FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2885453664> ?p ?o ?g. }

• W2885453664 abstract "Scientific Report20 August 2018free access Source DataTransparent process TMEM41B is a novel regulator of autophagy and lipid mobilization Francesca Moretti Novartis Institutes for BioMedical Research, Basel, Switzerland Search for more papers by this author Phil Bergman Novartis Institutes for BioMedical Research, Cambridge, MA, USA Search for more papers by this author Stacie Dodgson Harvard Medical School, Massachusetts General Hospital, Boston, MA, USA Search for more papers by this author David Marcellin Novartis Institutes for BioMedical Research, Basel, Switzerland Search for more papers by this author Isabelle Claerr Novartis Institutes for BioMedical Research, Basel, Switzerland Search for more papers by this author Jonathan M Goodwin Novartis Institutes for BioMedical Research, Cambridge, MA, USA Search for more papers by this author Rowena DeJesus Novartis Institutes for BioMedical Research, Cambridge, MA, USA Search for more papers by this author Zhao Kang Novartis Institutes for BioMedical Research, Cambridge, MA, USA Search for more papers by this author Christophe Antczak Novartis Institutes for BioMedical Research, Cambridge, MA, USA Search for more papers by this author Damien Begue Novartis Institutes for BioMedical Research, Basel, Switzerland Search for more papers by this author Debora Bonenfant Novartis Institutes for BioMedical Research, Basel, Switzerland Search for more papers by this author Alexandra Graff Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Christel Genoud Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author John S Reece-Hoyes Novartis Institutes for BioMedical Research, Cambridge, MA, USA Search for more papers by this author Carsten Russ Novartis Institutes for BioMedical Research, Cambridge, MA, USA Search for more papers by this author Zinger Yang Novartis Institutes for BioMedical Research, Cambridge, MA, USA Search for more papers by this author Gregory R Hoffman Novartis Institutes for BioMedical Research, Cambridge, MA, USA Search for more papers by this author Matthias Mueller Novartis Institutes for BioMedical Research, Basel, Switzerland Search for more papers by this author Leon O Murphy Novartis Institutes for BioMedical Research, Cambridge, MA, USA Search for more papers by this author Ramnik J Xavier Harvard Medical School, Massachusetts General Hospital, Boston, MA, USA Search for more papers by this author Beat Nyfeler Corresponding Author [email protected] orcid.org/0000-0003-0624-9571 Novartis Institutes for BioMedical Research, Basel, Switzerland Search for more papers by this author Francesca Moretti Novartis Institutes for BioMedical Research, Basel, Switzerland Search for more papers by this author Phil Bergman Novartis Institutes for BioMedical Research, Cambridge, MA, USA Search for more papers by this author Stacie Dodgson Harvard Medical School, Massachusetts General Hospital, Boston, MA, USA Search for more papers by this author David Marcellin Novartis Institutes for BioMedical Research, Basel, Switzerland Search for more papers by this author Isabelle Claerr Novartis Institutes for BioMedical Research, Basel, Switzerland Search for more papers by this author Jonathan M Goodwin Novartis Institutes for BioMedical Research, Cambridge, MA, USA Search for more papers by this author Rowena DeJesus Novartis Institutes for BioMedical Research, Cambridge, MA, USA Search for more papers by this author Zhao Kang Novartis Institutes for BioMedical Research, Cambridge, MA, USA Search for more papers by this author Christophe Antczak Novartis Institutes for BioMedical Research, Cambridge, MA, USA Search for more papers by this author Damien Begue Novartis Institutes for BioMedical Research, Basel, Switzerland Search for more papers by this author Debora Bonenfant Novartis Institutes for BioMedical Research, Basel, Switzerland Search for more papers by this author Alexandra Graff Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Christel Genoud Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author John S Reece-Hoyes Novartis Institutes for BioMedical Research, Cambridge, MA, USA Search for more papers by this author Carsten Russ Novartis