RESUMEN
The endoplasmic reticulum (ER) is an organelle with remarkable plasticity, capable of rapidly changing its structure to accommodate different functions based on intra- and extracellular cues. One of the ER structures observed in plants is known as "organized smooth endoplasmic reticulum" (OSER), consisting of symmetrically stacked ER membrane arrays. In plants, these structures were first described in certain specialized tissues, e.g. the sieve elements of the phloem, and more recently in transgenic plants overexpressing ER membrane resident proteins. To date, much of the investigation of OSER focused on yeast and animal cells but research into plant OSER has started to grow. In this update, we give a succinct overview of research into the OSER phenomenon in plant cells with case studies highlighting both native and synthetic occurrences of OSER. We also assess the primary driving forces that trigger the formation of OSER, collating evidence from the literature to compare two competing theories for the origin of OSER: that OSER formation is initiated by oligomerizing protein accumulation in the ER membrane or that OSER is the result of ER membrane proliferation. This has long been a source of controversy in the field and here we suggest a way to integrate arguments from both sides into a single unifying theory. Finally, we discuss the potential biotechnological uses of OSER as a tool for the nascent plant synthetic biology field with possible applications as a synthetic microdomain for metabolic engineering and as an extensive membrane surface for synthetic chemistry or protein accumulation.
Asunto(s)
Vías Biosintéticas , Retículo Endoplásmico Liso/fisiología , Retículo Endoplásmico Liso/ultraestructura , Membranas Intracelulares/fisiología , Membranas Intracelulares/ultraestructura , Células Vegetales/fisiología , Células Vegetales/ultraestructuraRESUMEN
Prevention of mitochondrial DNA (mtDNA) diseases may currently be possible using germline nuclear transfer (NT). However, scientific evidence to compare efficiency of different NT techniques to overcome mtDNA diseases is lacking. Here, we performed four types of NT, including first or second polar body transfer (PB1/2T), maternal spindle transfer (ST) and pronuclear transfer (PNT), using NZB/OlaHsd and B6D2F1 mouse models. Embryo development was assessed following NT, and mtDNA carry-over levels were measured by next generation sequencing (NGS). Moreover, we explored two novel protocols (PB2T-a and PB2T-b) to optimize PB2T using mouse and human oocytes. Chromosomal profiles of NT-generated blastocysts were evaluated using NGS. In mouse, our findings reveal that only PB2T-b successfully leads to blastocysts. There were comparable blastocyst rates among PB1T, PB2T-b, ST and PNT embryos. Furthermore, PB1T and PB2T-b had lower mtDNA carry-over levels than ST and PNT. After extrapolation of novel PB2T-b to human in vitro matured (IVM) oocytes and in vivo matured oocytes with smooth endoplasmic reticulum aggregate (SERa) oocytes, the reconstituted embryos successfully developed to blastocysts at a comparable rate to ICSI controls. PB2T-b embryos generated from IVM oocytes showed a similar euploidy rate to ICSI controls. Nevertheless, our mouse model with non-mutated mtDNAs is different from a mixture of pathogenic and non-pathogenic mtDNAs in a human scenario. Novel PB2T-b requires further optimization to improve blastocyst rates in human. Although more work is required to elucidate efficiency and safety of NT, our study suggests that PBT may have the potential to prevent mtDNA disease transmission.
