Format

Send to

Choose Destination
Autophagy. 2017 Sep 2;13(9):1602-1612. doi: 10.1080/15548627.2017.1341465. Epub 2017 Aug 16.

Local detection of PtdIns3P at autophagosome biogenesis membrane platforms.

Author information

1
a Cell Biology Department , Institut Necker-Enfants Malades (INEM) , INSERM U1151-CNRS UMR 8253, Paris , France.
2
b Université Paris Descartes-Sorbonne Paris Cité , Paris , France .

Abstract

Phosphatidylinositol 3-phosphate (PtdIns3P) is a key player of membrane trafficking regulation, mostly synthesized by the PIK3C3 lipid kinase. The presence of PtdIns3P on endosomes has been demonstrated; however, the role and dynamics of the pool of PtdIns3P dedicated to macroautophagy/autophagy remains elusive. Here we addressed this question by studying the mobilization of PtdIns3P in time and space during autophagosome biogenesis. We compared different dyes known to specifically detect PtdIns3P by fluorescence microscopy analysis, based on PtdIns3P-binding FYVE and PX domains, and show that these transfected dyes induce defects in endosomal dynamics as well as artificial and sustained autophagosome formation. In contrast, indirect use of recombinant FYVE enabled us to track and discriminate endosomal and autophagosomal pools of PtdIns3P. We used this method to analyze localization and dynamics of PtdIns3P subdomains on the endoplasmic reticulum, at sites of pre-autophagosome associated protein recruitment such as the PtdIns3P-binding ZFYVE1/DFCP1 and WIPI2 autophagy regulators. This approach thus revealed the presence of a specific pool of PtdIns3P at the site where autophagosome assembly is initiated.

KEYWORDS:

FYVE domain; PX domain; autophagosome; endosome; phosphatidylinositol-3-phosphate

PMID:
28813193
PMCID:
PMC5612047
DOI:
10.1080/15548627.2017.1341465
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Taylor & Francis Icon for PubMed Central
Loading ...
Support Center