Institutes for BioMedical Research, Cambridge, MA, USA Search for more papers by this author Zinger Yang Novartis Institutes for BioMedical Research, Cambridge, MA, USA Search for more papers by this author Gregory R Hoffman Novartis Institutes for BioMedical Research, Cambridge, MA, USA Search for more papers by this author Matthias Mueller Novartis Institutes for BioMedical Research, Basel, Switzerland Search for more papers by this author Leon O Murphy Novartis Institutes for BioMedical Research, Cambridge, MA, USA Search for more papers by this author Ramnik J Xavier Harvard Medical School, Massachusetts General Hospital, Boston, MA, USA Search for more papers by this author Beat Nyfeler Corresponding Author [email protected] orcid.org/0000-0003-0624-9571 Novartis Institutes for BioMedical Research, Basel, Switzerland Search for more papers by this author Author Information Francesca Moretti1, Phil Bergman2, Stacie Dodgson3, David Marcellin1, Isabelle Claerr1, Jonathan M Goodwin2,†, Rowena DeJesus2, Zhao Kang2, Christophe Antczak2, Damien Begue1, Debora Bonenfant1, Alexandra Graff4, Christel Genoud4, John S Reece-Hoyes2, Carsten Russ2, Zinger Yang2, Gregory R Hoffman2, Matthias Mueller1, Leon O Murphy2,†, Ramnik J Xavier3 and Beat Nyfeler *,1 1Novartis Institutes for BioMedical Research, Basel, Switzerland 2Novartis Institutes for BioMedical Research, Cambridge, MA, USA 3Harvard Medical School, Massachusetts General Hospital, Boston, MA, USA 4Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland †Present address: Casma Therapeutics, Cambridge, MA, USA *Corresponding author. Tel: +41 792612693; E-mail: [email protected] EMBO Rep (2018)19:e45889https://doi.org/10.15252/embr.201845889 See also: E Morel & P Codogno (September 2018) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Autophagy maintains cellular homeostasis by targeting damaged organelles, pathogens, or misfolded protein aggregates for lysosomal degradation. The autophagic process is initiated by the formation of autophagosomes, which can selectively enclose cargo via autophagy cargo receptors. A machinery of well-characterized autophagy-related proteins orchestrates the biogenesis of autophagosomes; however, the origin of the required membranes is incompletely understood. Here, we have applied sensitized pooled CRISPR screens and identify the uncharacterized transmembrane protein TMEM41B as a novel regulator of autophagy. In the absence of TMEM41B, autophagosome biogenesis is stalled, LC3 accumulates at WIPI2- and DFCP1-positive isolation membranes, and lysosomal flux of autophagy cargo receptors and intracellular bacteria is impaired. In addition to defective autophagy, TMEM41B knockout cells display significantly enlarged lipid droplets and reduced mobilization and β-oxidation of fatty acids. Immunostaining and interaction proteomics data suggest that TMEM41B localizes to the endoplasmic reticulum (ER). Taken together, we propose that TMEM41B is a novel ER-localized regulator of autophagosome biogenesis and lipid mobilization. Synopsis Autophagy maintains cellular homeostasis by targeting damaged organelles, pathogens or misfolded proteins for lysosomal degradation. The ER transmembrane protein TMEM41B is a novel regulator of autophagosome biogenesis, lipid droplet homeostasis and mitochondrial respiration. TMEM41B localizes to the endoplasmic reticulum. Knockout of TMEM41B impairs autophagosome biogenesis and lysosomal delivery of cargo. Absence of TMEM41B results in enlarged lipid droplets. TMEM41B is required for mobilization and mitochondrial β-oxidation of fatty acids. Introduction Lysosomal clearance of autophagic cargo is an essential catabolic cellular process that is compromised in several disease states 1. Autophagy is basally active in most cell types and can be strongly induced under stress such as nutrient starvation 2. mTOR complex 1 orchestrates cell growth response to nutrient availability and has been established as a key negative regulator of autophagy 3. Different sets of autophagy-related proteins control the stepwise progression of autophagy with initiation occurring at subdomains of the ER, termed omegasomes 4. As an early event, phosphorylation of phosphatidylinositol by VPS34 (PIK3C3) recruits PI(3)P effectors such