Asunto(s)
ADN Mitocondrial/genética , Enfermedades Mitocondriales/prevención & control , Terapia de Reemplazo Mitocondrial/métodos , Técnicas de Transferencia Nuclear , Cuerpos Polares/trasplante , Animales , Blastocisto/citología , Retículo Endoplásmico Liso/fisiología , Humanos , Ratones , Mitocondrias/genética , Enfermedades Mitocondriales/genética , Oocitos/crecimiento & desarrollo , Oocitos/trasplanteRESUMEN
C-type synaptic boutons (C-boutons) provide cholinergic afferent input to spinal cord motor neurons (MNs), which display an endoplasmic reticulum (ER)-related subsurface cistern (SSC) adjacent to their postsynaptic membrane. A constellation of postsynaptic proteins is clustered at C-boutons, including M2 muscarinic receptors, potassium channels, and σ-1 receptors. In addition, we previously found that neuregulin (NRG)1 is associated with C-boutons at postsynaptic SSCs, whereas its ErbB receptors are located in the presynaptic compartment. C-bouton-mediated regulation of MN excitability has been implicated in MN disease, but NRG1-mediated functions and the impact of various pathologic conditions on C-bouton integrity have not been studied in detail. Here, we investigated changes in C-boutons after electrical stimulation, pharmacological treatment, and peripheral nerve axotomy. SSC-linked NRG1 clusters were severely disrupted in acutely stressed MNs and after tunicamycin-induced ER stress. In axotomized MNs, C-bouton loss occurred in concomitance with microglial recruitment and was prevented by the ER stress inhibitor salubrinal. Activated microglia displayed a positive chemotaxis to C-boutons. Analysis of transgenic mice overexpressing NRG1 type I and type III isoforms in MNs indicated that NRG1 type III acts as an organizer of SSC-like structures, whereas NRG1 type I promotes synaptogenesis of presynaptic cholinergic terminals. Moreover, MN-derived NRG1 signals may regulate the activity of perineuronal microglial cells. Together, these data provide new insights into the molecular and cellular pathology of C-boutons in MN injury and suggest that distinct NRG1 isoform-mediated signaling functions regulate the complex matching between pre- and postsynaptic C-bouton elements.-Salvany, S., Casanovas, A., Tarabal, O., Piedrafita, L., Hernández, S., Santafé, M., Soto-Bernardini, M. C., Calderó, J., Schwab, M. H., Esquerda, J. E. Localization and dynamic changes of neuregulin-1 at C-type synaptic boutons in association with motor neuron injury and repair.
Asunto(s)
Células del Asta Anterior/fisiología , Fibras Nerviosas Amielínicas/fisiología , Regeneración Nerviosa/fisiología , Neurregulina-1/fisiología , Terminales Presinápticos/fisiología , Nervio Ciático/lesiones , Animales , Axotomía , Fibras Colinérgicas/fisiología , Cinamatos/farmacología , Estimulación Eléctrica , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/fisiología , Retículo Endoplásmico Liso/fisiología , Retículo Endoplásmico Liso/ultraestructura , Ratones , Ratones Transgénicos , Microglía/fisiología , Compresión Nerviosa , Neurregulina-1/genética , Terminales Presinápticos/efectos de los fármacos , Isoformas de Proteínas/fisiología , Nervio Ciático/fisiología , Transducción de Señal/fisiología , Fracciones Subcelulares/química , Tiourea/análogos & derivados , Tiourea/farmacología , Tunicamicina/toxicidad , Vacuolas/metabolismo , Vacuolas/ultraestructuraRESUMEN
Pigment aggregation in shrimp chromatophores is triggered by red pigment concentrating hormone (RPCH), a neurosecretory peptide whose plasma membrane receptor may be a G-protein coupled receptor (GPCR). While RPCH binding activates the Ca2+ /cGMP signaling cascades, a role for cyclic AMP (cAMP) in pigment aggregation is obscure, as are the steps governing Ca2+ release from the smooth endoplasmic reticulum (SER). A role for the antagonistic neuropeptide, pigment dispersing homone (α-PDH) is also unclear. In red, ovarian chromatophores from the freshwater shrimp Macrobrachium olfersi, we show that a G-protein antagonist (AntPG) strongly inhibits RPCH-triggered pigment aggregation, suggesting that RPCH binds to a GPCR, activating an inhibitory G-protein. Decreasing cAMP levels may cue pigment aggregation, since cytosolic cAMP titers, when augmented by cholera toxin, forskolin or vinpocentine, completely or partially impair pigment aggregation. Triggering opposing Ca2+ /cGMP and cAMP cascades by simultaneous perfusion with lipid-soluble cyclic nucleotide analogs induces a "tug-of-war" response, pigments aggregating in some chromatosomes with unpredictable, oscillatory movements in others. Inhibition of cAMP-dependent protein kinase accelerates aggregation and reduces dispersion velocities, suggesting a role in phosphorylation events, possibly regulating SER Ca2+ release and pigment aggregation. The second messengers IP3 and cADPR do not stimulate SER Ca2+ release. α-PDH does not sustain pigment dispersion, suggesting that pigment translocation in caridean chromatophores may be regulated solely by RPCH, since PDH is not required. We propose a working hypothesis to further unravel key steps in the mechanisms of pigment translocation within crustacean chromatophores that have remained obscure for nearly a century.
