOLGA2) and on centrosomal proteins, including CEP170 and components of γ-tubulin ring complex (γ-Turc), to facilitate the perinuclear concentration of Golgi membranes. Accordingly, SEPT1 depletion triggers a massive fragmentation of the Golgi ribbon, thereby compromising anterograde membrane traffic at the level of the Golgi.
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Autoantígenos/genética , Centrosoma/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/genética , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Septinas/genética , Células 3T3-L1 , Animales , Autoantígenos/metabolismo , Transporte Biológico , Compartimento Celular , Línea Celular , Centrosoma/ultraestructura , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Regulación de la Expresión Génica , Aparato de Golgi/ultraestructura , Células HEK293 , Células HeLa , Humanos , Células Jurkat/metabolismo , Células Jurkat/ultraestructura , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/ultraestructura , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Septinas/antagonistas & inhibidores , Septinas/metabolismo , Transducción de SeñalRESUMEN
Many disease-causing missense mutations affect intrinsically disordered regions (IDRs) of proteins, but the molecular mechanism of their pathogenicity is enigmatic. Here, we employ a peptide-based proteomic screen to investigate the impact of mutations in IDRs on protein-protein interactions. We find that mutations in disordered cytosolic regions of three transmembrane proteins (GLUT1, ITPR1, and CACNA1H) lead to an increased clathrin binding. All three mutations create dileucine motifs known to mediate clathrin-dependent trafficking. Follow-up experiments on GLUT1 (SLC2A1), the glucose transporter causative of GLUT1 deficiency syndrome, revealed that the mutated protein mislocalizes to intracellular compartments. Mutant GLUT1 interacts with adaptor proteins (APs) in vitro, and knocking down AP-2 reverts the cellular mislocalization and restores glucose transport. A systematic analysis of other known disease-causing variants revealed a significant and specific overrepresentation of gained dileucine motifs in structurally disordered cytosolic domains of transmembrane proteins. Thus, several mutations in disordered regions appear to cause "dileucineopathies."
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Transportador de Glucosa de Tipo 1/fisiología , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/fisiología , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión , Canales de Calcio Tipo T/genética , Canales de Calcio Tipo T/fisiología , Errores Innatos del Metabolismo de los Carbohidratos , Clatrina/metabolismo , Citoplasma/metabolismo , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/fisiología , Proteínas Intrínsecamente Desordenadas/metabolismo , Leucina/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas de Transporte de Monosacáridos/deficiencia , Mutación/genética , Péptidos , Unión Proteica , Proteómica/métodosRESUMEN
Clathrin-mediated endocytosis (CME) is a universal and evolutionarily conserved process that enables the internalization of numerous cargo proteins, including receptors for nutrients and signaling molecules, as well as synaptic vesicle reformation. Multiple genetic and chemical approaches have been developed to interfere with this process. However, many of these tools do not selectively block CME, for example by targeting components shared with clathrin-independent endocytosis pathways or by interfering with other cellular processes that indirectly affect CME.Clathrin, via interactions of endocytic proteins with its terminal domain (TD), serves as a central interaction hub for coat assembly in CME. Here, we describe an ELISA-based, high-throughput screening method used to identify small molecules that inhibit these interactions. In addition, we provide protocols for the purification of recombinant protein domains used for screening, e.g., the clathrin TD and the amphiphysin B/C domain. The screen has been applied successfully in the past, and ultimately led to the discovery of the Pitstop® family of inhibitors, but remains in use to evaluate the inhibitory potency of derivatives of these compounds, and to screen for completely novel inhibitor families.