as WIPI proteins to phagophores, also known as isolation membranes 5, 6. WIPI proteins in turn recruit LC3 and its conjugation machinery to growing isolation membranes, which can selectively encapsulate cargo via autophagy cargo receptors such as p62 (SQSTM1), TAX1BP1, or NDP52 (CALCOCO2) 2, 7, 8. The membrane elongation step to form a sealed autophagosome is incompletely understood and has been shown to require various cellular organelles and compartments including the ER, ER-Golgi intermediate compartment, plasma membrane, mitochondria, lipid droplets (LDs), and their corresponding contact sites 9-14. To identify novel regulators of mammalian autophagy, we have developed and validated FACS-based pooled CRISPR screening approaches to study the turnover of autophagy cargo receptors such as p62 15 or TAX1BP1 16. In this study, we dissected the regulation of the autophagy cargo receptors p62 and NDP52 by comparing basal and activated autophagy. This screening paradigm uncovered TMEM41B, which we characterized as a novel ER transmembrane protein required for autophagosome biogenesis and lipid mobilization. Results and Discussion We set out to uncover novel regulators of autophagy using the FACS-based pooled CRISPR screening paradigm outlined in Fig 1A. Neuroglioma H4 cells were chosen for their amenability to pooled CRISPR screening and well-profiled autophagy pathway at genomewide scale 15-17. We monitored two endogenous autophagy cargo receptors, namely p62 and NDP52, by immunofluorescence-based staining in Cas9-expressing H4 cells. A mini-pool library of the single most potent sgRNAs from our previous GFP-p62 genomewide screen 15 was screened basally or when autophagy was activated with the mTOR inhibitor AZD8055 18 (Dataset EV1). Core components of mammalian autophagy such as ATG3, ATG5, ATG7, ATG9A, ATG16L1, RB1CC1, or WIPI2 scored as robust regulators of both p62 and NDP52 (Fig 1B and C). Depletion of mTOR and Raptor (RPTOR) activated autophagy and reduced levels of p62 and NDP52 as expected, with fold changes being more pronounced under basal conditions. We identified the uncharacterized transmembrane protein TMEM41B as a strong hit for p62 turnover when autophagy was activated with AZD8055 (Fig 1B). TMEM41B was also among the top-scoring regulators of NDP52 (Fig 1C) and TAX1BP1 16. We validated this screening result in H4 Cas9 cells upon CRISPR-mediated depletion of TMEM41B. Since we were unable to identify an antibody to monitor endogenous levels of TMEM41B, we sequenced the TMEM41B genomic locus of the knockout (KO) cell population and confirmed editing in 63% of all sequences using the TIDE method 19. TMEM41B depletion did not alter basal levels of p62, but stalled its clearance upon autophagy induction with AZD8055, while NDP52 levels were significantly increased under basal as well as activated autophagy (Figs 1D and E, and EV1A). By quantifying the increase in protein levels upon lysosomal inhibition with the V-ATPase inhibitor Bafilomycin A1, we found that lysosomal flux of both p62 and NDP52 was significantly impaired when autophagy was activated with AZD8055 in TMEM41B-depleted cells (Fig 1D and F). Furthermore, we confirmed that the majority of LAMP1-positive organelles were filled with p62 upon treatment of control cells with a combination of AZD8055 and Bafilomycin A1, whereas a significant increase in empty luminal structures was detected in TMEM41B KO cells with p62 accumulating in close proximity (Fig 1G and H). The defect in autophagy cargo receptor turnover was also observed in HeLa cells and appears to be on-target as expression of untagged or Myc-tagged TMEM41B rescued changes in p62 and NDP52 in the TMEM41B KO cell populations (Fig EV1B and C). As a physiological autophagy cargo, we examined the fate of cytosolic Salmonella 20 and found that deletion of TMEM41B caused a robust replication advantage to Salmonella (Fig 1I), in line with their impaired lysosomal targeting and degradation. Figure 1. Sensitized CRISPR screens identify TMEM41B as a novel regulator of autophagy A. Schematic representation of pooled CRISPR screening workflow. H4 cells stably expressing Cas9 were transduced with a lentiviral sgRNA mini-pool library, selected for stable integration, expanded, and treated for 24 