Noor Gammoh

Macroautophagy mediates the degradation of long-lived proteins and organelles via the de novo formation of double-membrane autophagosomes that sequester cytoplasm and deliver it to the vacuole/lysosome; however, relatively little is known about autophagosome biogenesis. Atg8, a phosphatidylethanolamine-conjugated protein, was previously proposed to function in autophagosome membrane expansion, based on the observation that it mediates liposome tethering and hemifusion in vitro. We show here that with physiological concentrations of phosphatidylethanolamine, Atg8 does not act as a fusogen. Rather, we provide evidence for the involvement of exocytic Q/t-SNAREs in autophagosome formation, acting in the recruitment of key autophagy components to the site of autophagosome formation, and in regulating the organization of Atg9 into tubulovesicular clusters. Additionally, we found that the endosomal Q/t-SNARE Tlg2 and the R/v-SNAREs Sec22 and Ykt6 interact with Sso1-Sec9, and are required for normal Atg9 transport. Thus, multiple SNARE-mediated fusion events are likely to be involved in autophagosome biogenesis.

Autophagy, a catabolic pathway that delivers cellular components to lysosomes for degradation, can be activated by stressful conditions such as nutrient starvation and endoplasmic reticulum (ER) stress. We report that thapsigargin, an ER stressor widely used to induce autophagy, in fact blocks autophagy. Thapsigargin does not affect autophagosome formation but leads to accumulation of mature autophagosomes by blocking autophagosome fusion with the endocytic system. Strikingly, thapsigargin has no effect on endocytosis-mediated degradation of epidermal growth factor receptor. Molecularly, while both Rab7 and Vps16 are essential regulatory components for endocytic fusion with lysosomes, we found that Rab7 but not Vps16 is required for complete autophagy flux, and that thapsigargin blocks recruitment of Rab7 to autophagosomes. Therefore, autophagosomal-lysosomal fusion must be governed by a distinct molecular mechanism compared to general endocytic fusion.

