RESUMO
During macroautophagy, the human WIPI (WD-repeat protein interacting with phosphoinositides) proteins (WIPI1â»4) function as phosphatidylinositol 3-phosphate effectors at the nascent autophagosome. Likewise, the two WIPI homologues in Caenorhabditis elegans, ATG-18 and EPG-6, play important roles in autophagy, whereby ATG-18 is considered to act upstream of EPG-6 at the onset of autophagy. Due to its essential role in autophagy, ATG-18 was found to be also essential for lifespan extension in Caenorhabditis elegans; however, this has not yet been addressed with regard to EPG-6. Here, we wished to address this point and generated mutant strains that expressed the autophagy marker GFP::LGG-1 (GFP-LC3 in mammals) and harbored functional deletions of either atg-18 (atg18(gk378)), epg-6 (epg-6(bp242)) or both (atg-18(gk378);epg-6(bp242)). Using quantitative fluorescence microscopy, Western blotting, and lifespan assessments, we provide evidence that in the absence of either ATG-18 or EPG-6 autophagy was impaired, and while atg-18 mutant animals showed a short-lived phenotype, lifespan was significantly increased in epg-6 mutant animals. We speculate that the long-lived phenotype of epg-6 mutant animals points towards an autophagy-independent function of EPG-6 in lifespan control that warrants further mechanistic investigations in future studies.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/fisiologia , Longevidade/fisiologia , Proteínas de Membrana/metabolismo , Animais , Caenorhabditis elegans/embriologia , Embrião não Mamífero/citologia , Larva/fisiologia , Masculino , Modelos Biológicos , Análise de SobrevidaRESUMO
This chapter describes methods for the analysis of autophagy proteins in C. elegans aging. We discuss the strains to be considered, the methods for the delivery of double-stranded RNA, and the methods to measure autophagy levels, autophagic flux, and degradation by autophagy.
Assuntos
Envelhecimento/fisiologia , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/fisiologia , Bioensaio/métodos , Proteínas de Caenorhabditis elegans/metabolismo , Animais , Proteínas Relacionadas à Autofagia/análise , Proteínas Relacionadas à Autofagia/genética , Bioensaio/instrumentação , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/análise , Proteínas de Caenorhabditis elegans/genética , Técnicas de Silenciamento de Genes/instrumentação , Técnicas de Silenciamento de Genes/métodos , Longevidade/fisiologia , Modelos Animais , Interferência de RNARESUMO
Macroautophagy/autophagy involves the formation of an autophagosome, a double-membrane vesicle that delivers sequestered cytoplasmic cargo to lysosomes for degradation and recycling. Closely related, endocytosis mediates the sorting and transport of cargo throughout the cell, and both processes are important for cellular homeostasis. However, how endocytic proteins functionally intersect with autophagy is not clear. Mutations in the DAF-2/insulin-like IGF-1 (INSR) receptor at the permissive temperature result in a small increase in GFP::LGG-1 foci, i.e. autophagosomes, but a large increase at the nonpermissive temperature, allowing us to control the level of autophagy. In a RNAi screen for endocytic genes that alter the expression of GFP::LGG-1 in daf-2 mutants, we identified RAB-10, a small GTPase that regulates basolateral endocytosis. Loss of rab-10 in daf-2 mutants results in more GFP::LGG-1-positive foci at the permissive, but less GFP::LGG-1 or SQST-1::GFP foci at the nonpermissive temperature. As previously reported, loss of rab-10 alone resulted in an increase of GFP:LGG-1 foci. Exposure of rab-10 mutant animals to chloroquine, a known inhibitor of autophagic flux, failed to increase the number of GFP::LGG-1 foci. Moreover, colocalization between LMP-1::tagRFP and GFP::LGG-1 (the lysosome and autophagosome reporters) was decreased in daf-2; rab-10 dauers at the nonpermissive temperature. Intriguingly, RAB-10 was required to maintain the normal size of GFP::ATG-9-positive structures in daf-2 mutants at both the permissive and nonpermissive temperature. Finally, we found that RAB-10 GTPase cycling was required to control the size of GFP::ATG-9 foci. Collectively, our data support a model where rab-10 controls autophagic flux by regulating autophagosome formation and maturation.