Category: Home

Autophagy and autolysosome formation

Autophagy and autolysosome formation

Deacetylation of STX17 facilitates Snake venom antidote assembly of adn STX17—SNAP29—VAMP8 complex and also enhances auholysosome binding to Advanced antimicrobial technology HOPS complex, thus promoting autophagosome—lysosome fusion under stress conditions 65 Fig. The Antidepressant for premenstrual dysphoric disorder motor scaffolding protein JIP1 autolusosome known as MAPK8IP1 directs both plus-end and minus-end transport of autophagosomes in neurons. Article CAS PubMed Google Scholar Wang, Z. Systems Biology of Cell Death Mechanisms, German Cancer Research Center, Bioquant, Heidelberg, Germany. Small molecule probes for targeting autophagy. This work was supported by grants from the FRIBIO program of the Norwegian Research Council, the Norwegian Cancer Society, the Aakre Foundation and the Blix Foundation to T.


The mechanism of autophagy

Autophagy and autolysosome formation -

Through comparison of tandem-LC3 and GFP-Rab7 readouts it was possible to differentiate specific effects on the autophagic pathway from general changes in the endolysosomal degradation pathway.

Analysis of GFP-Rab7 by high-resolution imaging indicated that, similar to LC3, endolysosomal flux can be inferred by comparing the number of Rab7 vesicles in presence and absence of Baf Figure 2a. Under ND conditions low numbers of small GFP-Rab7 vesicles are detected. In response to Baf, the cytoplasm filled with larger GFP-Rab7 vesicles.

Western blot analysis confirmed that endogenous Rab7 turnover is Baf sensitive under both FM and ND conditions Figure 2b , with higher turnover under ND conditions. We further confirmed the specificity of Rab7 for the late endosomal and lysosomal compartments by cotransfecting stable GFP-Rab7 cells with either mCherry-Rab5 early endosomal marker or Lamp1-red fluorescent protein RFP lysosomal marker.

As expected, Rab7 localized to the Lamp1-RFP-positive lysosomal, rather than to the mCherry-Rab5 early endosomal compartments Additional file 5. Analogous to GFP-LC3, we measured the decrease of GFP-Rab7 fluorescence intensity under FM and ND conditions Figure 2c. ND increased the loss of GFP fluorescence intensity in a time-dependent manner, however with decreasing turnover rates after 6 h of incubation Figure 2d.

Similar to GFP-LC3, addition of Baf inhibited the decrease in GFP-Rab7 fluorescence intensity, but was not required to detect endolysosomal flux Figure 2 and Additional file 1. Quantitative detection of endolysosomal activity using flow cytometry. a Representative image of green fluorescent protein GFP -Rab7 cells, exposed to full medium FM or nutrient deprivation ND medium for 6 h ± bafilomycin A1 Baf.

b Western blot analysis of wild-type cells, exposed to FM or ND ± Baf for 6 h. Cell lysates were analyzed for Rab7 and β-actin. c Histograms show distribution of GFP-Rab7 fluorescence intensity after 6 h upper row or 16 h lower row incubation with FM or ND ± Baf.

In summary, flow cytometry detection of tandem-LC3 and GFP-Rab7 allows for detection of autophagic and endolysosomal flux, respectively. Importantly, quantitative flux measurements are determined without the need for lysosomal inhibitors, as flux could be inferred by comparing FM to ND conditions.

Furthermore, normalization of responses allows for relative comparison of autophagic and endolysosomal responses, as well as condition-dependent and time-dependent impacts.

As a benchmark, we next examined the relative impact of reported autophagy modulating compounds, under conditions of basal and ND-induced autophagy Figure 3.

Values represent fold-change for example, 0. An important consideration to the use of fluorescent proteins is the impact of cellular pH levels, protein synthesis, and compound autofluorescence. Thus, to compensate for non-specific changes to fluorescence intensities, experiments were performed under identical conditions in cells stably expressing control Ctr -tandem or Ctr-GFP.

By correcting each data point for either tandem-LC3 or GFP-Rab7 to the respective control value, changes unrelated to autophagy were minimized see Materials and methods for details. mCherry-green fluorescent protein GFP tandem -microtubule-associated protein 1 light chain 3 B LC3 a,b or GFP-Rab7 c,d cells were exposed to either full medium FM a,c or nutrient deprivation ND b,d conditions ± respective drugs rapamycin, 0.

Data was normalized as described in Materials and methods including normalization to control Ctr constructs. Values represent fold changes in relation to the respective control condition FM for a,c and ND for b,d. Cells were treated with autophagy inducers rapamycin inhibitor of mTOR; 0.

In addition, we tested the effect of three widely used autophagy inhibitors: Baf 0. See Additional files for high-resolution images of the respective drugs under FM Additional file 3 and ND Additional file 4 conditions, in presence and absence of Baf.

Rapamycin and resveratrol both induced a significant increase in autophagic activity compared to FM Figure 3a. Notably, under FM conditions, resveratrol enhanced autolysosomal degradation to levels greater than induced by ND conditions.

AICAR, which by activating AMPK inhibits mTOR [ 33 ], had no significant effect on autolysosome fusion or degradation. Similar to FM conditions, resveratrol further increased both autolysosome formation and degradation under ND conditions, with a more pronounced enhancing effect on autolysosomal degradation Figure 3b.

In contrast to FM conditions, rapamycin decreased both autolysosome formation and degradation under ND conditions. Rapamycin significantly increased endolysosomal flux under FM conditions, even above ND induced turnover Figure 3c. However, rapamycin had no significant effect on endolysosomal activity under ND conditions Figure 3d , indicating differential regulation for FM and ND conditions.

Both AICAR and resveratrol had no significant effect on endolysosomal turnover Figure 3c,d. Notably, Baf was the only compound inhibiting autolysosomal formation and degradation events and endolysosomal turnover under both FM and ND conditions Figure 3.

Under FM conditions, WM and 3-MA had no inhibitory effect on autophagy, with 3-MA even slightly increasing formation of autolysosomes Figure 3a. In contrast, both WM and 3-MA inhibited autolysosome formation and degradation under ND conditions, with WM being more efficient Figure 3b. In contrast, WM had no effects under FM, while inhibiting endolysosomal turnover under ND conditions, even more efficiently than Baf Figure 3c,d.

This indicates WM to be a more specific and potent inhibitor than 3-MA with respect to both autophagic and endolysosomal activity. Defective autophagy is implicated in many diseases [ 1 ], and therefore the identification of drugs specifically modulating autophagic flux, without interfering with other endolysosomal processes, is of great translational interest.

To that end, we modified the above workflow to screen the Prestwick Chemical Library, consisting of 1, FDA-approved compounds, for modulators of autophagy Additional file 7. Using the well plate format, drug impact was determined by measuring GFP-LC3 fluorescence intensity under basal FM autophagy conditions Figure 4a.

GFP-LC3 was used as a primary readout, since either upregulation or downregulation in autophagic activity would first be manifested in changed fluorescence intensity levels of GFP-LC3. Possible effects downstream of autolysosome formation are then identified in a secondary screen, using tandem-LC3 and GFP-Rab7 reporters, as well as LysoTracker Red LTR to assess lysosomal activity.

This early time point was determined sufficient to obtain significant differences without increasing the risk of long-term impacts, such as secondary effects influencing global autophagy or protein levels.

Based on changes to GFP-LC3 fluorescence intensity we identified 38 potential inducers and 36 potential inhibitors of autolysosomal formation Figure 4b , with a threshold applied at mean ± σ.

