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The discovery of recurrent mutations in subunits and regulators of the vacuolar-type H(+)-translocating ATPase (V-ATPase) in follicular lymphoma (FL) highlights a role for macroautophagy/autophagy, amino-acid, and nutrient-sensing pathways in the pathogenesis of this disease. Here, we report on novel mutations in the ER-resident chaperone VMA21, which is involved in V-ATPase assembly in 12% of FL. Mutations in a novel VMA21 hotspot (p.93X) result in the removal of a C-terminal non-canonical ER retrieval signal thus causing VMA21 mislocalization to lysosomes. The resulting impairment in V-ATPase activity prevents full lysosomal acidification and function, including impaired pH-dependent protein degradation as shown via lysosomal metabolomics and ultimately causes a degree of amino acid depletion in the cytoplasm. These deficiencies result in compensatory autophagy activation, as measured using multiple complementary assays in human and yeast cells. Of translational significance, the compensatory activation of autophagy creates a dependency for survival for VMA21-mutated primary human FL as shown using inhibitors to ULK1, the proximal autophagy-regulating kinase. Using high-throughput microscopy-based screening assays for autophagy-inhibiting compounds, we identify multiple clinical grade cyclin-dependent kinase inhibitors as promising drugs and thus provide new rationale for innovative clinical trials in FL harboring aberrant V-ATPase. Abbreviations: ALs: autolysosomes; APs: autophagosomes; ER: endoplasmic reticulum; FL: follicular lymphoma; GFP: green fluorescent protein; IP: immunoprecipitation; LE/LY: late endosomes/lysosomes; Lyso-IP: lysosomal immunoprecipitation; OST: oligosaccharide transferase; prApe1: precursor aminopeptidase I; SEP: super ecliptic pHluorin; V-ATPase: vacuolar-type H(+)-translocating ATPase

Supplementary Data 1 from Prostatic Acid Phosphatase Is Not a Prostate Specific Target

SUMMARYThe precursors to mammalian autophagosomes originate from pre-existing membranes contributed by a number of sources, and subsequently enlarge through intermembrane lipid transfer, then close to sequester the cargo, and merge with lysosomes to degrade the cargo. Using cellular and in vitro membrane fusion analyses coupled with proteomic and biochemical studies we show that autophagosomes are formed from a hybrid membrane compartment referred to as a prophagophore or HyPAS (hybrid preautophagosomal structure). HyPAS is initially LC3-negative and subsequently becomes an LC3-positive phagophore. The prophagophore emerges through fusion of RB1CC1/FIP200-containing vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes. A specialized Ca

Since the discovery of autophagy genes and proteins in the early1990s, numerous previously unknown physiological and pathological functions have been discovered for autophagy. At the same time, precise monitoring of autophagy has become important, and western blotting and fluorescence microscopy of the marker protein LC3 is widely used for this purpose. Here, we describe a modification of the widely used method, number of LC3 dots per cell. This protocol provides the proportion of vesicular LC3 staining over the total LC3 staining in the same cell. The approach is well suitable for quantification of endogenous LC3.

The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular and invitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS. A previously unrecognized apparatus defined here controls HyPAS biogenesis and mammalian autophagosomal precursor membranes. HyPAS can be modulated by pharmacological agents whereas its formation is inhibited upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or by expression of SARS-CoV-2nsp6. These findings reveal the origin of mammalian autophagosomal membranes, which emerge via convergence of secretory and endosomal pathways, and show that this process is targeted by microbial factors such as coronaviral membrane-modulating proteins.

Multi-tyrosine kinase inhibitors (MTKIs) have thus far had limited success in the treatment of castration-resistant prostate cancer (CRPC). Here, we report a phase I-cleared orally bioavailable MTKI, ESK981, with a novel autophagy inhibitory property that decreased tumor growth in diverse preclinical models of CRPC. The anti-tumor activity of ESK981 was maximized in immunocompetent tumor environments where it upregulated CXCL10 expression through the interferon gamma pathway and promoted functional T cell infiltration, which resulted in enhanced therapeutic response to immune checkpoint blockade. Mechanistically, we identify the lipid kinase PIKfyve as the direct target of ESK981. PIKfyve-knockdown recapitulated ESK981's anti-tumor activity and enhanced the therapeutic benefit of immune checkpoint blockade. Our study reveals that targeting PIKfyve via ESK981 turns tumors from cold into hot through inhibition of autophagy, which may prime the tumor immune microenvironment in advanced prostate cancer patients and be an effective treatment strategy alone or in combination with immunotherapies.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy‐related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders., This review provides an exhaustive overview of the contribution of autophagy to multiple pathological phenotypes in vivo, and discusses the therapeutic potential of autophagy modulation in disease prevention and treatment.

