RESUMEN
Autophagy is a central lysosomal degradation pathway required for maintaining cellular homeostasis and its dysfunction is associated with numerous human diseases. To identify players in autophagy, we tested â¼1200 chemically induced mutations on the X chromosome in Drosophila fat body clones and discovered that shibire (shi) plays an essential role in starvation-induced autophagy. shi encodes a dynamin protein required for fission of clathrin-coated vesicles from the plasma membrane during endocytosis. We showed that Shi is dispensable for autophagy initiation and autophagosome-lysosome fusion, but required for lysosomal/autolysosomal acidification. We also showed that other endocytic core machinery components like clathrin and AP2 play similar but not identical roles in regulating autophagy and lysosomal function as dynamin. Previous studies suggested that dynamin directly regulates autophagosome formation and autophagic lysosome reformation (ALR) through its excision activity. Here, we provide evidence that dynamin also regulates autophagy indirectly by regulating lysosomal function.
Asunto(s)
Autofagia , Proteínas de Drosophila/fisiología , Dinaminas/fisiología , Lisosomas/metabolismo , Complejo 2 de Proteína Adaptadora/fisiología , Animales , Autofagia/genética , Células Cultivadas , Clatrina/fisiología , Drosophila/genética , Drosophila/fisiología , Proteínas de Drosophila/genética , Dinamina II/fisiología , Dinaminas/genética , Mutación , RatasRESUMEN
Autophagy helps deliver sequestered intracellular cargo to lysosomes for proteolytic degradation and thereby maintains cellular homeostasis by preventing accumulation of toxic substances in cells. In a forward mosaic screen in Drosophila designed to identify genes required for neuronal function and maintenance, we identified multiple cacophony (cac) mutant alleles. They exhibit an age-dependent accumulation of autophagic vacuoles (AVs) in photoreceptor terminals and eventually a degeneration of the terminals and surrounding glia. cac encodes an α1 subunit of a Drosophila voltage-gated calcium channel (VGCC) that is required for synaptic vesicle fusion with the plasma membrane and neurotransmitter release. Here, we show that cac mutant photoreceptor terminals accumulate AV-lysosomal fusion intermediates, suggesting that Cac is necessary for the fusion of AVs with lysosomes, a poorly defined process. Loss of another subunit of the VGCC, α2δ or straightjacket (stj), causes phenotypes very similar to those caused by the loss of cac, indicating that the VGCC is required for AV-lysosomal fusion. The role of VGCC in AV-lysosomal fusion is evolutionarily conserved, as the loss of the mouse homologues, Cacna1a and Cacna2d2, also leads to autophagic defects in mice. Moreover, we find that CACNA1A is localized to the lysosomes and that loss of lysosomal Cacna1a in cerebellar cultured neurons leads to a failure of lysosomes to fuse with endosomes and autophagosomes. Finally, we show that the lysosomal CACNA1A but not the plasma-membrane resident CACNA1A is required for lysosomal fusion. In summary, we present a model in which the VGCC plays a role in autophagy by regulating the fusion of AVs with lysosomes through its calcium channel activity and hence functions in maintaining neuronal homeostasis.
Asunto(s)
Canales de Calcio Tipo N/genética , Canales de Calcio/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Endosomas/metabolismo , Lisosomas/metabolismo , Neuronas/metabolismo , Fagosomas/metabolismo , Animales , Autofagia/genética , Calcio/metabolismo , Canales de Calcio/deficiencia , Canales de Calcio Tipo N/deficiencia , Cerebelo/metabolismo , Cerebelo/ultraestructura , Proteínas de Drosophila/deficiencia , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Endosomas/ultraestructura , Femenino , Regulación de la Expresión Génica , Homeostasis/genética , Lisosomas/ultraestructura , Masculino , Fusión de Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/ultraestructura , Fagosomas/ultraestructura , Cultivo Primario de Células , Transmisión Sináptica , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestructuraRESUMEN
Invertebrate model systems are powerful tools for studying human disease owing to their genetic tractability and ease of screening. We conducted a mosaic genetic screen of lethal mutations on the Drosophila X chromosome to identify genes required for the development, function, and maintenance of the nervous system. We identified 165 genes, most of whose function has not been studied in vivo. In parallel, we investigated rare variant alleles in 1,929 human exomes from families with unsolved Mendelian disease. Genes that are essential in flies and have multiple human homologs were found to be likely to be associated with human diseases. Merging the human data sets with the fly genes allowed us to identify disease-associated mutations in six families and to provide insights into microcephaly associated with brain dysgenesis. This bidirectional synergism between fly genetics and human genomics facilitates the functional annotation of evolutionarily conserved genes involved in human health.