Eukaroytic cellular homeostasis is critically secured by autophagy, a catabolic pathway for the degradation of cytoplasmic material in the lysosomal compartment. Macroautophagy, one of the major autophagic pathway, is initiated upon PtdIns(3)P generation by activated PtdIns3KC3 in complex with Beclin 1, p150 and Atg14L. Subsequently, specific PtdIns(3)P-effector proteins permit the formation of double-membrane vesicles, autophagosomes, that sequester the cytoplasmic material. Autophagosomes then communicate and fuse with the lysosomal compartment for final cargo degradation. Members of the human WIPI family function as essential PtdIns(3)P-binding proteins during the initiation of macroautophagy downstream of PtdIns3KC3, and become membrane proteins of generated autophagosomes. Here, we discuss the function of human WIPIs and describe the WIPI puncta-formation analysis for the quantitative assessment of macroautophagy.
Autophagy (auto phagia:
The survival function of autophagy is based on the three major autophagic pathways, macroautophagy, microautophagy and chaperone-mediated autophagy (CMA) that coexist in eukaryotic cells . In the process of microautophagy, proteins or organells are non-selectively engulfed by the lysosome through lysosomal membrane invagination and vesicle scission . CMA specifically targets cytoplasmic proteins containing the KFERQ-like consensus motif for an Hsp70-assisted transport to the lysosomal compartment and an LAMP2-assisted import into the lysosomal lumen in higher eukaryotic cells . The process of macroautophagy is hallmarked by the formation of autophagosomes, double-membrane vesicles that sequester the cytoplasmic cargo (membranes, proteins, organells) and that communicate with the lysosomal compartment for final degradation. Constitutively active on a low basal level, macroautophagy stochastically clears the cytoplasm and promotes the recycling of its constituents. In addition, upon a variety of cellular insults that lead to organelle damage and protein aggregation, macroautophagy is specifically induced and engaged to counteract toxicity.
The cytoprotective function of the three major forms of autophagy critically prevent the development of a variety of age-related human diseases, including cancer and neurodegeneration. However, autophagic pathways also play a vital role in the manifestation of certain pathologies, thus it is of urgent interest to monitor and understand the differential contribution of autophagic pathways to both human health and disease .
2. The process of macroautophagy
Central to the process of macroautophagy is the formation of autophagosomes that sequester and carry the cytoplasmic cargo – membranes, proteins and organelles - to the lysosomal compartment for subsequent degradation and recycling (Figure 1). For decades, the membrane origin of autophagosomes was uncertain . Recently, a variety of independent studies provided evidence that multiple membrane sources should in fact become employed for the formation of autophagosomes . Upon a hierarchical recruitment of autophagy-related (Atg) proteins , membrane origins are thought to undergo membrane rearrangements, including the formation of ER-associated omegasome structures , from which autophagosomal precursor membranes (phagophores) emerge [12,13]. By communicating with the endosomal compartment, the phagophore membrane is proposed to elongate and close to form the autophagosome that robustly sequesters the cytoplasmic cargo within a double-membraned vesicular structure . Next, autophagosomes mature through communication with the endosomal/lysosomal compartment and the degradation of the sequestered cargo occurs in autolysosomes, fused vesicles of autophagosome and lysosomes . Interestingly,
The level of autophagosome formation is crucially balanced by the activity of the mTOR complex 1 (mTORC1), which inhibits macroautophagy when positioned at peripheral lysosomes and which releases its inhibitory role when positioned at perinuclear lysosomes  (Figure 2). The inhibition of mTORC1 mediates the activation of phosphatidylinositol 3-kinase class III (PtdIns3KC3 or Vps34) that phosphorylates PtdIns to generate PtdIns(3)P, an essential phospholipid for the forming autophagosomal membrane . PtdIns3KC3 functions in canonical macroautophagy in complex with Beclin 1 (Atg6 in yeast), p150 (or Vps15) and Atg14L , the latter recruiting PtdIns3KC3 to the ER  where membrane rearrangements are initialized by the PtdIns(3)P effector proteins DFCP1  and WIPIs [19,20]. Macroautophagy can also be induced by non-canonical entries, e.g. independent of Beclin 1 [20,21,22].
