RESUMO
Macroautophagy is an intracellular degradation system that utilizes the autophagosome to deliver cytoplasmic components to the lysosome. Measuring autophagic activity is critically important but remains complicated and challenging. Here, we have developed GFP-LC3-RFP-LC3ΔG, a fluorescent probe to evaluate autophagic flux. This probe is cleaved by endogenous ATG4 proteases into equimolar amounts of GFP-LC3 and RFP-LC3ΔG. GFP-LC3 is degraded by autophagy, while RFP-LC3ΔG remains in the cytosol, serving as an internal control. Thus, autophagic flux can be estimated by calculating the GFP/RFP signal ratio. Using this probe, we re-evaluated previously reported autophagy-modulating compounds, performed a high-throughput screen of an approved drug library, and identified autophagy modulators. Furthermore, we succeeded in measuring both induced and basal autophagic flux in embryos and tissues of zebrafish and mice. The GFP-LC3-RFP-LC3ΔG probe is a simple and quantitative method to evaluate autophagic flux in cultured cells and whole organisms.
Assuntos
Autofagia/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Lisossomos/efeitos dos fármacos , Sondas Moleculares/genética , Fagossomos/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Autofagia/genética , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Embrião não Mamífero , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Lisossomos/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Sondas Moleculares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fagossomos/metabolismo , Espectrometria de Fluorescência , Ubiquitina-Proteína Ligases , Peixe-ZebraRESUMO
Parkinson's disease (PD) is a major neurodegenerative chronic disease, most likely caused by a complex interplay of genetic and environmental factors. Information on various aspects of PD pathogenesis is rapidly increasing and needs to be efficiently organized, so that the resulting data is available for exploration and analysis. Here we introduce a computationally tractable, comprehensive molecular interaction map of PD. This map integrates pathways implicated in PD pathogenesis such as synaptic and mitochondrial dysfunction, impaired protein degradation, alpha-synuclein pathobiology and neuroinflammation. We also present bioinformatics tools for the analysis, enrichment and annotation of the map, allowing the research community to open new avenues in PD research. The PD map is accessible at http://minerva.uni.lu/pd_map .