Asunto(s)
Cromatóforos/fisiología , Palaemonidae/fisiología , Pigmentos Biológicos/metabolismo , Sistemas de Mensajero Secundario/fisiología , Transducción de Señal/fisiología , Animales , Calcio/metabolismo , Retículo Endoplásmico Liso/fisiología , Femenino , Regulación de la Expresión Génica/fisiologíaRESUMEN
The endoplasmic reticulum (ER) is a highly dynamic organelle. It is composed of four subcompartments including nuclear envelope (NE), rough ER (rER), smooth ER (sER) and transitional ER (tER). The subcompartments are interconnected, can fragment and dissociate and are able to reassemble again. They coordinate with cell function by way of protein regulators in the surrounding cytosol. The activity of the many associated molecular machines of the ER as well as the fluid nature of the limiting membrane of the ER contribute extensively to the dynamics of the ER. This review examines the properties of the ER that permit its isolation and purification and the physiological conditions that permit reconstitution both in vitro and in vivo in normal and in disease conditions.
Asunto(s)
Retículo Endoplásmico Rugoso/ultraestructura , Retículo Endoplásmico Liso/ultraestructura , Retículo Endoplásmico/fisiología , Retículo Endoplásmico/ultraestructura , Membrana Nuclear/ultraestructura , Animales , Fraccionamiento Celular , Citoesqueleto/metabolismo , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico Rugoso/metabolismo , Retículo Endoplásmico Rugoso/fisiología , Retículo Endoplásmico Liso/metabolismo , Retículo Endoplásmico Liso/fisiología , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/fisiología , Membranas Intracelulares/ultraestructura , Fusión de Membrana , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Membrana Nuclear/metabolismo , Membrana Nuclear/fisiología , Orgánulos/metabolismo , Orgánulos/fisiología , Ribosomas/metabolismo , Ribosomas/fisiología , Ribosomas/ultraestructura , Fracciones SubcelularesRESUMEN
In the present study, we described ultrastructural changes occurring in the neurons of the hypothalamic arcuate nucleus after food deprivation. Young male Wistar rats (5 months old, n = 12) were divided into three groups. The animals in Group I were used as control (normally fed), and the rats in Groups II and III were fasted for 48 hours and 96 hours, respectively. In both treated groups, fasting caused rearrangement of the rough endoplasmic reticulum forming lamellar bodies and membranous whorls. The lamellar bodies were rather short in the controls, whereas in the fasting animals they became longer and were sometimes participating in the formation of membranous whorls composed of the concentric layers of the smooth endoplasmic reticulum. The whorls were often placed in the vicinity of a very well developed Golgi complex. Some Golgi complexes displayed an early stage of whorl formation. Moreover, an increased serum level of 8-isoprostanes, being a reliable marker of total oxidative stress in the body, was observed in both fasting groups of rats as compared to the control.
Asunto(s)
Núcleo Arqueado del Hipotálamo/fisiología , Núcleo Arqueado del Hipotálamo/ultraestructura , Ayuno/fisiología , Neuronas/fisiología , Neuronas/ultraestructura , Estrés Oxidativo/fisiología , Factores de Edad , Envejecimiento/fisiología , Animales , Regulación del Apetito/fisiología , Biomarcadores/sangre , Peso Corporal/fisiología , Dinoprost/análogos & derivados , Dinoprost/sangre , Retículo Endoplásmico Rugoso/fisiología , Retículo Endoplásmico Rugoso/ultraestructura , Retículo Endoplásmico Liso/fisiología , Retículo Endoplásmico Liso/ultraestructura , Metabolismo Energético/fisiología , Privación de Alimentos/fisiología , Aparato de Golgi/fisiología , Aparato de Golgi/ultraestructura , Membranas Intracelulares/fisiología , Membranas Intracelulares/ultraestructura , Masculino , Ratas , Ratas WistarRESUMEN
The ER (endoplasmic reticulum) is composed of multiple domains including the nuclear envelope, ribosome-studded rough ER and the SER (smooth ER). The SER can also be functionally segregated into domains that regulate ER-Golgi traffic (transitional ER), ERAD (ER-associated degradation), sterol and lipid biosynthesis and calcium sequestration. The last two, as well as apoptosis, are critically regulated by the close association of the SER with mitochondria. Studies with AMFR (autocrine motility factor receptor) have defined an SER domain whose integrity and mitochondrial association can be modulated by ilimaquinone as well as by free cytosolic calcium levels in the normal physiological range. AMFR is an E3 ubiquitin ligase that targets its ligand directly to the SER via a caveolae/raft-dependent pathway. In the present review, we will address the relationship between the calcium-dependent morphology and mitochondrial association of the SER and its various functional roles in the cell.
