Fluorescence-Based Assays to Analyse Phosphatidylinositol 5-Phosphate in Autophagy.
View / Open Files
Authors
Vicinanza, M
Gratian, MJ
Bowen, M
Rubinsztein, DC
Publication Date
2017Journal Title
Methods Enzymol
ISSN
0076-6879
Publisher
Elsevier
Volume
587
Pages
311-330
Language
eng
Type
Article
Physical Medium
Print-Electronic
Metadata
Show full item recordCitation
Vicinanza, M., Gratian, M., Bowen, M., & Rubinsztein, D. (2017). Fluorescence-Based Assays to Analyse Phosphatidylinositol 5-Phosphate in Autophagy.. Methods Enzymol, 587 311-330. https://doi.org/10.1016/bs.mie.2016.09.062
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.
Keywords
Hela Cells, Humans, Phosphatidylinositol Phosphates, Homeodomain Proteins, Green Fluorescent Proteins, Tumor Suppressor Proteins, Receptors, Cytoplasmic and Nuclear, Recombinant Proteins, Microscopy, Confocal, Image Enhancement, Image Processing, Computer-Assisted, Autophagy, Autophagosomes
Sponsorship
Wellcome Trust (100140/Z/12/Z)
Wellcome Trust (095317/Z/11/Z)
Identifiers
External DOI: https://doi.org/10.1016/bs.mie.2016.09.062
This record's URL: https://www.repository.cam.ac.uk/handle/1810/284184
Rights
Licence:
http://www.rioxx.net/licenses/all-rights-reserved
Statistics
Total file downloads (since January 2020). For more information on metrics see the
IRUS guide.