h with the mTOR inhibitor AZD8055 (activated autophagy) or DMSO as vehicle control (basal autophagy). Endogenous p62 or NDP52 was visualized by immunostaining and cells separated by FACS into populations with high or low signal. Abundance of sgRNAs was quantified in cell populations by next-generation sequencing. B, C. Relative abundance of sgRNAs in cell populations with high versus low signal was visualized as log2 fold chances for basal as well as activated autophagy conditions for (B) p62 or (C) NDP52. Entire data are reported in Dataset EV1. D–F. Validation of TMEM41B. H4 Cas9 cells were infected with sgRNAs targeting TMEM41B or a non-targeting (NT) control, treated with 500 nM AZD8055, 50 nM Bafilomycin A1 (BafA1) or DMSO vehicle control for 24 h, and analyzed by immunoblotting. (E, F) p62 and NDP52 band intensities are depicted as total levels relative to vehicle control in H4 Cas9 NT cells (E), or as flux by calculating the ratio in BafA1-treated cells versus vehicle control (F). Data are presented as mean ± SD (n = 6 independent experiments) with paired t-test values. G. LAMP1 and p62 were co-stained and imaged using an LSM700 confocal microscope. Arrowheads point at p62-filled and empty LAMP1-positive organelles. Scale bar: 5 μm. H. LAMP1-positive organelles were manually quantified for containing a p62-filled or empty lumen. A total of 50–60 LAMP1-positive organelles were analyzed in each experiment. Data are presented as mean ± SD (n = 3 independent experiments) with paired t-test values. I. HeLa cells were transduced with Cas9 and TMEM41B or NT sgRNAs followed by infection with luciferase-expressing Salmonella typhimurium. Luciferase readings were taken up to 8.5 h post-infection. Results from one representative experiment are presented as mean ± SD (n = 8 technical replicates). Source data are available online for this figure. Source Data for Figure 1 [embr201845889-sup-0004-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Rescue of TMEM41B KO with sgRNA-resistant TMEM41B H4 Cas9 TMEM41B KO and NT control cells were probed for p62 and NDP52 by immunostaining and imaged with an automated CV7000 confocal microscope. Scale bar: 20 μm. Staining intensity was quantified with ImageJ and depicted as mean ± SD (n = 4 independent experiments) with paired t-test values. HeLa Cas9 cells were infected with sgRNAs targeting TMEM41B or NT control alongside lentiviruses expressing sgRNA-resistant Myc-tagged TMEM41B, treated with 500 nM AZD8055 or vehicle control for 24 h, and analyzed by immunoblotting 7 days post-infection. H4 Cas9 cells were infected with sgRNAs targeting TMEM41B or NT control alongside lentiviruses expressing sgRNA-resistant untagged or Myc-tagged TMEM41B, treated with 500 nM AZD8055 or vehicle control for 24 h, and analyzed by immunoblotting 7 days post-infection. Source data are available online for this figure. Download figure Download PowerPoint To investigate which step in the autophagic pathway is defective in TMEM41B KO cells, we analyzed LC3 as a marker for autophagosomal vesicles. TMEM41B-deficient cells displayed a significant build-up of lipidated LC3-II by immunoblot (Fig 2A and B) and increased LC3 puncta by immunostaining (Fig 2C and D). The LC3 puncta robustly co-localized with the PI(3)P effector protein WIPI2 (Fig 2E and F), which also accumulated as puncta in the absence of TMEM41B (Fig 2G). Furthermore, LC3 puncta co-localized with RFP-tagged DFCP1 (ZFYVE1; Fig 2H and I), another PI(3)P effector, which dynamically associates with forming autophagosomes 4. These data suggest that autophagy can be initiated in the absence of TMEM41B as evident by WIPI2 and DFCP1 being recruited and LC3 conjugated to isolation membranes, but that the process may stall and fail to efficiently form mature WIPI2- and DFCP1-negative autophagosomes. In support of this notion, we saw a significant reduction in the number of autophagosomes accumulating in TMEM41B-depleted cells when mCherry-GFP-tagged LC3 was monitored upon late-stage autophagy inhibition with Bafilomycin A1 (Fig 2J). Furthermore, ultrastructural analysis of TMEM41B KO cells by transmission electron microscopy revealed the presence of electron-dense structures, which had various shapes and were often found in close proximity to