In this article we discuss implications of the recent discovery of glycoRNAs found to be present at the cell surface of mammalian cells which was reported by Flynn et al. Cell 2021.

Biliary tract cancers (BTCs) are uncommon, but highly lethal, gastrointestinal malignancies. Gemcitabine/cisplatin is a standard-of-care systemic therapy, but has a modest impact on survival and harbors toxicities, including myelosuppression, nephropathy, neuropathy, and ototoxicity. Whereas BTCs are characterized by aberrations activating the cyclinD1/cyclin-dependent kinase (CDK)4/6/CDK inhibitor 2a/retinoblastoma pathway, clinical use of CDK4/6 inhibitors as monotherapy is limited by lack of validated biomarkers, diffident preclinical efficacy, and development of acquired drug resistance. Emerging studies have explored therapeutic strategies to enhance the antitumor efficacy of CDK4/6 inhibitors by the combination with chemotherapy regimens, but their mechanism of action remains elusive.Here, we report in vitro and in vivo synergy in BTC models, showing enhanced efficacy, reduced toxicity, and better survival with a combination comprising gemcitabine/cisplatin and CDK4/6 inhibitors. Furthermore, we demonstrated that abemaciclib monotherapy had only modest efficacy attributable to autophagy-induced resistance. Notably, triplet therapy was able to potentiate efficacy through elimination of the autophagic flux. Correspondingly, abemaciclib potentiated ribonucleotide reductase catalytic subunit M1 reduction, resulting in sensitization to gemcitabine.As such, these data provide robust preclinical mechanistic evidence of synergy between gemcitabine/cisplatin and CDK4/6 inhibitors and delineate a path forward for translation of these findings to preliminary clinical studies in advanced BTC patients.

Autophagy is a conserved catabolic process that delivers cytoplasmic components and organelles to lysosomes for degradation and recycling. This pathway serves to degrade nonfunctional organelles and aggregate-prone proteins, as well as to produce substrates for energy production and biosynthesis. Autophagy is especially important for the maintenance of stem cells, and for the survival and homeostasis of post-mitotic cells like neurons. Functional autophagy promotes longevity in several model organisms. Autophagy regulates immunity and inflammation at several levels and has both anti- and pro-tumorigenic roles in cancer. This review provides a concise overview of autophagy and its importance in cellular and organismal homeostasis, with emphasis on aging, stem cells, neuronal cells, immunity, inflammation, and cancer.

Primary dysfunction of autophagy due to Mendelian defects affecting core components of the autophagy machinery or closely related proteins have recently emerged as an important cause of genetic disease. This novel group of human disorders may present throughout life and comprises severe early-onset neurodevelopmental and more common adult-onset neurodegenerative disorders. Early-onset (or congenital) disorders of autophagy often share a recognizable "clinical signature," including variable combinations of neurological, neuromuscular and multisystem manifestations. Structural CNS abnormalities, cerebellar involvement, spasticity and peripheral nerve pathology are prominent neurological features, indicating a specific vulnerability of certain neuronal populations to autophagic disturbance. A typically biphasic disease course of late-onset neurodegeneration occurring on the background of a neurodevelopmental disorder further supports a role of autophagy in both neuronal development and maintenance. Additionally, an associated myopathy has been characterized in several conditions. The differential diagnosis comprises a wide range of other multisystem disorders, including mitochondrial, glycogen and lysosomal storage disorders, as well as ciliopathies, glycosylation and vesicular trafficking defects. The clinical overlap between the congenital disorders of autophagy and these conditions reflects the multiple roles of the proteins and/or emerging molecular connections between the pathways implicated and suggests an exciting area for future research. Therapy development for congenital disorders of autophagy is still in its infancy but may result in the identification of molecules that target autophagy more specifically than currently available compounds. The close connection with adult-onset neurodegenerative disorders highlights the relevance of research into rare early-onset neurodevelopmental conditions for much more common, age-related human diseases.