Among these hits were compounds previously reported as regulators of autophagy, including autophagy inducers resveratrol [ 30 ] and camptothecin [ 34 ], as well as autophagy inhibitors colchicine [ 35 ] and quinacrine [ 36 ] Figure 4b. Identification of potential activator and inhibitor of autophagy.

a Distribution of compounds. b Primary hits identified by flow cytometry based autophagy screen. Previously reported anti-cancer properties are indicated by filled squares.

Respective PubMed database identification numbers PMIDs can be found in Additional file 9. Remarkably, the 38 potential enhancers of autophagy included all of the 8 cardiac glycosides present in the Prestwick Chemical Library. Cardiac glycosides are commonly used in the clinical treatment of various heart conditions [ 37 ] and have recently emerged as potential cancer therapeutics [ 38 ].

Therefore, we selected the three most potent cardiac glycosides with clinical relevance digoxin, strophanthidin, and digoxigenin for validation and concentration-dependent analysis by measuring their effect on tandem-LC3, GFP-Rab7 and LTR see Additional file 8 for concentration dependent analysis of additional primary hits.

It is of clinical interest that at these concentrations, digoxin has been previously shown to induce apoptosis specifically in cancer cells and comparable concentrations can be found in plasma of cardiac patients [ 39 ].

Cardiac glycosides are novel and specific inducer of autophagic flux. Diagrams showing autophagic activity upper row , endolysosomal turnover middle row and lysosomal activity bottom row , determined by flow cytometric quantification of fluorescence intensities of mCherry-green fluorescent protein GFP tandem -microtubule-associated protein 1 light chain 3 B LC3 , GFP-Rab7 and LysoTracker Red LTR , respectively.

Values represent fold changes in relation to full medium FM control condition. Drugs have been used at indicated concentrations under FM for 6 h.

We further confirmed the induction of autophagic flux by high-resolution microscopy and western blotting. Likewise, all drugs increased the turnover of endogenous LC3-I and LC3-II as revealed under Baf treatment Figure 6b. All approaches identified digoxin as the more potent inducer of autophagy than strophanthidin and digoxigenin.

Confirmation of autophagic flux induction by cardiac glycosides. Cell lysates were analyzed for LC3 and β-actin levels by western blotting. Autophagy is of a highly dynamic nature and its interactions with the endolysosomal pathway are complex [ 9 — 12 ].

Therefore, analysis of autophagic activity requires detection of multiple pathway activities, including autophagosome formation and degradation, endolysosomal turnover and lysosomal degradative capacity. Here, we applied automated flow cytometry to quantitatively measure temporal, conditional, and drug-induced impacts on each of these individual steps.

Key to our approach was the population sampling of single live cells, which generated multiparametric datasets amenable to statistical analysis. Specifically, single cell discrimination of pH quenching of GFP fluorescence of tandem-LC3 reported autolysosome formation, while loss of mCherry fluorescence of tandem-LC3 reported autolysosomal degradation.

By comparing GFP and mCherry fluorescence intensities to the respective control conditions, autophagic flux was inferred without the need for lysosomal inhibitors Figure 1. Furthermore, we established GFP-Rab7 turnover as a robust indicator for general changes in endolysosomal activity Figure 2 , allowing for the distinction between specific autophagic and general endolysosomal activity.

Mean fluorescence intensities were sampled for both tandem-LC3 and GFP-Rab7, and outperformed western blot quantification in terms of sensitivity and accuracy Figures 1 and 2. Thereby, our assay allows for comparison of multiple autophagy parameters, with respect to concentration, temporal, and conditional dependencies.

Overall, combining multiparametric flow cytometry with high-content markers for autolysosomal degradation pathways improves standard screening methods [ 14 — 16 ] due to reduced risk of potential off-target effects by the addition of lysosomal inhibitors.

Moreover, our approach outperforms current flow-cytometry-based autophagy assays [ 26 , 40 ] through quantification of both autolysosomal formation and degradation as well as capturing changes in the endolysosomal pathway. The ability of our assay to facilitate the identification of specific regulators of autophagy is highlighted by the drug-specific chart of activities of different autophagic steps, obtained by individually monitoring the involved autolysosomal degradation pathways Figure 7.

The importance of such an approach was apparent in our initial benchmark where we analyzed compounds that are widely used to inhibit and activate autophagy. We tested the effect of these compounds under conditions of both basal FM and activated ND autophagy.

Activation of AMPK by AICAR had no effect on autophagy. This is in line with previous studies, reporting AMPK-independent effects of AICAR that block autophagy [ 41 , 42 ]. Drug profiling by multiparametric quantitative analysis of autolysosomal degradation pathways.

Strikingly, we found that drug impact can be strongly dependent on the underlying condition, with drugs having opposing effects if applied under FM or ND conditions. Rapamycin Figure 3 , commonly applied to induce autophagy [ 27 ], enhanced autophagy under FM, but had a surprising inhibitory effect under ND conditions.

ND is well established to strongly inhibit mTOR [ 43 ]. Thus, the addition of rapamycin under ND conditions may not lead to additional mTOR inhibition, but instead inhibit autophagy by non-specific effects. In contrast, 3-MA Figure 3 , commonly used as an autophagy inhibitor [ 32 ], decreased ND-induced autophagy, but activated autophagy if applied under FM conditions.

Autophagy is regulated by two PI3Ks; while class III PI3K is required for the induction of autophagy, class I PI3K negatively regulates autophagy. Depending on the condition, inhibition of PI3K by WM or 3-MA might therefore have either inhibitory or activating effects [ 44 ].

Furthermore, our results suggest both negative and positive regulation of endolysosomal activity by PI3K, similar to autophagic regulation. Thus, the sensitivity of our approach allows for the comparison of condition-dependent and relative potencies of autophagy modulators. Here, we showed condition-dependent effects of rapamycin and identified Baf as the most potent inhibitor under both FM and ND, followed by WM being more specific and potent than 3-MA.

We subsequently identified novel modulators of autophagy by screening 1, FDA approved, bioactive small compounds Figure 4 , demonstrating the translational potential of our approach.

GFP-LC3 was used as a primary readout for autolysosome formation in live cells, as previously described by Shvets et al. Selected hits were then subjected to detailed analysis by monitoring effects on tandem-LC3, GFP-Rab7, and LTR.

Thereby, we were able to identify defects downstream of autolysosome formation and confirm specific induction of the autophagic degradation pathway. Some of these hits have previously been reported as autophagic regulators, including resveratrol [ 30 ], camptothecin [ 34 ], colchicine [ 35 ] and quinacrine [ 36 ], demonstrating the accuracy of our approach for compound screening.

Strikingly, the novel hits of our screen contained a family of eight compounds, cardiac glycosides, which are commonly used to treat heart failure [ 37 ] and more recently as cancer therapeutics [ 38 ]. These findings demonstrate the potential of high-content autophagy screening for identifying dual-purpose compounds with the goal of minimizing damage to essential, non-proliferating cells, while targeting proliferating cancer cells.

For three cardiac glycosides, digoxin, strophanthidin and digoxigenin, we determined the optimal concentrations to specifically induce autolysosome formation and degradation, without affecting general endolysosomal activity Figure 5.

We further confirmed activation of autophagy by western blotting and imaging approaches Figure 6. Calcium can upregulate autophagy [ 21 , 45 ] and modulators of calcium are prominent among compounds identified in other autophagy screens [ 14 , 15 , 46 ].