Article9 January 2018Open Access Transparent process The WD40 domain of ATG16L1 is required for its non-canonical role in lipidation of LC3 at single membranes Katherine Fletcher Katherine Fletcher Signalling Programme, Babraham Institute, Cambridge, UK Search for more papers by this author Rachel Ulferts Rachel Ulferts Division of Virology, Department of Pathology, University of Cambridge, Cambridge, UK Search for more papers by this author Elise Jacquin Elise Jacquin Signalling Programme, Babraham Institute, Cambridge, UK Search for more papers by this author Talitha Veith Talitha Veith Division of Virology, Department of Pathology, University of Cambridge, Cambridge, UK Search for more papers by this author Noor Gammoh Noor Gammoh Edinburgh Cancer Research UK Centre, University of Edinburgh, Edinburgh, UK Search for more papers by this author Julia M Arasteh Julia M Arasteh Norwich Medical School, UEA, Norwich, UK Search for more papers by this author Ulrike Mayer Ulrike Mayer School of Biological Sciences, UEA, Norwich, UK Search for more papers by this author Simon R Carding Simon R Carding orcid.org/0000-0002-2383-9701 Quadrum Institute Bioscience, Norwich Research Park, Norwich, UK Search for more papers by this author Thomas Wileman Thomas Wileman Norwich Medical School, UEA, Norwich, UK Search for more papers by this author Rupert Beale Corresponding Author Rupert Beale [email protected] orcid.org/0000-0002-6705-8560 Division of Virology, Department of Pathology, University of Cambridge, Cambridge, UK Search for more papers by this author Oliver Florey Corresponding Author Oliver Florey [email protected] orcid.org/0000-0002-1075-7424 Signalling Programme, Babraham Institute, Cambridge, UK Search for more papers by this author Katherine Fletcher Katherine Fletcher Signalling Programme, Babraham Institute, Cambridge, UK Search for more papers by this author Rachel Ulferts Rachel Ulferts Division of Virology, Department of Pathology, University of Cambridge, Cambridge, UK Search for more papers by this author Elise Jacquin Elise Jacquin Signalling Programme, Babraham Institute, Cambridge, UK Search for more papers by this author Talitha Veith Talitha Veith Division of Virology, Department of Pathology, University of Cambridge, Cambridge, UK Search for more papers by this author Noor Gammoh Noor Gammoh Edinburgh Cancer Research UK Centre, University of Edinburgh, Edinburgh, UK Search for more papers by this author Julia M Arasteh Julia M Arasteh Norwich Medical School, UEA, Norwich, UK Search for more papers by this author Ulrike Mayer Ulrike Mayer School of Biological Sciences, UEA, Norwich, UK Search for more papers by this author Simon R Carding Simon R Carding orcid.org/0000-0002-2383-9701 Quadrum Institute Bioscience, Norwich Research Park, Norwich, UK Search for more papers by this author Thomas Wileman Thomas Wileman Norwich Medical School, UEA, Norwich, UK Search for more papers by this author Rupert Beale Corresponding Author Rupert Beale [email protected] orcid.org/0000-0002-6705-8560 Division of Virology, Department of Pathology, University of Cambridge, Cambridge, UK Search for more papers by this author Oliver Florey Corresponding Author Oliver Florey [email protected] orcid.org/0000-0002-1075-7424 Signalling Programme, Babraham Institute, Cambridge, UK Search for more papers by this author Author Information Katherine Fletcher1,‡, Rachel Ulferts2,‡, Elise Jacquin1, Talitha Veith2, Noor Gammoh3, Julia M Arasteh4, Ulrike Mayer5, Simon R Carding6, Thomas Wileman4, Rupert Beale *,2 and Oliver Florey *,1 1Signalling Programme, Babraham Institute, Cambridge, UK 2Division of Virology, Department of Pathology, University of Cambridge, Cambridge, UK 3Edinburgh Cancer Research UK Centre, University of Edinburgh, Edinburgh, UK 4Norwich Medical School, UEA, Norwich, UK 5School of Biological Sciences, UEA, Norwich, UK 6Quadrum Institute Bioscience, Norwich Research Park, Norwich, UK ‡These authors contributed equally to this work *Corresponding author. Tel: +44 1223 763421; Fax: +44 1223 336926; E-mail: [email protected] *Corresponding author. Tel: +44 1223 496431; Fax: +44 1223 496043; E-mail: [email protected] The EMBO Journal (2018)37:e97840https://doi.org/10.15252/embj.201797840 See also: D Fracchiolla & S Martens (February 2017) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract A hallmark of macroautophagy is the covalent lipidation of LC3 and insertion into the double-membrane phagophore, which is driven by the ATG16L1/ATG5-ATG12 complex. In contrast, non-canonical autophagy is a pathway through which LC3 is lipidated and inserted into single membranes, particularly endolysosomal vacuoles during cell engulfment events such as LC3-associated phagocytosis. Factors controlling the targeting of ATG16L1 to phagophores are dispensable for non-canonical autophagy, for which the mechanism of ATG16L1 recruitment is unknown. Here we show that the WD repeat-containing C-terminal domain (WD40 CTD) of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non-canonical autophagy, but dispensable for canonical autophagy. Using this strategy to inhibit non-canonical autophagy specifically, we show a reduction of MHC class II antigen presentation in dendritic cells from mice lacking the WD40 CTD. Further, we demonstrate activation of non-canonical autophagy dependent on the WD40 CTD during influenza A virus infection. This suggests dependence on WD40 CTD distinguishes between macroautophagy and non-canonical use of autophagy machinery. Synopsis Ligase complex component ATG16L1 is required for LC3 lipidation of double-membrane autophagsomes and of single membranes during phagocytosis and influenza infection, but only single membrane lipidation depends on ATG16L1's C-terminal WD40 domain. ATG16L1 recruitment to single-membrane compartments during non-canonical autophagy is dependent on the C-terminal WD40 domain (CTD). Modification of the ATG16L1 CTD offers a strategy to distinguish between canonical (macro)autophagy and non-canonical autophagy processes. Influenza infection activates non-canonical autophagy. Deletion of the ATG16L1 CTD impairs MHC class II antigen presentation in dendritic cells. Introduction Autophagy is a catabolic process where cytosolic components are degraded within the lysosome. Canonical autophagy (here referring to macroautophagy) involves the formation of double-membrane vesicles called autophagosomes that sequester intracellular material, including organelles or proteins, and target it to lysosomes (Feng et al, 2013). Canonical autophagy is activated by a variety of cellular stresses such as nutrient deprivation, and functions to maintain cellular energy metabolism and viability (Choi et al, 2013). The association of microtubule-associated protein 1 light chain 3 (LC3) has long been considered a defining hallmark of autophagosomes. LC3 is a cytosolic ubiquitin-like protein, which upon activation of canonical autophagy becomes covalently bound to phosphatidylethanolamine (PE) on autophagosomal membranes (Mizushima et al, 1998). It is now appreciated that the membrane remodelling machinery required for starvation-induced autophagy can be co-opted to a variety of different uses (Codogno et al, 2011). For example, LC3 can be conjugated to PE in the context of single-membrane, non-autophagosome compartments. We refer to these processes, which target LC3 to a single membrane, as “non-canonical autophagy” (Florey & Overholtzer, 2012). Examples of this non-canonical autophagy pathway include LC3-associated phagocytosis (LAP), where LC3 is lipidated at single-membrane phagosomes following the engulfment of bacterial and fungal pathogens or apoptotic and necrotic cells (Sanjuan et al, 2007; Florey et al, 2011; Martinez et al, 2011). A similar LAP-like LC3 lipidation event is seen during macropinocytosis and entosis, the latter a form of live cell engulfment (Florey et al, 2011). It has also recently been reported that a range of drugs possessing lysosomotropic or ionophore properties, including monensin, CCCP and chloroquine, are able to activate non-canonical autophagy and induce the lipidation of LC3 at single-membrane compartments of the endolysosomal system (Florey et al, 2015; Jacquin et al, 2017). While these processes associated with unconventional LC3 lipidation are not bona fide autophagic processes (Galluzzi et al, 2017), they are commonly referred to as non-canonical autophagy (Henault et al, 2012; Kim et al, 2013; Cadwell, 2016; Martinez et al, 2016; Jacquin et al, 2017). This should not be confused with ATG5- or Beclin1-independent autophagy (Codogno et al, 2011). Non-canonical autophagy has been implicated in regulating the degradation of material following macro-scale engulfments through the modulation of lysosome fusion with macroendocytic compartments. This is important for many immune responses such as pathogen clearance and antigen presentation (Sanjuan et al, 2007; Ma et al, 2012; Romao et al, 2013). LAP also plays a role in modulating the cytokine profile in macrophages following the engulfment of apoptotic cells (Martinez et al, 2011, 2016) resulting in a proinflammatory response. In the absence of LAP, mice develop an autoimmune phenotype that resembles systemic lupus erythematosus (Martinez et al, 2016). The exact molecular mechanisms underlying how non-canonical autophagy facilitates these events remain unknown. An important feature common to non-canonical autophagy processes is that the associated LC3 lipidation is independent from the upstream regulators of canonical autophagy, including the ULK1 complex containing ATG13 and FIP200, and nutrient status (Florey et al, 2011; Martinez et al, 2015). However, like canonical autophagy, non-canonical autophagy utilises the core ubiquitin-like conjugation machinery that consists of ATG3, 4, 5, 7, 10, 12, 16L1, which together act together to co-ordinate the lipidation of LC3 with PE. In vivo, ATG16L1 is responsible for the correct targeting of LC3 to forming autophagosome membranes (Fujita et al, 2008). It contains an N-terminal domain which associates with ATG5 and ATG12, a central FIP200 and WIPI2b binding domain, and a C-terminal WD40 domain (WD40 CTD). The ATG5-ATG12-ATG16L1 complex has an E3-Ubiquitin ligase-like enzymatic activity required for lipidation of LC3 and is essential for LC3 targeting to membranes during canonical and non-canonical autophagy (Fujita et al, 2008; Martinez et al, 2015). The central region in ATG16L1 (amino acids 229–242) encompassing binding sites for both FIP200 and WIPI2b is known as the FIP200 binding domain (FBD). Deletion of this domain prevents ATG16L1 recruitment to forming autophagosomes and inhibits the canonical autophagy response to both amino acid starvation and infection by cytosolic bacteria (Gammoh et al, 2013; Dooley et al, 2014). Recruitment of the ATG16L1 complex to forming autophagosomes is dependent on the generation of PI3P, via the type III phosphatidylinositol 3-kinase VPS34. Accordingly, inhibition of VPS34 with wortmannin and other inhibitors abrogates autophagosome formation and the associated LC3 lipidation (Itakura & Mizushima, 2010). Subsequently, the PI3P binding effector WIPI2b and FIP200, a member of the ULK1 complex, directly bind and recruit the ATG16L1 complex (Gammoh et al, 2013; Nishimura et al, 2013; Dooley et al, 2014). The C-terminal WD40 domain of ATG16L1 is not present in Atg16, the yeast homolog. The structure of the WD40 CTD has recently been solved, but its biological function remains unclear, although there is some evidence that the WD40 CTD can bind ubiquitin and other factors involved in lysophagy and some forms of xenophagy (Fujita et al, 2013; Boada-Romero et al, 2016; Bajagic et al, 2017). Considering the importance of ATG16L1 in LC3 lipidation during both canonical and non-canonical pathways, we sought to determine the mechanism by which ATG16L1 functions specifically during non-canonical autophagy. In this report, we reveal a critical role for the WD40 CTD of ATG16L1 in its recruitment to single-membrane endolysosomal compartments and for LC3 lipidation during non-canonical autophagy. Importantly, canonical autophagy does not appear to be affected by deletion of the WD40 CTD of ATG16L1. Thus, our results provide the first means to genetically distinguish between canonical and non-canonical autophagy. Influenza A virus (IAV) infection results in the lipidation of LC3 and its relocalisation to the plasma membrane and to perinuclear structures (Gannage et al, 2009; Beale et al, 2014). This depends on the viral M2 protein, a proton channel with multiple roles in the viral life cycle. Targeting of LC3 to the plasma membrane is promoted by a direct interaction between a LC3-interacting region (LIR) in the C-terminal tail of M2 and LC3. Here, using our strategy to distinguish canonical and non-canonical autophagy, we demonstrate that LC3 relocalisation during IAV infection depends on the proton channel activity of M2 and the WD40 CTD of ATG16L1, raising the possibility that activation of the non-canonical autophagy pathway can be triggered by loss of cellular pH gradients. Results ATG16L1 recruitment to membranes and LC3 lipidation during non-canonical autophagy does not require VPS34 and WIPI2b ATG16L1 in complex with ATG5 and ATG12 acts as an E3 enzyme that lipidates LC3 to PE in membranes. During canonical autophagy, ATG16L1 is responsible for targeting the complex to sites of forming autophagosomes. We reasoned that ATG16L1 may also direct LC3 lipidation during non-canonical autophagy, and to address this, first examined its recruitment. In agreement with published work (Fujita et al, 2008), we detect ATG16L1 colocalised with LC3 punctate autophagosome structures following activation of canonical autophagy by nutrient starvation (Fig 1A). To investigate non-canonical autophagy, we analysed LC3-associated phagocytosis (LAP), where LC3 is lipidated to zymosan-containing single-membrane phagosomes (Florey et al, 2011; Martinez et al, 2011; Romao et al, 2013). Interestingly, we detected ATG16L1 recruitment to LC3-positive phagosomes in the mouse macrophage cell line J7741.A (Fig 1B). To broaden this observation, we also analysed models of drug-induced non-canonical autophagy. We have previously reported activation of a non-canonical autophagy pathway by the sodium/proton ionophore monensin, which promotes LC3 lipidation to acidic single-membrane endolysosomal compartments, including those generated following entosis, a live cell engulfment process or engulfment of plain latex beads (Florey et al, 2015; Jacquin et al, 2017). Upon