Asunto(s)
Retículo Endoplásmico Liso/fisiología , Mitocondrias/fisiología , Animales , Células Cultivadas , Retículo Endoplásmico Liso/ultraestructura , Humanos , Mitocondrias/ultraestructuraRESUMEN
Simian Virus 40 (SV40) has been shown to enter host cells by caveolar endocytosis followed by transport via caveosomes to the endoplasmic reticulum (ER). Using a caveolin-1 (cav-1)-deficient cell line (human hepatoma 7) and embryonic fibroblasts from a cav-1 knockout mouse, we found that in the absence of caveolae, but also in wild-type embryonic fibroblasts, the virus exploits an alternative, cav-1-independent pathway. Internalization was rapid (t1/2 = 20 min) and cholesterol and tyrosine kinase dependent but independent of clathrin, dynamin II, and ARF6. The viruses were internalized in small, tight-fitting vesicles and transported to membrane-bounded, pH-neutral organelles similar to caveosomes but devoid of cav-1 and -2. The viruses were next transferred by microtubule-dependent vesicular transport to the ER, a step that was required for infectivity. Our results revealed the existence of a virus-activated endocytic pathway from the plasma membrane to the ER that involves neither clathrin nor caveolae and that can be activated also in the presence of cav-1.
Asunto(s)
Caveolas/fisiología , Caveolinas/fisiología , Clatrina/fisiología , Endocitosis/fisiología , Virus 40 de los Simios/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/fisiología , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Antígenos Virales de Tumores/metabolismo , Brefeldino A/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Proteínas de Unión al Calcio/genética , Caveolina 1 , Caveolina 2 , Caveolinas/análisis , Caveolinas/genética , Línea Celular , Línea Celular Tumoral , Colesterol/deficiencia , Colesterol/fisiología , Detergentes/química , Dinamina II/genética , Dinamina II/fisiología , Embrión de Mamíferos/citología , Endocitosis/efectos de los fármacos , Retículo Endoplásmico Liso/química , Retículo Endoplásmico Liso/fisiología , Fibroblastos/efectos de los fármacos , Fibroblastos/ultraestructura , Fibroblastos/virología , Expresión Génica , Genisteína/farmacología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Microdominios de Membrana/química , Microdominios de Membrana/fisiología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Microtúbulos/efectos de los fármacos , Microtúbulos/fisiología , Nocodazol/farmacología , Fosfoproteínas/genética , Virus de los Bosques Semliki/fisiología , Tiazoles/farmacología , Tiazolidinas , Transferrina/metabolismo , Vesículas Transportadoras/fisiología , Vesículas Transportadoras/ultraestructuraAsunto(s)
Bloqueadores de los Canales de Calcio/administración & dosificación , Carpa Dorada/fisiología , Neuronas/fisiología , Transmisión Sináptica/efectos de los fármacos , Verapamilo/administración & dosificación , Animales , Calcio/metabolismo , Canales de Calcio/fisiología , Retículo Endoplásmico Liso/fisiología , Neuronas/ultraestructuraRESUMEN
A two-dimensional intracellular Ca(2+) ([Ca(2+)](i)) imaging system was used to examine the relationship between [Ca(2+)](i) handling and the proliferation of MC3T3-E1 osteoblast-like cells. The resting [Ca(2+)](i) level in densely cultured cells was 1.5 times higher than the [Ca(2+)](i) level in sparsely cultured cells or in other cell types (mouse fibroblasts, rat vascular smooth muscle cells, and bovine endothelial cells). A high resting [Ca(2+)](i) level may be specific for MC3T3-E1 cells. MC3T3-E1 cells were stimulated with ATP (10 microM), caffeine (10 mM), thapsigargin (1 microM), or ionomycin (10 microM), and the effect on the [Ca(2+)](i) level of MC3T3-E1 cells was studied. The percentage of responding cells and the degree of [Ca(2+)](i) elevation were high in the sparsely cultured cells and low in densely cultured cells. The rank order for the percentage of responding cells and magnitude of the Ca(2+) response to the stimuli was ionomycin > thapsigargin = ATP > caffeine and suggests the existence of differences among the various [Ca(2+)](i) channels. All Ca(2+) responses in the sparsely cultured MC3T3-E1 cells, unlike in other cell types, disappeared after the cells reached confluence. Heptanol treatment of densely cultured cells restored the Ca(2+) response, suggesting that cell-cell contact is involved with the confluence-dependent disappearance of the Ca(2+) response. Immunohistological analysis of type 1 inositol trisphosphate receptors and electron microscopy showed distinct expression of inositol trisphosphate receptor proteins and smooth-surfaced endoplasmic reticulum in sparsely cultured cells but reduced levels in densely cultured cells. These results indicate that the underlying basis of confluence-dependent [Ca(2+)](i) regulation is down-regulation of smooth-surfaced endoplasmic reticulum by cell-cell contacts.