3. Human WIPIs
By screening for novel p53 inhibitory factors, we identified a partial, uncharacterized cDNA fragment  and subsequently cloned the corresponding full-length cDNA from normal human liver and testis . Using BLAST it became apparent that the isolated cDNA should be part of a novel human gene and protein family consisting of four members, which we subsequently cloned from normal human testis and placenta . We proposed to term this novel human family WIPI (WD-repeat protein interacting with phosphoinositides) based on the following findings. First, the primary amino acid sequence suggested that the WIPIs contain seven WD40 repeats [19,24] that should fold into 7-bladed beta propeller proteins with an open Velcro configuration, as shown by structural homology modeling . Second, WIPIs represent novel domains that specifically bind to PtdIns(3)P and PtdIns(3,5)P2 [19,24,25,26]. Third, a comprehensive bioinformatic analysis demonstrated that the human WIPI family identified belongs to an ancient protein family of 7-bladed beta propellers with two paralogous groups, one group containing human WIPI-1 and WIPI-2, and the other group containing WIPI-3 and WIPI-4 [19,26]. Jeffries and coworkers found that WIPI-1 (WIPI49) should function in mannose-6-phosphate receptor trafficking , and our own studies demonstrated that WIPI-1 functions during macroautophagy in human tumor cells .
All human WIPI genes are ubiquitiously expressed in normal human tissue, but show high levels in skeletal muscle and heart . Moreover, in a variety of human tumor types the abundance of all WIPI genes was shown to be aberrant when compared to matched normal samples from the same patient; WIPI-1 and WIPI-3 seemed to be more abundant, and WIPI-2 and WIPI-4 less abundant in the tumor . In human tumor cell lines the abundance of the four WIPIs also differs [19,26]. However, the contribution of WIPIs in tumor formation is as yet uncharacterized.
During the process of macroautophagy, essential PtdIns(3)P effector functions (Figure 2) have been assigned to members of the human WIPI family [10,19,22,26,27,28], according to the ancestral function of yeast Atg18 [25,29,30,31,32]. Moreover, human WIPIs were also shown to be involved in pathogen defense by promoting the degradation of internalized bacteria in the lysosomal compartment [33,34,35], and to further contribute to Parkin-mediated mitophagy .
Upon the initiation of autophagy (Figure 2) WIPI-1 and WIPI-2 specifically bind to generated PtdIns(3)P at phagophore membranes [10,19,26,37]. In addition, WIPI-1 and WIPI-2 also bind, although to a lesser extend, to PtdIns(3,5)P2 [24,26,37], however with unknown functional consequences. Phospholipid binding is mediated by evolutionarily conserved amino acids positioned in blade 5 and 6 of the beta-propeller structure of human WIPI proteins  and yeast homologs [38,39,40]. Further, WIPI-1 and WIPI-2 act as PtdIns(3)P effectors upstream of both the Atg12 and LC3 ubiquitin-like conjugation systems, hence regulate LC3 lipidation [10,22,26,37] which is required for the elongation of the phagophore. Moreover, both WIPI-1 and WIPI-2 become membrane proteins of formed autophagosomes and probably also of autolysosomes .
From the further specific localization of WIPI-1 and WIPI-2 upon the induction of macroautophagy, conclusions about the membrane origin of WIPI-positive autophagosomes can be concluded (Figure 2): i) as WIPI-1 specifically accumulates at the ER and at the plasma membrane (PM) upon starvation-induced macroautophagy, both of these membrane systems might contribute to phagophore and autophagosome formation, ii) as WIPI-2 also accumulates at the plasma membrane upon starvation, and in addition to membranes close to the Golgi, a differential engagement of particular membrane systems for autophagosome formation might be mediated by the different WIPIs .
