RESUMEN
Tunneling nanotubes (TNTs) are F-actin-based membrane tubes that form between cells in culture and in tissues. They mediate intercellular communication ranging from electrical signalling to the transfer of organelles. Here, we studied the role of TNTs in the interaction between apoptotic and healthy cells. We found that pheochromocytoma (PC) 12 cells treated with ultraviolet light (UV) were rescued when cocultured with untreated PC12 cells. UV-treated cells formed a different type of TNT with untreated PC12 cells, which was characterized by continuous microtubule localized inside these TNTs. The dynamic behaviour of mCherry-tagged end-binding protein 3 and the accumulation of detyrosinated tubulin in these TNTs indicate that they are regulated structures. In addition, these TNTs show different biophysical properties, for example, increased diameter allowing dye entry, prolonged lifetime and decreased membrane fluidity. Further studies demonstrated that microtubule-containing TNTs were formed by stressed cells, which had lost cytochrome c but did not enter into the execution phase of apoptosis characterized by caspase-3 activation. Moreover, mitochondria colocalized with microtubules in TNTs and transited along these structures from healthy to stressed cells. Importantly, impaired formation of TNTs and untreated cells carrying defective mitochondria were unable to rescue UV-treated cells in the coculture. We conclude that TNT-mediated transfer of functional mitochondria reverse stressed cells in the early stages of apoptosis. This provides new insights into the survival mechanisms of damaged cells in a multicellular context.
Asunto(s)
Apoptosis/fisiología , Comunicación Celular/fisiología , Mitocondrias , Nanotubos/química , Actinas , Animales , Técnicas de Cocultivo , Microtúbulos , Células PC12 , RatasAsunto(s)
Comunicación Celular/fisiología , Estructuras de la Membrana Celular/fisiología , Estructuras de la Membrana Celular/ultraestructura , Proteína p53 Supresora de Tumor/fisiología , Neoplasias de las Glándulas Suprarrenales/patología , Neoplasias de las Glándulas Suprarrenales/fisiopatología , Animales , Neoplasias Óseas/patología , Neoplasias Óseas/fisiopatología , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/fisiopatología , Células Madre Mesenquimatosas/patología , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteosarcoma/patología , Osteosarcoma/fisiopatología , Feocromocitoma/patología , Feocromocitoma/fisiopatología , Ratas , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genéticaRESUMEN
INTRODUCTION: Total gastrectomy (TG) is commonly performed for the treatment of patients with gastric cancer. However, reconstruction of the esophagojejunal (EJ) anastomosis can be technically demanding, with reported anastomotic leak rates in the Western world still approaching 10-15%. We report our experience using the transoral anvil delivery system (OrVil™) for creation of the EJ anastomosis after TG. METHODS: From 2007 to 2011, 48 consecutive patients with gastric cancer underwent open (n=31) or laparoscopic (n=17) TG. EJ reconstruction was performed with the transoral anvil deliver system (OrVil™) in an end-to-side fashion. Demographic, clinic, and perioperative data were obtained from a prospectively maintained database. RESULTS: Of the 48 patients, 83% were male. Median age at resection was 64 years. Median body mass index was 27.1 kg/m2. Seventy-nine percent (n=38) of patients had at least one comorbidity. Fifteen patients (31%) had at least one perioperative complication. There was one perioperative death (2%) following a duodenal stump leak. There were four EJ leaks (8%) and two EJ stenoses (independent of leak; 4%). There was one EJ leak (6%) and one EJ stenosis (6%) following a case that was first attempted laparoscopically. There were no deaths as a consequence of an EJ leak. CONCLUSIONS: The use of the transoral anvil delivery system during EJ reconstruction is a safe and effective option for reconstruction after open or laparoscopic TG with acceptable mortality and morbidity. The anastomotic leak rate appears to be comparable to that of other techniques.