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Retículo Endoplásmico Liso/fisiología , Osteoblastos/fisiología , Células 3T3 , Adenosina Trifosfato/farmacología , Animales , Cafeína/farmacología , Señalización del Calcio/efectos de los fármacos , Diferenciación Celular , División Celular , Retículo Endoplásmico Liso/efectos de los fármacos , Retículo Endoplásmico Liso/ultraestructura , Cinética , Ratones , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Tapsigargina/farmacologíaRESUMEN
Molecular chaperones assist in the biosynthesis and processing of proteins. Most chaperones are induced by physiological stresses. We have shown that dietary energy restriction decreases the mRNA and protein levels of many endoplasmic reticulum chaperones in the livers of mice. Here, we have investigated the response of chaperone mRNA to feeding. Control and 50% energy-restricted C3B10RF1 mice were deprived of food for 24 h, fed, and killed 0, 1.5, 5 or 12 h after feeding. Chaperone mRNAs were strongly induced as early as 1.5 h after feeding in control and energy-restricted mice. The integrated levels of these mRNA over 24 h were significantly lower in energy-restricted mice. The mRNA response to energy intake was mirrored over the course of days in the level of chaperone protein. A similar but smaller response to feeding was found in kidney and muscle. Puromycin and cycloheximide failed to inhibit the feeding response, suggesting that feeding releases chaperone expression from an unstable inhibitor. Studies with dibutyryl-cAMP- and glucagon-supplemented, normal and streptozotocin-diabetic mice suggest that glucagon and insulin may be mediators of the feeding response. Adrenalectomy enhanced the feeding induction, but dexamethasone administration had no effect. Thus, postprandial changes in insulin and glucagon may link chaperone gene expression to feeding, possibly in several tissues including liver.
Asunto(s)
Retículo Endoplásmico Liso/metabolismo , Ingestión de Energía/fisiología , Proteínas de Choque Térmico , Hígado/metabolismo , Chaperonas Moleculares/metabolismo , ARN Mensajero/aislamiento & purificación , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Chaperón BiP del Retículo Endoplásmico , Retículo Endoplásmico Liso/genética , Retículo Endoplásmico Liso/fisiología , Femenino , Privación de Alimentos/fisiología , Regulación de la Expresión Génica , Glucagón/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Insulina/metabolismo , Riñón/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Chaperonas Moleculares/genética , Músculos/metabolismo , Periodo PosprandialRESUMEN
Cellular cholesterol homoeostasis is regulated through proteolysis of the membrane-bound precursor sterol-regulatory-element-binding protein (SREBP) that releases the mature transcription factor form, which regulates gene expression. Our aim was to identify the nature and intracellular site of the putative sterol-regulatory pool which regulates SREBP proteolysis in hamster liver. Cholesterol metabolism was modulated by feeding hamsters control chow, or a cholesterol-enriched diet, or by treatment with simvastatin or with the oral acyl-CoA:cholesterol acyltransferase inhibitor C1-1011 plus cholesterol. The effects of the different treatments on SREBP activation were confirmed by determination of the mRNAs for the low-density lipoprotein receptor and hydroxymethylglutaryl-CoA (HMG-CoA) reductase and by measurement of HMG-CoA reductase activity. The endoplasmic reticulum was isolated from livers and separated into subfractions by centrifugation in self-generating iodixanol gradients. Immunodetectable SREBP-2 accumulated in the smooth endoplasmic reticulum of cholesterol-fed animals. Cholesterol ester levels of the smooth endoplasmic reticulum membrane (but not the cholesterol levels) increased after cholesterol feeding and fell after treatment with simvastatin or C1-1011. The results suggest that an increased cellular cholesterol load causes accumulation of SREBP-2 in the smooth endoplasmic reticulum and, therefore, that membrane cholesterol ester may be one signal allowing exit of the SREBP-2/SREBP-cleavage-regulating protein complex to the Golgi.