monensin treatment, we observed both ATG16L1 recruitment and LC3 lipidation to large entotic corpse-containing vacuoles (Fig 1C) and to latex bead-containing phagosomes (Fig 1D). These data demonstrate that, like double-membrane autophagosomes in canonical autophagy, ATG16L1 is recruited to single-membrane compartments during non-canonical autophagy. Figure 1. ATG16L1 is recruited to endolysosomal membranes during non-canonical autophagy A. Confocal images of control and starved HCT116 cells expressing GFP-LC3 and stained for ATG16L1. Arrows indicate autophagosome puncta double positive for LC3 and ATG16L1. Scale bar: 10 μm. B–D. Confocal images of ATG16L1 and GFP-LC3 on (B) zymosan-containing phagosomes in J774A.1 cells (arrows indicate phagosomes), scale bar: 5 μm; (C) monensin-treated entotic corpse vacuoles in MCF10A cells (asterisk indicate entotic corpse, arrows indicate entotic vacuoles), scale bar: 10 μm; and (D) latex bead-containing phagosomes in monensin-treated HCT116 cells (asterisk indicate bead-containing phagosomes, arrows indicate phagosome membranes), scale bar: 5 μm. E, F. Confocal images of GFP-LC3 in (E) starved cells or (F) entotic corpse vacuoles in monensin-treated MCF10A cells ± wortmannin pretreatment. Scale bars: 10 μm. G. Western blotting of LC3 in control, starved or monensin-treated HEK293 cells ± wortmannin. H. Quantification of LC3-II/LC3-I ratios from (G). I, J. Confocal images of WIPI2b staining and GFP-LC3 in (I) starved HCT116 cells. Arrows indicated double-positive autophagosome structures, and (J) entotic corpse vacuoles in monensin-treated MCF10A cells. Scale bars: 10 μm. Data information: In (H), data are presented as mean + SEM from three separate experiments. *P < 0.04 (Student's t-test). Download figure Download PowerPoint ATG16L1 recruitment to autophagosomes is dependent on PI3P generated by VPS34, and the PI3P effector WIPI2b that directly binds ATG16L1 (Dooley et al, 2014). In agreement with this, pretreatment of cells with the PI3 kinase inhibitor wortmannin abolishes canonical autophagy induced by starvation as measured by LC3 puncta formation and lipidation (Fig 1E and G). However, in line with our previous report (Florey et al, 2015), total levels of LC3 lipidation and localisation to entotic corpse vacuoles following treatment with monensin were not inhibited by wortmannin (Fig 1F–H). Consistent with the dispensability of VPS34 and PI3P in monensin-induced non-canonical autophagy, we found no evidence of WIPI2b recruitment to LC3-positive entotic corpse vacuoles (Fig 1J), while WIPI2b was observed at LC3-positive starvation-induced autophagosomes (Fig 1I). Together these data show that, upon activation of non-canonical autophagy, ATG16L1 can be recruited to single-membrane endolysosomal compartments independently of PI3P and WIPI2b, and thus through a mechanism distinct from canonical autophagy. ATG16L1 structure function in canonical autophagy To investigate the novel mechanisms underlying ATG16L1 recruitment to membranes during non-canonical autophagy, we sought to map the domain of ATG16L1 required to support endolysosomal LC3 lipidation. To do so, we re-expressed a set of ATG16L1 constructs (depicted in Fig 2A) in multiple independently generated ATG16L1-deficient cell lines. The ATG16L1 constructs consist of full-length ATG16L1 (FL), ATG16L1 lacking the region 219–242 that contains the WIPI2b and FIP200 binding sites (ΔFBD), and ATG16L1 lacking the WD40 CTD (ΔWD). We engineered ATG16L1-deficient clones of the human colon cancer cell line HCT116 and the human breast epithelial cell line MCF10A using CRISPR/Cas9. We also utilised ATG16L1-deficient mouse embryonic fibroblasts (MEFs) previously generated by traditional methods based on homologous recombination. In all three cases, we were able to generate stable cell lines deficient in ATG16L1 and in which either full-length or truncated ATG16L1 could be expressed (Fig 2B-D). Figure 2. The WD domain of ATG16L1 is not required for canonical autophagy A. Diagram of full-length (FL) 229–242 deletion (ΔFBD) and 1–336 (ΔWD) ATG16L1 constructs used in this study. B-D. Western blot analysis of ATG16L1 in (B) HCT116 ATG16L1−/−, (C) MEF Atg16L1−/− and (D) MCF10A ATG16L1−/− cells stably re-expressing ATG16L1 constructs. Arrows indicate specific ATG16L1 band. E. Western blotting for LC3 in complemented HCT116 cells ± PP242 (1 μM, 1 h). F. Quantification of fold differences of LC3-II/LC3-I ratios over controls from (E). G. Confocal images of GFP-LC3 in complemented MEF cells ± starvation (1 h). Scale bar: 10 μm. H. Quantification of GFP-LC3 puncta from 100 MEF cells per experiment cultured in full media (control) or EBSS (starve) for 1 h. I. Quantification of WIPI2b puncta in ATG16L1-complemented HCT116 cells. Puncta from 100 cells were counted per experiment. Data information: Data represent mean ± SEM from three separate experiments. (F) *P < 0.02. (H) ***P < 0.0001, **P < 0.001. (I) ***P < 0.0006, **P≪0.005 (Student's t-test). Download figure Download PowerPoint As expected, cells expressing full-length ATG16L1 exhibited an increase in lipidated LC3 (LC3-II) following activation of canonical autophagy by mTOR inhibition, using PP242 (Fig 2E and F). ΔWD cells displayed similar LC3 lipidation levels to full-length expressing cells (Fig 2E and F), indicating this domain is not required. In line with previous reports, however, we saw a reduction of LC3 lipidation in ΔFBD cells lacking the WIPI2b and FIP200 binding sites (Fig 2E and F), indicating this domain is required for canonical autophagy (Gammoh et al, 2013). Consistent with the Western blot results, we also observed increases in autophagosome number, as assessed by GFP-LC3 puncta, in full-length and ΔWD, but not ΔFBD MEFs (Fig 2G and H) and HCT116 cells (Fig EV1) following starvation. Formation of WIPI2b puncta lies upstream of ATG16L1 recruitment and thus LC3 lipidation in the canonical autophagy pathway. Consistent with this established hierarchy of autophagy proteins, all ATG16L1-expressing HCT116 cell lines supported increased WIPI2b puncta formation following starvation (Fig 2I). Click here to expand this figure. Figure EV1. The WD40 CTD of ATG16L1 is not required for canonical autophagy Confocal images of GFP-LC3-expressing HCT116 ATG16L1−/− complemented with full-length (FL), ΔFBD or ΔWD ATG16L1 under fed or starvation conditions. Scale bar: 10 μm. Quantification of GFP-LC3 puncta in ATG16L1-complemented HCT116 cells under fed or starvation conditions. Data information: In (B), data are presented as mean ± SEM from three independent experiments. ***P < 0.0004, **P < 0.002 (Student's t-test). Download figure Download PowerPoint These data demonstrate that our complemented ATG16L1 cell lines are competent for canonical autophagy induction and confirm previously reports data that the WIPI2b and FIP200 binding domain of ATG16L1 is required for LC3 lipidation and association with autophagosomal membranes in starvation-induced canonical autophagy, while the WD40 CTD is dispensable for this process. ATG16L1 structure function in monensin-induced non-canonical autophagy We next used our set of complemented ATG16L1 cell lines to study LC3 lipidation induced during monensin driven non-canonical autophagy. We have previously shown that in wild-type cells, the ionophore monensin increases lipidated LC3 (LC3-II) via two parallel pathways. Firstly, by inserting into membranes and facilitating the exchange of sodium and hydrogen ions, monensin raises lysosome pH thus blocking autophagosome flux (canonical). At the same time, monensin also induces osmotic imbalances within endolysosomal compartments, which are then targeted for lipidation with LC3 (non-canonical autophagy pathway; Florey et al, 2015). In contrast, the V-ATPase inhibitor bafilomycin A1 increases LC3-II levels solely by inhibiting autophagosome flux. Thus, comparing the levels of lipidated LC3 (LC3-II) induced by monensin versus bafilomycin A1 allows the distinction between these parallel effects. Accordingly, in HCT116 cells expressing full-length ATG16L1 monensin induces significantly more LC3-II than bafilomycin A1, (Fig 3A), indicating the activation of non-canonical autophagy. A similar pattern is observed in ΔFBD cells (Fig 3A), suggesting the FBD domain is dispensable in this context. Strikingly, we detected no difference in LC3-II levels between bafilomycin A1 and monensin treatment in ΔWD cells (Fig 3A). These data provide the first indication that, in contrast to canonical autophagy, the WD40 CTD of ATG16L1 is required for non-canonical autophagy. Figure 3. The WD domain of ATG16L1 is required for monensin-induced non-canonical autophagy Western blotting of LC3 in ATG16L1 FL, ΔFBD- or ΔWD-complemented HCT116 cells treated with bafilomycin (100 nM) or monensin (100 μM) for 1 h. Below is the quantification of LC3-II/LC3-I ratios. Western blotting of LC3 from ATG16L1-complemented HCT116 cells treated with wortmannin (67 μM), monensin (100 μM) or both for 1 h. Below is the quantification of LC3-II/LC3-I ratios. Confocal images of latex bead-containing phagosomes in control and monensin-treated GFP-LC3-expressing HCT116 cells complemented with ATG16L1 FL, ΔFBD or ΔWD. Samples were stained for LAMP1. Cropped images show bead phagosomes. Scale bar: 5 μm. Quantification of GFP-LC3 recruitment to LAMP-1-positive phagosomes. 100 phagosomes were counted per experiment. Data information: In (A, B, D), data are presented as mean ± SEM from three separate experiments. (A) *P < 0.02, ***P < 0.001. (B) **P < 0.0002 (Student's t-test). Download figure Download PowerPoint LC3 lipidation associated with non-canonical autophagy induced by monensin is resistant to wortmannin in wild-type cells (Fig 1D–G). We observe similar wortmannin-resistant LC3 lipidation in full-length and ΔFBD cells following monensin treatment (Fig 3B). However, in ΔWD cells, wortmannin significantly inhibited monensin driven LC3 lipidation. These data suggest that the lipidated LC3 observed in ΔWD cells derives only from a wortmannin-sensitive canonical autophagy pathway. In order to better differentiate between canonical versus non-canonical autophagy pathways, we next examined the localisation of LC3 in our set of complemented cells. Monensin mediated the recruitment of GFP-LC3 to LAMP1-positive latex bead-containing phagosomes in full-length and ΔFBD cells (Fig 3C and D). We have previously shown that the recruitment of GFP-LC3 in this model is lipidation dependent and not associated with canonical autophagy (Florey et al, 2015). In ΔWD cells, we could detect no GFP-LC3 recruitment to phagosomes (Fig 3C and D). A similar result was seen when examining entotic corpse vacuoles in complemented ATG16L1−/− MCF10A cells treated with monensin (Fig EV2A and B). This is consistent with monensin-induced LC3 lipidation being driven by continuous recruitment of the ATG16L1 complex to endolysosomal membranes rather than inhibition of ATG4 activity. This model is supported by fluorescence recovery after photobleaching (FRAP) data that shows GFP-LC3 localisation to monensin-treated entotic corpse vacuoles reappears following photobleaching (Fig EV2C and D). These experiments demonstrate that cells lacking the WD40 CTD of ATG16L1 are unable to support LC3 lipidation to endolysosomal compartments associated with monensin-induced non-canonical autophagy. Click here to expand this figure. Figure EV2. Monensin-induced LC3 lipidation to entotic corpse vacuole requires the WD40 CTD of ATG16L1 Quantification of GFP-LC3 recruitment to LAMP1-positive entotic corpse-containing vacuoles in MCF10A ATG16L1−/− cells complemented full-length (FL), ΔFBD or ΔWD ATG16L1 ± monensin (100 μM, 1 h). Representative confocal images of entotic corpse-containing vacuoles in GFP-expressing MCF10A cells treated with 100 μM monensin for 1 h and stained for LAMP1 (red) and DNA (blue). Scale bar: 10 μm. Representative sequence images from FRAP analysis of GFP-LC3 on entotic corpse-containing vacuoles treated with monensin (100 μM, 1 h). The region marked by a broken-line circle was photobleached, and the recovery of fluorescence at line 1 and 2 was monitored. Scale bar: 10 μm. Quantification of GFP fluorescence at line 1 and 2 from (C). Data information: In (A), data are presented as mean ± SEM from three independent experiments. Download figure Download PowerPoint ATG16L1 structure function in physiological non-canonical autophagy We next sought to test the requirement of the WD40 CTD of ATG16L1 in more physiological examples of non-canonical autophagy. LC3-associated phagocytosis (LAP) occurs during the phagocytic engulfment of apoptotic and necrotic cells, or the engulfment of some fungal and bacterial pathogens. LC3 is targeted to these single-membrane phagosomes independently of the canonical autophagy pathway, but dependent on the lipidation machinery that includes ATG16L1. MEF cells are able to engulf apoptotic cells (Gardai et al, 2005) and have previously been shown to be competent for LAP (Hubber et al, 2017). Using live cell imaging, we detected GFP-LC3 recruitment to phagosomes containing CellTracker Red-labelled apoptotic corpses in full-length and ΔFBD MEFs. However, consistent with our previous data using monensin, ΔWD cells did not support GFP-LC3 recruitment to apoptotic corpse-containing phagosomes (Fig 4A and B). These data demonstrate an essential requirement for the WD40 CTD of ATG16L1 during LC3-associated phagocytosis. Figure 4. The WD domain of ATG16L1 is essential for LC3-associated phagocytosis and other non-canonical autophagy-dependent processes Confocal images of GFP-LC3 in ATG16L1-complemented MEF cells phagocytosing red-labelled apoptotic cells. Scale bar: 10 μm. Quantification of GFP-LC3 recruitment to apoptotic corpse-containing phagosomes in (A). Twenty phagosomes were counted per experiment. Confocal images of red dextran-positive macropinosomes ATG16L1 complimented MEF cells. Cropped images show macropinosomes. Scale bar: 10 μm. Confocal images of GFP-LC3 in ATG16L1-complimented MEF cells treated with VacA toxin (10 μM, 4 h). Scale bar: 10 μm. Data information: In (B), data are presented as mean ± SEM from three separate experiments. ****P < 0.0001 (Student's t-test). Download figure Download PowerPoint Similar to LAP, LC3 has also been shown to be targeted to newly formed macropinosomes via a non-canonical autophagy pathway (Florey et al, 2011). Using red dextran as a fluid phase marker in PDGF stimulated MEFs, we found GFP-LC3 recruitment to red-labelled macropinosomes in full-length and ΔFBD cells but not in ΔWD cells (Fig 4C)