Asunto(s)
Anastomosis Quirúrgica , Esófago/cirugía , Gastrectomía , Yeyuno/cirugía , Laparoscopía , Neoplasias Gástricas/cirugía , Grapado Quirúrgico/instrumentación , Anciano , Fuga Anastomótica , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Procedimientos Quirúrgicos Mínimamente Invasivos , Pronóstico , Estudios Prospectivos , Procedimientos de Cirugía Plástica , Grapado Quirúrgico/métodosRESUMEN
INTRODUCTION: Spontaneous visceral artery dissection is an uncommon cause of acute abdominal pain. Complications are ischemia, aneurysm formation and rupture. We present a case with synchronous rupture of the splenic artery causing massive bleeding and demanding urgent surgery. To our knowledge, only 24 previous cases are reported in the literature. REPORT: The patient was a 56-year-old male smoker with no previous medical history who was treated surgically with exposure of the suprarenal aorta through left-sided medial visceral rotation and isolation of the celiac artery. The origin of the bleeding was identified as a longitudinal rupture of the splenic artery just distal to the hepatic artery. The artery was ligated and splenectomy was performed because of splenic infarction. The hepatic artery was patent and no reconstruction was needed. The postoperative course was uneventful, treatment with antiplatelets and antihypertensive drugs was initiated. The patient was discharged after ten days and at monthly follow-up the patient was in good condition. CT angiography was performed six months postoperative and the celiac trunk was patent but a small aneurysm had developed. DISCUSSION: Dissection of the celiac artery is uncommon and is rarely considered in the diagnosis of acute abdominal pain. The condition could be mistaken for a ruptured AAA. The condition may be underdiagnosed and it seems likely that more cases will be identified in the future as a result of the rapidly evolving vascular imaging modalities.
Asunto(s)
Abdomen Agudo/etiología , Disección Aórtica/diagnóstico , Arteria Celíaca , Hemorragia/etiología , Arteria Esplénica , Abdomen Agudo/diagnóstico por imagen , Abdomen Agudo/cirugía , Adulto , Anciano , Disección Aórtica/complicaciones , Disección Aórtica/diagnóstico por imagen , Disección Aórtica/cirugía , Antihipertensivos/uso terapéutico , Aortografía , Arteria Celíaca/diagnóstico por imagen , Arteria Celíaca/cirugía , Diagnóstico Diferencial , Femenino , Hemorragia/diagnóstico por imagen , Hemorragia/cirugía , Humanos , Ligadura , Masculino , Persona de Mediana Edad , Inhibidores de Agregación Plaquetaria/uso terapéutico , Rotura Espontánea , Esplenectomía , Arteria Esplénica/diagnóstico por imagen , Arteria Esplénica/cirugía , Tomografía Computarizada por Rayos X , Resultado del Tratamiento , Procedimientos Quirúrgicos VascularesRESUMEN
During the last 20 years remarkable achievements have been made in the understanding of the molecular basis of membrane traffic in the secretory pathway. A combination of morphological, biochemical and genetical approaches revealed the identity of various compartments and transport intermediates, and provided basic functional insights into membrane trafficking. Recently, live cell imaging approaches further refined our understanding of the underlying mechanisms of budding, transport and fusion of transport containers, led to the discovery of new pathways and triggered new concepts as to how membrane traffic is orchestrated. This multi-author review highlights recent advances in membrane traffic by focusing on transport vesicles as the central mediators of communication in the secretory pathway.
Asunto(s)
Transporte Biológico/fisiología , Membrana Celular/metabolismo , Endocitosis/fisiología , Células Eucariotas/fisiología , Proteínas de la Membrana/metabolismoRESUMEN
It emerges that myosin Va plays multiple roles in the trafficking of SGs (secretory granules). In addition to a function in the capture and transport of newly formed SGs in the F-actin-rich cortex, myosin Va is implicated in late transport events of these organelles, which precede their exocytosis. Consistent with these roles, interactions of myosin Va with an array of well-known proteins involved in regulated protein secretion have been documented.
Asunto(s)
Exocitosis/fisiología , Cadenas Pesadas de Miosina/fisiología , Miosina Tipo V/fisiología , Vesículas Secretoras/fisiología , Homeostasis , Humanos , Melanosomas/fisiología , Mutación , Cadenas Pesadas de Miosina/genética , Miosina Tipo V/genética , Transporte de ProteínasRESUMEN
Secretory granules store neuropeptides and hormones and exhibit regulated exocytosis upon appropriate cellular stimulation. They are generated in the trans-Golgi network as immature secretory granules, short-lived vesicular intermediates, which undergo a complex and poorly understood maturation process. Due to their short half-life and low abundance, real-time studies of immature secretory granules have not been previously possible. We describe here a pulse/chase-like system based on the expression of a human chromogranin B-GFP fusion protein in neuroendocrine PC12 cells, which permits direct visualization of the budding of immature secretory granules and their dynamics during maturation. Live cell imaging revealed that newly formed immature secretory granules are transported in a direct and microtubule-dependent manner within a few seconds to the cell periphery. Our data suggest that the cooperative action of microtubules and actin filaments restricts immature secretory granules to the F-actin-rich cell cortex, where they move randomly and mature completely within a few hours. During this maturation period, secretory granules segregate into pools of different motility. In a late phase of maturation, 60% of secretory granules were found to be immobile and about half of these underwent F-actin-dependent tethering.