Glycans have been shown to function as versatile molecular signals in cells. This prompted us to look at their roles in endocytosis, endolysosomal system and autophagy. We start by introducing the cell biological aspects of these pathways, the concept of the sugar code, and provide an overview on the role of glycans in the targeting of lysosomal proteins and in lysosomal functions. Moreover, we review evidence on the regulation of endocytosis and autophagy by glycans. Finally, we discuss the emerging concept that cytosolic exposure of luminal glycans, and their detection by endogenous lectins, provides a mechanism for the surveillance of the integrity of the endolysosomal compartments, and serves their eventual repair or disposal.

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The small GTPase RAB7A regulates late stages of the endocytic pathway and plays specific roles in neurons, controlling neurotrophins trafficking and signaling, neurite outgrowth and neuronal migration. Mutations in the RAB7A gene cause the autosomal dominant Charcot–Marie–Tooth type 2B (CMT2B) disease, an axonal peripheral neuropathy. As several neurodegenerative diseases are caused by alterations of endocytosis, we investigated whether CMT2B-causing mutations correlate with changes in this process. To this purpose, we studied the endocytic pathway in skin fibroblasts from healthy and CMT2B individuals. We found higher expression of late endocytic proteins in CMT2B cells compared to control cells, as well as higher activity of cathepsins and higher receptor degradation activity. Consistently, we observed an increased number of lysosomes, accompanied by higher lysosomal degradative activity in CMT2B cells. Furthermore, we found increased migration and increased RAC1 and MMP-2 activation in CMT2B compared to control cells. To validate these data, we obtained sensory neurons from patient and control iPS cells, to confirm increased lysosomal protein expression and lysosomal activity in CMT2B-derived neurons. Altogether, these results demonstrate that in CMT2B patient-derived cells, the endocytic degradative pathway is altered, suggesting that higher lysosomal activity contributes to neurodegeneration occurring in CMT2B. Electronic supplementary material The online version of this article (10.1007/s00018-020-03510-1) contains supplementary material, which is available to authorized users.

The autophagy protein Atg8 regulates autophagosome biogenesis and mediates cargo recruitment in different types of selective autophagy, but the molecular mechanisms mediating Atg8 functions during autophagosome biogenesis have been unclear. A study published in Nature Structural & Molecular Biology now shows that lipidated Atg8 possesses physicochemical activity that regulates membrane morphology, which underlies efficient autophagosome biogenesis.

The relationship between nutrient starvation and mitochondrial dynamics is poorly understood. We find that cells facing amino acid starvation display clear mitochondrial fusion as a means to evade mitophagy. Surprisingly, further supplementation of glutamine (Q), leucine (L), and arginine (R) did not reverse, but produced stronger mitochondrial hyperfusion. Interestingly, the hyperfusion response to Q + L + R was dependent upon mitochondrial fusion proteins Mfn1 and Opa1 but was independent of MTORC1. Metabolite profiling indicates that Q + L + R addback replenishes amino acid and nucleotide pools. Inhibition of fumarate hydratase, glutaminolysis, or inosine monophosphate dehydrogenase all block Q + L + R-dependent mitochondrial hyperfusion, which suggests critical roles for the tricarboxylic acid (TCA) cycle and purine biosynthesis in this response. Metabolic tracer analyses further support the idea that supplemented Q promotes purine biosynthesis by serving as a donor of amine groups. We thus describe a metabolic mechanism for direct sensing of cellular amino acids to control mitochondrial fusion and cell fate.

Lagotto Romagnolo breed dogs develop a progressive neurological disease with intracellular vacuolar storage when homozygous for a variant in the autophagy-related gene 4D ( ATG4D). A lysosomal enzyme deficiency has not been proven in this disease, despite its overlapping morphology with lysosomal storage diseases. Instead, basal autophagy was altered in fibroblasts from affected dogs. The aim of this study was to clarify the origin of the limiting membrane of the accumulating vacuoles and determine whether altered basal autophagy affects the extracellular release of vesicles in cells from diseased dogs. When assessed by immunoelectron microscopy, the membrane of the cytoplasmic vacuoles in affected tissues contained ATG4D, markers for autolysosomes (microtubule-associated protein 1A/B light chain 3 and lysosome-associated membrane protein 2) and for recycling endosomes (transferrin receptor 2), indicating that the vacuoles are hybrid organelles between endocytic and autophagic pathways. Ultracentrifugation, nanoparticle tracking analysis, and mass spectrometry were used to analyze the vesicles released from cultured fibroblasts of affected and control dogs. The amount of extracellular vesicles (EVs) released from affected fibroblasts was significantly increased during basal conditions in comparison to controls. This difference disappeared during starvation. The basal EV proteome of affected cells was enriched with cytosolic, endoplasmic reticulum, and mitochondrial proteins. Heat shock proteins and chaperones, some of which are known substrates of basal autophagy, were identified among the proteins unique to EVs of affected cells. An increased release of extracellular vesicles may serve as a compensatory mechanism in disposal of intracellular proteins during dysfunctional basal autophagy in this spontaneous disease.