Autophagy is a cellular catabolic pathway by which long-lived proteins and damaged organelles are targeted for degradation. Activation of autophagy enhances cellular tolerance to various stresses. Recent studies indicate that a class of anticancer agents, histone deacetylase (HDAC) inhibitors, can induce autophagy. One of the HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA), is currently being used for treating cutaneous T-cell lymphoma and under clinical trials for multiple other cancer types, including glioblastoma. Here, we show that SAHA increases the expression of the autophagic factor LC3, and inhibits the nutrient-sensing kinase mammalian target of rapamycin (mTOR). The inactivation of mTOR results in the dephosphorylation, and thus activation, of the autophagic protein kinase ULK1, which is essential for autophagy activation during SAHA treatment. Furthermore, we show that the inhibition of autophagy by RNAi in glioblastoma cells results in an increase in SAHA-induced apoptosis. Importantly, when apoptosis is pharmacologically blocked, SAHA-induced nonapoptotic cell death can also be potentiated by autophagy inhibition. Overall, our findings indicate that SAHA activates autophagy via inhibiting mTOR and up-regulating LC3 expression; autophagy functions as a prosurvival mechanism to mitigate SAHA-induced apoptotic and nonapoptotic cell death, suggesting that targeting autophagy might improve the therapeutic effects of SAHA.

Maintenance of protein homeostasis and organelle integrity and function is critical for cellular homeostasis and cell viability. Autophagy is the principal mechanism that mediates the delivery of various cellular cargoes to lysosomes for degradation and recycling. A myriad of studies demonstrate important protective roles for autophagy against disease. However, in cancer, seemingly opposing roles of autophagy are observed in the prevention of early tumour development versus the maintenance and metabolic adaptation of established and metastasizing tumours. Recent studies have addressed not only the tumour cell intrinsic functions of autophagy, but also the roles of autophagy in the tumour microenvironment and associated immune cells. In addition, various autophagy-related pathways have been described, which are distinct from classical autophagy, that utilize parts of the autophagic machinery and can potentially contribute to malignant disease. Growing evidence on how autophagy and related processes affect cancer development and progression has helped guide efforts to design anticancer treatments based on inhibition or promotion of autophagy. In this Review, we discuss and dissect these different functions of autophagy and autophagy-related processes during tumour development, maintenance and progression. We outline recent findings regarding the role of these processes in both the tumour cells and the tumour microenvironment and describe advances in therapy aimed at autophagy processes in cancer. Autophagy can serve both tumour-suppressive and tumour-promoting roles, often depending on disease stage and mutational background. Recent findings have advanced our understanding of these seemingly opposing roles of autophagy in cancer cells themselves and in the tumour microenvironment.