ER tubules (Fig EV2). These structures were virtually absent in H4 Cas9 cells and we hypothesize that they may represent accumulating isolation membranes. Noteworthy, Shoemaker et al independently identified TMEM41B as a novel autophagy regulator by CRISPR-based screening of tandem fluorescent autophagy reporters and showed that accumulated LC3-II remained largely trypsin-sensitive in a protease protection assay of TMEM41B KO cell extracts [preprint: 21]. This is consistent with our data and suggests that TMEM41B is required to form mature and sealed autophagosomes. Figure 2. Knockout of TMEM41B impairs autophagosome biogenesis H4 Cas9 cells were infected with sgRNAs targeting ATG7, TMEM41B or NT control, treated with 500 nM AZD8055 or vehicle control for 24 h, and analyzed by immunoblotting. Ratio of LC3-II to LC3-I band intensities is depicted as mean ± SD (n = 6 independent experiments) with Wilcoxon test values. H4 Cas9 TMEM41B and NT control cells were probed by LC3B immunostaining and imaged with an automated CV7000 confocal microscope. Scale bar: 20 μm. The number of LC3 puncta per cell was quantified using Yokogawa Analysis Software (YAS) and depicted as mean ± SD (n = 3 technical replicates). LC3 and WIPI2 were co-stained and imaged using an LSM700 confocal microscope. Arrowheads point at co-localized LC3 and WIPI2 puncta. Scale bar: 5 μm. The number of WIPI2 puncta positive for LC3 was quantified using ImageJ and depicted as mean ± SD (n = 5 technical replicates). The total number of WIPI2 puncta per cell was quantified using the Harmony software on images acquired with an automated Operetta microscope. Data are depicted as mean ± SD (n = 3 technical replicates). H4 Cas9 TMEM41B and NT control cells were infected with a RFP-DFCP1 expression construct for 72 h, fixed, stained with LC3 antibodies, and imaged with an automated CV7000 confocal microscope. Arrowheads point at co-localized LC3 and RFP-DFCP1 puncta. Scale bar: 5 μm. The number of RFP-DFCP1 puncta positive for LC3 was quantified using ImageJ and depicted as mean ± SD (n = 10 technical replicates). mCherry-GFP-LC3 was expressed in H4 Cas9 TMEM41B KO and NT control cells which were treated with 50 nM BafA1 or vehicle control for 24 h, fixed and imaged. Scale bar: 20 μm. The number of mCherry- and GFP-positive puncta per cell was quantified using YAS and is depicted as mean ± SD (n = 3 technical replicates). Source data are available online for this figure. Source Data for Figure 2 [embr201845889-sup-0005-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Ultrastructural analysis of TMEM41B KO cells A, B. H4 Cas9 (A) and H4 Cas9 TMEM41B KO clone 6 (B) cells were analyzed by transmission electron microscopy. Representative images are shown at the level of the entire cell, the juxtanuclear space as well as an inlet thereof. Mitochondria (M), lipid droplets (LD), and electron-dense structures (white arrowheads) are indicated. Scale bar: 10 μm. Download figure Download PowerPoint What is the underlying cause of the autophagy defect in TMEM41B KO cells? To tackle this question, we screened a panel of fluorescent probes and observed a striking accumulation of BODIPY 493, BODIPY FL C12, and NBD cholesterol probes in large puncta in TMEM41B-depleted cells (Fig 3A). These probes visualize neutral lipids and cholesterol, key components of cellular lipid droplets (LDs) 22. The accumulation of neutral lipids and cholesterol appeared specific as fluorescent probes for other lipids such as sphingomyelin and ceramide were unchanged upon TMEM41B depletion, and organelle markers such as ER-Tracker or LysoTracker looked intact (Fig EV3A). To better characterize a potential role of TMEM41B in regulating LDs, we analyzed H4 cells basally or upon treatment with BSA-conjugated oleic acid by probing with HCS LipidTox Green Neutral Lipid Stain, a probe with high affinity for neutral LDs. TMEM41B-depleted cells showed a significant increase in LDs both basally and upon oleic acid treatment (Fig 3B). LDs mainly increased in size and intensity, while the overall number was not significantly increased under basal conditions and rather decreased upon oleic acid treatment in TMEM41B KO cells (Fig 3C). LDs strongly co-localized with ADRP (Perilipin-2; Fig EV3B), a protein