Asunto(s)
Acetatos , Ésteres del Colesterol/metabolismo , Proteínas de Unión al ADN/metabolismo , Retículo Endoplásmico Liso/fisiología , Factores de Transcripción/metabolismo , Acetamidas , Animales , Colesterol/análisis , Cricetinae , Proteínas de Unión al ADN/genética , Dieta Aterogénica , Inhibidores Enzimáticos/farmacología , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Reductasas/metabolismo , Membranas Intracelulares/química , Hígado/metabolismo , Mesocricetus , ARN Mensajero/biosíntesis , Receptores de LDL/biosíntesis , Receptores de LDL/genética , Simvastatina/farmacología , Esterol O-Aciltransferasa/antagonistas & inhibidores , Proteína 2 de Unión a Elementos Reguladores de Esteroles , Sulfonamidas , Ácidos Sulfónicos/farmacología , Factores de Transcripción/genética , Triglicéridos/análisisRESUMEN
Transitional endoplasmic reticulum (tER) consists of confluent rough and smooth endoplasmic reticulum (ER) domains. In a cell-free incubation system, low-density microsomes (1.17 g cc(-1)) isolated from rat liver homogenates reconstitute tER by Mg(2+)GTP- and Mg(2+)ATP-hydrolysis-dependent membrane fusion. The ATPases associated with different cellular activities protein p97 has been identified as the relevant ATPase. The ATP depletion by hexokinase or treatment with either N-ethylmaleimide or anti-p97 prevented assembly of the smooth ER domain of tER. High-salt washing of low-density microsomes inhibited assembly of the smooth ER domain of tER, whereas the readdition of purified p97 with associated p47 promoted reconstitution. The t-SNARE syntaxin 5 was observed within the smooth ER domain of tER, and antisyntaxin 5 abrogated formation of this same membrane compartment. Thus, p97 and syntaxin 5 regulate assembly of the smooth ER domain of tER and hence one of the earliest membrane differentiated components of the secretory pathway.
Asunto(s)
Adenosina Trifosfatasas/fisiología , Retículo Endoplásmico Rugoso/fisiología , Retículo Endoplásmico Liso/fisiología , Proteínas de la Membrana/fisiología , Proteínas Nucleares/fisiología , Adenosina Trifosfatasas/inmunología , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/química , Animales , Anticuerpos/farmacología , Sistema Libre de Células/metabolismo , Retículo Endoplásmico Rugoso/metabolismo , Retículo Endoplásmico Rugoso/ultraestructura , Retículo Endoplásmico Liso/efectos de los fármacos , Retículo Endoplásmico Liso/ultraestructura , Inhibidores Enzimáticos/farmacología , Etilmaleimida/farmacología , Guanosina Trifosfato/química , Hexoquinasa/metabolismo , Membranas Intracelulares/ultraestructura , Fusión de Membrana , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/ultraestructura , Microscopía Electrónica , Microsomas Hepáticos/metabolismo , Microsomas Hepáticos/ultraestructura , Proteínas Nucleares/inmunología , Proteínas Nucleares/metabolismo , Proteínas Qa-SNARE , RatasRESUMEN
The release of chemical transmitter from nerve terminals is critically dependent on a transient increase in intracellular Ca2+. The increase in Ca2+ may be due to influx of Ca2+ from the extracellular fluid or release of Ca2+ from intracellular stores such as mitochondria. Whether Ca2+ utilized in transmitter release is liberated from organelles other than mitochondria is uncertain. Smooth endoplasmic reticulum is known to release Ca2+, e.g., on activation by inositol trisphosphate or cyclic adenosine diphosphate-ribose, so the possibility exists that Ca2+ from this source may be involved in the events leading to exocytosis. We examined this hypothesis by testing whether inositol trisphosphate and cyclic adenosine diphosphate-ribose modified transmitter release. We used liposomes to deliver these agents into the cytoplasmic compartment and binomial analysis to determine their effects on the quantal components of transmitter release. Administration of inositol trisphosphate (10(-4)M) caused a rapid, 25% increase in the number of quanta released. This was due to an increase in the number of functional release sites, as the other quantal parameters were unaffected. The effect was reversed with 40 min of wash. Virtually identical results were obtained with cyclic adenosine diphosphate-ribose (10(-4)M). Inositol trisphosphate caused a 10% increase in quantal size, whereas cyclic adenosine diphosphate-ribose had no effect. The results suggest that quantal transmitter release can be increased by Ca2+ released from smooth endoplasmic reticulum upon stimulation by inositol trisphosphate or cyclic adenosine diphosphate-ribose. This may involve priming of synaptic vesicles at the release sites or mobilization of vesicles to the active zone. Inositol trisphosphate may have an additional action to increase the content of transmitter within the vesicles. These findings raise the possibility of a role of endogenous inositol phosphate and smooth endoplasmic reticulum in the regulation of cytoplasmic Ca2+ and transmitter release.
