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.

  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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 · 4.82 Impact Factor
  • Source
    • "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]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Invading pathogens provoke the autophagic machinery and, in a process termed xenophagy, the host cell survives because autophagy is employed as a safeguard for pathogens that escaped phagosomes. However, some pathogens can manipulate the autophagic pathway and replicate within the niche of generated autophagosome-like vesicles. By automated fluorescence-based high content analyses, we demonstrate that Staphylococcus aureus strains (USA300, HG001, SA113) stimulate autophagy and become entrapped in intracellular PtdIns(3)P-enriched vesicles that are decorated with human WIPI-1, an essential PtdIns(3)P effector of canonical autophagy and membrane protein of both phagophores and autophagosomes. Further, agr-positive S. aureus (USA300, HG001) strains were more efficiently entrapped in WIPI-1 positive autophagosome-like vesicles when compared to agr-negative cells (SA113). By confocal and electron microscopy we provide evidence that single- and multiple-Staphylococci entrapped undergo cell division. Moreover, the number of WIPI-1 positive autophagosome-like vesicles entrapping Staphylococci significantly increased upon (i) lysosomal inhibition by bafilomycin A(1) and (ii) blocking PIKfyve-mediated PtdIns(3,5)P(2) generation by YM201636. In summary, our results provide evidence that the PtdIns(3)P effector function of WIPI-1 is utilized during xenophagy of Staphylococcus aureus. We suggest that invading S. aureus cells become entrapped in autophagosome-like WIPI-1 positive vesicles targeted for lysosomal degradation in nonprofessional host cells.
    International Journal of Cell Biology 07/2012; 2012:179207. DOI:10.1155/2012/179207
  • Source
    • "). 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). "
    [Show abstract] [Hide abstract]
    ABSTRACT: PtdIns(3)P plays critical roles in the autophagy pathway. However, little is known about how PtdIns(3)P effectors act with autophagy proteins in autophagosome formation. Here we identified an essential autophagy gene in C. elegans, epg-6, which encodes a WD40 repeat-containing protein with PtdIns(3)P-binding activity. EPG-6 directly interacts with ATG-2. epg-6 and atg-2 regulate progression of omegasomes to autophagosomes, and their loss of function causes accumulation of enlarged early autophagic structures. Another WD40 repeat PtdIns(3)P effector, ATG-18, plays a distinct role in autophagosome formation. We also established the hierarchical relationship of autophagy genes in degradation of protein aggregates and revealed that the UNC-51/Atg1 complex, EPG-8/Atg14, and binding of lipidated LGG-1 to protein aggregates are required for omegasome formation. Our study demonstrates that autophagic PtdIns(3)P effectors play distinct roles in autophagosome formation and also provides a framework for understanding the concerted action of autophagy genes in protein aggregate degradation.
    Developmental Cell 08/2011; 21(2):343-57. DOI:10.1016/j.devcel.2011.06.024 · 10.37 Impact Factor
Show more


Available from