Fluorescence-Based Assays to Analyse Phosphatidylinositol 5-Phosphate in Autophagy
| Autor: | Mariella Vicinanza, David C. Rubinsztein, M J Gratian, M Bowen |
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| Přispěvatelé: | Rubinsztein, David [0000-0001-5002-5263], Apollo - University of Cambridge Repository |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Autophagosome Green Fluorescent Proteins Receptors Cytoplasmic and Nuclear Context (language use) Biology 03 medical and health sciences PIKFYVE Super-resolution structured illumination microscopy Phosphatidylinositol Phosphates Autophagy Image Processing Computer-Assisted Humans Phosphatidylinositol 5-phosphate Homeodomain Proteins Microscopy Confocal PI(5)P Tumor Suppressor Proteins Autophagosomes Image Enhancement Recombinant Proteins Chromatin Cell biology 030104 developmental biology Biochemistry Signal transduction Biogenesis HeLa Cells |
| DOI: | 10.17863/cam.31552 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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