Biliary tract cancers (BTCs) are uncommon but highly lethal gastrointestinal malignancies. Gemcitabine/cisplatin is a standard-of-care (SOC) systemic therapy, but has a modest impact on survival and harbor toxicities including myelosuppression, nephropathy, neuropathy and ototoxicity. While BTCs are characterized by aberrations activating the cyclinD1-CDK4/6-CDKN2A-RB pathway, clinical use of CDK4/6 inhibitors as monotherapy is limited by lack of validated biomarkers, diffident pre-clinical efficacy and development of acquired drug resistance. Emerging studies have explored therapeutic strategies to enhance the anti-tumor efficacy of CDK4/6 inhibitors by combination with chemotherapy-regimens but their mechanism of action remains elusive. Here, we report in vitro and in vivo synergy in BTC models, showing enhanced efficacy, reduced toxicity and better survival with a combination comprising gemcitabine/cisplatin and CDK4/6 inhibitors. Furthermore, we demonstrated that abemaciclib monotherapy had only modest efficacy due to autophagy induced resistance. Notably, triplettherapy was able to potentiate efficacy through elimination of the autophagic flux. Correspondingly, abemaciclib potentiated RRM1 reduction, resulting in sensitization to gemcitabine. Conclusions: As such, these data provide robust pre-clinical mechanistic evidence of synergy between gemcitabine/cisplatin and CDK4/6 inhibitors, and delineate a path forward for translation of these findings to preliminary clinical studies in advanced BTC patients.

Combined_supplemental_materials-Syrja_et_al for Altered Basal Autophagy Affects Extracellular Vesicle Release in Cells of Lagotto Romagnolo Dogs With a Variant ATG4D by Pernilla Syrjä, Mari Palviainen, Tarja Jokinen, Kaisa Kyöstilä, Hannes Lohi, Petra Roosje, Linda Anderegg, Tosso Leeb, Antti Sukura and Eeva-Liisa Eskelinen in Veterinary Pathology

Background: Toll like receptor-9 (TLR9) is an intracellular, innate immune system DNA-receptor, which is widely expressed in cancers. We previously demonstrated that low tumor TLR9 protein expression is associated with poor prognosis among triple negative breast cancer (TNBC) patients. A similar result is now detected also in larger, mRNA expression based datasets (Kmplot.com), which extend our initial observation to basal and luminal B clinical subtypes. The reason for TLR9 association with poor prognosis is not known.Aim: Since poor outcomes in early breast cancer are due to adjuvant treatment failures, we studied the role of TLR9 in breast cancer chemotherapy responses, growth inhibition and autophagy. Materials and Methods: Chemotherapy effects on the growth of control shRNA and TLR9 shRNA TNBC MDA-MB-231 and estrogen receptor expressing (ER+) T47-D cells were studied with cell confluency assays in vitro and in orthotopic mouse models in vivo. Autophagy was assessed with LC3I and II Western blots. Association of tumor TLR9 status with autophagy will be further studied in a cohort of neo-adjuvant treated patients, by comparing pre-treatment TLR9 expression with post-treatment LC3 vesicular expression, with immunohistochemistry. Autophagy gene expression will also be studied with RNAseq. Results: TLR9 shRNA cells were significantly less sensitive to the growth inhibitory effects of standard breast cancer chemotherapies than the corresponding control shRNA cells in vitro. The TLR9 shRNA T47-D cells were also significantly less sensitive to tamoxifen, than corresponding control shRNA cells. Docetaxel inhibited the growth of tumors formed by control shRNA cells, but not by TLR9 shRNA MDA-MB-231 cells in an orthotopic mouse model in vivo. Further chemotherapies and autophagy responses are being tested in this model as well. Standard chemotherapies induced accumulation of LC3IIB in control shRNA cells, but not in TLR9 cells, as demonstrated with Western blots. Conclusions: Decreased expression of tumor TLR9 is associated with 1) poor prognosis among basal and luminal B subtype patient samples, and in 2) decreased growth inhibitory and 3) decreased autophagy responses to common breast cancer chemotherapies and tamoxifen. Our results suggest that treatment responses, and thereby survival of patients with low-TLR9 tumors, may be improved by enhancing TLR9-associated autophagy. Citation Format: Katri Selander, Juha Klaper, Nataliia Petruk, Mauricio Ramm, Arja Jukkola, Eeva-Liisa Eskelinen. Low tumor TLR9 expression results in resistance to growth inhibitory and autophagy responses to common breast cancer treatments [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-13.

