RESUMEN
Here we introduce zapalog-mediated endoplasmic reticulum trap (zapERtrap), which allows one to use light to precisely trigger forward trafficking of diverse integral membrane proteins from internal secretory organelles to the cell surface with single cell and subcellular spatial resolution. To demonstrate its utility, we use zapERtrap in neurons to dissect where synaptic proteins emerge at the cell surface when processed through central (cell body) or remote (dendrites) secretory pathways. We reveal rapid and direct long-range trafficking of centrally processed proteins deep into the dendritic arbor to synaptic sites. Select proteins were also trafficked to the plasma membrane of the axon initial segment, revealing a novel surface trafficking hotspot. Proteins locally processed through dendritic secretory networks were widely dispersed before surface insertion, challenging assumptions for precise trafficking at remote sites. These experiments provide new insights into compartmentalized secretory trafficking and showcase the tunability and spatiotemporal control of zapERtrap, which will have broad applications for regulating cell signaling and function.
Asunto(s)
Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Neuronas/metabolismo , Vías Secretoras/genética , Sinapsis/metabolismo , Transmisión Sináptica/genética , Animales , Animales Recién Nacidos , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Membrana Celular/ultraestructura , Retículo Endoplásmico/ultraestructura , Femenino , Colorantes Fluorescentes/química , Expresión Génica , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Hipocampo/citología , Hipocampo/metabolismo , Luz , Masculino , Imagen Molecular/métodos , Neuronas/citología , Cultivo Primario de Células , Transporte de Proteínas , Ratas , Ratas Sprague-Dawley , Receptores AMPA/genética , Receptores AMPA/metabolismo , Sinapsis/ultraestructura , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
The tyrosinase gene family is currently composed of three members, tyrosinase and two tyrosinase-related proteins, TRP-1 and TRP-2. These three gene products have all been found to act in the synthesis of melanin pigments with the enzyme tyrosinase catalyzing the initial rate-limiting steps. Thus far these genes have primarily been analyzed in higher vertebrates. We have used degenerate PCR primers to isolate a large fragment of an axolotl tyrosinase-related protein. Sequence analysis of the entire 1,057-bp fragment isolated indicates a high degree of similarity to the mouse TRP-1, the product of the brown locus. Phylogenetic analysis supports the conclusion that the fragment isolated corresponds to the axolotl TRP-1 homolog. This is the first TRP-1 gene to be identified in an amphibian species.
Asunto(s)
Ambystoma mexicanum/genética , Glicoproteínas de Membrana , Oxidorreductasas , Filogenia , Reacción en Cadena de la Polimerasa/métodos , Proteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Clonación Molecular/métodos , Cobre/metabolismo , Cartilla de ADN , Humanos , Datos de Secuencia Molecular , Neurospora/genética , Biosíntesis de Proteínas , Proteínas/química , Homología de Secuencia de Aminoácido , Streptococcus/genéticaRESUMEN
It has been proposed that cell wall loosening during plant cell growth may be mediated by the endotransglycosylation of load-bearing polymers, specifically of xyloglucans, within the cell wall. A xyloglucan endotransglycosylase (XET) with such activity has recently been identified in several plant species. Two cell wall proteins capable of inducing the extension of plant cell walls have also recently been identified in cucumber hypocotyls. In this report we examine three questions: (1) Does XET induce the extension of isolated cell walls? (2) Do the extension-inducing proteins possess XET activity? (3) Is the activity of the extension-inducing proteins modulated by a xyloglucan nonasaccharide (Glc4-Xyl3-Gal2)? We found that the soluble proteins from growing cucumber (cucumis sativum L.) hypocotyls contained high XET activity but did not induce wall extension. Highly purified wall-protein fractions from the same tissue had high extension-inducing activity but little or no XET activity. The XET activity was higher a pH 5.5 than at pH 4.5, while extension activity showed the opposite sensitivity to pH. Reconstituted wall extension was unaffected by the presence of a xyloglucan nonasaccharide (Glc4-Xyl3-Gal2), an oligosaccharide previously shown to accelerate growth in pea stems and hypothesized to facilitate growth through an effect on XET-induced cell wall loosening. We conclude that XET activity alone is neither sufficient nor necessary for extension of isolated walls from cucumber hypocotyls.
Asunto(s)
Pared Celular/enzimología , Glucanos , Glicosiltransferasas/metabolismo , Plantas/enzimología , Xilanos , Secuencia de Carbohidratos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Proteínas de Plantas/metabolismo , Polisacáridos/metabolismoRESUMEN
In a prospective randomized trial, moxalactam administered to 66 children was compared with ampicillin or chloramphenicol given to 68 children for the treatment of Haemophilus influenzae type b meningitis. Acute morbidity and mortality rates were equivalent between the two treatment groups. At 2 years after discharge, the results of psychologic tests (Bayley Scales of Infant Development or McCarthy Scales of Children's Abilities) were also equivalent between the two treatment groups for patients remaining in the study.
Asunto(s)
Ampicilina/uso terapéutico , Cloranfenicol/uso terapéutico , Meningitis por Haemophilus/tratamiento farmacológico , Moxalactam/uso terapéutico , Preescolar , Ensayos Clínicos como Asunto , Discapacidades del Desarrollo/etiología , Quimioterapia Combinada , Estudios de Seguimiento , Haemophilus influenzae/efectos de los fármacos , Humanos , Lactante , Meningitis por Haemophilus/complicaciones , Meningitis por Haemophilus/psicología , Estudios Prospectivos , Distribución AleatoriaRESUMEN
In a prospective, randomized study, moxalactam in 44 children was compared with ampicillin or chloramphenicol in 47 children for the treatment of Haemophilus influenzae type b meningitis. Both groups were comparable in terms of clinical and laboratory findings at admission. The hospital course, neurologic sequelae including deafness, and number of deaths were the same for both groups. The incidence of adverse reactions also was the same except that diarrhea and thrombocytosis occurred significantly (P less than or equal to 0.04) more frequently in children given moxalactam. Moxalactam was equivalent to ampicillin or chloramphenicol in the treatment of H. influenzae type b meningitis in children.