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Fluorescence-Based Assays to Analyse Phosphatidylinositol 5-Phosphate in Autophagy.

Accepted version
Peer-reviewed

Type

Article

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Authors

Vicinanza, M 
Gratian, MJ 
Bowen, M 
Rubinsztein, DC 

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.

Description

Keywords

Autophagy, PI(5)P, Super-resolution structured illumination microscopy, Autophagosomes, Autophagy, Green Fluorescent Proteins, HeLa Cells, Homeodomain Proteins, Humans, Image Enhancement, Image Processing, Computer-Assisted, Microscopy, Confocal, Phosphatidylinositol Phosphates, Receptors, Cytoplasmic and Nuclear, Recombinant Proteins, Tumor Suppressor Proteins

Journal Title

Methods Enzymol

Conference Name

Journal ISSN

0076-6879
1557-7988

Volume Title

587

Publisher

Elsevier
Sponsorship
Wellcome Trust (100140/Z/12/Z)
Wellcome Trust (095317/Z/11/Z)