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Human WIPI-1 puncta-formation: A novel assay to assess mammalian autophagy

Autophagy Laboratory, Department of Molecular Biology, University of Tuebingen, Auf der Morgenstelle 15, 72076 Tuebingen, Germany.
FEBS Letters (Impact Factor: 3.34). 08/2007; 581(18):3396-404. DOI: 10.1016/j.febslet.2007.06.040
Source: PubMed

ABSTRACT Autophagy depends on the activity of phosphoinositide-3 kinase class III to generate PI(3)P. We identified the human WIPI protein family of PI(3)P-binding factors and showed that WIPI-1 (Atg18) is linked to autophagy in human cells. Induction of autophagy by rapamycin, gleevec, thapsigargin and amino acid deprivation led to an accumulation of WIPI-1 at LC3-positive membrane structures (WIPI-1 puncta-formation), suggested to represent autophagosomal isolation membranes. WIPI-1 puncta-formation is inhibited by wortmannin and LY294002, and PI(3)P-binding-deficient WIPI-1 is puncta-formation-incompetent. Quantification of WIPI-1 puncta should be suitable to assay mammalian autophagy.

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    • "TgPIKfyve harbours a chaperonin domain (CPN) (green) known to be engaged in regulatory interactions, a CHK domain (Cys, His and Lys) (blue) and the 5-kinase catalytic domain (black) at the C-terminus responsible for the lipid kinase activity. PX4 contains 5 WD40 (blue) motifs known to coordinate multi-protein complex assemblies and binding to PI(3,5)P2 (Proikas-Cezanne et al., 2007). Scale bar represents 200 or 400 a.a respectively. "
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    • "Thereby, endogenous WIPI-1 can be visualized by indirect immunofluorescence or alternatively by introducing GFP-WIPI-1 as conducted in the present study. Fluorescent WIPI-1 puncta reflect the accumulation of WIPI-1 at membranes via its specific binding to PtdIns(3)P was found to represent phagophores and autophagosomes [10] [11]. In addition, WIPI-1 binds to PtdIns(3)P at the endoplasmic reticulum and at the plasma membrane upon the induction of autophagy, indicative for membrane origins where phagophore/autophagosome formation is initiated by unknown mechanisms [10]. "
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    • "). The basic sequence Phe-Arg-Arg-Gly (FRRG), located at the junction between the fifth and sixth b-propeller blades, is essential for PtdInsP binding of yeast Atg18 and mammalian WIPI1 (Dove et al., 2004; Proikas-Cezanne et al., 2007). At the corresponding position, ATG-18 (and WIPI1/2) has FRRG (amino acids 227–230) and EPG-6 (and WIPI3/4) has LRRG (amino acids 258–261). "
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