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Clatrina/antagonistas & inhibidores , Descubrimiento de Drogas , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Animales , Clatrina/química , Clatrina/genética , Clatrina/aislamiento & purificación , Vesículas Cubiertas por Clatrina/efectos de los fármacos , Vesículas Cubiertas por Clatrina/metabolismo , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas/métodos , Endocitosis/efectos de los fármacos , Endocitosis/fisiología , Ensayo de Inmunoadsorción Enzimática , Expresión Génica , Humanos , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
OBJECTIVE: Intracellular vesicle trafficking maintains cellular structures and functions. The assembly of cargo-laden vesicles at the trans-Golgi network is initiated by the ARF family of small GTPases. Here, we demonstrate the role of the trans-Golgi localized monomeric GTPase ARFRP1 in endosomal-mediated vesicle trafficking of mature adipocytes. METHODS: Control (Arfrp1flox/flox) and inducible fat-specific Arfrp1 knockout (Arfrp1iAT-/-) mice were metabolically characterized. In vitro experiments on mature 3T3-L1 cells and primary mouse adipocytes were conducted to validate the impact of ARFRP1 on localization of adiponectin and the insulin receptor. Finally, secretion and transferrin-based uptake and recycling assays were performed with HeLa and HeLa M-C1 cells. RESULTS: We identified the ARFRP1-based sorting machinery to be involved in vesicle trafficking relying on the endosomal compartment for cell surface delivery. Secretion of adiponectin from fat depots was selectively reduced in Arfrp1iAT-/- mice, and Arfrp1-depleted 3T3-L1 adipocytes revealed an accumulation of adiponectin in Rab11-positive endosomes. Plasma adiponectin deficiency of Arfrp1iAT-/- mice resulted in deteriorated hepatic insulin sensitivity, increased gluconeogenesis and elevated fasting blood glucose levels. Additionally, the insulin receptor, undergoing endocytic recycling after ligand binding, was less abundant at the plasma membrane of adipocytes lacking Arfrp1. This had detrimental effects on adipose insulin signaling, followed by insufficient suppression of basal lipolytic activity and impaired adipose tissue expansion. CONCLUSIONS: Our findings suggest that adiponectin secretion and insulin receptor surface targeting utilize the same post-Golgi trafficking pathways that are essential for an appropriate systemic insulin sensitivity and glucose homeostasis.
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Adiponectina/metabolismo , Endosomas/metabolismo , Receptor de Insulina/metabolismo , Vías Secretoras , Red trans-Golgi/metabolismo , Células 3T3-L1 , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Adipocitos/metabolismo , Animales , Células Cultivadas , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Transporte de ProteínasRESUMEN
Septins constitute a family of GTP-binding proteins, which assemble into non-polar filaments in a nucleotide-dependent manner. These filaments can be recruited to negatively charged membrane surfaces. When associated with membranes septin filaments can act as diffusion barriers, which confine subdomains of distinct biological functions. In addition, they serve scaffolding roles by recruiting cytosolic proteins and other cytoskeletal elements. Septins have been implicated in a large variety of membrane-dependent processes, including cytokinesis, signaling, cell migration, and membrane traffic, and several family members have been implicated in disease. However, surprisingly little is known about the molecular mechanisms underlying their biological functions. This review summarizes evidence in support of regulatory roles of septins during endo-lysosomal sorting, with a particular focus on phosphoinositides, which serve as spatial landmarks guiding septin recruitment to distinct subcellular localizations.
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Contact sites between the endoplasmic reticulum and the plasma membrane mediate receptor signalling. How this function is controlled physically and functionally is poorly understood. Extended synaptotagmins are now shown to shuttle the lipid metabolite diacylglycerol from the plasma membrane to the endoplasmic reticulum in receptor-stimulated cells.
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Retículo Endoplásmico , Sinaptotagminas , Animales , Transporte Biológico , Membrana Celular , LípidosRESUMEN
Phosphoinositides are a minor class of short-lived membrane phospholipids that serve crucial functions in cell physiology ranging from cell signalling and motility to their role as signposts of compartmental membrane identity. Phosphoinositide 4-phosphates such as phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are concentrated at the plasma membrane, on secretory organelles, and on lysosomes, whereas phosphoinositide 3-phosphates, most notably phosphatidylinositol 3-phosphate (PI(3)P), are a hallmark of the endosomal system. Directional membrane traffic between endosomal and secretory compartments, although inherently complex, therefore requires regulated phosphoinositide conversion. The molecular mechanism underlying this conversion of phosphoinositide identity during cargo exit from endosomes by exocytosis is unknown. Here we report that surface delivery of endosomal cargo requires hydrolysis of PI(3)P by the phosphatidylinositol 3-phosphatase MTM1, an enzyme whose loss of function leads to X-linked centronuclear myopathy (also called myotubular myopathy) in humans. Removal of endosomal PI(3)P by MTM1 is accompanied by phosphatidylinositol 4-kinase-2α (PI4K2α)-dependent generation of PI(4)P and recruitment of the exocyst tethering complex to enable membrane fusion. Our data establish a mechanism for phosphoinositide conversion from PI(3)P to PI(4)P at endosomes en route to the plasma membrane and suggest that defective phosphoinositide conversion at endosomes underlies X-linked centronuclear myopathy caused by mutation of MTM1 in humans.