Cardiac glycosides have been suggested for cancer therapy, due to their potential to induce tumor specific cell death [ 38 ]. It is remarkable that many of the identified autophagy activators have both anti-cancer and cardioprotective properties Figure 4b and Additional file 9 , indicating potential drugability of autophagic cell death-associated mechanisms.

Conversely, many of the reported autophagy inhibitors induce cardiotoxicity and have anti-cancer properties. The role of autophagy inhibition in the efficiency of cell death induction during chemotherapy warrants further study [ 47 ].

Our study demonstrates the suitability of high-content screening for the characterization of localized-drug impact on autophagy. Future work might employ inducible expression systems, potentially also including additional sensors, such as GFP-p62 [ 48 ], thereby further increasing the sensitivity of our approach and facilitating the portability to other cell types.

In summary, we present a multiparametric screening approach, validated against common imaging and biochemical assays, which allows for quantitative measurements of the entire autolysosomal pathway, independent of lysosomal inhibitors. The ability to measure relative impacts on different pathway events revealed striking conditional differences between the most commonly used drug modulators of autophagy.

In addition, this approach was highly scalable, allowing for quantitative drug screening of 1, small compounds. From within a total number of 74 hits, cardiac glycosides were identified and 3 were further validated as novel inducers of autophagy.

Thus high content autophagy screening is effective for identifying drugs of interest for highly relevant disease type and thereby suggesting clear treatment strategies for in vivo confirmation.

Transient transfection mCherry-Rab5 and Lamp1-RFP was carried out with Effectene Qiagen. Cells were incubated in full medium FM or nutrient deprivation ND conditions as indicated. Cells were exposed to ND conditions by replacing FM with modified Krebs-Henseleit balanced salt solution mM NaCl, 4.

To generate mCherry-GFP-LC3B, LCB3 CDS was amplified from pEGFP-LC3 [ 24 ] inserted into pmCherry-C1. Subsequently enhanced GFP EGFP was inserted, in frame, between mCherry and LC3B. For flow cytometric analysis, culture medium was removed and cells were incubated in 50 μl Trypsin-ethylenediaminetetraacetic acid EDTA Invitrogen for 5 min.

Flow cytometric analysis was carried out with a modified Beckman-Coulter Krefeld, Germany FCMPL, allowing direct sampling from well plates and simultaneous excitation and detection of green nm and red nm fluorescent proteins. At least 1, events were collected for each well. Color compensation was carried out for multicolor detection using matched single fluorescent proteins compensation controls.

To minimize non-specific compound effects, each experiment was carried out in parallel with both the sensor construct and the respective fluorescent protein alone, that is, mCherry-GFP Ctr-tandem or GFP Ctr-GFP.

For each sample, cell number and mean fluorescence intensity were reported. All measurements were normalized to FM conditions and then corrected for unspecific changes by normalization to respective Ctr constructs, measured under identical conditions.

The mean of the normalized values was expressed as fold changes to either FM or ND, as indicated. Positive values indicate activation and negative values indicate inhibition. Each experiment was carried out at least three times independently. For staining of lysosomes, cells were incubated with LTR Invitrogen at a concentration of 50 nM for the last 30 min of treatment, washed once with the respective medium and then processed for analysis.

Rapamycin 0. Analysis by flow cytometry was carried out as described above. Bafilomycin A1 Baf , rapamycin, wortmannin WM , 3-MA, AICAR, resveratrol, and epoxomicin were purchased from Calbiochem Darmstadt, Germany.

Digoxin, digoxigenin, strophanthidin, doxorubicin, daunorubicin and mitoxantrone were purchased from Sigma-Aldrich.

Drugs were diluted in dimethylsulfoxide DMSO. Statistical significance was determined using a one-tailed Student's t test. Values are expressed as mean ± SEM. High-content imaging was carried out using a DeltaVision RT deconvolution microscope Applied Precision, Issaquah, WA, USA equipped with a 60 × oil immersion objectives and a CCD digital camera Hamamatsu, Herrsching, Germany.

Images were deconvolved to maximize spatial resolution and processed using ImageJ software [ 49 ]. Images shown are maximum projections of Z stacks of representative cells selected from at least three independent experiments. After indicated time points, whole cell lysates were prepared with radioimmunoprecipitation assay RIPA lysis buffer Upstate, Charlottesville, VA, USA containing Protease Inhibitor Cocktail Roche, Mannheim, Germany.

Protein concentrations were measured by Coomassie assay Sigma-Aldrich , adjusted to obtain equal loading and mixed with NuPage sample buffer master mix NuPage LDS buffer and reducing reagent, Invitrogen.

Immunodetection was carried out using primary antibodies against β-actin Abcam, Cambridge, UK , LC3B Cell Signaling, Danvers, MA, USA , and Rab7 Cell Signaling. Membranes were prepared with horseradish peroxidase HRP -linked secondary antibodies Cell Signaling and chemiluminescence was detected using a Chemiluminescence Detection System Intas, Göttingen, Germany.

Immunoblots shown are representative of at least three independent experiments. Quantification of immunoblots Figure 1a was performed on three independent samples using ImageJ software [ 49 ]. Intensity of respective bands LC3-II were quantified and normalized to the loading control β-actin.

Mizushima N, Levine B, Cuervo AM, Klionsky DJ: Autophagy fights disease through cellular self-digestion.

Article PubMed Central CAS PubMed Google Scholar. Mathew R, Karantza-Wadsworth V, White E: Role of autophagy in cancer. Nat Rev Cancer. Mathew R, Karp CM, Beaudoin B, Vuong N, Chen G, Chen H-Y, Bray K, Reddy A, Bhanot G, Gelinas C, Dipaola RS, Karantza-Wadsworth V, White E: Autophagy suppresses tumorigenesis through elimination of p Komatsu M, Kurokawa H, Waguri S, Taguchi K, Kobayashi A, Ichimura Y, Sou Y-S, Ueno I, Sakamoto A, Tong KI, Kim M, Nishito Y, Iemura S-i, Natsume T, Ueno T, Kominami E, Motohashi H, Tanaka K, Yamamoto M: The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1.

Nat Cell Biol. CAS PubMed Google Scholar. Kroemer G, Mariño G, Levine B: Autophagy and the integrated stress response. Mol Cell. Buss SJ, Muenz S, Riffel JH, Malekar P, Hagenmueller M, Weiss CS, Bea F, Bekeredjian R, Schinke-Braun M, Izumo S, Katus HA, Hardt SE: Beneficial effects of Mammalian target of rapamycin inhibition on left ventricular remodeling after myocardial infarction.

J Am Coll Cardiol. Article CAS PubMed Google Scholar. Baur JA, Sinclair DA: Therapeutic potential of resveratrol: the in vivo evidence. Nat Rev Drug Discov. Easton JB, Houghton PJ: mTOR and cancer therapy.

Berg TO, Fengsrud M, Strømhaug PE, Berg T, Seglen PO: Isolation and characterization of rat liver amphisomes. Evidence for fusion of autophagosomes with both early and late endosomes.

J Biol Chem. Raiborg C, Stenmark H: The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins. Lee J-A, Beigneux A, Ahmad ST, Young SG, Gao F-B: ESCRT-III dysfunction causes autophagosome accumulation and neurodegeneration.

Curr Biol. Fader CM, Colombo MI: Autophagy and multivesicular bodies: two closely related partners. Cell Death Differ.

Balgi AD, Fonseca BD, Donohue E, Tsang TCF, Lajoie P, Proud CG, Nabi IR, Roberge M: Screen for chemical modulators of autophagy reveals novel therapeutic inhibitors of mTORC1 signaling. PLoS ONE. Article PubMed Central PubMed Google Scholar.