Recently a noncanonical activity of autophagy proteins has been discovered that targets lipidation of microtubule-associated protein 1 light chain 3 (LC3) onto macroendocytic vacuoles, including macropinosomes, phagosomes, and entotic vacuoles. While this pathway is distinct from canonical autophagy, the mechanism of how these nonautophagic membranes are targeted for LC3 lipidation remains unclear. Here we present evidence that this pathway requires activity of the vacuolar-type H(+)-ATPase (V-ATPase) and is induced by osmotic imbalances within endolysosomal compartments. LC3 lipidation by this mechanism is induced by treatment of cells with the lysosomotropic agent chloroquine, and through exposure to the Heliobacter pylori pore-forming toxin VacA. These data add novel mechanistic insights into the regulation of noncanonical LC3 lipidation and its associated processes, including LC3-associated phagocytosis (LAP), and demonstrate that the widely and therapeutically used drug chloroquine, which is conventionally used to inhibit autophagy flux, is an inducer of LC3 lipidation.

Autophagy is a finely orchestrated cellular catabolic process that requires multiple autophagy-related gene products (ATG proteins). The ULK1 complex functions to integrate upstream signals to downstream ATG proteins through an unknown mechanism. Here we have identified an interaction between mammalian FIP200 and ATG16L1, essential components of the ULK1 and ATG5 complexes, respectively. Further analyses show this is a direct interaction mediated by a short domain of ATG16L1 that we term the FIP200-binding domain (FBD). The FBD is not required for ATG16L1 self-dimerization or interaction with ATG5. Notably, an FBD-deleted ATG16L1 mutant is defective in mediating amino acid starvation-induced autophagy, which requires the ULK1 complex. However, this mutant retains its function in supporting glucose deprivation-induced autophagy, a ULK1 complex-independent process. This study therefore identifies a previously uncharacterized interaction between the ULK1 and ATG5 complexes that can distinguish ULK1-dependent and -independent autophagy processes.

Human papillomaviruses (HPVs) are the causative agents of a number of human cancers, of which cervical cancer is the most important. This occurs following persistent infection with a limited number of viral subtypes and is characterized by continued expression of the viral E6 and E7 oncoproteins. A unique characteristic of the cancer-causing HPV types is the presence of a PDZ recognition motif on the carboxy terminus of the E6 oncoprotein. Through this motif, E6 directs the proteasome-mediated degradation of cellular proteins involved in the regulation of cell polarity and in cell proliferation control. These include components of the Scrib and Par polarity complexes, as well as a number of other PDZ domain-containing substrates. Thus, PVs are now providing novel insights into the functioning of many of these cellular proteins, and into which of these functions, in particular, are relevant for maintaining normal cellular homeostasis. In this review, we discuss the biological consequences of papillomaviral targeting of these cell polarity regulators, both with respect to the viral life cycle and, most importantly, to the development of HPV-induced malignancy.

The function of macroautophagy/autophagy during tumor initiation or in established tumors can be highly distinct and context-dependent. To investigate the role of autophagy in gliomagenesis, we utilized a KRAS-driven glioblastoma mouse model in which autophagy is specifically disrupted via RNAi against Atg7, Atg13 or Ulk1. Inhibition of autophagy strongly reduced glioblastoma development, demonstrating its critical role in promoting tumor formation. Further supporting this finding is the observation that tumors originating from Atg7-shRNA injections escaped the knockdown effect and thereby still underwent functional autophagy. In vitro, autophagy inhibition suppressed the capacity of KRAS-expressing glial cells to form oncogenic colonies or to survive low serum conditions. Molecular analyses revealed that autophagy-inhibited glial cells were unable to maintain active growth signaling under growth-restrictive conditions and were prone to undergo senescence. Overall, these results demonstrate that autophagy is crucial for glioma initiation and growth, and is a promising therapeutic target for glioblastoma treatment.

Despite recently uncovered connections between autophagy and the endocytic pathway, the role of autophagy in regulating endosomal function remains incompletely understood. Here, we find that the ablation of autophagy-essential players disrupts EGF-induced endocytic trafficking of EGFR. Cells lacking ATG7 or ATG16L1 exhibit increased levels of phosphatidylinositol-3-phosphate (PI(3)P), a key determinant of early endosome maturation. Increased PI(3)P levels are associated with an accumulation of EEA1-positive endosomes where EGFR trafficking is stalled. Aberrant early endosomes are recognised by the autophagy machinery in a TBK1- and Gal8-dependent manner and are delivered to LAMP2-positive lysosomes. Preventing this homeostatic regulation of early endosomes by autophagy reduces EGFR recycling to the plasma membrane and compromises downstream signalling and cell survival. Our findings uncover a novel role for the autophagy machinery in maintaining early endosome function and growth factor sensing.

Membrane targeting of autophagy-related complexes is an important step that regulates their activities and prevents their aberrant engagement on non-autophagic membranes. ATG16L1 is a core autophagy protein implicated at distinct phases of autophagosome biogenesis. In this study, we dissected the recruitment of ATG16L1 to the pre-autophagosomal structure (PAS) and showed that it requires sequences within its coiled-coil domain (CCD) dispensable for homodimerisation. Structural and mutational analyses identified conserved residues within the CCD of ATG16L1 that mediate direct binding to phosphoinositides, including phosphatidylinositol 3-phosphate (PI3P). Mutating putative lipid binding residues abrogated the localisation of ATG16L1 to the PAS and inhibited LC3 lipidation. On the other hand, enhancing lipid binding of ATG16L1 by mutating negatively charged residues adjacent to the lipid binding motif also resulted in autophagy inhibition, suggesting that regulated recruitment of ATG16L1 to the PAS is required for its autophagic activity. Overall, our findings indicate that ATG16L1 harbours an intrinsic ability to bind lipids that plays an essential role during LC3 lipidation and autophagosome maturation.

Abstract Adult neural stem cells (NSCs) must tightly regulate quiescence and proliferation. Single-cell analysis has suggested a continuum of cell states as NSCs exit quiescence. Here we capture and characterize in vitro primed quiescent NSCs and identify LRIG1 as an important regulator. We show that BMP-4 signaling induces a dormant non-cycling quiescent state (d-qNSCs), whereas combined BMP-4/FGF-2 signaling induces a distinct primed quiescent state poised for cell cycle re-entry. Primed quiescent NSCs (p-qNSCs) are defined by high levels of LRIG1 and CD9, as well as an interferon response signature, and can efficiently engraft into the adult subventricular zone (SVZ) niche. Genetic disruption of Lrig1 in vivo within the SVZ NSCs leads an enhanced proliferation. Mechanistically, LRIG1 primes quiescent NSCs for cell cycle re-entry and EGFR responsiveness by enabling EGFR protein levels to increase but limiting signaling activation. LRIG1 is therefore an important functional regulator of NSC exit from quiescence.

The high-risk HPV E6 proteins have been shown to direct the degradation of a variety of cellular proteins that contain PDZ domains. Although some of these proteins are involved in regulating processes of cell growth and polarity in Drosophila, little is known about their function in higher eukaryotic epithelial cells. In HPV-containing cells derived from cervical tumours, we find that the patterns of expression of the E6 targets hDlg (discs large), hScrib (Scribble), and MUPP1 are consistent with their being substrates for E6-induced degradation. It is also clear that, in the case of hDlg, E6 is specifically targeting nuclear pools of the protein rather than membrane-bound forms. We have also analysed the activity of a subset of E6 target proteins in the suppression of oncogene-induced cell transformation. Interestingly, Dlg, MAGI-1 and MUPP1 efficiently suppressed cell transformation, while MAGI-2 and MAGI-3 were ineffective in this assay. These results suggest that in the context of HPV-induced transformation Dlg, MAGI-1 and MUPP1 can function as tumour suppressors.

Ubiquitin-proteasome system and autophagy are the two major mechanisms for protein degradation in eukaryotic cells. LC3, a ubiquitin-like protein, plays an essential role in autophagy through its ability to be conjugated to phosphatidylethanolamine. In this study, we discovered a novel LC3-processing activity, and biochemically purified the 20S proteasome as the responsible enzyme. Processing of LC3 by the 20S proteasome is ATP- and ubiquitin-independent, and requires both the N-terminal helices and the ubiquitin fold of LC3; and addition of the N-terminal helices of LC3 to the N terminus of ubiquitin renders ubiquitin susceptible to 20S proteasomal activity. Further, the 20S proteasome processes LC3 in a stepwise manner, it first cleaves LC3 within its ubiquitin fold and thus disrupt the conjugation function of LC3; subsequently and especially at high concentrations of the proteasome, LC3 is completely degraded. Intriguingly, proteolysis of LC3 by the 20S proteasome can be inhibited by p62, an LC3-binding protein that mediates autophagic degradation of polyubiquitin aggregates in cells. Therefore, our study implicates a potential mechanism underlying interplay between the proteasomal and autophagic pathways. This study also provides biochemical evidence suggesting relevance of the controversial ubiquitin-independent proteolytic activity of the 20S proteasome.

Autophagy requires the formation of membrane vesicles, known as autophagosomes, that engulf cellular cargoes and subsequently recruit lysosomal hydrolases for the degradation of their contents. A number of autophagy-related proteins act to mediate the de novo biogenesis of autophagosomes and vesicular trafficking events that are required for autophagy. Of these proteins, ATG16L1 is a key player that has important functions at various stages of autophagy. Numerous recent studies have begun to unravel novel activities of ATG16L1, including interactions with proteins and lipids, and how these mediate its role during autophagy and autophagy-related processes. Various domains have been identified within ATG16L1 that mediate its functions in recognising single and double membranes and activating subsequent autophagy-related enzymatic activities required for the recruitment of lysosomes. These recent findings, as well as the historical discovery of ATG16L1, pathological relevance, unresolved questions and contradictory observations, will be discussed here.

Metastasis is the major reason for the death of patients suffering from malignant diseases such as human hepatocellular carcinoma (HCC). Among the complex metastatic process, resistance to anoikis is one of the most important steps. Previous studies demonstrate that microRNA-26a (miR-26a) is an important tumor suppressor that inhibits the proliferation and invasion of HCC cells by targeting multiple oncogenic proteins. However, whether miR-26a can also influence anoikis has not been well established. Here, we discovered that miR-26a promotes anoikis of HCC cells both in vitro and in vivo. With a combinational analysis of bioinformatics and public clinical databases, we predicted that alpha5 integrin (ITGA5), an integrin family member, is a putative target of miR-26a. Furthermore, we provide experimental evidence to confirm that ITGA5 is a bona fide target of miR-26a. Through gain- and loss-of-function studies, we demonstrate that ITGA5 is a functional target of miR-26a-induced anoikis in HCC cells. Collectively, our findings reveal that miR-26a is a novel player during anoikis and a potential therapeutic target for the treatment of metastatic HCC.