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Endosomas/metabolismo , Exocitosis , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositoles/metabolismo , 1-Fosfatidilinositol 4-Quinasa/metabolismo , Transporte Biológico , Línea Celular , Membrana Celular/metabolismo , Células HeLa , Humanos , Hidrólisis , Fusión de Membrana , Miopatías Estructurales Congénitas/enzimología , Miopatías Estructurales Congénitas/genética , Miopatías Estructurales Congénitas/patología , Monoéster Fosfórico Hidrolasas/deficiencia , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/deficiencia , Proteínas Tirosina Fosfatasas no Receptoras/genética , Proteínas Tirosina Fosfatasas no Receptoras/metabolismoRESUMEN
Septins constitute a family of GTP-binding proteins that are involved in a variety of biological processes. Several isoforms have been implicated in disease, but the molecular mechanisms underlying pathogenesis are poorly understood. Here, we show that depletion of SEPT9 decreases surface levels of epidermal growth factor receptors (EGFRs) by enhancing receptor degradation. We identify a consensus motif within the SEPT9 N-terminal domain that supports its association with the adaptor protein CIN85 (also known as SH3KBP1). We further show CIN85-SEPT9 to be localized exclusively to the plasma membrane, where SEPT9 is recruited to EGF-engaged receptors in a CIN85-dependent manner. Finally, we demonstrate that SEPT9 negatively regulates EGFR degradation by preventing the association of the ubiquitin ligase Cbl with CIN85, resulting in reduced EGFR ubiquitylation. Taken together, these data provide a mechanistic explanation of how SEPT9, though acting exclusively at the plasma membrane, impairs the sorting of EGFRs into the degradative pathway.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Receptores ErbB/metabolismo , Regulación de la Expresión Génica , Septinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencias de Aminoácidos/genética , Membrana Celular/metabolismo , Receptores ErbB/genética , Células HeLa , Humanos , Redes y Vías Metabólicas , Unión Proteica , Proteolisis , ARN Interferente Pequeño , Septinas/genética , Ubiquitina/metabolismoRESUMEN
Bin-Amphiphysin-Rvs (BAR) domain-containing proteins form oligomeric assemblies that aid membrane remodeling. In this issue of Developmental Cell, Pang et al. (2014) show that the BAR domain of ACAP1, although architecturally similar to other BAR domains, cooperates with its neighboring pleckstrin homology domain to deform membranes and facilitate endosomal recycling.
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Membrana Celular/metabolismo , Proteínas Activadoras de GTPasa/química , HumanosRESUMEN
Synaptic vesicle recycling has long served as a model for the general mechanisms of cellular trafficking. We used an integrative approach, combining quantitative immunoblotting and mass spectrometry to determine protein numbers; electron microscopy to measure organelle numbers, sizes, and positions; and super-resolution fluorescence microscopy to localize the proteins. Using these data, we generated a three-dimensional model of an "average" synapse, displaying 300,000 proteins in atomic detail. The copy numbers of proteins involved in the same step of synaptic vesicle recycling correlated closely. In contrast, copy numbers varied over more than three orders of magnitude between steps, from about 150 copies for the endosomal fusion proteins to more than 20,000 for the exocytotic ones.
