Vacuolar protein sorting 41 (VPS41), a component of the homotypic fusion and protein sorting (HOPS) complex for lysosomal fusion, is essential for the trafficking of lysosomal membrane proteins via lysosome-associated membrane protein (LAMP) carriers from the trans-Golgi network (TGN) to endo/lysosomes. However, the molecular mechanisms underlying this pathway and VPS41's role herein remain poorly understood. Here, we investigated the effects of ectopically localizing VPS41 to mitochondria on LAMP distribution. Using electron microscopy, we identified that mitochondrial-localized VPS41 recruited LAMP1- and LAMP2A-positive vesicles resembling LAMP carriers. The retention using selective hooks (RUSH) system further revealed that newly synthesized LAMPs were specifically recruited by mitochondrial VPS41, a function not shared by other HOPS subunits. Notably, we identified the small GTPase Arl8b as a critical factor for LAMP carrier trafficking. Arl8b was present on LAMP carriers and bound to the WD40 domain of VPS41, enabling their recruitment. These findings reveal a unique role of VPS41 in recruiting TGN-derived LAMP carriers and expand our understanding of VPS41-Arl8b interactions beyond endosome-lysosome fusion, providing new insights into lysosomal trafficking mechanisms.
© 2025 Sanzà et al.
Product Citations: 164
VPS41 recruits biosynthetic LAMP-positive vesicles through interaction with Arl8b.
In The Journal of Cell Biology on 3 April 2025 by Sanzà, P., van der Beek, J., et al.
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Cell Biology
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Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation.
In The Journal of Cell Biology on 6 January 2025 by Tey, P. Y., Dufner, A., et al.
The immune checkpoint regulator CTLA4 is an unusually short-lived membrane protein. Here, we show that its lysosomal degradation is dependent on ubiquitylation at lysine residues 203 and 213. Inhibition of the v-ATPase partially restores CTLA4 levels following cycloheximide treatment, but also reveals a fraction that is secreted in exosomes. The endosomal deubiquitylase, USP8, interacts with CTLA4, and its loss enhances CTLA4 ubiquitylation in cancer cells, mouse CD4+ T cells, and cancer cell-derived exosomes. Depletion of the USP8 adapter protein, HD-PTP, but not ESCRT-0 recapitulates this cellular phenotype but shows distinct properties vis-à-vis exosome incorporation. Re-expression of wild-type USP8, but neither a catalytically inactive nor a localization-compromised ΔMIT domain mutant can rescue delayed degradation of CTLA4 or counteract its accumulation in clustered endosomes. UbiCRest analysis of CTLA4-associated ubiquitin chain linkages identifies a complex mixture of conventional Lys63- and more unusual Lys27- and Lys29-linked polyubiquitin chains that may underly the rapidity of protein turnover.
© 2024 Tey et al.
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Cell Biology
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Preprint on Research Square on 31 December 2024 by Palau, F., Cantarero, L., et al.
Abstract Pathogenic variants of GDAP1 cause Charcot-Marie-Tooth disease (CMT), an inherited neuropathy characterized by axonal degeneration. GDAP1, an atypical glutathione S-transferase, localizes to the outer mitochondrial membrane (OMM), regulating this organelle's dynamics, transport, and membrane contact sites (MCSs). It has been proposed that GDAP1 functions as a cellular redox sensor. However, its precise contribution to redox homeostasis remains poorly understood, as does the possible redox regulation at mitochondrial MCSs. Given the relationship between the peroxisomal redox state and overall cellular redox balance, we investigated the role of GDAP1 in peroxisomal function and mitochondrial MCSs maintenance by using high-resolution microscopy, live cell imaging with pH-sensitive fluorescent probes, and transcriptomic and lipidomic analyses in the Gdap1-/- mice and patient-derived fibroblasts. We demonstrate that GDAP1 deficiency disrupts mitochondria-peroxisome MCSs and leads to peroxisomal abnormalities, which are reversible upon pharmacological activation of PPARγ or glutathione supplementation. These results identify GDAP1 as a new tether of mitochondria-peroxisome MCSs that maintain peroxisomal number and integrity. The supply of glutathione (GSH-MEE) or GDAP1 overexpression suffices to rescue these MCSs. Furthermore, GDAP1 may regulate the redox state within the microdomain of mitochondrial MCSs, as suggested by decreased pH at mitochondria-lysosome contacts in patient-derived fibroblasts, highlighting the relationship between GDAP1 and redox-sensitive targets. Finally, in vivo analysis of sciatic nerve tissue in Gdap1-/- mice revealed significant axonal structural abnormalities, including nodes of Ranvier disruption and defects in the distribution and morphology of mitochondria, lysosomes, and peroxisomes, emphasizing the importance of GDAP1 in sustaining axon integrity in the peripheral nervous system. Taken together, this study positions GDAP1 as a multifunctional protein that mediates mitochondrial interaction with cellular organelles of diverse functions, contributes to redox state sensing, and helps maintain axonal homeostasis. In addition, we identify PPAR as a novel therapeutic target, based on knowledge of the underlying pathogenetic mechanisms.
