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: 3.17). 08/2007; 581(18):3396-404. DOI: 10.1016/j.febslet.2007.06.040
Source: PubMed


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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    ABSTRACT: Phosphoinositides regulate numerous cellular processes, by recruiting cytosolic effector proteins and acting as membrane signaling entities. The cellular metabolism and localization of phosphoinositides are tightly regulated by distinct lipid kinases and phosphatases. Here, we identify and characterize a unique phosphatidylinositol 3-Kinase (PI3K) in Toxoplasma gondii, a protozoan parasite belonging to the phylum Apicomplexa. Conditional depletion of this enzyme and subsequently of its product, PI(3)P, drastically alters the morphology and inheritance of the apicoplast, an endosymbiontic organelle of algal origin that is a unique feature of many Apicomplexa. We searched the T. gondii genome for PI(3)P binding proteins and identified in total six PX and FYVE-domain containing proteins including a PIKfyve lipid kinase, which phosphorylates PI(3)P into PI(3,5)P2 . While depletion of putative PI(3)P binding proteins shows that they are not essential for parasite growth and apicoplast biology, conditional disruption of PIKfyve induces enlarged apicoplasts, as observed upon the loss of PI(3)P. A similar defect of apicoplast homeostasis was also observed by knocking-down the PIKfyve regulatory protein ArPIKfyve, suggesting that in T. gondii, PI(3)P-related function for the apicoplast might mainly be to serve as a precursor for the synthesis of PI(3,5)P2 . Accordingly, PI3K is conserved in all apicomplexan parasites whereas PIKfyve and ArPIKfyve are absent in Cryptosporidium species which lack an apicoplast, supporting a direct role of PI(3,5)P2 in apicoplast homeostasis. This study enriches the already diverse functions attributed to PI(3,5)P2 in eukaryotic cells and highlights these parasite lipid kinases as potential drug targets.
    Cellular Microbiology 10/2014; 17(4). DOI:10.1111/cmi.12383 · 4.92 Impact Factor
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    • "WIPI1 is a PI3P-binding protein that is involved in phagophore formation37. We then examined WIPI1 expression levels to analyze the kinase activity of PIK3C3 in RNF2 KO cells. "
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    ABSTRACT: WASH (Wiskott-Aldrich syndrome protein (WASP) and SCAR homolog) was identified to function in endosomal sorting via Arp2/3 activation. We previously demonstrated that WASH is a new interactor of BECN1 and present in the BECN1-PIK3C3 complex with AMBRA1. The AMBRA1-DDB1-CUL4A complex is an E3 ligase for K63-linked ubiquitination of BECN1, which is required for starvation-induced autophagy. WASH suppresses autophagy by inhibition of BECN1 ubiquitination. However, how AMBRA1 is regulated during autophagy remains elusive. Here, we found that RNF2 associates with AMBRA1 to act as an E3 ligase to ubiquitinate AMBRA1 via K48 linkage. RNF2 mediates ubiquitination of AMBRA1 at lysine 45. Notably, RNF2 deficiency enhances autophagy induction. Upon autophagy induction, RNF2 potentiates AMBRA1 degradation with the help of WASH. WASH deficiency impairs the association of RNF2 with AMBRA1 to impede AMBRA1 degradation. Our findings reveal another novel layer of regulation of autophagy through WASH recruitment of RNF2 for AMBRA1 degradation leading to downregulation of autophagy.Cell Research advance online publication 1 July 2014; doi:10.1038/cr.2014.85.
    Cell Research 07/2014; 24(8). DOI:10.1038/cr.2014.85 · 12.41 Impact Factor
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    • "As mentioned previously, PI3P plays an important role in recruiting PI3P effectors to the site of AP biogenesis. WD repeat domain phosphoinositide-interacting proteins (WIPI-1 and 2), mammalian orthologues of yeast Atg18, are a major family of PI3P-binding effectors that localize to the site of nucleation and promote AP formation; therefore, they are often used to monitor PI3P synthesis during autophagy [16,53]. Here, GFP-WIPI-1 was transiently transfected in control and Vps34 KO MEFs and its localization examined under normal media and starvation conditions (HBSS for 90min). "
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    ABSTRACT: Synthesis of phosphatidylinositol-3-phosphate (PI3P) by Vps34, a class III phosphatidylinositol 3-kinase (PI3K), is critical for the initial steps of autophagosome (AP) biogenesis. Although Vps34 is the sole source of PI3P in budding yeast, mammalian cells can produce PI3P through alternate pathways, including direct synthesis by the class II PI3Ks; however, the physiological relevance of these alternate pathways in the context of autophagy is unknown. Here we generated Vps34 knockout mouse embryonic fibroblasts (MEFs) and using a higher affinity 4x-FYVE finger PI3P-binding probe found a Vps34-independent pool of PI3P accounting for (~)35% of the total amount of this lipid species by biochemical analysis. Importantly, WIPI-1, an autophagy-relevant PI3P probe, still formed some puncta upon starvation-induced autophagy in Vps34 knockout MEFs. Additional characterization of autophagy by electron microscopy as well as protein degradation assays showed that while Vps34 is important for starvation-induced autophagy there is a significant component of functional autophagy occurring in the absence of Vps34. Given these findings, class II PI3Ks (α and β isoforms) were examined as potential positive regulators of autophagy. Depletion of class II PI3Ks reduced recruitment of WIPI-1 and LC3 to AP nucleation sites and caused an accumulation of the autophagy substrate, p62, which was exacerbated upon the concomitant ablation of Vps34. Our studies indicate that while Vps34 is the main PI3P source during autophagy, class II PI3Ks also significantly contribute to PI3P generation and regulate AP biogenesis.
    PLoS ONE 10/2013; 8(10):e76405. DOI:10.1371/journal.pone.0076405 · 3.23 Impact Factor
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