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Encéfalo/metabolismo , Terminales Presinápticos/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptosomas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Encéfalo/ultraestructura , Exocitosis , Imagenología Tridimensional , Immunoblotting/métodos , Espectrometría de Masas/métodos , Microscopía Electrónica/métodos , Modelos Neurológicos , Terminales Presinápticos/química , Terminales Presinápticos/ultraestructura , Transporte de Proteínas , Ratas , Ratas Wistar , Vesículas Sinápticas/química , Sinaptosomas/química , Sinaptosomas/ultraestructura , Proteínas de Transporte Vesicular/análisisRESUMEN
Endosomal membrane traffic serves crucial roles in cell physiology, signaling, and development. Sorting between endosomes and the trans-Golgi network (TGN) is regulated among other factors by the adaptor AP-1, an essential component of multicellular organisms. Membrane recruitment of AP-1 requires phosphatidylinositol 4-phosphate [PI(4)P], though the precise mechanisms and PI4 kinase isozyme (or isozymes) involved in generation of this PI(4)P pool remain unclear. The Wnt pathway is a major developmental signaling cascade and depends on endosomal sorting in Wnt-sending cells. Whether TGN/endosomal sorting modulates signaling downstream of Frizzled (Fz) receptors in Wnt-receiving cells is unknown. Here, we identify PI4-kinase type 2ß (PI4K2ß) as a regulator of TGN/endosomal sorting and Wnt signaling. PI4K2ß and AP-1 interact directly and are required for efficient sorting between endosomes and the TGN. Zebrafish embryos depleted of PI4K2ß or AP-1 lack pectoral fins due to defective Wnt signaling. Rescue experiments demonstrate requirements for PI4K2ß-AP-1 complex formation and PI4K2ß-mediated PI(4)P synthesis. Furthermore, PI4K2ß binds to the Fz-associated component Dishevelled (Dvl) and regulates endosomal recycling of Fz receptors and Wnt target gene expression. These data reveal an evolutionarily conserved role for PI4K2ß and AP-1 in coupling phosphoinositide metabolism to AP-1-mediated sorting and Wnt signaling.
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Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Factor de Transcripción AP-1/genética , Vía de Señalización Wnt , Proteínas de Pez Cebra/genética , Animales , Línea Celular , Endosomas/metabolismo , Receptores Frizzled/metabolismo , Humanos , Ratones , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Transporte de Proteínas , Ratas , Factor de Transcripción AP-1/metabolismo , Pez Cebra , Proteínas de Pez Cebra/metabolismo , Red trans-Golgi/metabolismoRESUMEN
Polyamines are important regulators of basal cellular functions but also subserve highly specific tasks in the mammalian brain. With this respect, polyamines and the synthesizing and degrading enzymes are clearly differentially distributed in neurons versus glial cells and also in different brain areas. The synthesis of the diamine putrescine may be driven via two different pathways. In the "classical" pathway urea and carbon dioxide are removed from arginine by arginase and ornithine decarboxylase. The alternative pathway, first removing carbon dioxide by arginine decarboxlyase and then urea by agmatinase, may serve the same purpose. Furthermore, the intermediate product of the alternative pathway, agmatine, is an endogenous ligand for imidazoline receptors and may serve as a neurotransmitter. In order to evaluate and compare the expression patterns of the two gate keeper enzymes arginase and arginine decarboxylase, we generated polyclonal, monospecific antibodies against arginase-1 and arginine decarboxylase. Using these tools, we immunocytochemically screened the rat brain and compared the expression patterns of both enzymes in several brain areas on the regional, cellular and subcellular level. In contrast to other enzymes of the polyamine pathway, arginine decarboxylase and arginase are both constitutively and widely expressed in rat brain neurons. In cerebral cortex and hippocampus, principal neurons and putative interneurons were clearly labeled for both enzymes. Labeling, however, was strikingly different in these neurons with respect to the subcellular localization of the enzymes. While with antibodies against arginine decarboxylase the immunosignal was distributed throughout the cytoplasm, arginase-like immunoreactivity was preferentially localized to Golgi stacks. Given the apparent congruence of arginase and arginine decarboxylase distribution with respect to certain cell populations, it seems likely that the synthesis of agmatine rather than putrescine may be the main purpose of the alternative pathway of polyamine synthesis, while the classical pathway supplies putrescine and spermidine/spermine in these neurons.