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Nup358 restricts ER-mitochondria connectivity by modulating mTORC2/Akt/GSK3β signalling.
In EMBO Reports on 1 October 2024 by Kalarikkal, M., Saikia, R., et al.
ER-mitochondria contact sites (ERMCSs) regulate processes, including calcium homoeostasis, energy metabolism and autophagy. Previously, it was shown that during growth factor signalling, mTORC2/Akt gets recruited to and stabilizes ERMCSs. Independent studies showed that GSK3β, a well-known Akt substrate, reduces ER-mitochondria connectivity by disrupting the VAPB-PTPIP51 tethering complex. However, the mechanisms that regulate ERMCSs are incompletely understood. Here we find that annulate lamellae (AL), relatively unexplored subdomains of ER enriched with a subset of nucleoporins, are present at ERMCSs. Depletion of Nup358, an AL-resident nucleoporin, results in enhanced mTORC2/Akt activation, GSK3β inhibition and increased ERMCSs. Depletion of Rictor, a mTORC2-specific subunit, or exogenous expression of GSK3β, was sufficient to reverse the ERMCS-phenotype in Nup358-deficient cells. We show that growth factor-mediated activation of mTORC2 requires the VAPB-PTPIP51 complex, whereas, Nup358's association with this tether restricts mTORC2/Akt signalling and ER-mitochondria connectivity. Expression of a Nup358 fragment that is sufficient for interaction with the VAPB-PTPIP51 complex suppresses mTORC2/Akt activation and disrupts ERMCSs. Collectively, our study uncovers a novel role for Nup358 in controlling ERMCSs by modulating the mTORC2/Akt/GSK3β axis.
© 2024. The Author(s).
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Cell Biology
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In Nature Communications on 26 September 2024 by Schiffelers, L. D., Tesfamariam, Y. M., et al.
Inflammasome activation results in the cleavage of gasdermin D (GSDMD) by pro-inflammatory caspases. The N-terminal domains (GSDMDNT) oligomerize and assemble pores penetrating the target membrane. As methods to study pore formation in living cells are insufficient, the order of conformational changes, oligomerization, and membrane insertion remained unclear. We have raised nanobodies (VHHs) against human GSDMD and find that cytosolic expression of VHHGSDMD-1 and VHHGSDMD-2 prevents oligomerization of GSDMDNT and pyroptosis. The nanobody-stabilized GSDMDNT monomers partition into the plasma membrane, suggesting that membrane insertion precedes oligomerization. Inhibition of GSDMD pore formation switches cell death from pyroptosis to apoptosis, likely driven by the enhanced caspase-1 activity required to activate caspase-3. Recombinant antagonistic nanobodies added to the extracellular space prevent pyroptosis and exhibit unexpected therapeutic potential. They may thus be suitable to treat the ever-growing list of diseases caused by activation of (non-) canonical inflammasomes.
© 2024. The Author(s).
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In Signal Transduct Target Ther on 11 May 2024 by Shin, H. J., Lee, W., et al.
Fig.3.D
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Tyrosine kinase inhibitors targeting EGFR signalling pathway reduces SCoV2 propagation. a A scheme for primary screening of the antiviral effect of EGFR inhibitors against SCoV2 propagation. At 4 h post-infection, HEK293T cells infected with SCoV2 at an MOI of 1 were washed with fresh cell cultur...