During neurotransmission, synaptic vesicles undergo multiple rounds of exo-endocytosis, involving recycling and/or degradation of synaptic proteins. While ubiquitin signaling at synapses is essential for neural function, it has been assumed that synaptic proteostasis requires the ubiquitin-proteasome system (UPS). We demonstrate here that turnover of synaptic membrane proteins via the endolysosomal pathway is essential for synaptic function. In both human and mouse, hypomorphic mutations in the ubiquitin adaptor protein PLAA cause an infantile-lethal neurodysfunction syndrome with seizures. Resulting from perturbed endolysosomal degradation, Plaa mutant neurons accumulate K63-polyubiquitylated proteins and synaptic membrane proteins, disrupting synaptic vesicle recycling and neurotransmission. Through characterization of this neurological intracellular trafficking disorder, we establish the importance of ubiquitin-mediated endolysosomal trafficking at the synapse.

ABSTRACT In order to ensure a productive life cycle, human papillomaviruses (HPVs) require fine regulation of their gene products. Uncontrolled activity of the viral oncoproteins E6 and E7 results in the immortalization of the infected epithelial cells and thus prevents the production of mature virions. Ectopically expressed E2 has been shown to suppress transcription of the HPV E6 and E7 region in cell lines where the viral DNA is integrated into the host genome, resulting in growth inhibition. However, it has been demonstrated that growth control of these cell lines can also occur independently of HPV E2 transcriptional activity in high-risk HPV types. In addition, E2 is unable to suppress transcription of the same region in cell lines derived from cervical tumors that harbor only episomal copies of the viral DNA. Here we show that HPV type 16 (HPV-16) E2 is capable of inhibiting HPV-16 E7 cooperation with an activated ras oncogene in the transformation of primary rodent cells. Furthermore, we demonstrate a direct interaction between the E2 and E7 proteins which requires the hinge region of E2 and the zinc-binding domain of E7. These viral proteins interact in vivo, and E2 has a marked effect upon both the stability of E7 and its cellular location, where it is responsible for recruiting E7 onto mitotic chromosomes at the later stages of mitosis. These results demonstrate a direct role for E2 in regulating the function of E7 and suggest an important role for E2 in directing E7 localization during mitosis.

Vesicular trafficking events play key roles in the compartmentalization and proper sorting of cellular components. These events have crucial roles in sensing external signals, regulating protein activities and stimulating cell growth or death decisions. Although mutations in vesicle trafficking players are not direct drivers of cellular transformation, their activities are important in facilitating oncogenic pathways. One such pathway is the sensing of external stimuli and signalling through receptor tyrosine kinases (RTKs). The regulation of RTK activity by the endocytic pathway has been extensively studied. Compelling recent studies have begun to highlight the association between autophagy and RTK signalling. The influence of this interplay on cellular status and its relevance in disease settings will be discussed here.

Human papillomaviruses are the causative agents of cervical cancer. Previous studies have shown that loss of the viral E2 protein during malignant progression is an important feature of HPV-induced malignancy due to the resulting uncontrolled expression of the viral oncoproteins E6 and E7. We now show however that the viral E2 and E6 proteins are both capable of regulating each other's activity. When coexpressed, E2 and E6 induce marked changes in the pattern of each other's expression, with preferential accumulation in nuclear speckles. The two proteins interact directly, resulting in changes in the substrate specificities of E6 and the biochemical activities of E2. Thus, while E6 efficiently degrades its PDZ domain-containing substrates in the absence of E2, this activity is greatly diminished when E2 is present. Likewise, E2 alone drives both viral DNA replication and viral gene expression. However, in the presence of E6, viral DNA replication is inhibited while the transcriptional activity of E2 is elevated. These studies define a far more complex pattern of interaction between E2 and E6 than was previously thought and redefines the possible consequences of loss of E2 with respect to uncontrolled E6 activity and consequent malignant progression.

Glioblastoma is the most common and aggressive adult brain tumour, with poor median survival and limited treatment options. Following surgical resection and chemotherapy, recurrence of the disease is inevitable. Genomic studies have identified key drivers of glioblastoma development, including amplifications of receptor tyrosine kinases, which drive tumour growth. To improve treatment, it is crucial to understand survival response processes in glioblastoma that fuel cell proliferation and promote resistance to treatment. One such process is autophagy, a catabolic pathway that delivers cellular components sequestered into vesicles for lysosomal degradation. Autophagy plays an important role in maintaining cellular homeostasis and is upregulated during stress conditions, such as limited nutrient and oxygen availability, and in response to anti-cancer therapy. Autophagy can also regulate pro-growth signalling and metabolic rewiring of cancer cells in order to support tumour growth. In this review, we will discuss our current understanding of how autophagy is implicated in glioblastoma development and survival. When appropriate, we will refer to findings derived from the role of autophagy in other cancer models and predict the outcome of manipulating autophagy during glioblastoma treatment.