Asunto(s)
Actinas/metabolismo , Microtúbulos/metabolismo , Transporte de Proteínas/fisiología , Vesículas Secretoras/fisiología , Animales , Antineoplásicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Cromograninas/genética , Cromograninas/metabolismo , Furina , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Modelos Biológicos , Nocodazol/farmacología , Orgánulos/química , Orgánulos/metabolismo , Células PC12 , Transporte de Proteínas/efectos de los fármacos , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Subtilisinas/metabolismo , Tiazoles/farmacología , Tiazolidinas , TransfecciónRESUMEN
Chromogranin B, a soluble acidic secretory protein, is widely distributed in neuroendocrine and neuronal cells, although not in other cell types. To identify the elements governing such widespread, yet selective, expression of the gene, we characterized the isolated mouse chromogranin B promoter. 5'-Promoter deletions localized neuroendocrine cell type-specific expression to the proximal chromogranin B promoter (from -216 to -91 bp); this region contains an E box (at [-206 bp]CACCTG[-201 bp]), four G/C-rich regions (at [-196 bp]CCCCGC[-191 bp], [-134 bp]CCGCCCGC[-127 bp], [-125 bp]GGCGCCGCC[-117 bp], and [-115 bp]CGGGGC[-110 bp]), and a cAMP response element (CRE; at [-102 bp]TGACGTCA[-95 bp]). A 60-bp core promoter region, defined by an internal deletion from - 134 to -74 bp upstream of the cap site and spanning the CRE and three G/C-rich regions, directed tissue-specific expression of the gene. The CRE motif directed cell type-specific expression of the chromogranin B gene in neurons, whereas three of the G/C-rich regions played a crucial role in neuroendocrine cells. Both the endogenous chromogranin B gene and the transfected chromogranin B promoter were induced by preganglionic secretory stimuli (pituitary adenylyl cyclase-activating polypeptide, vasoactive intestinal peptide, or a nicotinic cholinergic agonist), establishing stimulus-transcription coupling for this promoter. The adenylyl cyclase activator forskolin, nerve growth factor, and retinoic acid also activated the chromogranin B gene. Secretagogue-inducible expression of chromogranin B also mapped onto the proximal promoter; inducible expression was entirely lost upon internal deletion of the 60-bp core (from 134 to -74 bp). We conclude that CRE and G/C-rich domains are crucial determinants of both cell type-specific and secretagogue-inducible expression of the chromogranin B gene.
Asunto(s)
Cromograninas/genética , Regulación de la Expresión Génica , Sistemas Neurosecretores/fisiología , Regiones Promotoras Genéticas/fisiología , Animales , Secuencia de Bases/genética , Cromogranina B , Eliminación de Gen , Ratones , Datos de Secuencia Molecular , Mutación/fisiología , Factor de Crecimiento Nervioso/farmacología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Sistemas Neurosecretores/citología , Células PC12 , Fenotipo , Regiones Promotoras Genéticas/genética , Ratas , Estereoisomerismo , Estimulación Química , TransfecciónRESUMEN
Neuropeptides and hormones, in contrast to constitutive secretory proteins, are sorted to and stored in secretory granules and released upon a stimulus. During the last two decades, signals and mechanisms involved in their sorting to the regulated pathway of protein secretion have been addressed in numerous studies. Taken together these studies revealed three important features of regulated secretory proteins: aggregation, sorting signal motifs and membrane binding. Here we try to dissect the sorting process with regard to these features and discuss their relevance in the context of current sorting models. We especially address the question where in the secretory pathway sorting takes place and discuss a possible role of sorting receptors.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Hormonas/metabolismo , Neuropéptidos/metabolismo , Transducción de Señal/fisiología , Animales , Exocitosis/fisiología , Aparato de Golgi/fisiología , Humanos , Fusión de Membrana/fisiologíaRESUMEN
The generation of spectral mutants of the green fluorescent protein (GFP) set the stage for multiple-color imaging in living cells. However, the use of this technique has been limited by a spectral overlap of the available GFP mutants and/or by insufficient resolution in both time and space. Using a new setup for dual-color imaging, we demonstrate here the visualization of small, fast moving vesicular structures with a high time resolution. Two GFP-fusion proteins were generated: human chromogranin B, a secretory granule matrix protein, and phogrin, a secretory granule membrane protein. They were tagged with enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP), respectively. Both fusion proteins were cotransfected in Vero cells, a cell line from green monkey kidney. EYFP and ECFP were excited sequentially at high time rates using a monochromator. Charged coupled device (CCD)-based image acquisition resulted in 5-8 dual-color images per second, with a resolution sufficient to detect transport vesicles in mammalian cells. Under these conditions, a fully automated time-resolved analysis of the movement of color-coded objects was achieved. The development of specialized software permitted the analysis of the extent of colocalization between the two differentially labeled sets of cellular structures over time. This technical advance will provide an important tool to study the dynamic interactions of subcellular structures in living cells.
Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Proteínas de la Membrana , Microscopía Fluorescente/métodos , Animales , Chlorocebus aethiops , Cromograninas/genética , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Glicoproteínas de Membrana/genética , Microscopía Fluorescente/instrumentación , Microscopía por Video/instrumentación , Microscopía por Video/métodos , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas Clase 8 Similares a Receptores , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , Transfección , Células VeroAsunto(s)
Cromograninas/metabolismo , Vesículas Secretoras/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico Activo , Cromogranina A , Cromograninas/química , Cromograninas/genética , Exones , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Ratas , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
The existence of specific sorting signals which direct regulated secretory proteins to secretory granules (SGs) was hypothesized two decades ago and since then has been addressed in numerous studies. The discovery that aggregation of regulated secretory proteins is involved in their sorting to SGs questioned the existence of specific sorting signals. In this short review we summarize the identification of a specific sorting signal for chromogranin B (CgB), a regulated secretory protein which undergoes Ca2+/pH-dependent aggregation. This signal is represented by the N-terminal disulfide-bonded loop of CgB encoded by exon 3 and is necessary to direct CgB to SGs. Its essential role was revealed only by the expression of a loopless deletion mutant in the absence of endogenous protein synthesis to preclude aggregative sorting of the former with the latter. The signal is also sufficient to direct a reporter protein to SGs, but only its multiple presence on the reporter leads to high sorting efficiency. Importantly, the identified signal functions at the level of the TGN by binding to membrane components that give rise to SGs. Furthermore, these studies lead to further insights into the mechanism of sorting. First, conclusive evidence is provided that regulated secretory proteins lacking a specific signal, can be sorted via coaggregation with proteins containing a specific sorting signal. Second, the data support an additional function of aggregation in the TGN which is multimerization of sorting signals per sorting unit leading to highly efficient sorting to SGs.
Asunto(s)
Cromograninas/química , Cromograninas/genética , Gránulos Citoplasmáticos/metabolismo , Señales de Clasificación de Proteína/metabolismo , Secuencia de Aminoácidos , Animales , Cromograninas/metabolismo , Disulfuros , Exocitosis , Exones , Humanos , Datos de Secuencia Molecular , Señales de Clasificación de Proteína/químicaRESUMEN
HYPOTHESIS: We hypothesize that magnetic resonance cholangiopancreatography (MRCP) is comparable to endoscopic retrograde cholangiopancreatographic (ERCP) as a diagnostic tool in patients with malignant biliary obstruction. DESIGN: Eighteen patients with suspected pancreaticobiliary malignancy were evaluated by ERCP and MRCP in 8 months (March 1, 1996, to October 31, 1996). Magnetic resonance cholangiopancreatography was performed with a 1.5-T scanner using 4-mm slices. Images were obtained in a 14- to 28-second breath-hold. Images from MRCP were retrospectively evaluated by a radiologist for image quality, ductal dilation, level of obstruction, and overall diagnostic impression. Images from ERCP were retrospectively evaluated by a biliary endoscopist (L.H.S.) and served as the standard for calculating sensitivity, specificity, and positive predictive values. In addition, intraoperative findings were compared with MRCP results in all patients explored. RESULTS: Diagnostic-quality MR images were obtained in 18 patients (100%). Diagnostic-quality endoscopic images were obtained in 16 (89%) of 18 attempted biliary cannulations and 11 (78%) of 14 attempted pancreatic cannulations. Magnetic resonance CP accurately delineated the level of extrahepatic biliary ductal obstruction in 13 (87%) of 15 patients. More important, MRCP provided valuable staging information in most patients. Findings from MRCP correlated with operative findings (size and location of tumor and mesenteric vascular involvement) in 8 (80%) of 10 patients who underwent surgery, while failing in 2 patients (20%) with carcinomatosis. CONCLUSIONS: Magnetic resonance CP is a sensitive study for detecting the presence and level of biliary ductal obstruction in patients with cancer. The results are comparable to those of ERCP; however, MRCP provides additional data regarding extent of disease that is not available from ERCP alone.