which coats the surface of LDs 23. Analysis of ADRP in TMEM41B-depleted cells confirmed the increase in lipid droplets both by immunostaining (Fig 3D and E) and by Western blot (Fig 3F and G). Also our electron microscopy analysis showed enlarged LDs upon TMEM41B KO (Fig EV2B, LD). We conclude that TMEM41B-deficient cells display enlarged LDs pointing to a potential role of TMEM41B in lipid mobilization, trafficking, and/or metabolism. Figure 3. TMEM41B-deficient cells display enlarged lipid droplets H4 Cas9 cells stably expressing NT, TMEM41B, or ATG7 sgRNAs were stained with BODIPY 493, NBD cholesterol, or BODIPY FL C12 probes for 2 h at 37°C and imaged live with an automated CV7000 confocal microscope. Puncta area per cell was quantified using YAS and depicted as mean ± SD (n = 4 technical replicates). Scale bar: 20 μm. H4 Cas9 TMEM41B KO and NT control cells were treated overnight with 400 μM BSA-conjugated oleic acid or 0.1% BSA as vehicle control, stained for 2 h with HCS LipidTox Green Neutral Lipid Stain, and imaged with an automated Operetta microscope. Scale bar: 20 μm. Lipid droplet size, number, and mean fluorescence intensity were quantified using Harmony software. Data are presented as mean ± SD (n = 3 technical replicates). An average of 1,500 cells was analyzed per replicate. H4 Cas9 TMEM41B KO and NT control cells were probed by ADRP immunostaining and imaged with an automated CV7000 confocal microscope. Scale bar: 20 μm. Size of ADRP droplets was quantified with YAS and depicted as mean ± SD (n = 3 technical replicates). Protein level of ADRP was probed by immunoblotting in H4 Cas9 TMEM41B KO and NT control cells. ADRP band intensities were quantified and depicted as mean ± SD (n = 4 independent experiments) with paired t-test values. Source data are available online for this figure. Source Data for Figure 3 [embr201845889-sup-0006-SDataFig3.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Analysis of additional lipid and organelle markers H4 Cas9 TMEM41B KO, ATG7 KO, and NT control cells were stained with NBD C6 ceramide, BODIPY FL C12 sphingomyelin, ER-Tracker, or LysoTracker probes. After 2 h incubation at 37°C, cells were imaged live with an automated CV7000 confocal microscope using a 60× objective. Representative images are shown, and scale bar represents 20 μm. H4 Cas9 TMEM41B KO and NT control cells were stained with ADRP antibodies and HCS LipidTox Deep Red Neutral Lipid Stain and imaged with an automated CV7000 confocal microscope. Representative images are shown, and scale bar represents 20 μm. Download figure Download PowerPoint Lipid droplets constitute the primary storage site for fatty acids (FAs) and can be targeted for lysosomal breakdown via lipophagy, a process that has been shown to depend on autophagy core components such as ATG7 24. Since deletion of ATG7 did not result in a significant accumulation of BODIPY 493, BODIPY FL C12, and NBD cholesterol probes in H4 cells (Fig 3A), we looked into lipophagy-independent processes. More recently, LDs have been shown to be mobilized and consumed for autophagy initiation in both yeast 13 and mammalian cells 25. Furthermore, cells mobilize FAs in LDs to produce ATP via mitochondrial β-oxidation 26, 27, and enlarged lipid droplets have been linked to defective β-oxidation in worms 28. To evaluate a role of TMEM41B in lipid mobilization, we assessed the flux of FAs from LDs to mitochondria by pulsing cells with a red fluorescent FA analog (Red C12) 27 and chased it in complete medium (CM) or upon serum deprivation (SD). In control cells, serum deprivation re-localized the red fluorescent FA probe from LDs to mitochondria, while most of the Red C12 signal remained in LDs in TMEM41B KO cells and showed little overlap with mitochondria (Fig 4A). Quantification of Red C12 fluorescence intensity within the MitoTracker staining revealed a significant reduction in TMEM41B KO cells (Fig 4B), consistent with a role of TMEM41B in mobilizing lipids from LDs. We next assessed the metabolic capacity of TMEM41B-depleted cells by Seahorse analysis. TMEM41B KO cells displayed a small but significant decrease in basal mitochondrial oxygen consumption, whereas extracellular acidification rates were increased (Fig 4C). These data suggest that