Asunto(s)
Adenosina Difosfato Ribosa/análogos & derivados , Inositol 1,4,5-Trifosfato/farmacología , Neuronas Motoras/metabolismo , Terminaciones Nerviosas/metabolismo , Neurotransmisores/metabolismo , Adenosina Difosfato Ribosa/farmacología , Animales , ADP-Ribosa Cíclica , Retículo Endoplásmico Liso/fisiología , Rana pipiensRESUMEN
Administration of tellurium (Te) in weaning rats causes a well-established demyelinating neuropathy induced by the inhibition in myelinating Schwann cells (SC) of the synthesis of cholesterol, a major component of the myelin sheath, at the level of squalene epoxidase. We have used this experimental model of Te neuropathy to study the biogenesis and reorganization of the endomembranes of the nuclear envelope and endoplasmic reticulum (ER) in response to Te treatment by ultrastructural analysis and in situ hybridization for the detection of HMG CoA reductase and synthase mRNA, which encode key enzymes in cholesterol synthesis. The adaptive response of myelinating SC to cholesterol depletion includes cell hypertrophy, the formation of tubular invaginations of proliferating nuclear membranes giving rise to peculiar nuclear inclusions termed crystalloids, and, at the cytoplasmic level, the formation of lamellar bodies of rough ER, proliferation of the smooth ER, and overexpression of HMG CoA reductase and synthase mRNAs. The changes revert after withdrawal of Te treatment. Our results show that the biogenesis and structural organization of both endomembrane systems change dynamically upon Te-induced cholesterol depletion, indicating that this constituent plays a critical role in the organization of nuclear envelope and ER compartments in SC. The results also suggest that the HMG CoA reductase, an integral membrane protein of ER, provides the signal for the extensive membrane assembly. While the physiological meaning of crystalloid remains to be clarified, the hypertrophy of the smooth ER may represent a cytoprotective mechanism involved in detoxification of the neurotoxic agent or its metabolic derivates.
Asunto(s)
Núcleo Celular/metabolismo , Retículo Endoplásmico Liso/fisiología , Células de Schwann/efectos de los fármacos , Células de Schwann/fisiología , Telurio/farmacología , Animales , Citoplasma/fisiología , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Sintasa/genética , Cuerpos de Inclusión/ultraestructura , Metabolismo de los Lípidos , Masculino , Microesferas , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
To determine whether there are anatomical correlates for intraterminal Ca2+ stores to regulate exocytosis of dense-cored vesicles (DCVs) and whether these stores can modulate exocytosis of synaptic vesicles, we studied the spatial distributions of DCVs, smooth endoplasmic reticulum (SER), and mitochondria in 19 serially reconstructed nerve terminals in bullfrog sympathetic ganglia. On average, each bouton had three active zones, 214 DCVs, 26 SER fragments (SERFs), and eight mitochondria. DCVs, SERFs and mitochondria were located, on average, 690, 624, and 526 nm, respectively, away from active zones. Virtually no DCVs were within "docking" (i.e., < or = 50 nm) distances of the active zones. Thus, it is unlikely that DCV exocytosis occurs at active zones via mechanisms similar to those for exocytosis of synaptic vesicles. Because there were virtually no SERFs or mitochondria within 50 nm of any active zone, Ca2+ modulation by these organelles is unlikely to affect ACh release evoked by a single action potential. In contrast, 30% of DCVs and 40% of SERFs were located within 50 nm of the nonspecialized regions of the plasma membrane. Because each bouton had at least one SERF within 50 nm of the plasma membrane and most of these SERFs had DCVs, but not mitochondria, near them, it is possible for Ca2+ release from the SER to provide the Ca2+ necessary for DCV exocytosis. The fact that 60% of the mitochondria had some part within 50 nm of the plasma membrane means that it is possible for mitochondrial Ca2+ buffering to affect DCV exocytosis.