Neurodegenerative disorders of ageing such as Alzheimer disease, Parkinson disease and Huntington disease are characterized by the presence of neurotoxic misfolded and aggregated proteins. One reason underlying the accumulation of these proteins is insufficient clearance by intracellular and extracellular pathways such as the autophagic–lysosomal network and the glymph system. This article reviews the potential for therapeutically enhancing the clearance of neurotoxic proteins to curtail the onset and slow the progression of neurodegenerative disorders of ageing. Neurodegenerative disorders of ageing (NDAs) such as Alzheimer disease, Parkinson disease, frontotemporal dementia, Huntington disease and amyotrophic lateral sclerosis represent a major socio-economic challenge in view of their high prevalence yet poor treatment. They are often called 'proteinopathies' owing to the presence of misfolded and aggregated proteins that lose their physiological roles and acquire neurotoxic properties. One reason underlying the accumulation and spread of oligomeric forms of neurotoxic proteins is insufficient clearance by the autophagic–lysosomal network. Several other clearance pathways are also compromised in NDAs: chaperone-mediated autophagy, the ubiquitin–proteasome system, extracellular clearance by proteases and extrusion into the circulation via the blood–brain barrier and glymphatic system. This article focuses on emerging mechanisms for promoting the clearance of neurotoxic proteins, a strategy that may curtail the onset and slow the progression of NDAs.

Introduction: Follicular lymphoma (FL) constitutes the second most common non-Hodgkin lymphoma in the Western world. FL carries a high incidence of gene mutations in various V-ATPase subunits and regulators (ATP6V1B2, ATP6AP1, and VMA21) accounting for a combined 25-30% of FL cases. We have previously demonstrated that an FL-associated mutation in the V-ATPase V1 subunit ATP6V1B2 induces autophagic flux, thus conferring a survival addiction on mutated FL B cells. Here, we report results from functional analysis of the common hotspot truncating mutations p.R93* in the V-ATPase assembly factor VMA21 in FL. Methods: We measured autophagic flux using a variety of complementary assays in recombinant cell lines expressing inducible VMA21 WT and MUT (p.R93*). Lysosome acidification was measured using pH-sensitive dyes linked to dextran and flow-cytometry. The subcellular localization of VAM21 was investigated using confocal microscopy. The VMA21 WT and MUT-associated proteome was analyzed using immunoprecipitation followed by quantitative mass spectrometry-based protein profiling (IP-MS). Results: The inducible expression of VMA21 p.R93* activated autophagic flux as measured through various assays, including: i) increased autolysosome numbers measured via electron microscopy, ii) elevated LC3-II and elevated free GFP in HEK293T and lymphoma cells expressing a GFP-LC3-RFP probe, iii) elevated autophagosomes and autolysosomes detected via confocal microscopy in HEK293T cells, which were ablated by ULK1 and PIK3C3/VPS34 inhibitors, iv) elevated autophagic flux in various yeast assays. The inducible expression of mutant VMA21 partially impaired lysosomal acidification, implicating inhibitory effects on the V-ATPase holoenzyme. Analysis of subcellular localization of WT and MUT VMA21 using lysosomal-IPs and confocal microscopy showed that VMA21 MUT mislocalized to lysosomes and did not shuttle back to the ER. Using quantitative IP-MS, we detected elevated amounts of V-ATPase components in the VMA21 MUT immunoprecipitates, indicating increased complex formation between MUT VMA21 and fully assembled V-ATPase. Studies are underway measuring V-ATPase activity in lysosomes in VMA21 WT and MUT cells and effects on the lysosomal metabolome. In addition, we are testing the effects of small molecule autophagy inhibitors on the survival of VMA21 MUT primary FL cells. Conclusion: Our data support the hypothesis that the FL-associated VMA21 p.R93* has altered subcellular trafficking. As a consequence, more MUT VMA21 associates with the V-ATPase holoenzyme resulting in partial impairment of V-ATPase activity. Similar to our previous findings on ATP6V1B2 mutations, the MUT VMA21 aberrantly activates autophagic flux possibly as compensation to impaired V-ATPase function. This study supports future investigations into therapeutic targeting of aberrantly activated autophagy in FL. Citation Format: Fangyang Wang, Ying Yang, Gabriel Boudagh, Eeva-Liisa Eskelinen, Daniel J. Klionsky, Sami N. Malek. Follicular Lymphoma-associated mutations in the V-ATPase assembly factor VMA21 activate autophagic flux [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1939.