TMEM41B-depleted cells may have increased glycolysis to compensate for a defect in mitochondrial respiration. To more specifically evaluate the β-oxidation of endogenous FAs, we used substrate-limited medium and measured oxygen consumption in the presence or absence of etomoxir, which is an inhibitor of mitochondrial FA import by blocking carnitine palmitoyl transferase-1 (CPT-1) 29. This experimental setup confirmed the basal decrease in mitochondrial oxygen consumption in TMEM41B-deficient cells and demonstrated that etomoxir abolishes the difference in oxygen consumption between TMEM41B KO and control cells (Fig 4D). By calculating etomoxir-dependent oxygen consumption, we found that TMEM41B-depleted cells display significantly lower endogenous FA utilization rates (Fig 4E). We propose that TMEM41B supports mobilization and mitochondrial β-oxidation of fatty acids. Figure 4. TMEM41B is required for mobilization and utilization of endogenous fatty acids A. H4 Cas9 TMEM41B KO and NT control cells were pulsed with the fatty acid analog Red C12, chased in complete medium or upon serum deprivation, and stained with MitoTracker. Cells were imaged live with an automated CV7000 confocal microscope. Scale bar: 20 μm. B. Fatty acid Red C12 intensity was measured within MitoTracker signal, and ratio between SD and CM conditions was calculated. Data are presented as mean ± SD (n = 3 technical replicates). C–E. H4 Cas9 TMEM41B KO and NT control cells were plated in XF96 plates and analyzed using a Seahorse Bioscience XF96. (C) Basal oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured in cells grown in complete medium. OCR/ECAR ratio is also reported. Data are presented as mean ± SEM (n = 30–32 technical replicates) from one representative experiment. (D) OCR was measured in cells grown in substrate-limited medium and treated in the absence or presence of Etomoxir (Eto). Oligomycin (OA), FCCP (FC), and a mixture of rotenone and antimycin A (RA) were added as indicated. Data are shown as mean ± SEM (n = 69 technical replicates). (E) Endogenous FA utilization was calculated by subtracting basal normalized OCR values of Eto-treated cells from untreated cells. Data are shown as mean ± SEM (n = 69 technical replicates). Download figure Download PowerPoint TMEM41B has six predicted transmembrane domains, but its intracellular localization has not been univocally determined. Expression and immunostaining of N- or C-terminally Myc-tagged TMEM41B showed a tubular pattern, which co-localized with the ER marker calnexin (Fig 5A) and KDEL (Fig EV4B). An antibody directed against TMEM41B was unable to recognize the endogenous protein but confirmed a tubular ER pattern upon expression of untagged TMEM41B (Fig EV4A). To confirm that overexpression of TMEM41B does not perturb its intracellular localization, we tagged the endogenous TMEM41B locus with a C-terminal Myc epitope by CRISPR and validated the knock-in (KI) by immunoblot and genomic PCR (Fig 5B–D). This KI approach in HeLa cells confirmed that TMEM41B robustly co-localizes with the ER marker calnexin (Fig 5B). TMEM41B has a predicted “SNARE-associated Golgi protein” domain, which shares homology to bacterial DedA proteins 30, yeast TVP38, and mammalian TMEM41A, TMEM64, and VMP1 (IPR032816, 31). The homology to VMP1 is interesting as this ER-localized trans" @default.
• W2885453664 created "2018-08-22" @default.
• W2885453664 creator A5002128457 @default.
• W2885453664 creator A5009011090 @default.
• W2885453664 creator A5010478641 @default.
• W2885453664 creator A5010674430 @default.
• W2885453664 creator A5013656815 @default.
• W2885453664 creator A5014249484 @default.
• W2885453664 creator A5017219096 @default.
• W2885453664 creator A5019627876 @default.
• W2885453664 creator A5027087200 @default.
• W2885453664 creator A5027605302 @default.
• W2885453664 creator A5039723259 @default.
• W2885453664 creator A5044241549 @default.
• W2885453664 creator A5045437170 @default.
• W2885453664 creator A5056408708 @default.
• W2885453664 creator A5062206481 @default.
• W2885453664 creator A5068333465 @default.
• W2885453664 creator A5073448033 @default.
• W2885453664 creator A5074186441 @default.
• W2885453664 creator A5076590452 @default.
• W2885453664 creator A5086338711 @default.