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Tyrosine kinase inhibitors targeting EGFR signalling pathway reduces SCoV2 propagation. a A scheme for primary screening of the antiviral effect of EGFR inhibitors against SCoV2 propagation. At 4 h post-infection, HEK293T cells infected with SCoV2 at an MOI of 1 were washed with fresh cell culture media 5 times and subsequently treated with the indicated EGFR inhibitors (10 μM) for 44 h. Cell culture supernatant and pellet were used for further analyses (b, c). b Extracellular RNA was isolated from culture supernatants of SCoV2-infected HEK293T cells and used for the quantification of SCoV2 RNA levels by real-time qRT-PCR. Data shown are the representative of two independent experiments (mean ± SD; n = 2). c Whole cell lysates of SCoV2-infected HEK293T cells were analysed by immunoblotting with antibody specific to SCoV2 N protein. β-actin, an internal loading control. d Rescue of SCoV2-induced mitochondrial translocation of EGFR by vandetanib treatment. Cytosolic (Cyto) and mitochondrial (Mito) fractions isolated from uninfected and SCoV2-infected HEK293T cells (MOI of 1) treated with vandetanib (10 μM) were analysed by immunoblotting with EGFR antibody. Fractions: purified cytoplasm, Cyto; purified mitochondria, Mito. Organelle marker: TOM20, mitochondria; GAPDH, cytoplasm. Infection marker: SCoV2 nucleocapsid (N). e Rescue of SCoV2-induced elevation in intracellular ATP level by vandetanib treatment. At 1 day post-treatment of vandetanib (10 μM), intracellular ATP level in SCoV2-infected HEK293T cells (MOI of 1) was analysed as described in Materials and Methods. Data shown are the average of two independent experiments (mean ± SD; n = 2; *p < 0.05). f, Western blot analysis of mitochondrial respiratory chain complex enzyme expression. At 1 day post-treatment of vandetanib (10 μM), the expression level of complex I, II, III, IV and V enzymes was analysed by immunoblotting with anti-Hu total OXPHOS complex antibody. C, complex; β-actin, an internal loading control; SCoV2 N, infection marker. g–h, HEK293T cells infected with SCoV2 for 4 h at an MOI of 1 were washed with fresh cell culture media 5 times and then further cultured for 20 h in the presence of vandetanib (10 μM) for RNAseq analysis. g Heat maps of relative mRNA expression of the indicated mitochondrial OXPHOS genes isolated from SCoV2-infected and vandetanib-treated SCoV2-infected cells. Each box indicates an average of three independent experiments. Colour indicates log2 fold-change for SCoV2-infected vs. uninfected cells and vandetanib-treated SCoV2-infected vs. SCoV2-infected cells, respectively. h Read coverage across the SCoV2 genome in the presence and absence of vandetanib. The graph represents the number of viral reads per position of the SCoV2 genome in HEK293T cells (SCoV2, dark blue; SCoV2/vandetanib, orange). A scaled model of the SCoV2 genome and its genes is portrayed below
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Collected and cropped from Signal Transduct Target Ther by CiteAb, provided under a CC-BY license
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In Signal Transduct Target Ther on 11 May 2024 by Shin, H. J., Lee, W., et al.
Fig.2.E
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SCoV2 promotes EGFR-mediated cell survival signal cascade and mitochondrial EGFR translocation. a Quantification of SCoV2-infected cell viability. Total number of viable HEK293T (left) or Calu-3 (right) cells infected with SCoV2 at an MOI of 1 was measured at the indicated time points as describe...
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SCoV2 promotes EGFR-mediated cell survival signal cascade and mitochondrial EGFR translocation. a Quantification of SCoV2-infected cell viability. Total number of viable HEK293T (left) or Calu-3 (right) cells infected with SCoV2 at an MOI of 1 was measured at the indicated time points as described in Materials and Methods. Data shown are the representative of three independent experiments (mean ± SD; n = 2). b Quantitative analysis of EGFR gene expression in SCoV2-infected cells. At 1 day post-infection, the expression level of EGFR mRNA in HEK293T cells infected with SCoV2 at an MOI of 1 were analysed by real-time qRT-PCR (mean ± SD; n = 3; *p < 0.05). Data shown are the representative of two independent experiments. c Western blot analysis showing SCoV2-induced activation in EGFR-mediated signal cascade in SCoV2-infected cells. At 1 day post-infection, whole cell lysates (WCL) of HEK293T (left) or Calu-3 (right) cells infected with SCoV2 at an MOI of 1 were analysed by immunoblotting with antibodies specific to p-EGFR, EGFR, p-Akt (Thr308) and Akt. SCoV2 nucleocapsid (N) protein, infection control; β-actin, an internal loading control. d Western blot analysis