4. WIPI-1 puncta-formation analysis
The specific protein localization of WIPI-1 at both phagophores and autophagosomes has been employed for the quantitative assessment of macroautophagy in mammalian cells  and extended for usage of automated fluorescent image acquisition and analysis [22,34,42]. Upon the induction of macroautophagy, e.g. by rapamycin administration or starvation (Figure 3), WIPI-1 accumulates at autophagosomal membranes, termed puncta. Upon the inhibition of autophagy, e.g. by wortmannin treatment, WIPI-1 is distributed throughout the cytoplasm. Under nutrient-rich conditions few WIPI-1 puncta-positive cells are observed and this assessment reflects basal macroautophagy. To visualize endogenous WIPI-1, indirect immunofluorescence with specific anti-WIPI-1 antibodies is conducted. Alternatively, overexpressed WIPI-1 fusion proteins, e.g. tagged to GFP, can also be employed to quantify the status of macroautophagy. Both, the number of cells displaying WIPI-1 puncta and the number of WIPI-1 puncta per cell can be used to assess macroautophagy [43,44].
The notion that human WIPIs function as essential PtdIns(3)P effectors in macroautophagy needs to be addressed in more molecular detail as follows: i) analysing the individual contribution of the WIPIs to phagophore formation, ii) defining the function of WIPIs at autophagosomes and autolysosomes and iii) identification of WIPI interacting proteins and the signaling network regulating the PtdIns(3)P effector function of WIPIs. As WIPIs are aberrantly expressed in human tumors, the role of WIPIs during tumorigenesis, in particular the regulation of gene expression in normal and tumor cells is of further current interest. Moreover, the identification of compounds that permit a direct interference with the specific binding of WIPIs to PtdIns(3)P might become suitable in the future to specifically modulate macroautophagy in anti-tumor therapies.
ATG, autophagy related; CMA, chaperone-mediated autophagy; PtdIns(3)P, phosphatidylinositol 3-phosphate; PtdIns(3,5)P2, phosphatidylinositol 3,5-bisphosphate; PtdIns3KC3, phosphatidylinositol 3-kinase class III; mTOR, mammalian target of rapamycin; mTORC1, mTOR complex 1; WIPI, WD-repeat protein interacting with phosphoinositides.
AcknowledgmentsWe thank the German Research Foundation (DFG, SFB 773) for grant support to TP-C, and the Forschungsschwerpunktprogramm Baden-Wuerttemberg (Kapitel 1403 Tit. Gr. 74) for supporting the doctoral thesis of DB.
Levine B (2005) Eating oneself and uninvited guests: autophagy-related pathways in cellular defense. Cell 120: 159-162.
Yang Z, Klionsky DJ (2010) Eaten alive: a history of macroautophagy. Nat Cell Biol 12: 814-822.
Melendez A, Talloczy Z, Seaman M, Eskelinen EL, Hall DH, et al. (2003) Autophagy genes are essential for dauer development and life-span extension in C. elegans. Science 301: 1387-1391.
Kuma A, Hatano M, Matsui M, Yamamoto A, Nakaya H, et al. (2004) The role of autophagy during the early neonatal starvation period. Nature 432: 1032-1036.
Mizushima N, Levine B, Cuervo AM, Klionsky DJ (2008) Autophagy fights disease through cellular self-digestion. Nature 451: 1069-1075.
Mijaljica D, Prescott M, Devenish RJ (2011) Microautophagy in mammalian cells: revisiting a 40-year-old conundrum. Autophagy 7: 673-682.
Kaushik S, Cuervo AM (2012) Chaperone-mediated autophagy: a unique way to enter the lysosome world. Trends Cell Biol 22: 407-417.
Juhasz G, Neufeld TP (2006) Autophagy: a forty-year search for a missing membrane source. PLoS Biol 4: e36.
Mari M, Tooze SA, Reggiori F (2011) The puzzling origin of the autophagosomal membrane. F1000 Biol Rep 3: 25.