Asunto(s)
Colangiopancreatografia Retrógrada Endoscópica , Colestasis/diagnóstico , Imagen por Resonancia Magnética , Conductos Pancreáticos/diagnóstico por imagen , Conductos Pancreáticos/patología , Adulto , Anciano , Colestasis/etiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
Recent development of in vivo microscopy techniques, including green fluorescent proteins, has allowed the visualization of a wide range of dynamic processes in living cells. For quantitative and visual interpretation of such processes, new concepts for time-resolved image analysis and continuous time-space visualization are required. Here, we describe a versatile and fully automated approach consisting of four techniques, namely highly sensitive object detection, fuzzy logic-based dynamic object tracking, computer graphical visualization, and measurement in time-space. Systematic model simulations were performed to evaluate the reliability of the automated object detection and tracking method. To demonstrate potential applications, the method was applied to the analysis of secretory membrane traffic and the functional dynamics of nuclear compartments enriched in pre-mRNA splicing factors.
Asunto(s)
Fenómenos Fisiológicos Celulares , Microscopía/métodos , Modelos Biológicos , Animales , Biología/métodos , Chlorocebus aethiops , Cromograninas/análisis , Cromograninas/genética , Simulación por Computador , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Proteínas Recombinantes de Fusión/análisis , Factores de Tiempo , Transfección , Células VeroRESUMEN
BACKGROUND: Since 1996 patients in stable condition who need therapeutic endoscopic retrograde cholangiopancreatography (ERCP) at our institution have been treated as outpatients whenever possible. We reviewed our institution's experience and compared outpatient versus inpatient therapeutic ERCP for endobiliary stent placement in the care of patients with malignant common bile duct obstruction. METHODS: A retrospective review of all therapeutic ERCPs for the palliation of malignant common bile duct obstruction with endobiliary stents was performed from March 1, 1996, through December 1, 1997. RESULTS: One hundred nine therapeutic ERCPs were performed on 84 patients to place a polyethylene endobiliary stent for malignant common bile duct obstruction. Forty-three procedures were performed on 31 outpatients, 66 on 53 inpatients. There was no significant difference between outpatient and inpatient groups with regard to age, gender, procedure success rate, complication rate, need for endoscopic sphincterotomy, or whether the procedure was for initial stent placement or stent exchange. Inpatients had no procedure-related complications; outpatients had two. There was no procedure-related mortality in either group. CONCLUSION: Therapeutic ERCP for palliation of malignant common bile duct obstruction can be safely and successfully performed on an outpatient basis for selected patients. This should result in better quality of life for these patients with advanced cancer and substantial cost savings.