Zhang L, Yu J, Pan H, Hu P, Hao Y, Cai W, Zhu H, Yu AD, Xie X, Ma D, Yuan J: Small molecule regulators of autophagy identified by an image-based high-throughput screen. Proc Natl Acad Sci USA. Williams A, Sarkar S, Cuddon P, Ttofi EK, Saiki S, Siddiqi FH, Jahreiss L, Fleming A, Pask D, Goldsmith P, O'Kane CJ, Floto RA, Rubinsztein DC: Novel targets for Huntington's disease in an mTOR-independent autophagy pathway.

Nat Chem Biol. Farkas T, Høyer-Hansen M, Jäättelä M: Identification of novel autophagy regulators by a luciferase-based assay for the kinetics of autophagic flux. Klionsky DJ, Cuervo AM, Seglen PO: Methods for monitoring autophagy from yeast to human.

Jäger S, Bucci C, Tanida I, Ueno T, Kominami E, Saftig P, Eskelinen E-L: Role for Rab7 in maturation of late autophagic vacuoles. J Cell Sci. Article PubMed Google Scholar. Luzio JP, Pryor PR, Bright NA: Lysosomes: fusion and function. Nat Rev Mol Cell Biol.

FEBS J. Tagawa Y, Yoshimori T, Moriyama Y, Masaki R, Tashiro Y: Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, HII-E cells.

Cell Struct Funct. Shaner NC, Steinbach PA, Tsien RY: A guide to choosing fluorescent proteins. Nat Methods. Kimura S, Noda T, Yoshimori T: Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3.

Shvets E, Fass E, Elazar Z: Utilizing flow cytometry to monitor autophagy in living mammalian cells. Blommaart EF, Luiken JJ, Blommaart PJ, van Woerkom GM, Meijer AJ: Phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes.

Narkar VA, Downes M, Yu RT, Embler E, Wang Y-X, Banayo E, Mihaylova MM, Nelson MC, Zou Y, Juguilon H, Kang H, Shaw RJ, Evans RM: AMPK and PPARdelta agonists are exercise mimetics. Corton JM, Gillespie JG, Hawley SA, Hardie DG: 5-aminoimidazolecarboxamide ribonucleoside.

A specific method for activating AMP-activated protein kinase in intact cells?. Eur J Biochem. Opipari AW, Tan L, Boitano AE, Sorenson DR, Aurora A, Liu JR: Resveratrol-induced autophagocytosis in ovarian cancer cells. Cancer Res.

Blommaart EF, Krause U, Schellens JP, Vreeling-Sindelárová H, Meijer AJ: The phosphatidylinositol 3-kinase inhibitors wortmannin and LY inhibit autophagy in isolated rat hepatocytes. Kimura N, Tokunaga C, Dalal S, Richardson C, Yoshino K-i, Hara K, Kemp BE, Witters LA, Mimura O, Yonezawa K: A possible linkage between AMP-activated protein kinase AMPK and mammalian target of rapamycin mTOR signalling pathway.

Genes Cells. Abedin MJ, Wang D, McDonnell MA, Lehmann U, Kelekar A: Autophagy delays apoptotic death in breast cancer cells following DNA damage. Köchl R, Hu XW, Chan EYW, Tooze SA: Microtubules facilitate autophagosome formation and fusion of autophagosomes with endosomes. Gupta A, Roy S, Lazar AJF, Wang W-L, McAuliffe JC, Reynoso D, McMahon J, Taguchi T, Floris G, Debiec-Rychter M, Schoffski P, Trent JA, Debnath J, Rubin BP: Autophagy inhibition and antimalarials promote cell death in gastrointestinal stromal tumor GIST.

Gheorghiade M, van Veldhuisen DJ, Colucci WS: Contemporary use of digoxin in the management of cardiovascular disorders. Prassas I, Diamandis EP: Novel therapeutic applications of cardiac glycosides. By contrast, in advanced tumours or during cancer therapy, autophagy enables tumour cells to survive harsh conditions such as hypoxia and metabolic stresses.

In line with this, elevated lysosomal activity resulting from increased activity of TFEB and TFE3 — which may be caused by chromosomal rearrangements, gene amplification or upregulation, and enhanced nuclear transport — is associated with the development or metastasis of various tumours, such as renal cell carcinomas, prostate cancer, pancreatic cancer, non-small-cell lung cancer NSCLC and breast cancer 88 , It is important to note that TFE3 and TFEB may also promote cancer progression, at least in part independently of their function in autophagy, by activating signalling pathways implicated in tumorigenesis, such as WNT and TGFβ signalling However, increased expression and nuclear import of TFE3 and TFEB have been demonstrated to maintain a high autophagy level to sustain intracellular amino acid pools in the pathogenesis of pancreatic ductal adenocarcinoma Enhanced TFEB activity can also promote lysosome exocytosis, releasing proteolytic enzymes, such as cathepsins, into the cell microenvironment, which fuels extracellular matrix remodelling, thereby stimulating cancer cell invasion and metastasis Perturbation of autophagosome maturation machinery may also contribute to the occurrence and development of tumours.

Alterations in EPG5 have been suggested to be involved in breast and prostate cancers , EPG5 expression is significantly lower in NSCLC clinical samples, and EPG5 knockdown promotes NSCLC cell proliferation and tumorigenesis WDR45 is genetically altered in patients with uterine corpus endometrial carcinoma and downregulated in cervical cancer development , However, owing to the heterogeneity and redundancy in the autophagosome maturation machinery highlighted above, changes in individual components are probably not sufficient to drive tumorigenesis and tumorigenic progression, and it is likely that alterations in autophagosome maturation coexist with other oncogenic lesions, further aggravating pathology.

Autophagy responds to invading pathogens by capturing them and delivering them to lysosomes for degradation a process known as xenophagy ; this facilitates antigen presentation for activation of innate and adaptive immune responses 15 , 16 , 17 , Certain pathogens even block autophagosome maturation and subvert the resulting vesicles for their own benefit.

Positive-strand RNA viruses belonging to the family Picornaviridae , such as poliovirus, rhinovirus, coxsackievirus B3 and enterovirus D68, utilize double-membrane vesicles DMVs as membrane scaffolds for replication and transcription , , DMVs formed in virus-infected cells are smaller than regular autophagosomes.

The betacoronaviruses, including mouse hepatitis virus MHV , Middle East respiratory syndrome coronavirus, severe acute respiratory syndrome coronavirus SARS-CoV and SARS-CoV-2, also induce the formation of DMVs for anchoring the viral replication and transcription complexes 32 , , , The viral RNA products are localized in the DMV lumen and transported to the cytosol for translation and virion assembly via double-membrane-spanning molecular pores However, the canonical autophagic machinery, such as the LC3 lipidation system, is not required for DMV formation or coronavirus replication , Certain autophagy proteins, however, are required for coronavirus infection.

LC3 decorates the DMVs and is required for MHV replication Unlike on autophagic structures, where LC3 is conjugated with phosphatidylethanolamine, non-lipidated LC3 is present on the DMVs in MHV-infected cells Autophagy proteins involved in the generation of PtdIns3P are required for SARS-CoV-2 infection , The ER-localized transmembrane autophagy proteins EPG3 also known as VMP1 and TMEM41B are essential for autophagosome formation 39 , , , , EPG3 and TMEM41B are also essential for replication of coronaviruses such as SARS-CoV-2 refs , , but the step at which these proteins act during the viral life cycle has yet to be identified.