A missense variant in the autophagy-related ATG4D-gene has been associated with a progressive degenerative neurological disease in Lagotto Romagnolo (LR) dogs. In addition to neural lesions, affected dogs show an extraneural histopathological phenotype characterized by severe cytoplasmic vacuolization, a finding not previously linked with disturbed autophagy in animals. Here we aimed at testing the hypothesis that autophagy is altered in the affected dogs, at reporting the histopathology of extraneural tissues and at excluding lysosomal storage diseases. Basal and starvation-induced autophagy were monitored by Western blotting and immunofluorescence of microtubule associated protein 1A/B light chain3 (LC3) in fibroblasts from 2 affected dogs. The extraneural findings of 9 euthanized LRs and skin biopsies from 4 living affected LRs were examined by light microscopy, electron microscopy, and immunohistochemistry (IHC), using antibodies against autophagosomal membranes (LC3), autophagic cargo (p62), and lysosomal membranes (LAMP2). Biochemical screening of urine and fibroblasts of 2 affected dogs was performed. Under basal conditions, the affected fibroblasts contained significantly more LC3-II and LC3-positive vesicles than did the controls. Morphologically, several cells, including serous secretory epithelium, endothelial cells, pericytes, plasma cells, and macrophages, contained cytoplasmic vacuoles with an ultrastructure resembling enlarged amphisomes, endosomes, or multivesicular bodies. IHC showed strong membranous LAMP2 positivity only in sweat glands. The results show that basal but not induced autophagy is altered in affected fibroblasts. The ultrastructure of affected cells is compatible with altered autophagic and endo-lysosomal vesicular traffic. The findings in this spontaneous disease provide insight into possible tissue-specific roles of basal autophagy.

Autophagy is an evolutionarily conserved degradation pathway for cells to maintain homeostasis, produce energy, degrade misfolded proteins and damaged organelles, and fight against intracellular pathogens. The process of autophagy entails the isolation of cytoplasmic cargo into double membrane bound autophagosomes that undergo maturation by fusion with endosomes and lysosomes in order to obtain degradation capacity. RAB proteins regulate intracellular vesicle trafficking events including autophagy. RAB24 is an atypical RAB protein that is required for the clearance of late autophagic vacuoles under basal conditions. RAB24 has also been connected to several diseases including ataxia, cancer and tuberculosis. This review gives a short summary on autophagy and RAB proteins, and an overview on the current knowledge on the roles of RAB24 in autophagy and disease.

Mammalian homologs of yeast Atg8 protein (mAtg8s) are important in autophagy, but their exact mode of action remains ill‐defined. Syntaxin 17 (Stx17), a SNARE with major roles in autophagy, was recently shown to bind mAtg8s. Here, we identified LC3‐interacting regions (LIRs) in several SNAREs that broaden the landscape of the mAtg8‐SNARE interactions. We found that Syntaxin 16 (Stx16) and its cognate SNARE partners all have LIR motifs and bind mAtg8s. Knockout of Stx16 caused defects in lysosome biogenesis, whereas a Stx16 and Stx17 double knockout completely blocked autophagic flux and decreased mitophagy, pexophagy, xenophagy, and ribophagy. Mechanistic analyses revealed that mAtg8s and Stx16 control several properties of lysosomal compartments including their function as platforms for active mTOR. These findings reveal a broad direct interaction of mAtg8s with SNAREs with impact on membrane remodeling in eukaryotic cells and expand the roles of mAtg8s to lysosome biogenesis.