Itakura E, Mizushima N (2010) Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins. Autophagy 6: 764-776.
Axe EL, Walker SA, Manifava M, Chandra P, Roderick HL, et al. (2008) Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J Cell Biol 182: 685-701.
Hayashi-Nishino M, Fujita N, Noda T, Yamaguchi A, Yoshimori T, et al. (2009) A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation. Nat Cell Biol 11: 1433-1437.
Yla-Anttila P, Vihinen H, Jokitalo E, Eskelinen EL (2009) 3D tomography reveals connections between the phagophore and endoplasmic reticulum. Autophagy 5: 1180-1185.
Jahreiss L, Menzies FM, Rubinsztein DC (2008) The itinerary of autophagosomes: from peripheral formation to kiss-and-run fusion with lysosomes. Traffic 9: 574-587.
Korolchuk VI, Saiki S, Lichtenberg M, Siddiqi FH, Roberts EA, et al. (2011) Lysosomal positioning coordinates cellular nutrient responses. Nat Cell Biol 13: 453-460.
Obara K, Ohsumi Y (2011) PtdIns 3-Kinase Orchestrates Autophagosome Formation in Yeast. J Lipids 2011: 498768.
Matsunaga K, Saitoh T, Tabata K, Omori H, Satoh T, et al. (2009) Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages. Nat Cell Biol 11: 385-396.
Matsunaga K, Morita E, Saitoh T, Akira S, Ktistakis NT, et al. (2010) Autophagy requires endoplasmic reticulum targeting of the PI3-kinase complex via Atg14L. J Cell Biol 190: 511-521.
Proikas-Cezanne T, Waddell S, Gaugel A, Frickey T, Lupas A, et al. (2004) WIPI-1alpha (WIPI49), a member of the novel 7-bladed WIPI protein family, is aberrantly expressed in human cancer and is linked to starvation-induced autophagy. Oncogene 23: 9314-9325.
Codogno P, Mehrpour M, Proikas-Cezanne T (2012) Canonical and non-canonical autophagy: variations on a common theme of self-eating? Nat Rev Mol Cell Biol 13: 7-12.
Gao P, Bauvy C, Souquere S, Tonelli G, Liu L, et al. (2010) The Bcl-2 homology domain 3 mimetic gossypol induces both Beclin 1-dependent and Beclin 1-independent cytoprotective autophagy in cancer cells. J Biol Chem 285: 25570-25581.
Mauthe M, Jacob A, Freiberger S, Hentschel K, Stierhof YD, et al. (2011) Resveratrol-mediated autophagy requires WIPI-1-regulated LC3 lipidation in the absence of induced phagophore formation. Autophagy 7: 1448-1461.
Waddell S, Jenkins JR, Proikas-Cezanne T (2001) A "no-hybrids" screen for functional antagonizers of human p53 transactivator function: dominant negativity in fission yeast. Oncogene 20: 6001-6008.
Jeffries TR, Dove SK, Michell RH, Parker PJ (2004) PtdIns-specific MPR pathway association of a novel WD40 repeat protein, WIPI49. Mol Biol Cell 15: 2652-2663.
Dove SK, Piper RC, McEwen RK, Yu JW, King MC, et al. (2004) Svp1p defines a family of phosphatidylinositol 3,5-bisphosphate effectors. EMBO J 23: 1922-1933.
Polson HE, de Lartigue J, Rigden DJ, Reedijk M, Urbe S, et al. (2010) Mammalian Atg18 (WIPI2) localizes to omegasome-anchored phagophores and positively regulates LC3 lipidation. Autophagy 6.
Lu Q, Yang P, Huang X, Hu W, Guo B, et al. (2011) The WD40 repeat PtdIns(3)P-binding protein EPG-6 regulates progression of omegasomes to autophagosomes. Dev Cell 21: 343-357.
Vergne I, Roberts E, Elmaoued RA, Tosch V, Delgado MA, et al. (2009) Control of autophagy initiation by phosphoinositide 3-phosphatase Jumpy. EMBO J 28: 2244-2258.