In betacoronavirus-infected cells, the replicated viruses are transported inside lysosomes and released through the exocytic pathway Autophagosomes also can sequestrate and mediate non-lytic extracellular release of poliovirus, coxsackievirus B3 and enterovirus D68 refs , , Bacteria invade host cells via phagocytosis and reside in bacterium-containing vacuoles.

If the vacuolar membrane is damaged, bacteria can escape into the cytosol. Autophagy captures bacteria in the cytosol or in damaged vacuoles and delivers them to lysosomes for destruction via xenophagy 16 , Bacteria use different secretion systems to deliver effectors or toxins to evade autophagy surveillance and even to exploit autophagic vacuoles for intracellular survival and growth.

Bacterial virulence factors can block autophagy by inhibiting the autophagy induction signal, impairing autophagy recognition, or directly attenuating the function of autophagy proteins 16 , For example, the Legionella pneumophila effector protein RavZ inhibits host autophagy by functioning as a cysteine protease that uncouples lipid-conjugated ATG8 proteins RavZ cleaves ATG8 proteins between the carboxy-terminal glycine and the penultimate aromatic residue, producing ATG8 proteins that cannot be reconjugated SopF, the effector of Salmonella enterica subsp.

enterica serovar Typhimurium, impairs initiation of xenophagy by interfering with the binding of the V-ATPase on damaged bacterium-containing vacuoles to ATG16L Maturation of bacterium-containing autophagic vacuoles autophagosomes or fused vesicles of bacterium-containing vacuoles and autophagosomes into degradative autolysosomes by deposition of lytic enzymes can also be inhibited.

In macrophages, autophagic vacuoles containing the virulent strain of Mycobacterium tuberculosis H37Rv fail to recruit RAB7 for maturation into autolysosomes , Vacuoles containing Mycobacterium marinum and Yersinia pestis exhibit features of non-degradative autolysosomes that are devoid of lysosomal enzymes , Some bacteria for example, Serratia marcescens , Staphylococcus aureus , Anaplasma phagocytophilum and Coxiella burnetii even exploit autophagic vacuoles as replication niches for intracellular growth and proliferation , , , , , In line with this, replication of these pathogens is promoted by autophagy induction and is blocked by autophagy inhibition , , Viral proteinase 3C of coxsackievirus B3 and enterovirus D68 mediates cleavage of SNAP29, separating the two SNARE motifs and thus impairing the formation of the SNARE complex , The phosphoprotein P of human parainfluenza virus type 3 binds to SNAP29 to inhibit its interaction with STX17 ref.

Tethering factors can also be targeted by viral effectors. PLEKHM1 is proteolytically targeted by proteinase 3C of coxsackievirus B3 to separate the HOPS complex-binding and LC3-binding amino terminus from the RAB7-interacting carboxy terminus, thus abolishing its tethering function Fig.

Cells infected with SARS-CoV-2, or expressing the viral accessory protein ORF3a, sequester components of the HOPS complex on late endosomes This prevents functional HOPS complex from interacting with STX17 and consequently inhibits assembly of the STX17—SNAP29—VAMP8 complex 32 Fig.

M2 protein of influenza virus A inhibits autophagosome maturation by interfering with the beclin 1-containing and UVRAG-containing VPS34 complex Fig. Bacterial virulence factors also block the maturation and elimination of bacterium-containing autophagic vacuoles, but the mechanisms are less well understood.

Examples are shown in Fig. Autophagy can capture invading pathogens and deliver them to lysosomes for destruction. Pathogens including viruses and bacteria therefore use various mechanisms to block autophagosome—lysosome fusion to escape autophagy clearance. a Viral proteinase 3C of coxsackievirus B3 CVB3 and enterovirus D68 EVD68 cleaves SNAP29 to reduce SNARE assembly , Proteinase 3C of CVB3 also cleaves the tether protein PLEKHM1.

Phosphoprotein P of human parainfluenza virus type 3 HPIV3 competes with STX17 for SNAP29 binding ORF3a of severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 sequestrates the HOPS complex on late endosomes, thus impairing SNARE complex assembly.

M2 protein of influenza virus A IVA dampens the activity of the VPS34 complex to prevent autophagosome maturation. b Streptolysin O SLO damages the membrane of group A Streptococcus GAS -containing endosomes to trigger their engulfment by autophagosomes.

Translocation of the co-toxin NAD-glycohydrolase NADase into the cytoplasm blocks the fusion of GAS-containing autophagic vacuoles with lysosomes The virulence factor IsaB of methicillin-resistant Staphylococcus aureus MRSA blocks lysosomal acidification to suppress the function of autolysosomes The virulence factor VacA of Helicobacter pylori prevents TRPML1-mediated calcium efflux from endosomes to disrupt endolysosomal trafficking and thus autophagosome maturation Different steps of the autophagy pathway are potential targets for therapeutic interventions , Inhibitors and activators of autophagosome formation, including VPS34 and ULK1 inhibitors or the activating peptide Tat—beclin 1 , are potent modulators of autophagy, but are not yet available for clinical use , By contrast, lysosomotropic agents , which inhibit the activity of lysosomes and block their fusion with autophagic vesicles, have been used in several clinical trials , , Modulation of autophagosome maturation through targeting key components of the maturation machinery or through controlling autolysosomal activity by targeting the transcriptional programme of lysosome and autophagosome biogenesis is also a potential therapeutic option to regulate autophagic flux, but such approaches require further optimization if they are to be used in a clinical setting.

Inhibiting and stimulating autophagosome maturation are important therapeutic avenues to explore. In many neurodegenerative diseases, autophagosome maturation is blocked , , so restoring the autophagy flux would be an important therapeutic option. When inhibition of autophagy is required, as, for example, in cancer cells, it is important to consider the step at which the autophagic pathway must be interrupted.

Different outcomes can be observed when autophagy is blocked at autophagosome formation or autophagosome maturation. In line with this, it has been shown that blocking maturation of autophagosomes favours cell death via necroptosis in human cancer cells in response to TNF-related apoptosis-inducing ligand TRAIL , whereas blocking the formation of autophagosomes triggers TRAIL-dependent apoptosis in the same cells Targeting the machinery that controls autophagosome maturation such as ESCRT, SNAREs and the HOPS complex or targeting post-translational modifications of these proteins would be ideal to either increase or decrease autophagy flux , , Inhibitors of O -GlcNAc transferase and those of the opposing enzyme, O -GlcNAcase, can stimulate or inhibit autophagosome maturation, respectively, by modulating the O -GlcNAcylation of SNAP29 refs 66 , , Table 1.

As many cellular proteins are O -GlcNAcylated, blocking O -GlcNAc sites on a specific protein would require the development of methods to increase the selectivity of targeting, such as the use of aptamers or nanobodies Another strategy to inhibit autophagosome maturation would be to use the small molecule TCH to specifically activate the proteasomal degradation of SNAP29 and STX17 ref.

Inhibiting Rubicon is a potential approach to promote autophagy; however, pharmacological Rubicon inhibitors have yet to be developed. This strategy must be carefully evaluated because Rubicon is a positive modulator of LC3-associated phagocytosis, a process known to have a protective effect in many inflammatory diseases , , It has been shown that TFEB overexpression has beneficial effects in ameliorating LSDs and obesity by stimulating lipophagy , , However, chronic overexpression of TFEB favours the progression of pancreatic tumours and NSCLCs Thus, developing small molecules to acutely stimulate TFEB and harness the autophagy—lysosomal pathway would be beneficial in ameliorating diseases in which autophagy has a defensive role Small molecules can activate TFEB indirectly by modulating its upstream kinases or phosphatases Table 1.