The NIH-funded center for autophagy research named Autophagy, Inflammation, and Metabolism (AIM) Center of Biomedical Research Excellence, located at the University of New Mexico Health Science Center is now completing its second year as a working center with a mission to promote autophagy research locally, nationally, and internationally. The center has thus far supported a cadre of 6 junior faculty (mentored PIs; mPIs) at a near-R01 level of funding. Two mPIs have graduated by obtaining their independent R01 funding and 3 of the remaining 4 have won significant funding from NIH in the form of R21 and R56 awards. The first year and a half of setting up the center has been punctuated by completion of renovations and acquisition and upgrades for equipment supporting autophagy, inflammation and metabolism studies. The scientific cores usage, and the growth of new studies is promoted through pilot grants and several types of enablement initiatives. The intent to cultivate AIM as a scholarly hub for autophagy and related studies is manifested in its Vibrant Campus Initiative, and the Tuesday AIM Seminar series, as well as by hosting a major scientific event, the 2019 AIM symposium, with nearly one third of the faculty from the International Council of Affiliate Members being present and leading sessions, giving talks, and conducting workshop activities. These and other events are often videostreamed for a worldwide scientific audience, and information about events at AIM and elsewhere are disseminated on Twitter and can be followed on the AIM web site. AIM intends to invigorate research on overlapping areas between autophagy, inflammation and metabolism with a number of new initiatives to promote metabolomic research. With the turnover of mPIs as they obtain their independent funding, new junior faculty are recruited and appointed as mPIs. All these activities are in keeping with AIM's intention to enable the next generation of autophagy researchers and help anchor, disseminate, and convey the depth and excitement of the autophagy field.

Live-cell imaging has been widely used to study autophagosome biogenesis and maturation. When combined with correlative electron microscopy, this approach can be extended to reveal ultrastructural details in three dimensions. The resolution of electron microscopy is needed when membrane contact sites and tubular connections between organelles are studied.

Live-cell imaging has been widely used to study autophagosome biogenesis and maturation. When combined with correlative electron microscopy, this approach can be extended to reveal ultrastructural details in three dimensions. The resolution of electron microscopy is needed when membrane contact sites and tubular connections between organelles are studied.

DRIVE is supported by a MarieSkłodowska-Curie ETN grant under theEuropean Union’s Horizon 2020 Research and Innovation Programme(Grant Agreement No 765912). P.B. is also supported by the Ministerio de Ciencia, Innovación y Universidades (BFU2015-65623) and by Redes de laComunidad de Madrid 2017/BMD-3813. Work in P.C. laboratory is sup-ported by institutional fundings from INSERM, CNRS and University ParisDescartes and by grants from Agence Nationale pour la Recherche (ANR) andFondation pour la Recherche Médicale(FRM). Z.E. research is supported bythe Israel Science Foundation ISF, the Legacy Heritage Fund and theWeizmann Institute Minerva center. E.-L.E. is also supported by TheAcademy of Finland project grant (286787), by Magnus EhrnroothFoundation, and by COST Action 15138 Transautophagy. V.K. is also sup-ported by the CRUK programme grant C2739/A22897. C.K. is also supportedby funding from the Vienna Science and Technology Fund (WWTF, VRG10-001) from the FWF Austrian Science Fund (P28113-B28), from the EuropeanResearch Council (ERC) under the European Union’s Horizon 2020 Researchand Innovation Programme (grant agreement No 769065), and from theEMBO Young Investigator Program. H.J. is supported by a grant fromAction Medical Research (Grant reference GN 2446), United Kingdom. O.P.is also supported by the German Federal Ministry of Education and Research(BMBF) (01EK1608B and 13GW0128B). T.P.-C. is also supported by theGerman Research Foundation (Deutsche Forschungsgemeinschaft, DFG);SFB/TRR 209 (project B02); FOR 2625 (project 1). A.S. is also supported bythe Research Council of Norway (project # 221831 and through its Centres ofExcellence funding scheme project # 262652) and the Norwegian CancerSociety (project #190251). F.R. is also supported by ZonMW VICI(016.130.606), ZonMW TOP (91217002), ALW Open Programme(ALWOP.310) and Marie Skłodowska-Curie Cofund (713660) grant, DRIVEis supported by a MarieSkłodowska-Curie ETN grant under the European Union’s Horizon 2020 Research and Innovation Programme(Grant Agreement No 765912). P.B. is also supported by the Ministerio deCiencia, Innovación y Universidades (BFU2015-65623) and by Redes de la Comunidad de Madrid 2017/BMD-3813. Work in P.C. laboratory is sup-ported by institutional fundings from INSERM, CNRS and University ParisDescartes and by grants from Agence Nationale pour la Recherche (ANR) andFondation pour la Recherche Médicale(FRM). Z.E. research is supported bythe Israel Science Foundation ISF, the Legacy Heritage Fund and theWeizmann Institute Minerva center. E.-L.E. is also supported by The Academy of Finland project grant (286787), by Magnus EhrnroothFoundation, and by COST Action 15138 Transautophagy. V.K. is also sup-ported by the CRUK programme grant C2739/A22897. C.K. is also supportedby funding from the Vienna Science and Technology Fund (WWTF, VRG10-001) from the FWF Austrian Science Fund (P28113-B28), from the EuropeanResearch Council (ERC) under the European Union’s Horizon 2020 Researchand Innovation Programme (grant agreement No 769065), and from the EMBO Young Investigator Program. H.J. is supported by a grant fromAction Medical Research (Grant reference GN 2446), United Kingdom. O.P.is also supported by the German Federal Ministry of Education and Research(BMBF) (01EK1608B and 13GW0128B). T.P.-C. is also supported by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG);SFB/TRR 209 (project B02); FOR 2625 (project 1). A.S. is also supported by the Research Council of Norway (project # 221831 and through its Centres ofExcellence funding scheme project # 262652) and the Norwegian CancerSociety (project #190251). F.R. is also supported by ZonMW VICI(016.130.606), ZonMW TOP (91217002), ALW Open Programme(ALWOP.310) and Marie Skłodowska-Curie Cofund (713660) grants.