Guan J, Stromhaug PE, George MD, Habibzadegah-Tari P, Bevan A, et al. (2001) Cvt18/Gsa12 is required for cytoplasm-to-vacuole transport, pexophagy, and autophagy in Saccharomyces cerevisiae and Pichia pastoris. Mol Biol Cell 12: 3821-3838.
Barth H, Meiling-Wesse K, Epple UD, Thumm M (2001) Autophagy and the cytoplasm to vacuole targeting pathway both require Aut10p. FEBS Lett 508: 23-28.
Stromhaug PE, Reggiori F, Guan J, Wang CW, Klionsky DJ (2004) Atg21 is a phosphoinositide binding protein required for efficient lipidation and localization of Atg8 during uptake of aminopeptidase I by selective autophagy. Mol Biol Cell 15: 3553-3566.
Efe JA, Botelho RJ, Emr SD (2007) Atg18 regulates organelle morphology and Fab1 kinase activity independent of its membrane recruitment by phosphatidylinositol 3,5-bisphosphate. Mol Biol Cell 18: 4232-4244.
Kageyama S, Omori H, Saitoh T, Sone T, Guan JL, et al. (2011) The LC3 recruitment mechanism is separate from Atg9L1-dependent membrane formation in the autophagic response against Salmonella. Mol Biol Cell 22: 2290-2300.
Mauthe M, Yu W, Krut O, Kronke M, Gotz F, et al. (2012) WIPI-1 Positive Autophagosome-Like Vesicles Entrap Pathogenic Staphylococcus aureus for Lysosomal Degradation. Int J Cell Biol 2012: 179207.
Ogawa M, Yoshikawa Y, Kobayashi T, Mimuro H, Fukumatsu M, et al. (2011) A Tecpr1-dependent selective autophagy pathway targets bacterial pathogens. Cell Host Microbe 9: 376-389.
Itakura E, Kishi-Itakura C, Koyama-Honda I, Mizushima N (2012) Structures containing Atg9A and the ULK1 complex independently target depolarized mitochondria at initial stages of Parkin-mediated mitophagy. J Cell Sci 125: 1488-1499.
Proikas-Cezanne T, Ruckerbauer S, Stierhof YD, Berg C, Nordheim A (2007) Human WIPI-1 puncta-formation: a novel assay to assess mammalian autophagy. FEBS Lett 581: 3396-3404.
Krick R, Busse RA, Scacioc A, Stephan M, Janshoff A, et al. (2012) Structural and functional characterization of the two phosphoinositide binding sites of PROPPINs, a beta-propeller protein family. Proc Natl Acad Sci U S A 109: E2042-2049.
Baskaran S, Ragusa MJ, Boura E, Hurley JH (2012) Two-site recognition of phosphatidylinositol 3-phosphate by PROPPINs in autophagy. Mol Cell 47: 339-348.
Watanabe Y, Kobayashi T, Yamamoto H, Hoshida H, Akada R, et al. (2012) Structure-based Analyses Reveal Distinct Binding Sites for Atg2 and Phosphoinositides in Atg18. J Biol Chem 287: 31681-31690.
Proikas-Cezanne T, Robenek H (2011) Freeze-fracture replica immunolabelling reveals human WIPI-1 and WIPI-2 as membrane proteins of autophagosomes. J Cell Mol Med 15: 2007-2010.
Pfisterer SG, Mauthe M, Codogno P, Proikas-Cezanne T (2011) Ca2+/calmodulin-dependent kinase (CaMK) signaling via CaMKI and AMP-activated protein kinase contributes to the regulation of WIPI-1 at the onset of autophagy. Mol Pharmacol 80: 1066-1075.
Proikas-Cezanne T, Pfisterer SG (2009) Assessing mammalian autophagy by WIPI-1/Atg18 puncta formation. Methods Enzymol 452: 247-260.
Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena A, et al. (2012) Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8: 445-544.