showing SCoV2-induced activation in EGFR-mediated signal cascade in lung specimen of SCoV2-infected hACE2 transgenic mouse. At 1 day post-infection, whole tissue lysates (WTL) of lung specimens of SCoV2-infected hACE2 transgenic mouse were analysed by immunoblotting with antibodies specific to p-EGFR, EGFR, p-Akt (Thr308) and Akt. SCoV2 nucleocapsid (N) protein, infection control; β-actin, an internal loading control. e Western blot analysis of SCoV2-induced mitochondrial translocation of EGFR. HEK293T cells were infected with SCoV2 at an MOI of 1. At 2 days post-infection, cytosolic (Cyto) and mitochondrial (Mito) fractions isolated from uninfected and SCoV2-infected HEK293T cells were evaluated by immunoblotting with antibodies specific to p-EGFR and EGFR. Fractions: purified cytoplasm, Cyto; purified mitochondria, Mito. Organelle marker: TOM20, mitochondria; GAPDH, cytoplasm. Infection marker: SCoV2 nucleocapsid (N) antigen. The relative intensity of EGFR normalised to TOM20 and p-EGFR normalised to EGFR, respectively, was analysed by ImageJ. f Confocal microscopy showing mitochondrial translocation of EGFR in SCoV2-infected cells. Uninfected (upper) and SCoV2-infected (lower) HEK293T cells prestained with MitoTracker (red) were immunostained with antibodies specific to EGFR (green) and SCoV2-N (blue). Nuclei are demarcated with white circles. Infected (+) and uninfected (-) cells are marked. Yellow scale bar, 5 μm (upper), 10 μm (lower). In the zoomed images, the white arrow indicate endogenous EGFR recruited to the mitochondria in SCoV2-infected cells (yellow). g Western blot analysis of mitochondrial EGFR translocation in the lung tissues of hACE2 transgenic mouse infected with SCoV2. Mitochondrial fractions (Mito) isolated from lung tissue of uninfected and SCoV2-infected hACE2 transgenic mouse were analysed by immunoblotting with EGFR antibody. Organelle marker: TOM20, mitochondria. Infection marker: SCoV2 nucleocapsid (N) antigen. The relative intensity of EGFR expression was normalised to TOM20 expression
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Collected and cropped from Signal Transduct Target Ther by CiteAb, provided under a CC-BY license
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In Signal Transduct Target Ther on 11 May 2024 by Shin, H. J., Lee, W., et al.
Fig.1.E
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SCoV2 induces aberrant mitochondrial elongation and alterations in mitochondrial oxidative phosphorylation process. a Quantification of intracellular ATP level of SCoV2-infected HEK293T cells at the indicated time points (MOI of 1). Data shown are the average of two independent experiments (mean ...
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SCoV2 induces aberrant mitochondrial elongation and alterations in mitochondrial oxidative phosphorylation process. a Quantification of intracellular ATP level of SCoV2-infected HEK293T cells at the indicated time points (MOI of 1). Data shown are the average of two independent experiments (mean ± SD; n = 4; *p < 0.05). b Quantification of ΔΨm change in HEK293T (left) and Calu-3 (right) cells infected with SCoV2 and IAV (H1N1, PR8 strain), respectively (MOI of 1). ΔΨm in SCoV2 or IAV-infected cells were monitored by TMRE assay at 1 day post-infection. ΔΨm in FCCP-treated cells were monitored by TMRE assay at 2 h post-treatment. Data shown are the representative of three independent experiments (mean ± SD; n = 3; *p < 0.05, **p < 0.001, ***p < 0.0001). ΔΨm, mitochondrial membrane potential. FCCP, synthetic mitochondrial uncoupler, a control for ΔΨm loss. The accompanying diagram (right) represents the SCoV2-induced increase in ΔΨm. c Quantification of ΔΨm increase by SCoV2 RNA-N cluster. HEK293T cells were transfected with DNA plasmid encoding SCoV2 nucleocapsid (N) gene. ΔΨm in cells were monitored by TMRE assay at 24 h post-transfection (left panel). HEK293T cells transfected with DNA plasmid encoding SCoV2 nucleocapsid (N) gene for 24 h were transfected with SCoV2 RNA (1 μg/ml, middle panel), poly(I:C) (1 μg/ml, middle panel), human tRNA (1 μg/ml, right panel) and IAV RNA (1 μg/ml, right panel), respectively. ΔΨm in cells were monitored by TMRE assay at 4 h post-transfection. Data shown are the representative of three independent experiments (mean ± SD; n = 4 or n = 8; *p < 0.05, **p < 0.001). poly(I:C), synthetic analogue of double-stranded RNA, a control for ΔΨm loss; human tRNA, a control for endogenous sense RNA; TF ctrl., transfection reagent only; mock, pcDNA3.1 plasmid DNA. The accompanying diagram (right) represents the SCoV2 RNA-N cluster-induced increase in ΔΨm. d Confocal microscopy showing the elongated shape of mitochondria in SCoV2-infected cells. SCoV2-infected HEK293T cells were immunostained with SCoV2-S (red) and TOM20 (green) antibodies. Infected (+) and uninfected (-) cells are marked. The white arrows indicate the expression of SCoV2 spike (S) antigen as an infection marker. Nuclei are demarcated with white circles. Yellow scale bar, 10 μm. The zoomed images reveal elongated shape of mitochondria in SCoV2-infected cells (right) compared with typical mitochondrial tubular network in uninfected cells (left). The accompanying graph represents the quantification of mitochondrial length by MBF ImageJ. e Western blot analysis of MFN1/2 expression in SCoV2-infected cells. Mitochondrial fraction (Mito) isolated from HEK293T cells infected with SCoV2 at an MOI of 1 was analysed by immunoblotting with anti-MFN1/2 antibody. SCoV2 nucleocapsid (N) protein, infection control; TOM20, an internal loading control. f Western blot analysis of Drp1, phospho-Drp1 (S616) and mitochondrial fission factor (MFF) expression in SCoV2-infected cells. Whole cell lysates extracted from HEK293T cells infected with SCoV2 at an MOI of 1 was analysed by immunoblotting with antibodies specific to Drp1, phospho-Drp1 (Ser616), and MFF, respectively. SCoV2 nucleocapsid (N) protein, infection control; β-actin, an internal loading control. g Real-time qPCR data showing the increase in mitochondrial DNA of SCoV2-infected HEK293T cells. At 1 day post-infection, the expression level of mitochondrial ND2 and COX2 DNA was analysed by real-time qPCR. GAPDH was used to normalise changes in ND2 and COX2 expression. h Western blot analysis of mitochondrial respiratory chain complex enzyme expression in HEK293T cells infected with SCoV2 at an MOI of 1. At 1 day post-infection, the expression level of complex I, II, III, IV and V enzymes was analysed by immunoblotting with anti-Hu total OXPHOS complex antibody. C, complex; SCoV2 spike (S) protein, infection control; β-actin, an internal loading control. i Heat maps of relative mRNA of the indicated mitochondrial OXPHOS genes isolated from uninfected and SCoV2-infected HEK293T cells. Each box indicates an average of three independent experiments. Colour indicates log2 fold-change for uninfected vs. SCoV2-infected cells. j Western blot analysis of mitochondrial respiratory chain complex enzyme expression in lung tissue isolated hACE2 transgenic mouse infected with SCoV2. At 1 day post-infection, the expression level of complex I, II, III, IV and V enzymes was analysed by immunoblotting with anti-Rodent total OXPHOS complex antibody. C, complex; SCoV2 nucleocapsid (N) protein, infection control; β-actin, an internal loading control
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Collected and cropped from Signal Transduct Target Ther by CiteAb, provided under a CC-BY license
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In Int J Biol Sci on 12 July 2022 by Jung, S., Myagmarjav, D., et al.
Fig.6.D
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Enhanced TRIP-Br1-mediated mitophagy via up-regulation of lysosomal proteases cathepsins B and D. A-B MCF7WT-TRIP-Br1 and MCF7KD-TRIP-Br1 cells were treated with 0.1 µM STS for 3 h and 24 h. The precursor and mature forms of cathepsins B and D were determined via immunoblotting analysis. β-actin...
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Enhanced TRIP-Br1-mediated mitophagy via up-regulation of lysosomal proteases cathepsins B and D. A-B MCF7WT-TRIP-Br1 and MCF7KD-TRIP-Br1 cells were treated with 0.1 µM STS for 3 h and 24 h. The precursor and mature forms of cathepsins B and D were determined via immunoblotting analysis. β-actin was used as an internal control. Data are presented as mean ± SD (n = 3). C MCF7WT-TRIP-Br1 and MCF7KD-TRIP-Br1 cells were cultured in confocal dishes for 24 h and then incubated with Lysotracker (Red) for 90 min. These cells were captured using a confocal microscope. Quantification of red fluorescence intensity of Lysotracker is shown. Values are expressed as mean ± SD of three independent experiments with each count of no less than 100 cells. D Both cell lines were cultured for 24 h and then treated with 0.1 μM STS for 3 h. Cells were harvested and lysosomal fractions were isolated as described in Materials and Methods. γ-Tubulin was used as a cytosolic marker.
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Collected and cropped from Int J Biol Sci by CiteAb, provided under a CC-BY license
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In Proc Natl Acad Sci U S A on 21 June 2022 by Simonetti, B., Daly, J. L., et al.
Fig.1.D
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Development of a methodology to biotinylate TGN-resident proteins. (A) Schematic of HRP-TGN46 biotinylation labeling of endogenous TGN-resident proteins. In the presence of biotin-phenol and hydrogen peroxide, HRP catalyses the formation of membrane-impermeable biotin-phenoxyl that covalently lab...