For example, TFEB nuclear transport is promoted by rapamycin via inhibiting mTORC1 activity or by compounds isolated from the herb Euphorbia peplus Linn via the PKC—GSK3β cascade Chloroquine CQ , hydroxychloroquine HCQ and their derivatives are the only clinically approved drugs that act on autophagosome maturation Table 1.

They are used alone or in combination with other drugs, mostly in ongoing oncology trials, in general with the goal of optimizing therapies by blocking autophagy induced by cancer treatments , , The new-generation dimeric CQ derivatives Ly05 and DQ are active at lower concentrations than CQ and HCQ , , CQ and HCQ block autophagy flux by inhibiting the hydrolytic capacity of autolysosomes.

They increase the pH in autolysosomal compartments and hence block the activity of acidic proteases and other enzymes Thus, inhibition of PPT1 results in autophagy inhibition. Of note, lysosomotropic agents target all acidic compartments and also other pathways , , and thus in some cases the beneficial effects of lysosomotropic agents can be attributed to mechanisms other than a blockade of autophagy , For example, these drugs inhibit tumour progression, independently of the autophagy blockade, by altering the trafficking of signalling molecules that is, NOTCH1 in the endocytic pathway and by other mechanisms , It is also worth mentioning that the activity of CQ and HCQ observed in vitro may not be the same in vivo due to different parameters.

Moreover, the acidic environment in tumours can protonate the lysosomotropic agent and greatly reduce its cellular uptake Autophagosome maturation is an essential step in the autophagy pathway that ensures the formation of degradative autolysosomes.

It adds another layer of complexity and provides an extra node to integrate nutrient status and stresses for regulation of autophagic degradation.

The distinct organization and trafficking of the endolysosomal compartment in different cell types and growth conditions add complexity at the intersection of the autophagy and endocytic pathways.

Thus, the trans -SNARE complexes and tethering factors act coordinately with context-specific factors to mediate fusion of autophagosomes with endocytic vesicles and lysosomes.

Further investigations are needed to elucidate how different signalling pathways and stresses coordinate autophagosome initiation and maturation to ensure efficient progression of autophagic flux and how these processes are adapted in different cell types or pathophysiological contexts.

Autophagosome maturation is widely manipulated by pathogens to escape from destruction and for replication and growth. Pathogens that use autophagic vacuoles for replication can both activate autophagosome initiation and block maturation to achieve their maximal accumulation.

Understanding how viral proteins and bacterial virulence factors modulate host autophagy will help us to develop strategies to interfere with the pathogen—host interaction and even to restore autophagy as a defence mechanism. Such strategies are urgently required with the evolution of multidrug-resistant bacteria.

Elucidating the underlying mechanisms for autophagosome maturation defects and deregulation of the function of the autophagosome—lysosome system is also key for us to understand the pathogenesis of various human diseases. Targeting autophagosome maturation — via modulation of SNAREs, tethers and their regulators as well as lysosome biogenesis and function — offers an effective strategy for the treatment of these diseases.

Biomarkers and methods that reliably monitor autophagy flux in vivo are needed to examine temporal changes of autophagy activity and to evaluate interventions that target autophagosome maturation.

A combination of assays has been used to measure autophagy flux and to monitor autophagosome maturation However, many of these assays are difficult to implement in humans. Several methods have recently been developed to serve as reliable autophagy biomarkers in humans Analysis of autophagy flux in isolated peripheral blood mononuclear cells is used to measure autophagy activity in human blood samples , Positron emission tomography can be used with hypoxia tracers to correlate hypoxia and autophagy in tumours and also to gauge the level of specific autophagy substrates in tissues by the use of positron emission tomography ligands that bind to autophagy substrates , The levels of specific molecules in biological fluids can also be used to determine autophagy flux in tissues.

For instance, the blood level of arginase 1 reflects autophagy activity in the liver Thus, to screen drugs targeting autophagy, there is an urgent need for reliable, high-throughput clinical biomarkers to measure autophagic activity by the identification of tissue-specific circulating autophagy by-products and the development of flux probes for use in imaging techniques.

Feng, Y. The machinery of macroautophagy. Cell Res. Article CAS PubMed Google Scholar. Lamb, C. The autophagosome: origins unknown, biogenesis complex. Cell Biol. Nakatogawa, H. Mechanisms governing autophagosome biogenesis. Mizushima, N. The role of Atg proteins in autophagosome formation.

Cell Dev. Stolz, A. Cargo recognition and trafficking in selective autophagy. Takahashi, Y. et al. An autophagy assay reveals the ESCRT-III component CHMP2A as a regulator of phagophore closure.

Article PubMed PubMed Central CAS Google Scholar. VPS37A directs ESCRT recruitment for phagophore closure. Article CAS PubMed PubMed Central Google Scholar. Zhen, Y. ESCRT-mediated phagophore sealing during mitophagy.

Autophagy 16 , — Ktistakis, N. Digesting the expanding mechanisms of autophagy. Trends Cell Biol. Zhao, Y. Formation and maturation of autophagosomes in higher eukaryotes: a social network. Autophagosome maturation: an epic journey from the ER to lysosomes. Pu, J. Mechanisms and functions of lysosome positioning.

Cell Sci. CAS PubMed PubMed Central Google Scholar. Jiang, P. Autophagy and human diseases. Choi, Y. Autophagy during viral infection — a double-edged sword.

Deretic, V. Autophagy in infection, inflammation and immunity. Kimmey, J. Bacterial pathogens versus autophagy: implications for therapeutic interventions.

Trends Mol. Levine, B. Autophagy in immunity and inflammation. Nature , — Filimonenko, M. Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease.

Razi, M. Early endosomes and endosomal coatomer are required for autophagy. Jahn, R. SNAREs — engines for membrane fusion. Langemeyer, L. Rab GTPase function in endosome and lysosome biogenesis. Yu, I. Tethering factors as organizers of intracellular vesicular traffic. Nguyen, T. Vaites, L.

Ji, C. β-Propeller proteins WDR45 and WDR45B control autophagosome maturation into autolysosomes in neural cells. e6 Itakura, E.

Cell , — Matsui, T. Autophagosomal YKT6 is required for fusion with lysosomes independently of syntaxin Takats, S. Autophagosomal syntaxin dependent lysosomal degradation maintains neuronal function in Drosophila.

Tsuboyama, K. The ATG conjugation systems are important for degradation of the inner autophagosomal membrane. Science , — Kumar, S. Mechanism of Stx17 recruitment to autophagosomes via IRGM and mammalian Atg8 proteins. Diao, J. ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes.

Miao, G. ORF3a of the COVID virus SARS-CoV-2 blocks HOPS complex-mediated assembly of the SNARE complex required for autolysosome formation. Cell 56 , — e Interaction of the HOPS complex with syntaxin 17 mediates autophagosome clearance in Drosophila.

Cell 25 , — Article PubMed PubMed Central Google Scholar. The HOPS complex mediates autophagosome-lysosome fusion through interaction with syntaxin Stroupe, C. Purification of active HOPS complex reveals its affinities for phosphoinositides and the SNARE Vam7p.

EMBO J. Gao, J. Molecular mechanism to target the endosomal Mon1-Ccz1 GEF complex to the pre-autophagosomal structure. eLife 7 , e Hegedus, K. The Ccz1-Mon1-Rab7 module and Rab5 control distinct steps of autophagy.