Summary The dynamics and coordination between autophagy machinery and selective receptors during mitophagy are unknown. Also unknown is whether mitophagy depends on pre-existing membranes or is triggered on the surface of damaged mitochondria. Using a ubiquitin-dependent mitophagy inducer, the lactone ivermectin, we have combined genetic and imaging experiments to address these questions. Ubiquitination of mitochondrial fragments is required the earliest, followed by auto-phosphorylation of TBK1. Next, early essential autophagy proteins FIP200 and ATG13 act at different steps, whereas ULK1 and ULK2 are dispensable. Receptors act temporally and mechanistically upstream of ATG13 but downstream of FIP200. The VPS34 complex functions at the omegasome step. ATG13 and optineurin target mitochondria in a discontinuous oscillatory way, suggesting multiple initiation events. Targeted ubiquitinated mitochondria are cradled by endoplasmic reticulum (ER) strands even without functional autophagy machinery and mitophagy adaptors. We propose that damaged mitochondria are ubiquitinated and dynamically encased in ER strands, providing platforms for formation of the mitophagosomes., Graphical Abstract, Highlights • Mitophagy requires coordination of machineries for target recognition and engulfment • IVM-induced mitophagy depends on TRAF2/CIAP1/CIAP2 for ubiquitination of mitochondria • Ubiquitinated mitochondria within ER regions facilitate targeting and autophagy • ULK complex is required, but not for initiation, with FIP200 acting before ATG13, Zachari et al. use genetic and imaging experiments to define the ordered steps of mitophagy induction in response to the drug ivermectin. Their findings suggest that damaged mitochondria are ubiquitinated and dynamically encased in ER strands that then provide the platform for the formation of mitophagosomes.

Recently, NIH has funded a center for autophagy research named the Autophagy, Inflammation, and Metabolism (AIM) Center of Biomedical Research Excellence, located at the University of New Mexico Health Science Center (UNM HSC), with aspirations to promote autophagy research locally, nationally, and internationally. The center has 3 major missions: (i) to support junior faculty in their endeavors to develop investigations in this area and obtain independent funding; (ii) to develop and provide technological platforms to advance autophagy research with emphasis on cellular approaches for high quality reproducible research; and (iii) to foster international collaborations through the formation of an International Council of Affiliate Members and through hosting national and international workshops and symposia. Scientifically, the AIM center is focused on autophagy and its intersections with other processes, with emphasis on both fundamental discoveries and applied translational research.