• W2885453664 creator A5088024162 @default.
• W2885453664 date "2018-08-20" @default.
• W2885453664 modified "2023-10-15" @default.
• W2885453664 title "<scp>TMEM</scp> 41B is a novel regulator of autophagy and lipid mobilization" @default.
• W2885453664 cites W1526993239 @default.
• W2885453664 cites W1546144313 @default.
• W2885453664 cites W1925393555 @default.
• W2885453664 cites W1964552652 @default.
• W2885453664 cites W1967743942 @default.
• W2885453664 cites W1984160651 @default.
• W2885453664 cites W1984680116 @default.
• W2885453664 cites W1987210736 @default.
• W2885453664 cites W1993902882 @default.
• W2885453664 cites W2002439304 @default.
• W2885453664 cites W2002636911 @default.
• W2885453664 cites W2007505836 @default.
• W2885453664 cites W2015832516 @default.
• W2885453664 cites W2019079031 @default.
• W2885453664 cites W2035310767 @default.
• W2885453664 cites W2048519887 @default.
• W2885453664 cites W2048705639 @default.
• W2885453664 cites W2060905615 @default.
• W2885453664 cites W2062056725 @default.
• W2885453664 cites W2065031087 @default.
• W2885453664 cites W2077757969 @default.
• W2885453664 cites W2078013685 @default.
• W2885453664 cites W2081962941 @default.
• W2885453664 cites W2083187008 @default.
• W2885453664 cites W2094241924 @default.
• W2885453664 cites W2096308078 @default.
• W2885453664 cites W2101871339 @default.
• W2885453664 cites W2106070783 @default.
• W2885453664 cites W2107554012 @default.
• W2885453664 cites W2114847797 @default.
• W2885453664 cites W2126397038 @default.
• W2885453664 cites W2127767424 @default.
• W2885453664 cites W2132763961 @default.
• W2885453664 cites W2135581618 @default.
• W2885453664 cites W2135855797 @default.
• W2885453664 cites W2144718635 @default.
• W2885453664 cites W2154836325 @default.
• W2885453664 cites W2156975935 @default.
• W2885453664 cites W2159814827 @default.
• W2885453664 cites W2161002942 @default.
• W2885453664 cites W2461558445 @default.
• W2885453664 cites W2549196136 @default.
• W2885453664 cites W2557496587 @default.
• W2885453664 cites W2560888990 @default.
• W2885453664 cites W2617561678 @default.
• W2885453664 cites W2751061998 @default.
• W2885453664 cites W2752949980 @default.
• W2885453664 cites W86630917 @default.
• W2885453664 doi "https://doi.org/10.15252/embr.201845889" @default.
• W2885453664 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6123663" @default.
• W2885453664 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/30126924" @default.
• W2885453664 hasPublicationYear "2018" @default.
• W2885453664 type Work @default.
• W2885453664 sameAs 2885453664 @default.
• W2885453664 citedByCount "123" @default.
• W2885453664 countsByYear W28854536642018 @default.
• W2885453664 countsByYear W28854536642019 @default.
• W2885453664 countsByYear W28854536642020 @default.
• W2885453664 countsByYear W28854536642021 @default.
• W2885453664 countsByYear W28854536642022 @default.
• W2885453664 countsByYear W28854536642023 @default.
• W2885453664 crossrefType "journal-article" @default.
• W2885453664 hasAuthorship W2885453664A5002128457 @default.
• W2885453664 hasAuthorship W2885453664A5009011090 @default.
• W2885453664 hasAuthorship W2885453664A5010478641 @default.
• W2885453664 hasAuthorship W2885453664A5010674430 @default.
• W2885453664 hasAuthorship W2885453664A5013656815 @default.
• W2885453664 hasAuthorship W2885453664A5014249484 @default.
• W2885453664 hasAuthorship W2885453664A5017219096 @default.
• W2885453664 hasAuthorship W2885453664A5019627876 @default.
• W2885453664 hasAuthorship W2885453664A5027087200 @default.
• W2885453664 hasAuthorship W2885453664A5027605302 @default.
• W2885453664 hasAuthorship W2885453664A5039723259 @default.
• W2885453664 hasAuthorship W2885453664A5044241549 @default.

• next