Report26 April 2020Open Access Source DataTransparent process Trichoplein binds PCM1 and controls endothelial cell function by regulating autophagy Andrea Martello University/BHF Centre for Cardiovascular Science, QMRI, University of Edinburgh, Edinburgh, UK Search for more papers by this author Angela Lauriola Department of Biomedical, Metabolic and Neural Sciences, University of Modena & Reggio Emilia, Modena, Italy Search for more papers by this author David Mellis University/BHF Centre for Cardiovascular Science, QMRI, University of Edinburgh, Edinburgh, UK Search for more papers by this author Elisa Parish University/BHF Centre for Cardiovascular Science, QMRI, University of Edinburgh, Edinburgh, UK Search for more papers by this author John C Dawson Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Lisa Imrie Centre for Synthetic and Systems Biology (SynthSys), University of Edinburgh, Edinburgh, UK Search for more papers by this author Martina Vidmar University/BHF Centre for Cardiovascular Science, QMRI, University of Edinburgh, Edinburgh, UK Search for more papers by this author Noor Gammoh orcid.org/0000-0001-9402-9581 Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Tijana Mitić University/BHF Centre for Cardiovascular Science, QMRI, University of Edinburgh, Edinburgh, UK Search for more papers by this author Mairi Brittan University/BHF Centre for Cardiovascular Science, QMRI, University of Edinburgh, Edinburgh, UK Search for more papers by this author Nicholas L Mills University/BHF Centre for Cardiovascular Science, QMRI, University of Edinburgh, Edinburgh, UK Usher Institute, University of Edinburgh, Edinburgh, UK Search for more papers by this author Neil O Carragher Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Domenico D'Arca Corresponding Author [email protected] orcid.org/0000-0002-7240-6005 Department of Biomedical, Metabolic and Neural Sciences, University of Modena & Reggio Emilia, Modena, Italy Search for more papers by this author Andrea Caporali Corresponding Author [email protected] orcid.org/0000-0003-2905-3096 University/BHF Centre for Cardiovascular Science, QMRI, University of Edinburgh, Edinburgh, UK Search for more papers by this author Andrea Martello University/BHF Centre for Cardiovascular Science, QMRI, University of Edinburgh, Edinburgh, UK Search for more papers by this author Angela Lauriola Department of Biomedical, Metabolic and Neural Sciences, University of Modena & Reggio Emilia, Modena, Italy Search for more papers by this author David Mellis University/BHF Centre for Cardiovascular Science, QMRI, University of Edinburgh, Edinburgh, UK Search for more papers by this author Elisa Parish University/BHF Centre for Cardiovascular Science, QMRI, University of Edinburgh, Edinburgh, UK Search for more papers by this author John C Dawson Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Lisa Imrie Centre for Synthetic and Systems Biology (SynthSys), University of Edinburgh, Edinburgh, UK Search for more papers by this author Martina Vidmar University/BHF Centre for Cardiovascular Science, QMRI, University of Edinburgh, Edinburgh, UK Search for more papers by this author Noor Gammoh orcid.org/0000-0001-9402-9581 Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Tijana Mitić University/BHF Centre for Cardiovascular Science, QMRI, University of Edinburgh, Edinburgh, UK Search for more papers by this author Mairi Brittan University/BHF Centre for Cardiovascular Science, QMRI, University of Edinburgh, Edinburgh, UK Search for more papers by this author Nicholas L Mills University/BHF Centre for Cardiovascular Science, QMRI, University of Edinburgh, Edinburgh, UK Usher Institute, University of Edinburgh, Edinburgh, UK Search for more papers by this author Neil O Carragher Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Domenico D'Arca Corresponding Author [email protected] orcid.org/0000-0002-7240-6005 Department of Biomedical, Metabolic and Neural Sciences, University of Modena & Reggio Emilia, Modena, Italy Search for more papers by this author Andrea Caporali Corresponding Author [email protected] orcid.org/0000-0003-2905-3096 University/BHF Centre for Cardiovascular Science, QMRI, University of Edinburgh, Edinburgh, UK Search for more papers by this author Author Information Andrea Martello1, Angela Lauriola2, David Mellis1, Elisa Parish1, John C Dawson3, Lisa Imrie4, Martina Vidmar1, Noor Gammoh3, Tijana Mitić1, Mairi Brittan1, Nicholas L Mills1,5, Neil O Carragher3, Domenico D'Arca *,2 and Andrea Caporali *,1 1University/BHF Centre for Cardiovascular Science, QMRI, University of Edinburgh, Edinburgh, UK 2Department of Biomedical, Metabolic and Neural Sciences, University of Modena & Reggio Emilia, Modena, Italy 3Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK 4Centre for Synthetic and Systems Biology (SynthSys), University of Edinburgh, Edinburgh, UK 5Usher Institute, University of Edinburgh, Edinburgh, UK *Corresponding author. Tel: +39 059 2055610; E-mail: [email protected] *Corresponding author. Tel: +44 131 2426760; E-mail: [email protected] EMBO Rep (2020)21:e48192https://doi.org/10.15252/embr.201948192 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Autophagy is an essential cellular quality control process that has emerged as a critical one for vascular homeostasis. Here, we show that trichoplein (TCHP) links autophagy with endothelial cell (EC) function. TCHP localizes to centriolar satellites, where it binds and stabilizes PCM1. Loss of TCHP leads to delocalization and proteasome-dependent degradation of PCM1, further resulting in degradation of PCM1's binding partner GABARAP. Autophagic flux under basal conditions is impaired in THCP-depleted ECs, and SQSTM1/p62 (p62) accumulates. We further show that TCHP promotes autophagosome maturation and efficient clearance of p62 within lysosomes, without affecting their degradative capacity. Reduced TCHP and high p62 levels are detected in primary ECs from patients with coronary artery disease. This phenotype correlates with impaired EC function and can be ameliorated by NF-κB inhibition. Moreover, Tchp knock-out mice accumulate of p62 in the heart and cardiac vessels correlating with reduced cardiac vascularization. Taken together, our data reveal that TCHP regulates endothelial cell function via an autophagy-mediated mechanism. Synopsis The interaction of TCHP with PCM1 regulates basal autophagy through controlling GABARAP stability. Loss of TCHP function results in impaired autophagy, the accumulation of SQSTM1/p62 and endothelial dysfunction. TCHP localizes to centriolar satellites, where it binds and stabilizes PCM1. Loss of TCHP results in degradation of PCM1's binding partner GABARAP and impairment of autophagic flux. TCHP regulates endothelial cell function via an autophagy-mediated mechanism. Introduction Autophagy is an essential quality control function for the cell to maintain its homeostasis, through selectively degrading harmful protein aggregates or damaged organelles. Moreover, autophagy is a vital intracellular process for recycling nutrients and generating energy for maintenance of cell viability in most tissues and adverse conditions 1. Basal autophagy mediates proper cardiovascular function 2. Variety of cardiovascular risk factors can cause defective autophagy in vascular cells, producing high levels of metabolic stress and impairing the functionality of endothelial cells (ECs) 3. Autophagy has been shown to regulate angiogenic activity and the release of von Willebrand factor from ECs 4. Also, endothelial-specific deficiency of autophagy is pro-inflammatory and pro-senescent, as it promoted atherogenic phenotype in a murine model of atherosclerosis 5. Specific autophagic receptors are responsible for selective autophagy by tethering cargo to the site of autophagosomal engulfment 6. The recognition of ubiquitinated substrates is provided by molecular adaptors including p62/SQSTM1 (p62), which bind on one side to ubiquitin and, on the other end, to autophagosome-specific proteins (like members of the LC3/GABARAP/Gate16 family). The interaction between p62 and LC3/GABARAP bridges the autophagic machinery with its cargo, thereby fostering the selective engulfment by the autophagosome 7. In mammalian cells, six ATG8 orthologues exist that are divided into the LC3 and GABARAP subfamilies which have a non-redundant function during autophagosome biogenesis. Specifically, LC3 subfamily members promote elongation of phagophore membranes, whereas GABARAP is critical in the closure of the phagophore membrane 8, and fusion of autophagosomes with lysosomes 9. Recent studies demonstrated that a pool of GABARAP exists in the centrosome and peri-centrosomal region and regulates autophagosome formation during amino acid starvation 10. The levels of p62 are regulated transcriptionally and through continuous degradation during basal autophagy. The defective autophagy, however, induces accumulation of p62, followed by the formation of aggregates 11. Accumulation of p62 is further observed in human ECs in the cerebral cavernous malformation disease 12 and in human smooth muscle cells whereby p62 accumulation accelerated the development of stress-induced premature senescence 13. Besides its role in autophagy, p62 is a scaffolding hub for the cellular signalling pathways involving NF-kB activation, nerve growth factor signalling and caspase activation 14. Trichoplein (TCHP) is cytosolic ubiquitously expressed 62 kDa protein identified as a keratin filament binding protein 15. So far, the function of TCHP is deemed dependent on its partner proteins and their cellular localization. In proliferating cells, TCHP serves as a scaffold protein not only for appendage-associated Ninein, involved in microtubule anchoring at the mother centriole 16, but also for the centriole-associated Aurora kinase A activity, implicated in the destabilization of cilia 17. Alternatively, in differentiated, non-dividing epithelial cells, TCHP translocates from the centrioles to keratin filaments, and desmosomes 15. TCHP was reported to reside on the outer mitochondrial membrane (OMM), where it binds mitofusin2 (Mfn2), regulating the ER–mitochondria tethering and promoting mitochondria fission 18, 19. Moreover, increased levels of TCHP enable decorin evoked mitophagy 20. It is still unknown what role TCHP plays in EC function, particularly regarding its localization and mechanisms of action. We report here that TCHP localizes in centriolar satellites and it has an unexpected role in controlling autophagy in ECs. Results and Discussion Lack of TCHP impairs endothelial cell function Accumulating evidence links the intact autophagic responses with the preservation of cardiovascular homeostasis in several physiological and pathological settings 21. To examine the impact of TCHP on endothelial function, we performed a Matrigel tubule formation assay. TCHP down-regulation (Fig EV1A) severely affected the tubule forming capacity of HUVECs in vitro (Fig EV1B) and the formation of vessels in vivo using Matrigel plugs (Fig EV1C). In agreement, a reduced level of TCHP also affects ECs migration as measured in the wound healing (Fig EV1D). To further dissect the phenotype of ECs lacking TCHP, we analysed the expression of a subset of genes controlling angiogenesis, inflammation and cell cycle. TCHP knock-down cells showed an increase of IL1β, IL6, IL8, MCP1, CDKN2A/p16 and CDNKNB/p14 expression (Fig EV1E) and displayed a senescent-associated phenotype as seen by the increase of CDKN2A/p16 (Fig EV1F), β-galactosidase activity (β-Gal) (Fig EV1G) and the accumulation of aggresomes at 7 days postlentiviral transduction (Fig EV1H). Click here to expand this figure. Figure EV1. TCHP knock-down affects endothelial cells function Western blot anti-TCHP following knock-down of TCHP. Endothelial network formation on Matrigel was analysed by quantification of the total length of tube-like structures and number of meshes (unpaired t-test, **P = 0.0018 and **P = 0.0014 vs. control, respectively); right panels, representative microphotographs from Matrigel assay; scale bars, 100 μm. Left panels: representative images are showing the new microvessels positive for CD31 (green), in the implanted plugs. Scale bars, 50 and 25 μm for the inset. Right panel: quantification of the area of CD31 coverage in the Matrigel plugs mixed with TCHP siRNA or control oligos at 21 days after implantation; (n = 5 mice per group, unpaired t-test, **P = 0.0004 vs. control). Effect of TCHP knock-down on HUVEC migration speed measured by electric cell-substrate impedance sensing (ECIS) (unpaired t-test, **P = 0.0013 vs. control). Relative mRNA levels of TCHP and subset of genes. Graphs represent transcripts measured at 3 and 7 days post-TCHP knock-down (unpaired t-test vs. control; IL-1β: **P = 0.0048; IL-6: *P = 0.015; IL-8: *P = 0.0365—3 days; *P = 0.0168—7 days; MCP1: *P = 0.0223; p16: *P = 0.0133; p14: **P = 0.0073; TCHP: *P = 0.0185—3 days; *P = 0.0018—7 days). Western Blot anti-p16. β-Galactosidase activity as reveal by the chromogenic β-Gal substrate X-Gal. Scale bars 25 μm. Lower panel: quantification (unpaired t-test, **P < 0.0001 vs. control). Aggresome staining and quantification of protein aggregates; scale bars, 25 μm. Lower panel: quantification (unpaired t-test, **P < 0.0001 vs. control). Data information: Statistical analyses were performed on at least three independent experiments. Data are represented as mean ± SD. Download figure Download PowerPoint TCHP binds PCM1 to regulate its localization To identify TCHP interacting partners, we performed co-immunoprecipitation (Co-IP) coupled with mass spectrometry analysis using FLAG-tagged TCHP as bait in HEK293 cells. The centriolar satellite protein PCM1 was identified as the most enriched protein in anti-FLAG pull-down in comparison with the control experiment (Table EV1 and Appendix Fig S1A). We validated the mass spectrometry results showing that in HEK293 cells, two different FLAG-tagged versions (N- and C- terminal) of TCHP co-immunoprecipitated with endogenous PCM1 (Fig 1A). TCHP displays two coiled-coil regions, at the N terminus (1–136 AA), which are necessary and sufficient for centriolar localization and function 22. Using FLAG-tagged TCHP deletion mutants (Appendix Fig S1B), we found that the residues corresponding to the second coiled-coil region (41–136 AA) are critical for the binding to PCM1 (Fig 1B). Moreover, we demonstrated that the interaction between TCHP and PCM1 is conserved in HUVECs since TCHP is co-immunoprecipitated with endogenous PCM1 (Fig 1C). Figure 1. TCHP interacts directly with PCM1 A, B. (A) Immunoblot analysis showed HEK293 transfected for 48 h with expression vectors for TCHP, and FLAG-TCHP at N- and C-terminal. (B) Immunoblot analysis showed HEK293 transfected for 48 h with expression vectors for TCHP-FLAG C-terminal or constructs with deletions of the coiled-coil domain 1 (TCHP Δ1) and 2 (TCHP Δ2), as indicated in the scheme. For (A and B), total lysates were immunoprecipitated with anti-FLAG antibodies, and blots were probed sequentially with anti-PCM1, anti-FLAG and anti-ACTIN antibodies. IgG light chains are indicated with Red Ponceau staining. The input totals were analysed by parallel immunoblotting as a control for the level of expression. C. Anti-PCM1 immunoprecipitation from HUVECs cells and TCHP and PCM1 immunoblot. D. Co-localization of TCHP-V5 and centriolar satellite proteins. HUVECs were transduced with TCHP-V5 lentivirus and stained for anti-PCM1, anti-CEP290, anti-CEP72 and anti-V5 antibodies. Scale bars, 25 and 5 μm in the inset. Right panel: the two-channel intensity correlation of pixels corresponding to regions identified with TCHP-V5 and satellite markers (n = 90 cells; Pearson co-localization coefficient). E. TCHP knock-down or control cells were stained for anti-PCM1 antibody. Panel below: quantification (n = 50 cells; unpaired t-test; **P = 0.004 vs. control). Scale bars, 50 and 5 μm in the inset. Data information: Statistical analyses were performed on at least three independent experiments. Data are represented as mean ± SD. Download figure Download PowerPoint Endogenous or expressed TCHP showed a dynamic localization in cells that could be due to a different repositioning of TCHP during different cell cycle stages or under the effect of cellular stressor or stimuli 15, 17. Moreover, THCP localization in different subcellular compartments may mirror different functional roles played by the same protein. We next tested the localization of PCM1 and TCHP in ECs. PCM1 was the first satellite protein identified 23 and is acting as satellite assembly scaffold for other centriolar satellite proteins such as Cep290 24 and Cep72 25. We established that TCHP extensively co-localized with PCM1 in the pericentriolar matrix and satellite region (Fig 1D) and localized close to the nucleus in the same compartment occupied by the centriolar satellite proteins CEP290 and CEP72 (Fig 1D). Interestingly, depletion of TCHP had a significant effect on PCM1 localization, showing loss of PCM1 accumulation at the perinuclear region and dispersion throughout the cytoplasm (Fig 1E). Overall, these data demonstrated for the first time that TCHP binds PCM1 and localizes in centriolar satellites. During the revision of this manuscript, an independent study was published reporting the spatial and proteomic profiling of 22 human satellite proteins using proximity-dependent biotin identification, including PCM1, CEP290 and CEP72 26. Consistently with these results, this unbiased approach has identified TCHP as part of the centriolar satellite protein network, directly interacting with PCM1, CEP290 and CEP72. TCHP regulates PCM1 and GABARAP stability Since the constant turnover of PCM1 is regulated by the proteolytic degradation 27, we analysed PCM1 protein levels and degradation rate in the TCHP-depleted cells. Like centrosomes and cytoskeleton-associated proteins 28, PCM1 was enriched in the Triton X-100 insoluble fraction, and TCHP depletion reduced PCM1 at the steady state (Fig 2A). Next, we used cycloheximide (CHX) and MG132 to block the translation and proteasomal degradation, respectively. Expression of PCM1 and GABARAP increased in TCHP knock-down cells compared to the control cells (Appendix Fig S2A and B). In TCHP-depleted cells treated with CHX, PCM1 protein degradation was enhanced compared to control cells, while inhibited by MG132, thus revealing the augmented proteasome-dependent turnover of PCM1 (Fig 2A). Figure 2. TCHP regulates PCM1 and GABARAP stability A, B. (A) Control or TCHP knock-down HUVECs were subjected to cycloheximide (CHX) and MG132 treatments for the indicated number of hours before immunoblotting. PCM1 levels were analysed in the insoluble fraction. Laminin has been used as a loading control. Below panel: quantification of PCM1 degradation (one-way ANOVA; *P = 0.0180 vs. control time 0). (B) Condition as in (A), Western blot for anti-GABARAP and anti-ACTIN antibodies. Below panel: quantification of GABARAP degradation (one-way ANOVA; *P = 0.0215 vs. control time 0). C, D. (C) Control or TCHP overexpressing HUVECs were subjected to CHX treatment for the indicated number of hours prior to immunoblotting. Western blot was probed for anti-PCM1 and anti-V5 antibodies. Laminin has been used as a loading control. (D) Condition as in (C), Western blot was probed for anti-V5, anti-GABARAP and anti-ACTIN antibodies. Below panels: quantification of (C) (one-way ANOVA; **P < 0.0001 vs. control time 0; ##P = 0.0003 vs. control CHX) and (D) (one-way ANOVA; **P < 0.0001 vs. control time 0; ##P = 0.0056 vs. control CHX). Data information: Statistical analyses were performed on at least three independent experiments. Data are represented as mean ± SD. Source data are available online for this figure. Source Data for Figure 2 [embr201948192-sup-0005-SDataFig2.tif] Download figure Download PowerPoint PCM1 binds directly to GABARAP through a canonical LIR motif regulating GABARAP-specific autophagosome formation 10. Although PCM1 depletion did not affect autophagy per se, it has destabilized GABARAP, but not LC3 29, through proteasomal degradation 10. Recently, the PCM1-GABARAP interaction has been further dissected through the analysis of the crystal structure of the PCM1 LIR motif bound to GABARAP, demonstrating that the manipulation of the key sites in either the PCM1 LIR motif or sequences flanking the LIR motif can alter the binding specificity of autophagy adaptors and receptors to ATG8 proteins 30. The preference in PCM1 binding might explain the non-redundant functions of LC3 and GABARAP subfamilies. GABARAP protein is essential for maturation and expansion 8 and autophagosome fusion with lysosomes 9. Accordingly, we confirmed in ECs, a proteasome-dependent reduction of GABARAP during the 6 h of treatment with CHX. Without TCHP, in line with reduced stability of PCM1, the rate of GABARAP degradation was enhanced (Fig 2B). Conversely, ectopic expression of TCHP-V5 increased PCM1 (Fig 2C) and GABARAP protein levels (Fig 2D) extending their stability. Altogether, these data suggest that TCHP regulates PCM1 and GABARAP by proteasomal degradation. Whether TCHP affects the stability of other centriolar satellites components or whether the loss of TCHP had a more severe effect on the integrity of centriolar satellites remains to be determined. TCHP down-regulation impairs autophagic homeostasis We next set to establish what role TCHP plays in autophagy. Transmission electron microscope (TEM) revealed a significant increase in the number of autophagic vesicles when TCHP is depleted in HUVECs (Fig EV2A). Click here to expand this figure. Figure EV2. Autophagic features in TCHP-depleted endothelial cells Representative pictures from transmission electron microscopy analysis. Scale bars, 500 nm. Right panel: quantification of autophagic vacuoles (n = 16 cells, unpaired t-test; **P = 0.0010 vs. control). Representative pictures and quantification of HUVECs after Alexa 488 Click-iT® l-azidohomoalanine (AHA) labelling. Right panel: quantification (n = 50 cells, one-way ANOVA; **P = 0.0037 vs. control, ##P = 0.0005 vs. shTCHP). Scale bars, 25 μm. TCHP knock-down or control HUVECs were transduced with TCHP-V5 or control vectors and were stained for anti-PCM1 antibody. Panel below: quantification (n = 50 cells; unpaired t-test; **P = 0.0003 vs. control; #P = 0.0166 vs. shTCHP). Scale bars, 25 μm. Western blot for anti-V5 and anti-p62 antibody (one-way ANOVA; **P = 0.0044; #P = 0.0439 vs. TCHP-V5). Data information: Statistical analyses were performed on at least three independent experiments. Data are mean ± SD. Download figure Download PowerPoint Alongside the reduction in GABARAP, immunocytochemistry staining revealed an increased number of LC3- and p62-positive puncta (Fig 3A). We next set to determine the regulation of the autophagic flux by analysing the levels of p62 and LC3 in basal and Hank's buffered (HBSS) starved cells with or without bafilomycin A1 (BafA1). In a full medium, Western blot analysis confirmed an increased level of the lipidated form of LC3 (LC3-II) band and an increase of p62 protein levels in TCHP knock-down cells compared with control (Fig 3B). When autophagic flux was blocked with BafA1 at basal conditions, there was a higher accumulation of p62 in the control cells compared with TCHP knock-down cells. On the other hand, the LC3 lipidation rate increased more in control cell than in the TCHP knock-down cells after autophagy stimulation. Finally, the treatment with HBSS re-activated the autophagic flux in TCHP knock-down cells as demonstrated by substantial degradation of LC3-II and reduction of p62 (Fig 3B). Figure 3. Analysis of autophagy in TCHP-depleted endothelial cells Immunofluorescent staining for LC3 and p62 in TCHP knock-down and control cells. Scale bars, 25 μm. Lower panel: quantification (n = 90 cells, unpaired t-test; LC3: **P < 0.0001 vs. control; p62: **P < 0.0001 vs. control). Western blot of p62 and LC3 during under normal culture condition or starved condition (HBSS) or the presence of BafA1 (2 h) in TCHP knock-down or control cells. Lower panels: p62 quantification (one-way ANOVA; **P = 0.0003 vs. control, ##P = 0.0093 vs. shTCHP) and LC3 quantification (one-way ANOVA; **P = 0.0008 vs. control, ##P = 0.0011 vs. shTCHP). HUVECs were transduced with the tandem mCherry-EGFP-LC3 and with shRNA TCHP or control vectors. Left panels: representative picture of mCherrry-EGFP-LC3 reporters. Scale bars, 25 μm. Right panel: quantification of the number of mCherry-only (red bars, autolysosomes) or double-positive (mCherry+/EGFP+; yellow bars, autophagosomes) (n = 80 cells, one-way ANOVA; *P = 0.0447 vs. control; ##P = 0.0007 vs. shTCHP). Ratiometric flow cytometric analysis of mCherrry-EGFP-LC3 reporters as in C (one-way ANOVA; *P = 0.0247 vs. control; ##P = 0.0088 vs. shTCHP). Quantification of long-lived protein degradation assay in TCHP knock-down and control cells by flow cytometry (one-way ANOVA; **P = 0.0018 vs. control, ##P < 0.0001 vs. shTCHP). HUVECs were transduced with TCHP-V5 and control vectors. Western blot for anti-V5 and anti-p62 antibody during under normal culture condition or HBSS or BafA1 or torin-1 treatment. Lower panel: quantification (one-way ANOVA; **P = 0.0044; #P = 0.0117 vs. TCHP-V5). Data information: Statistical analyses were performed on at least three independent experiments. Data are represented as mean ± SD. Download figure Download PowerPoint The reduced autophagic flux was further analysed using mCherry-EGFP-LC3 assays as a complementary approach 31. The mCherry fluorescence was lower in TCHP knock-down cells compared with the control, attesting to a decrease in autolysosome formation and a slower autophagic flux in cells lacking TCHP (Fig 3C). There was not a significant difference in the percentage or a total number of mature autolysosomes following starvation in HBSS medium (Fig 3C). These results were also confirmed by quantitative ratiometric flow cytometry analysis showing a decrease in mCherry/GFP fluorescence ratio in TCHP knock-down cells compared with the control, while HBSS treatment increased the ratio in both conditions (Fig 3D). To further confirm the impairment of autophagic flux in TCHP knock-down cells, we performed a non-radioactive pulse-chase protocol using l-azidohomoalanine (AHA) labelling to quantify long-lived protein degradation during autophagy 32 (Figs 3E and EV2B). TCHP knock-down cells conserved a substantial amount of cellular fluorescence intensity as compared with the control sample. Conversely, after autophagy stimulation, under amino acid starvation for 2 h, both TCHP knock-down and control cells had reduced fluorescence intensity (Figs 3E and EV2B). Finally, overexpression of TCHP reduced the basal level of p62 in ECs and the accumulation of p62 after BafA1 treatment under full growth medium (Fig 3F) and increased the degradation of p62 during nutrient starvation or treatment with torin-1 (Fig 3F). Moreover, exogenous TCHP partially rescued PCM1 localization in the centriolar satellites (Fig EV2C) and decreased p62 accumulation in ECs lacking TCHP (Fig EV2D). In an attempt to validate the rescue experiments using TCHP mutants, we observed that exogenously expressed TCHPΔ1,2, unlike wild-type TCHP or TCHPΔ1, failed to co-localize with PCM1, at least in HeLa cells. Moreover, a scattered PCM1 staining pattern was observed, similar to that seen after TCHP knock-down. The latter finding would suggest that the TCHPΔ,1,2 mutant could act as a dominant-negative for TCHP or perhaps other centriolar satellite proteins. Further studies will be required to elucidate the independent role of TCHPΔ,1,2 mutant in ECs. Overall, these results demonstrated a reduced autophagic flux in TCHP knock-down cells. Nevertheless, although the autophagic flux is reduced in TCHP knock-down cells, stress-induced autophagy appears to be functional, suggesting that TCHP-dependent reduction of basal autophagy is reversible and could be pharmacologically re-activated. The depletion of TCHP inhibits autophagosome maturation and efficient delivery of p62 to the lysosomes Since GABARAP is critical for autophagosome expansion and maturation 9, we performed the proteinase K protection assay 33 to assess the efficiency of cargo receptor loading during autophagosome biogenesis and maturation. Autophagic vesicles were isolated by cytoplasm differential centrifugation and treated with proteinase K to d

