title: Fluorescence-Based Assays to Analyse Phosphatidylinositol 5-Phosphate in Autophagy.
abstract: Autophagosome formation is stimulated by VPS34-dependent PI(3)P formation and by alternative VPS34-independent pathways. We recently described that PI(5)P regulates autophagosome biogenesis and rescues autophagy in VPS34-inactivated cells, suggesting that PI(5)P contributes to canonical autophagy. Our analysis revealed a hitherto unknown functional interplay between PIKfyve and PIPK type II in controlling PI(5)P levels in the context of autophagy. Among phosphoinositides, visualization of PI(5)P in intact cells has remained difficult. While PI(5)P has been implicated in signaling pathways, chromatin organization, bacterial invasion, and cytoskeletal remodeling, our study is the first report showing PI(5)P localization on autophagosomes and early autophagosomal structures when autophagy is induced by nutrient deprivation (amino acids or glucose starvation). We provided a detailed analysis of PI(5)P distribution by the use of super-resolution structured illuminated microscopy. Here, we present a set of tools for detection of PI(5)P during autophagy by confocal microscopy, live-cell imaging, and super-resolution microscopy.
authors.affiliation: Cambridge Institute for Medical Research, Wellcome/MRC Building, Cambridge Biomedical Campus, Cambridge, United Kingdom.
authors.forename: M
authors.initials: M
authors.lastname: Vicinanza
authors.affiliation: Cambridge Institute for Medical Research, Wellcome/MRC Building, Cambridge Biomedical Campus, Cambridge, United Kingdom.
authors.forename: M J
authors.initials: MJ
authors.lastname: Gratian
authors.affiliation: Cambridge Institute for Medical Research, Wellcome/MRC Building, Cambridge Biomedical Campus, Cambridge, United Kingdom.
authors.forename: M
authors.initials: M
authors.lastname: Bowen
authors.affiliation: Cambridge Institute for Medical Research, Wellcome/MRC Building, Cambridge Biomedical Campus, Cambridge, United Kingdom. Electronic address: dcr1000@cam.ac.uk.
authors.forename: D C
authors.initials: DC
authors.lastname: Rubinsztein
mesh_terms: D000071182:Autophagosomes / Q000378:metabolism; D001343:Autophagy / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007089:Image Enhancement / Q000379:methods; D007091:Image Processing, Computer-Assisted / Q000379:methods; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods*; D018129:Phosphatidylinositol Phosphates / Q000032:analysis* / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D018398:Homeodomain Proteins; C444654:ING2 protein, human; D018129:Phosphatidylinositol Phosphates; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; C113567:phosphatidylinositol 5-phosphate; D049452:Green Fluorescent Proteins
keywords: Autophagy; PI(5)P; Super-resolution structured illumination microscopy