Cell 27 , — Manil-Segalen, M. The C. elegans LC3 acts downstream of GABARAP to degrade autophagosomes by interacting with the HOPS subunit VPS Cell 30 , — Article CAS Google Scholar.

Tian, Y. elegans screen identifies autophagy genes specific to multicellular organisms. Wang, Z. Cell 63 , — Zhao, H. Mice deficient in Epg5 exhibit selective neuronal vulnerability to degeneration. McEwan, D.

Cell 57 , 39—54 The authors demonstrate that PLEKHM1 tethers autophagosomes and lysosomes by simultaneously binding to autophagosomal LC3 and lysosome-localized RAB7, and also promotes autophagosome—lysosome fusion by recruiting the HOPS complex. Wijdeven, R.

Cholesterol and ORP1L-mediated ER contact sites control autophagosome transport and fusion with the endocytic pathway. Wetzel, L. TECPR1 promotes aggrephagy by direct recruitment of LC3C autophagosomes to lysosomes.

Chen, D. A mammalian autophagosome maturation mechanism mediated by TECPR1 and the AtgAtg5 conjugate. Cell 45 , — Jahreiss, L. The itinerary of autophagosomes: from peripheral formation to kiss-and-run fusion with lysosomes.

Traffic 9 , — Korolchuk, V. Lysosomal positioning coordinates cellular nutrient responses. Bonifacino, J. Moving and positioning the endolysosomal system. Jia, R. BORC coordinates encounter and fusion of lysosomes with autophagosomes. Autophagy 13 , — Pankiv, S. FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end-directed vesicle transport.

Johansson, M. Activation of endosomal dynein motors by stepwise assembly of Rab7-RILP-pGlued, ORP1L, and the receptor betalll spectrin. Rocha, N. Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7-RILP-p Glued and late endosome positioning.

van der Kant, R. Late endosomal transport and tethering are coupled processes controlled by RILP and the cholesterol sensor ORP1L. PubMed Google Scholar. Lee, S.

Maday, S. Autophagosomes initiate distally and mature during transport toward the cell soma in primary neurons. This work reveals that in primary neurons, autophagosomes formed at the distal tip of the axon undergo retrograde transport to the cell soma, accompanied by gradual maturation and acidification.

Fu, M. LC3 binding to the scaffolding protein JIP1 regulates processive dynein-driven transport of autophagosomes. Cell 29 , — Cason, S. Sequential dynein effectors regulate axonal autophagosome motility in a maturation-dependent pathway. Article Google Scholar.

Russell, R. Autophagy regulation by nutrient signaling. Furuya, T. Negative regulation of Vps34 by Cdk mediated phosphorylation. Cell 38 , — Nazio, F. mTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6.

Fine-tuning of ULK1 mRNA and protein levels is required for autophagy oscillation. Liu, C. Cul3-KLHL20 ubiquitin ligase governs the turnover of ULK1 and VPS34 complexes to control autophagy termination. Cell 61 , 84—97 Jean, S. Starvation-induced MTMR13 and RAB21 activity regulates VAMP8 to promote autophagosome-lysosome fusion.

EMBO Rep. Fraldi, A. Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders. Shen, Q. Acetylation of STX17 syntaxin 17 controls autophagosome maturation. Autophagy 17 , — Guo, B.

O-GlcNAc-modification of SNAP regulates autophagosome maturation. Hanover, J. The hexosamine signaling pathway: O-GlcNAc cycling in feast or famine.

Acta , 80—95 A brief history of autophagy from cell biology to physiology and disease. Funderburk, S. The beclin 1-VPS34 complex — at the crossroads of autophagy and beyond.

Matsunaga, K. Two beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages. Cheng, X. Pacer mediates the function of class III PI3K and HOPS complexes in autophagosome maturation by engaging Stx Cell 65 , — Liang, C.

Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking. Sun, Q. Rubicon controls endosome maturation as a Rab7 effector.

Natl Acad. USA , — Zhong, Y. Distinct regulation of autophagic activity by Atg14L and Rubicon associated with beclin 1-phosphatidylinositolkinase complex. Kim, Y. mTORC1 phosphorylates UVRAG to negatively regulate autophagosome and endosome maturation.

Cell 57 , — Judith, D. ATG9A shapes the forming autophagosome through arfaptin 2 and phosphatidylinositol 4-kinase III beta.

Wang, H. GABARAPs regulate PI4P-dependent autophagosome: lysosome fusion. Del Bel, L. Sac1, a lipid phosphatase at the interface of vesicular and nonvesicular transport.

Traffic 19 , — Article PubMed CAS Google Scholar. Cell 77 , — e5 Jaber, N. Vps34 regulates Rab7 and late endocytic trafficking through recruitment of the GTPase-activating protein Armus.

Carroll, B. Cell 25 , 15—28 Piper, R. Late endosomes: sorting and partitioning in multivesicular bodies. Traffic 2 , — Baba, T. Phosphatidylinositol 4,5-bisphosphate controls Rab7 and PLEKHM1 membrane cycling during autophagosome-lysosome fusion.

Raben, N. TFEB and TFE3: linking lysosomes to cellular adaptation to stress. Sardiello, M. A gene network regulating lysosomal biogenesis and function. Settembre, C. TFEB links autophagy to lysosomal biogenesis.

Ballabio, A. Lysosomes as dynamic regulators of cell and organismal homeostasis. Puertollano, R. The complex relationship between TFEB transcription factor phosphorylation and subcellular localization. A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB. Li, Y.

Protein kinase C controls lysosome biogenesis independently of mTORC1. Medina, D. Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB. This study shows that upon activation by lysosomal calcium release through MCOLN1, the phosphatase calcineurin dephosphorylates TFEB and promotes its nuclear translocation to induce autophagy and lysosomal biogenesis.

Martina, J. Protein phosphatase 2A stimulates activation of TFEB and TFE3 transcription factors in response to oxidative stress. Banani, S. Biomolecular condensates: organizers of cellular biochemistry.

Shin, Y. Liquid phase condensation in cell physiology and disease. Science , eaaf Zhang, H. Liquid-liquid phase separation in biology: mechanisms, physiological functions and human diseases. China Life Sci 63 , — Article PubMed Google Scholar. Boija, A. Transcription factors activate genes through the phase-separation capacity of their activation domains.

Sabari, B. Biomolecular condensates and gene activation in development and disease. Cell 55 , 84—96 Inositol polyphosphate multikinase inhibits liquid-liquid phase separation of TFEB to negatively regulate autophagy activity.

Cell 55 , — This study demonstrates that the TFEB condensate formed via LLPS is involved in transcription and its formation is negatively regulated by the nuclear-localized protein IPMK. Tabata, K. Fujita, N. Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy.

eLife 6 , e The RBGRBG-2 complex modulates autophagy activity by regulating lysosomal biogenesis and function in C. Nixon, R. Neurodegenerative lysosomal disorders: a continuum from development to late age.

Autophagy 4 , — Otomo, A. Dysregulation of the autophagy-endolysosomal system in amyotrophic lateral sclerosis and related motor neuron diseases. Puls, I. Mutant dynactin in motor neuron disease.

Parkinson, N. ALS phenotypes with mutations in CHMP2B charged multivesicular body protein 2B. Neurology 67 , — Mauvezin, C. Autophagosome-lysosome fusion is independent of V-ATPase-mediated acidification. McBrayer, M. A block of autophagy in lysosomal storage disorders.