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Development of a methodology to biotinylate TGN-resident proteins. (A) Schematic of HRP-TGN46 biotinylation labeling of endogenous TGN-resident proteins. In the presence of biotin-phenol and hydrogen peroxide, HRP catalyses the formation of membrane-impermeable biotin-phenoxyl that covalently label nearby endogenous proteins. (B) Transmission electron micrograph of HRP-TGN46–expressing HeLa cells incubated with DAB and H2O2 for 10 min. (Scale bar, 500 nm; zoom scale bar, 100 nm.) ER, endoplasmic reticulum; GA, Golgi apparatus; M, mitochondrion; N, nucleus; NE, nuclear envelope. (C) STED microscopy of HRP-TGN46–expressing HeLa cells following biotinylation, stained with fluorescent streptavidin and immunofluorescence labeling of Golgin-97. (Scale bars, 5 µm; Insets, 2 µm.) (D) Streptavidin affinity isolation of biotinylated proteins from total cell lysate of HRP-TGN46–expressing cells incubated with H2O2 in the presence or absence of BP, and their corresponding known subcellular localization. (E) Scatterplot of all HRP-TGN46–labeled proteins, with known mannose-6-phosphate–tagged proteins overlaid in red, and example TGN retrograde cargoes highlighted by blue labels; n = 5 independent repeats. (F–G) Graphical representation of overrepresentation of gene ontology cellular compartments (F) and biological processes (G) within the HRP-TGN46–proteome according to their calculated P value. Overrepresented gene ontology terms are colored green, and underrepresented terms are colored red. The numbers of identified proteins belonging to each category are provided within parentheses.
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Collected and cropped from Proc Natl Acad Sci U S A by CiteAb, provided under a CC-BY license
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In Nat Commun on 19 May 2022 by Mece, O., Houbaert, D., et al.
Fig.5.A
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Autophagy regulates CPT1A expression and acetyl-CoA levels.Representative blots for indicated proteins (a) and densitometric quantification (b) of CPT1A levels in si CTRL or si ATG5 LEC. Mean ± SD, N = 3 biological replicates analyzed using unpaired Student’s t test (two-tailed), ***p = 0.0001. c...
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Autophagy regulates CPT1A expression and acetyl-CoA levels.Representative blots for indicated proteins (a) and densitometric quantification (b) of CPT1A levels in si CTRL or si ATG5 LEC. Mean ± SD, N = 3 biological replicates analyzed using unpaired Student’s t test (two-tailed), ***p = 0.0001. c RT-qPCR analysis of si CTRL and si ATG5 LEC. mRNA expression of CPT1A (relative to HPRT). Mean ± SD, N = 3 biological replicates analyzed using one sample t test (two-tailed), *p = 0.03. d Relative Citric acid levels in si CTRL and si ATG5 LEC measured via mass spectrometry. Mean ± SD, N = 3 biological replicates analyzed using one sample t test (two-tailed), *p = 0.03. e Relative acetyl CoA/CoA ratio measured via mass spectrometry in si CTRL, si ATG5, or etomoxir (Eto, 100 µM, 48 h) treated LEC. Mean ± SD, N = 3 biological replicates analyzed using one way ANOVA with Tukey’s test for multiple comparisons, *p < 0.05 vs si CTRL. Representative blots (f) and densitometric quantification (g) for H3K9 ac levels relative to total H3 of si CTRL and si ATG5 LEC. Mean ± SD, N = 5 biological replicates analyzed using unpaired Student’s t test (two-tailed), *p = 0.02. h Representative blots for indicated proteins with GAPDH as loading control of si CTRL, si ATG5, si ATG7, or si ULK1 LEC. Densitometric quantification is indicated beneath the blots (H3K9 ac levels relative to total H3 and ATG5, ULK1, and ATG7 levels relative to GAPDH). Mean ± SD, N ≥ 3 biological replicates analyzed using one way ANOVA with Tukey’s test for multiple comparisons, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs si CTRL.
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
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In Cell Discov on 3 May 2022 by Yu, F., Zhang, Q., et al.
Fig.5.A
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O-GlcNAcylation-dependent ferritinophagy and mitophagy contribute to ferroptosis.a U2OS cells were treated with OSMI-1 for the indicated time and cell lysates were subjected to immunoblotting with indicated antibodies. b–d U2OS cells were incubated with OSMI-1 for 12 h, and treated with or withou...
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O-GlcNAcylation-dependent ferritinophagy and mitophagy contribute to ferroptosis.a U2OS cells were treated with OSMI-1 for the indicated time and cell lysates were subjected to immunoblotting with indicated antibodies. b–d U2OS cells were incubated with OSMI-1 for 12 h, and treated with or without RSL3 for 6 h. Cells were stained with MitoTracker and subjected to immunofluorescence microscopy with antibodies against TOM20 and HSP60 (b). The percentage of dead cells (c) and fragmented mitochondria (d) were quantified. e U2OS cells were transfected with control or NCOA4 siRNAs for 48 h and treated with DMSO or OSMI-1 for 12 h. Cells were subjected to immunofluorescence microscopy with antibodies against ferritin and LAMP1. f U2OS cells were transfected with control or combination of PINK1 and NCOA4 siRNAs for 48 h and treated with DMSO or OSMI-1 for 12 h, and then induced with RSL3 for 3 h. Cells were stained with PI and assessed by flow cytometry. g U2OS cells were treated as indicated for 12 h and then induced with RSL3 for 3 h. Cells were stained with Trypan blue solution and the percentages of dead cells were quantified. Scale bars, 10 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant. Error bars indicate SD.