Asunto(s)
Atención Ambulatoria , Colangiopancreatografia Retrógrada Endoscópica , Colestasis Extrahepática/terapia , Neoplasias del Conducto Colédoco/terapia , Conducto Colédoco/diagnóstico por imagen , Stents , Anciano , Distribución de Chi-Cuadrado , Colangiopancreatografia Retrógrada Endoscópica/estadística & datos numéricos , Colestasis Extrahepática/diagnóstico por imagen , Colestasis Extrahepática/etiología , Neoplasias del Conducto Colédoco/complicaciones , Neoplasias del Conducto Colédoco/diagnóstico por imagen , Femenino , Humanos , Masculino , Persona de Mediana Edad , Cuidados Paliativos/estadística & datos numéricos , Polietilenos , Estudios Retrospectivos , Stents/estadística & datos numéricos , Resultado del TratamientoRESUMEN
Secretogranin II, an acidic protein in the chromogranin/secretogranin family, is widely distributed in neuroendocrine secretory granules. What factors govern such widespread, yet selective, expression? The 5' deletions localized neuroendocrine cell type-specific expression to the proximal mouse secretogranin II promoter: such expression was abolished after deletion past the cAMP response element (CRE; [-67 bp]TGACGTCA[-60 bp]), and transfer of the CRE to a neutral promoter conferred 3.4- to 5.3-fold neuroendocrine selectivity. Thus, the CRE is, at least partly, sufficient to confer tissue-specific expression. Substantial (48-59%) loss of cell type-specific expression also occurred upon deletion past the serum response element (SRE; [-302 bp]GATGTCC[-296 bp]), and transfer of the SRE to a neutral promoter also conferred neuroendocrine selectivity. Expression of both the endogenous gene and the transfected secretogranin II promoter was up-regulated after secretagogues, and the degree of trans-activation of the transfected promoter (2.2- to 5.4-fold) paralleled activation of the endogenous gene (1.8- to 3.2-fold). The 5' promoter deletions revealed complete loss of secretagogue responses after deletion past the CRE. Transfer of the CRE to a neutral promoter conferred secretagogue responses (by 2.2- to 18.6-fold). Substantial (59-74%) falls in secretagogue responses also occurred after deletion past the promoter's SRE. Transfer of the SRE to a neutral promoter conferred secretagogue responses (by 2.7- to 8.3-fold). We conclude that the CRE is a crucial determinant of cell type-specific constitutive and secretagogue-inducible expression of the secretogranin II gene and that the SRE also plays a substantial role in both processes.
Asunto(s)
Sangre/metabolismo , AMP Cíclico/fisiología , Regulación de la Expresión Génica/fisiología , Sistemas Neurosecretores/fisiología , Proteínas/genética , Elementos de Respuesta/fisiología , Animales , Secuencia de Bases/genética , Cromograninas , Eliminación de Gen , Ratones , Datos de Secuencia Molecular , Mutación/genética , Sistemas Neurosecretores/citología , Células PC12 , Regiones Promotoras Genéticas/genética , Ratas , TransfecciónRESUMEN
The disulfide-bonded loop of chromogranin B (CgB), a regulated secretory protein with widespread distribution in neuroendocrine cells, is known to be essential for the sorting of CgB from the trans-Golgi network (TGN) to immature secretory granules. Here we show that this loop, when fused to the constitutively secreted protein alpha1-antitrypsin (AT), is sufficient to direct the fusion protein to secretory granules. Importantly, the sorting efficiency of the AT reporter protein bearing two loops (E2/3-AT-E2/3) is much higher compared with that of AT with a single disulfide-bonded loop. In contrast to endogenous CgB, E2/3-AT-E2/3 does not undergo Ca2+/pH-dependent aggregation in the TGN. Furthermore, the disulfide-bonded loop of CgB mediates membrane binding in the TGN and does so with 5-fold higher efficiency if two loops are present on the reporter protein. The latter finding supports the concept that under physiological conditions, aggregates of CgB are the sorted units of cargo which have multiple loops on their surface leading to high membrane binding and sorting efficiency of CgB in the TGN.
Asunto(s)
Cromograninas/metabolismo , Gránulos Citoplasmáticos/metabolismo , Disulfuros/química , Aparato de Golgi/metabolismo , alfa 1-Antitripsina/metabolismo , Secuencia de Aminoácidos , Animales , Calcio/farmacología , Cromogranina B , Cromograninas/química , Cromograninas/genética , Exocitosis/genética , Concentración de Iones de Hidrógeno , Inmunohistoquímica , Cinética , Microscopía Fluorescente , Datos de Secuencia Molecular , Células PC12 , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Transfección/genética , alfa 1-Antitripsina/genéticaRESUMEN
Since the advent of green-fluorescent protein (GFP) technology there has been an explosion of interest in applying this molecule to cell biology. This review summarizes new insights in secretory membrane traffic obtained by the use of GFP fusion proteins. Transport steps between the endoplasmic reticulum and the Golgi apparatus, intra-Golgi traffic, and transport from the Golgi to the plasma membrane are discussed. In addition, insights into the dynamics of the Golgi compartment in plant cells and in mitotic mammalian cells have been included. We conclude that membrane traffic in the secretory pathway appears to be much more dynamic and diverse than previously thought and that GFP promises to be a powerful means to unravel these complex processes.