Asunto(s)
Calcio/metabolismo , Orgánulos/fisiología , Orgánulos/ultraestructura , Terminales Presinápticos/fisiología , Terminales Presinápticos/ultraestructura , Animales , Fraccionamiento Celular , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Retículo Endoplásmico Liso/fisiología , Retículo Endoplásmico Liso/ultraestructura , Exocitosis , Ganglios Simpáticos/fisiología , Ganglios Simpáticos/ultraestructura , Microscopía Electrónica , Mitocondrias/fisiología , Mitocondrias/ultraestructura , Rana catesbeiana , Vesículas Sinápticas/fisiología , Vesículas Sinápticas/ultraestructuraRESUMEN
Endoplasmic reticulum structure was studied on Mauthner neurons of adult fish and fry. Two-fold decrease of extension of rough endoplasmic reticulum components and similar increase of extension of smooth endoplasmic reticulum components were found to occur following long-term stimulation of Mauthner neurons. The significance of transformation of rough endoplasmic reticulum into smooth one as a result of neuronal compensatory response was discussed.
Asunto(s)
Adaptación Fisiológica , Retículo Endoplásmico Rugoso/ultraestructura , Retículo Endoplásmico Liso/ultraestructura , Neuronas/ultraestructura , Animales , Retículo Endoplásmico Rugoso/fisiología , Retículo Endoplásmico Liso/fisiología , Peces , Carpa Dorada , Microscopía Electrónica/métodos , Neuronas/fisiología , Estimulación Física , Puente/fisiología , Puente/ultraestructuraRESUMEN
Autocrine motility factor receptor (AMF-R) is a cell surface receptor that is also localized to a smooth subdomain of the endoplasmic reticulum, the AMF-R tubule. By postembedding immunoelectron microscopy, AMF-R concentrates within smooth plasmalemmal vesicles or caveolae in both NIH-3T3 fibroblasts and HeLa cells. By confocal microscopy, cell surface AMF-R labeled by the addition of anti-AMF-R antibody to viable cells at 4 degreesC exhibits partial colocalization with caveolin, confirming the localization of cell surface AMF-R to caveolae. Labeling of cell surface AMF-R by either anti-AMF-R antibody or biotinylated AMF (bAMF) exhibits extensive colocalization and after a pulse of 1-2 h at 37 degreesC, bAMF accumulates in densely labeled perinuclear structures as well as fainter tubular structures that colocalize with AMF-R tubules. After a subsequent 2- to 4-h chase, bAMF is localized predominantly to AMF-R tubules. Cytoplasmic acidification, blocking clathrin-mediated endocytosis, results in the essentially exclusive distribution of internalized bAMF to AMF-R tubules. By confocal microscopy, the tubular structures labeled by internalized bAMF show complete colocalization with AMF-R tubules. bAMF internalized in the presence of a 10-fold excess of unlabeled AMF labels perinuclear punctate structures, which are therefore the product of fluid phase endocytosis, but does not label AMF-R tubules, demonstrating that bAMF targeting to AMF-R tubules occurs via a receptor-mediated pathway. By electron microscopy, bAMF internalized for 10 min is located to cell surface caveolae and after 30 min is present within smooth and rough endoplasmic reticulum tubules. AMF-R is therefore internalized via a receptor-mediated clathrin-independent pathway to smooth ER. The steady state localization of AMF-R to caveolae implicates these cell surface invaginations in AMF-R endocytosis.