The proper function of the Scribble tumour suppressor complex is dependent upon the correct localisation of its components. Previously we observed dynamic relocalisation of the hDlg component under conditions of osmotic stress. We now show that the other two components of the complex, hScrib and Hugl-1 display similar patterns of expression. We demonstrate, by shRNA ablation of hScrib expression, that hDlg and Hugl-1 are in part dependent upon hScrib for their correct localization. However under conditions of osmotic stress this apparent dependency no longer exists: hDlg and Hugl-1 localise to cell membranes independently of hScrib. We also demonstrate an interaction between the three components of the hScrib complex and the tSNARE syntaxin 4, and show that correct localization of the Scrib complex is in part tSNARE dependent. This is the first detailed analysis of the co-localisation and function of the hScrib complex in mammalian cells and demonstrates a direct link between the control of the hScrib complex and vesicle transport pathways.

Cells respond to cytotoxicity by activating a variety of signal transduction pathways. One pathway frequently upregulated during cytotoxic response is macroautophagy (hereafter referred to as autophagy). Previously, we demonstrated that pan-histone deacetylase (HDAC) inhibitors, such as the anticancer agent suberoylanilide hydroxamic acid (SAHA, Vorinostat), can induce autophagy. In this study, we show that HDAC inhibition triggers autophagy by suppressing MTOR and activating the autophagic kinase ULK1. Furthermore, autophagy inhibition can sensitize cells to both apoptotic and nonapoptotic cell death induced by SAHA, suggesting the therapeutic potential of autophagy targeting in combination with SAHA therapy. This study also raised a series of questions: What is the role of HDACs in regulating autophagy? Do individual HDACs have distinct functions in autophagy? How do HDACs regulate the nutrient-sensing kinase MTOR? Since SAHA-induced nonapoptotic cell death is not driven by autophagy, what then is the mechanism underlying the apoptosis-independent death? Tackling these questions should lead to a better understanding of autophagy and HDAC biology and contribute to the development of novel therapeutic strategies.

Human papillomavirus (HPV) E7 is essential in inducing S-phase progression in differentiating epithelial cells. We have previously shown that HPV-16 E7 activity can be controlled by a direct interaction with the viral transcriptional activator E2, thereby inhibiting transforming potential of E7. We have extended these analyses to show that E2 induces a generalized re-localization of E7 within the cell nucleus, one potential consequence of which is the inhibition of E7-induced degradation of pRb. Most importantly, we show that E2 can also inhibit the ability of E7 to induce centrosome abnormalities, thus preventing aberrant mitoses. Taken together, these studies highlight the central importance of E2 in controlling the functions of E7, independently of the ability of E2 to regulate transcription.

ABSTRACT The regulation of human papillomavirus (HPV) gene expression by the E2 protein is a critical feature of the viral life cycle. Previous studies have shown an important role in transcription for the ubiquitin-proteasome pathway, but its role in HPV gene expression has not been addressed. We now show that HPV E2 requires an active proteasome for its optimal transcriptional activator function. This involves an interaction with the Mdm2 ubiquitin ligase, which together with E2 acts synergistically to activate the HPV type 16 promoter. We also show that HPV E2 recruits Mdm2 onto HPV promoter sequences, providing an explanation for this cooperative activity.

Autophagosomes are vital organelles required to facilitate the lysosomal degradation of cytoplasmic cargo, thereby playing an important role in maintaining cellular homeostasis. A number of autophagy‐related (ATG) protein complexes are recruited to the site of autophagosome biogenesis where they act to facilitate membrane growth and maturation. Regulated recruitment of ATG complexes to autophagosomal membranes is essential for their autophagic activities and is required to ensure the efficient engulfment of cargo destined for lysosomal degradation. In this review, we discuss our current understanding of the spatiotemporal hierarchy between ATG proteins, examining the mechanisms underlying their recruitment to membranes. A particular focus is placed on the relevance of phosphatidylinositol 3‐phosphate and the extent to which the core autophagy players are reliant on this lipid for their localisation to autophagic membranes. In addition, open questions and potential future research directions regarding the membrane recruitment and displacement of ATG proteins are discussed here.

The role of macroautophagy (hereafter autophagy) in cancer biology and response to clinical intervention is complex. It is clear that autophagy is dysregulated in a wide variety of tumor settings, both during tumor initiation and progression, and in response to therapy. However, the pleiotropic mechanistic roles of autophagy in controlling cell behavior make it difficult to predict in a given tumor setting what the role of autophagy, and, by extension, the therapeutic outcome of targeting autophagy, might be. In this review we summarize the evidence in the literature supporting pro- and anti-tumorigenic and -therapeutic roles of autophagy in cancer. This overview encompasses roles of autophagy in nutrient management, cell death, cell senescence, regulation of proteotoxic stress and cellular homeostasis, regulation of tumor-host interactions and participation in changes in metabolism. We also try to understand, where possible, the mechanistic bases of these roles for autophagy. We specifically expand on the emerging role of genetically-engineered mouse models of cancer in shedding light on these issues in vivo.We also consider how any or all of the above functions of autophagy proteins might be targetable by extant or future classes of pharmacologic agents. We conclude by briefly exploring non-canonical roles for subsets of the key autophagy proteins in cellular processes, and how these might impact upon cancer.

Abstract Autophagy is an essential cellular quality control process that emerged critical for vascular homeostasis. Here we describe, the role for Trichoplein (TCHP) protein in linking autophagy with endothelial cells (ECs) function. The depletion of TCHP in ECs impairs migration and sprouting. TCHP directly binds PCM1, to regulate degradation of GABARAP, thus leading to a defective autophagy. Mechanistically, TCHP is indispensable for autophagosome maturation and its depletion resulted in the accumulation of SQSTM1/p62 (p62) and unfolded protein aggregates in ECs. The latter process is coupled to TCHP-mediated NF-kB activation. Of note, low levels of TCHP and high p62 levels were detected in primary ECs from patients with coronary artery disease. In addition, Tchp knock-out mice showed accumulation of p62 in the heart and cardiac vessels and reduced cardiac vascularization. Here, we reveal an autophagy-mediated mechanism for TCHP down-regulation, which poses a plausible target for regulation of endothelial function.

Recently a noncanonical activity of autophagy proteins has been discovered that targets lipidation of microtubule-associated protein 1 light chain 3 (LC3) onto macroendocytic vacuoles, including macropinosomes, phagosomes, and entotic vacuoles. While this pathway is distinct from canonical autophagy, the mechanism of how these nonautophagic membranes are targeted for LC3 lipidation remains unclear. Here we present evidence that this pathway requires activity of the vacuolar-type H⁺-ATPase (V-ATPase) and is induced by osmotic imbalances within endolysosomal compartments. LC3 lipidation by this mechanism is induced by treatment of cells with the lysosomotropic agent chloroquine, and through exposure to the <i>Heliobacter pylori</i> pore-forming toxin VacA. These data add novel mechanistic insights into the regulation of noncanonical LC3 lipidation and its associated processes, including LC3-associated phagocytosis (LAP), and demonstrate that the widely and therapeutically used drug chloroquine, which is conventionally used to inhibit autophagy flux, is an inducer of LC3 lipidation.

Recently a noncanonical activity of autophagy proteins has been discovered that targets lipidation of microtubule-associated protein 1 light chain 3 (LC3) onto macroendocytic vacuoles, including macropinosomes, phagosomes, and entotic vacuoles. While this pathway is distinct from canonical autophagy, the mechanism of how these nonautophagic membranes are targeted for LC3 lipidation remains unclear. Here we present evidence that this pathway requires activity of the vacuolar-type H⁺-ATPase (V-ATPase) and is induced by osmotic imbalances within endolysosomal compartments. LC3 lipidation by this mechanism is induced by treatment of cells with the lysosomotropic agent chloroquine, and through exposure to the <i>Heliobacter pylori</i> pore-forming toxin VacA. These data add novel mechanistic insights into the regulation of noncanonical LC3 lipidation and its associated processes, including LC3-associated phagocytosis (LAP), and demonstrate that the widely and therapeutically used drug chloroquine, which is conventionally used to inhibit autophagy flux, is an inducer of LC3 lipidation.

Ubiquitin‐proteasome system and autophagy are the two major mechanisms for protein degradation in eukaryotic cells. LC3, a ubiquitin‐like protein, plays an essential role in autophagy through its ability to be conjugated to phosphatidylethanolamine. In this study, we discovered a novel LC3‐processing activity, and biochemically purified the 20S proteasome as the responsible enzyme. Processing of LC3 by the 20S proteasome is ATP‐ and ubiquitin‐independent, and requires both the N‐terminal helices and the ubiquitin fold of LC3. The 20S proteasome processes LC3 in a stepwise manner, it first cleaves LC3 within its ubiquitin fold and thus disrupt the conjugation function of LC3; subsequently and especially at high concentrations of the proteasome, LC3 is completely degraded. Intriguingly, proteolysis of LC3 by the 20S proteasome can be inhibited by p62, an LC3‐binding protein that mediates autophagic degradation of polyubiquitin aggregates in cells. Therefore, our study implicates a potential mechanism underlying interplay between the proteasomal and autophagic pathways. This study also provides biochemical evidence suggesting cellular relevance of the controversial ubiquitin‐independent proteolytic activity of the 20S proteasome.

In order to ensure a productive life cycle, Human Papillomaviruses (HPVs) require fine regulation of their gene products. Uncontrolled activity of the viral oncoproteins, E6 and E7, results in the immortalisation of the infected epithelial cells and thus prevents the production of mature virions. Here, we investigate the regulation of HPV-16 E7 activities through its interaction with both viral and cellular gene products. First, we show that HPV-16 E7 and E2 can interact directly and the region mediating this interaction is defined on each protein. The expression of E2 inhibits some of E7 oncogenic activities including primary cell transformation, induction of centrosome abnormalities and pRB degradation. In addition, E2 can stabilise E7 and redirect its localisation where it can associate with some of E2’s activities such as transcriptional activation and mitotic chromosome binding. Secondly, we provide evidence that E7 can be phosphorylated by CDK2 in vitro preferentially on its N-terminal domain, and we hypothesise that this occurs on more than one residue on E7. In vivo, we show that the activity of CDK2, as well as CKII, is necessary for the stability of E7. Finally, we identified an interaction between HPV-16 E2 and E7 with the cellular oncoprotein, Mdm2. Mdm2 appears to destabilise E7 targeting it to proteasome-mediated degradation at PML bodies. The stability of E7 in cells that have reduced expression of Mdm2 is markedly increased indicating that the expression of Mdm2 indeed destabilises E7. In the case of the Mdm2 interaction with E2, we observe that E2 inhibits Mdm2 mediated degradation of p53 and pRB and that the expression of Mdm2 enhances E2’s transcriptional activity and induces its re-localisation at specific structures within the nucleus. Overall, our findings expand our knowledge of the regulation of virally encoded proteins both through direct protein-protein interactions between themselves and through their interactions with cellular proteins.

Wilfling et al., 2020Wilfling F. Lee C.-W. Erdmann P. Zheng Y. Sherpa D. Jentsch S. Pfander B. Schulman B.A. Baumeister W. A selective autophagy pathway for phase-separated endocytic protein deposits.Mol. Cell. 2020; 80 (this issue): 764-778Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar characterize a selective autophagy pathway in yeast for early clathrin-mediated endocytosis (CME) proteins facilitated by the phase separation of the CME protein, Ede1, which acts as an intrinsic autophagy receptor for the degradation of Ede1-dependent endocytic protein deposits (ENDs).