Vergarajauregui, S. Autophagic dysfunction in mucolipidosis type IV patients. Decressac, M. TFEB-mediated autophagy rescues midbrain dopamine neurons from alpha-synuclein toxicity. USA , E—E Cortes, C. Polyglutamine-expanded androgen receptor interferes with TFEB to elicit autophagy defects in SBMA.

This work reveals that the polyglutamine-expanded androgen receptor physically binds to and inactivates TFEB, thereby impairing autophagy function and contributing to the pathogenesis of spinal and bulbar muscular atrophy. Flavin, W. Endocytic vesicle rupture is a conserved mechanism of cellular invasion by amyloid proteins.

Impaired endo-lysosomal membrane integrity accelerates the seeding progression of alpha-synuclein aggregates. Google Scholar. Cullup, T. Recessive mutations in EPG5 cause Vici syndrome, a multisystem disorder with defective autophagy.

Byrne, S. Vici syndrome: a review. Orphanet J. Rare Dis. Role of Epg5 in selective neurodegeneration and Vici syndrome. Autophagy 9 , — Haack, T. Exome sequencing reveals de novo WDR45 mutations causing a phenotypically distinct, X-linked dominant form of NBIA. Saitsu, H.

De novo mutations in the autophagy gene WDR45 cause static encephalopathy of childhood with neurodegeneration in adulthood. Autophagy 11 , — Suleiman, J.

WDR45B-related intellectual disability, spastic quadriplegia, epilepsy, and cerebral hypoplasia: a consistent neurodevelopmental syndrome. Role of Wdr45b in maintaining neural autophagy and cognitive function. Jiao, J.

Skeletal muscle autophagy and its role in sarcopenia and organismal aging. Dowling, J. X-linked myopathy with excessive autophagy: a failure of self-eating.

Myerowitz, R. Impaired autophagy: the collateral damage of lysosomal storage disorders. EBioMedicine 63 , Nishino, I. Primary LAMP-2 deficiency causes X-linked vacuolar cardiomyopathy and myopathy Danon disease. Tanaka, Y. Accumulation of autophagic vacuoles and cardiomyopathy in LAMPdeficient mice.

Saraste, A. No cardiomyopathy in X-linked myopathy with excessive autophagy. Tresse, E. Autophagy 6 , — Ju, J. Valosin-containing protein VCP is required for autophagy and is disrupted in VCP disease. Towers, C. Autophagy and cancer: modulation of cell death pathways and cancer cell adaptations.

Dikic, I. Mechanism and medical implications of mammalian autophagy. Astanina, E. Multifaceted activities of transcription factor EB in cancer onset and progression. Perera, R. Transcriptional control of autophagy-lysosome function drives pancreatic cancer metabolism.

The authors demonstrate that pancreatic ductal adenocarcinoma cells contain higher levels of nuclear MITF, TFE3 and TFEB than normal cells, which in turn activate autophagy to promote tumour maligancy. Kundu, S. TMEMB drives lung cancer metastasis by inducing TFEB-dependent lysosome synthesis and secretion of cathepsins.

Sjoblom, T. The consensus coding sequences of human breast and colorectal cancers. Wu, B. Intratumoral heterogeneity and genetic characteristics of prostate cancer. Cancer , — Li, H. Theranostics 9 , — Bai, M. Analysis of deubiquitinase OTUD5 as a biomarker and therapeutic target for cervical cancer by bioinformatic analysis.

PeerJ 8 , e Lebovitz, C. Cross-cancer profiling of molecular alterations within the human autophagy interaction network. Huang, J. Bacteria-autophagy interplay: a battle for survival. Jackson, W.

Subversion of cellular autophagosomal machinery by RNA viruses. PLoS Biol. Wong, H. Wong, J. Autophagosome supports coxsackievirus B3 replication in host cells.

Corona, A. Enteroviruses remodel autophagic trafficking through regulation of host SNARE proteins to promote virus replication and cell exit. Cell Rep. Mohamud, Y. Enteroviral infection inhibits autophagic flux via disruption of the SNARE complex to enhance viral replication.

Kemball, C. Coxsackievirus infection induces autophagy-like vesicles and megaphagosomes in pancreatic acinar cells in vivo. Knoops, K. SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum.

Miller, K. Coronavirus interactions with the cellular autophagy machinery. Snijder, E. A unifying structural and functional model of the coronavirus replication organelle: tracking down RNA synthesis. Wolff, G. A molecular pore spans the double membrane of the coronavirus replication organelle.

Schneider, W. Genome-scale identification of SARS-CoV-2 and pan-coronavirus host factor networks. Cell , — e14 Zhao, Z. Coronavirus replication does not require the autophagy gene ATG5. Autophagy 3 , — Reggiori, F. Coronaviruses hijack the LC3-I-positive EDEMosomes, ER-derived vesicles exporting short-lived ERAD regulators, for replication.

Cell Host Microbe 7 , — Using MHV infection as a model, the authors show that coronaviruses hijack LC3-labelled EDEMsomes as DMVs for their replication.

Dniloski, Z. Identification of required host factors for SARS-CoV-2 infection in human cells. Cell , 92— e16 Wang, R.

Genetic screens identify host factors for SARS-CoV-2 and common cold coronaviruses. Morita, K. Genome-wide CRISPR screen identifies TMEM41B as a gene required for autophagosome formation.

Cell 67 , — Moretti, F. TMEM41B is a novel regulator of autophagy and lipid mobilization. Shoemaker, C. CRISPR screening using an expanded toolkit of autophagy reporters identifies TMEM41B as a novel autophagy factor.

Hoffmann, H. TMEM41B is a pan-flavivirus host factor. Ghosh, S. β-Coronaviruses use lysosomes for egress instead of the biosynthetic secretory pathway. This is the first demonstration that betacoronaviruses exploit lysosomal exocytosis for egress, accompanied by blockage of lysosomal acidification, inactivation of lysosomal degradation and impaired antigen presentation.

Bird, S. Nonlytic viral spread enhanced by autophagy components. Choy, A. The Legionella effector RavZ inhibits host autophagy through irreversible Atg8 deconjugation. Xu, Y. A bacterial effector reveals the V-ATPase-ATG16L1 axis that initiates xenophagy.

This study demonstrates that the Salmonella Typhimurium T3SS effector SopF blocks V-ATPase on damaged bacterium-containing vacuoles from recruiting ATG16L1 to initiate xenophagy. Chandra, P. Mycobacterium tuberculosis inhibits RAB7 recruitment to selectively modulate autophagy flux in macrophages.

Romagnoli, A. ESX-1 dependent impairment of autophagic flux by Mycobacterium tuberculosis in human dendritic cells. Autophagy 8 , — Lerena, M. Mycobacterium marinum induces a marked LC3 recruitment to its containing phagosome that depends on a functional ESX-1 secretion system.

Cell Microbiol.

BMC Biology Advanced antimicrobial technology 9Body toning and self-confidence number: 38 Cite this article. Metrics details. Formxtion Advanced antimicrobial technology lysosomal autolysosoms of autolysksome components. Recent work has associated autolyysosome dysfunction with pathologies, including cancer and cardiovascular disease. To date, the identification of clinically-applicable drugs that modulate autophagy has been hampered by the lack of standardized assays capable of precisely reporting autophagic activity. We developed and implemented a high-content, flow-cytometry-based screening approach for rapid, precise, and quantitative measurements of pharmaceutical control over autophagy. Autophagy and autolysosome formation

Author: Zulumi

2 thoughts on “Autophagy and autolysosome formation

Leave a comment

Yours email will be published. Important fields a marked *

Design by