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Collected and cropped from Cell Discov by CiteAb, provided under a CC-BY license
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In PLoS Pathog on 1 March 2022 by Glon, D., Vilmen, G., et al.
Fig.1.E
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BHRF1 induces the formation of mito-aggresomes in a MT-dependent manner.(A-C) HeLa cells were transfected for 24 h with BHRF1-HA plasmid or with EV as a control. Mitochondria were labeled with MitoTracker and cells immunostained for BHRF1. Nuclei were stained with DAPI. (A) Confocal images with i...
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BHRF1 induces the formation of mito-aggresomes in a MT-dependent manner.(A-C) HeLa cells were transfected for 24 h with BHRF1-HA plasmid or with EV as a control. Mitochondria were labeled with MitoTracker and cells immunostained for BHRF1. Nuclei were stained with DAPI. (A) Confocal images with insets (3X) on mitochondrial phenotype. Values of mitochondrial CI are indicated on representative cells. Scale bars: 10 μm and 2 μm for insets. (B) Left, assessment of mitochondrial aggregation by calculation of CI. Right, percentage of cells presenting a mito-aggresome (n = 20 cells per condition). (C) Quantification of mitochondrial fission parameters, aspect ratio (AR; left panel) and form factor (FF, right panel). (D) Confocal images of HeLa cells transfected with BHRF1-HA plasmid (or EV) and immunostained for pericentrin and BHRF1 (upper panel) or mitochondria (lower panel). Nuclei were stained with DAPI. Scale bar: 20 μm. Arrowheads show the centrosomal localization of pericentrin. (E-F) MT network depolymerization assay. (E) Representative images where BHRF1 expression was visualized by HA immunostaining (gray), the MT network by α-tubulin immunostaining (green), and mitochondria are immunostained with an anti-TOM20 antibody (red). Values of mitochondrial CI are indicated on representative cells. Scale bars: 10 μm. (F) Percentage of BHRF1-HA-positive cells presenting a mito-aggresome (n = 30 cells per condition) in cells treated with nocodazole and after MT regrowth. Data represent the mean ± SEM of three independent experiments. ** P < 0.01; *** P < 0.001 (Student’s t-test).
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Collected and cropped from PLoS Pathog by CiteAb, provided under a CC-BY license
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In Nat Commun on 2 June 2021 by Della-Flora Nunes, G., Wilson, E. R., et al.
Fig.4.I
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Mus musculus (House mouse)
Mitochondria of Phb1-SCKO are dysfunctional.a The mitochondrial DNA (mtDNA) content is decreased in Phb1-SCKO mice (red) compared to controls (blue) starting at postnatal day 40 (P40). N = 4–6 animals per genotype. Unpaired two-tailed t-test corrected for multiple comparisons using the Holm-Sidak...
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Mitochondria of Phb1-SCKO are dysfunctional.a The mitochondrial DNA (mtDNA) content is decreased in Phb1-SCKO mice (red) compared to controls (blue) starting at postnatal day 40 (P40). N = 4–6 animals per genotype. Unpaired two-tailed t-test corrected for multiple comparisons using the Holm-Sidak method [P20 (t = 1.114, df = 10, p = 0.29), P40 (t = 3.068, df = 10, p = 0.012), P90 (t = 2.174, df = 7, p = 0.066)]. b Mitochondrial dynamics is affected by deletion of Phb1 in primary mouse SCs. Top: PhAM fluorescence was photoconverted using a focal laser stimulation and dynamics of the photoconverted mitochondria (arrows) were observed for 30 min. While the PhAM (photoconverted) signal quickly dissipated in the control SCs, it remained stagnant in Phb1-SCKO SCs. Inset: position 0 represents the center of the stimulated area. Bottom: quantification of PhAM (photoconverted) signal around the stimulated area at different time points. Mean (line) and SEM (shaded area) of the signal is reported. A.u. = arbitrary units. N = 6–7 cells per genotype. c Processing of the mitochondrial fusion protein Opa1 is increased in Phb1-SCKO mice. N = 6 animals per genotype. Unpaired two-tailed t-test [P20 (t = 16.664, df = 10, p < 0.000001), P40 (t = 17.015, df = 10, p < 0.000001), P90 (t = 5.335, df = 10, p = 0.00033)]. d Ablation of Phb1 leads to reduced mitochondrial membrane potential in primary SCs of P40 PHB1-SCKO mice as compared to controls. e Quantification of (d). N = 6 wells per genotype. Unpaired two-tailed t-test (t = 4.583, df = 10, p = 0.001). Asterisk: fibroblast. f–h In primary rat SCs, Seahorse analyses indicate that Phb1 knockdown with either sh-Phb #1 (red) or sh-Phb #2 (green) does not change basal mitochondrial respiration (One-way ANOVA. F (2, 21) = 2.195, p = 0.1363) (g), but impairs the spare respiratory capacity (h). Timing of injection of oligomycin (Olig), FCCP, and rotenone (Rot) + antimycin A (AA) are indicated in (f). i Western blot illustrating the reduction in levels of PHB1 upon treatment with shRNAs (63% and 21% reduction in PHB1/β-tubulin ratio for sh-Phb #1 and sh-Phb #2, respectively). N = 8 wells per condition. One-way ANOVA. F (2,21) = 5.594, p = 0.0113; psh-Phb1#1 = 0.0068; psh-Phb1#2 = 0.0456. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. n.s. non-significant.
