RESUMEN
Synaptic vesicle (SV) exo- and endocytosis are tightly coupled to sustain neurotransmission in presynaptic terminals, and both are regulated by Ca(2+). Ca(2+) influx triggered by voltage-gated Ca(2+) channels is necessary for SV fusion. However, extracellular Ca(2+) has also been shown to be required for endocytosis. The intracellular Ca(2+) levels (<1 microM) that trigger endocytosis are typically much lower than those (>10 microM) needed to induce exocytosis, and endocytosis is inhibited when the Ca(2+) level exceeds 1 microM. Here, we identify and characterize a transmembrane protein associated with SVs that, upon SV fusion, localizes at periactive zones. Loss of Flower results in impaired intracellular resting Ca(2+) levels and impaired endocytosis. Flower multimerizes and is able to form a channel to control Ca(2+) influx. We propose that Flower functions as a Ca(2+) channel to regulate synaptic endocytosis and hence couples exo- with endocytosis.
Asunto(s)
Canales de Calcio/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endocitosis , Exocitosis , Vesículas Sinápticas/metabolismo , Animales , Canales de Calcio/análisis , Proteínas de Drosophila/análisis , Drosophila melanogaster/citología , Isoformas de Proteínas/análisis , Isoformas de Proteínas/metabolismo , Vesículas Sinápticas/químicaRESUMEN
Neurodegeneration can be triggered by genetic or environmental factors. Although the precise cause is often unknown, many neurodegenerative diseases share common features such as protein aggregation and age dependence. Recent studies in Drosophila have uncovered protective effects of NAD synthase nicotinamide mononucleotide adenylyltransferase (NMNAT) against activity-induced neurodegeneration and injury-induced axonal degeneration. Here we show that NMNAT overexpression can also protect against spinocerebellar ataxia 1 (SCA1)-induced neurodegeneration, suggesting a general neuroprotective function of NMNAT. It protects against neurodegeneration partly through a proteasome-mediated pathway in a manner similar to heat-shock protein 70 (Hsp70). NMNAT displays chaperone function both in biochemical assays and cultured cells, and it shares significant structural similarity with known chaperones. Furthermore, it is upregulated in the brain upon overexpression of poly-glutamine expanded protein and recruited with the chaperone Hsp70 into protein aggregates. Our results implicate NMNAT as a stress-response protein that acts as a chaperone for neuronal maintenance and protection. Our studies provide an entry point for understanding how normal neurons maintain activity, and offer clues for the common mechanisms underlying different neurodegenerative conditions.
Asunto(s)
Amida Sintasas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/enzimología , Chaperonas Moleculares/metabolismo , Degeneración Nerviosa , Enfermedades Neurodegenerativas/prevención & control , Nicotinamida-Nucleótido Adenililtransferasa/metabolismo , Amida Sintasas/genética , Animales , Ataxina-1 , Ataxinas , Encéfalo/metabolismo , Células COS , Chlorocebus aethiops , Modelos Animales de Enfermedad , Proteínas de Drosophila/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Chaperonas Moleculares/genética , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/toxicidad , Enfermedades Neurodegenerativas/enzimología , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Nicotinamida-Nucleótido Adenililtransferasa/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/toxicidad , Ataxias Espinocerebelosas/enzimología , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/patología , Ataxias Espinocerebelosas/prevención & controlRESUMEN
Neurons establish specific synaptic connections with their targets, a process that is highly regulated. Numerous cell adhesion molecules have been implicated in target recognition, but how these proteins are precisely trafficked and targeted is poorly understood. To identify components that affect synaptic specificity, we carried out a forward genetic screen in the Drosophila eye. We identified a gene, named ric1 homologue (rich), whose loss leads to synaptic specificity defects. Loss of rich leads to reduction of N-Cadherin in the photoreceptor cell synapses but not of other proteins implicated in target recognition, including Sec15, DLAR, Jelly belly, and PTP69D. The Rich protein binds to Rab6, and Rab6 mutants display very similar phenotypes as the rich mutants. The active form of Rab6 strongly suppresses the rich synaptic specificity defect, indicating that Rab6 is regulated by Rich. We propose that Rich activates Rab6 to regulate N-Cadherin trafficking and affects synaptic specificity.
Asunto(s)
Cadherinas/metabolismo , Proteínas de Drosophila/metabolismo , Células Fotorreceptoras de Invertebrados/fisiología , Transducción de Señal/fisiología , Sinapsis/fisiología , Proteínas de Unión al GTP rab/metabolismo , Proteínas ras/genética , Animales , Drosophila , Proteínas de Drosophila/genética , Mutación , Células Fotorreceptoras de Invertebrados/metabolismo , Sinapsis/genética , Sinapsis/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas ras/metabolismoRESUMEN
Cell fate decisions mediated by the Notch signalling pathway require direct cell-cell contact between adjacent cells. In Drosophila melanogaster, an external sensory organ (ESO) develops from a single sensory organ precursor (SOP) and its fate specification is governed by differential Notch activation. Here we show that mutations in actin-related protein-3 (Arp3) compromise Notch signalling, leading to a fate transformation of the ESO. Our data reveal that during ESO fate specification, most endocytosed vesicles containing the ligand Delta traffic to a prominent apical actin-rich structure (ARS) formed in the SOP daughter cells. Using immunohistochemistry and transmission electron microscopy (TEM) analyses, we show that the ARS contains numerous microvilli on the apical surface of SOP progeny. In Arp2/3 and WASp mutants, the surface area of the ARS is substantially reduced and there are significantly fewer microvilli. More importantly, trafficking of Delta-positive vesicles from the basal area to the apical portion of the ARS is severely compromised. Our data indicate that WASp-dependent Arp2/3 actin polymerization is crucial for apical presentation of Delta, providing a mechanistic link between actin polymerization and Notch signalling.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/fisiología , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Proteínas de la Membrana/metabolismo , Microvellosidades/metabolismo , Órganos de los Sentidos/embriología , Proteína del Síndrome de Wiskott-Aldrich/fisiología , Complejo 2-3 Proteico Relacionado con la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Animales , Transporte Biológico/genética , Transporte Biológico/fisiología , Western Blotting , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Drosophila melanogaster/genética , Drosophila melanogaster/ultraestructura , Endocitosis/genética , Endocitosis/fisiología , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Microvellosidades/ultraestructura , Órganos de los Sentidos/metabolismo , Órganos de los Sentidos/ultraestructura , Proteína del Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
Adipose tissue development and function play a central role in the pathogenesis and pathophysiology of metabolic syndromes. Here, we show that chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) plays a pivotal role in adipogenesis and energy homeostasis. COUP-TFII is expressed in the early stages of white adipocyte development. COUP-TFII heterozygous mice (COUP-TFII(+/-)) have much less white adipose tissue (WAT) than wild-type mice (COUP-TFII(+/+)). COUP-TFII(+/-) mice display a decreased expression of key regulators for WAT development. Knockdown COUP-TFII in 3T3-L1 cells resulted in an increased expression of Wnt10b, while chromatin immunoprecipitation analysis revealed that Wnt10b is a direct target of COUP-TFII. Moreover, COUP-TFII(+/-) mice have increased mitochondrial biogenesis in WAT, and COUP-TFII(+/-) mice have improved glucose homeostasis and increased energy expenditure. Thus, COUP-TFII regulates adipogenesis by regulating the key molecules in adipocyte development and can serve as a target for regulating energy metabolism.