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
Image 1 of 42
In Sci Rep on 3 December 2020 by Bhalla, K., Jaber, S., et al.
Fig.1.C
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WB
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Homo sapiens (Human)
Regulation in SIRT3 axis in response to ATM deficiency in DLBCL. (A) Gene clustering illustrated by a heatmap of gene expression across samples (n = 3/group) indicate glutamate receptor and glutamine synthesis gene targets as top regulated pathways in shATM cell line compared to its non-target co...
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Regulation in SIRT3 axis in response to ATM deficiency in DLBCL. (A) Gene clustering illustrated by a heatmap of gene expression across samples (n = 3/group) indicate glutamate receptor and glutamine synthesis gene targets as top regulated pathways in shATM cell line compared to its non-target control (NT). Genes associated with top regulated pathways identified by IPA are indicated in heat map by red arrows. Gene expression values come from log2 raw counts generated by htseq-count. Heat map was created using the R library Complex Heatmap, which is part of Bioconductor software (3.1): http://bioconductor.org/packages/release/bioc/html/ComplexHeatmap.html. (B,C) Western blot analysis and quantitation of SIRT3 expression from fractionated cellular extracts (B) from total cell lysate (C) from mitochondrial cell lysates obtained from ABC-DLBCL cell lines HLY and SUDHL2 (L2) and GCB-DLBCL cell lines SUDHL6 (L6). Quantitation of SIRT3 protein expression is depicted below. The western analysis data and bar chart illustrating relative quantitation presented in (B) and (C) are the representative experiment of three independent experiments that were initiated from independent cell culture preparations. (D) SIRT3 expression in CRISPR-knockout (CKO) ATM DLBCL cell line, HLY. Size of mitochondrial isoform of SIRT3 (28 kDa) is depicted. The expression level of SIRT3 in CKO-ATM DLBCL cell line as determined by western blot analysis depicted here is a representative experiment of three independent experiments.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
Image 1 of 42
In Cancers (Basel) on 27 October 2020 by Valença, I., Ferreira, A. R., et al.
Fig.1.A
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Peroxisome and mitochondria protein and metabolic changes in prostate cancer (PCa). (A) Western blot analysis showing the expression levels of the peroxisomal proteins 70-kDa peroxisomal membrane protein (PMP70), acyl-CoA-binding domain-containing protein 5 (ACBD5), catalase (CAT), peroxin 5 (PEX...
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Peroxisome and mitochondria protein and metabolic changes in prostate cancer (PCa). (A) Western blot analysis showing the expression levels of the peroxisomal proteins 70-kDa peroxisomal membrane protein (PMP70), acyl-CoA-binding domain-containing protein 5 (ACBD5), catalase (CAT), peroxin 5 (PEX5), peroxin 19 (PEX19), acyl-CoA oxidase 1 (ACOX1), acyl-CoA oxidase 3 (ACOX3), 3-ketoacyl-CoA thiolase (ACAA1), sterol carrier protein x (SCPx), acyl-CoA synthetase 4 (ACSL4), 3,2-trans-enoyl-CoA isomerase (PECI), and ATP binding cassette subfamily D member 1 (ABCD1), and the mitochondrial proteins voltage-dependent anion-selective channel protein 1 (VDAC1), carnitine palmitoyl transferase 1 (CPT1), cytochrome c oxidase subunit 4 (COXIV), ATP synthase subunit alpha and beta (ATP5A, ATP5B), mitochondrial import receptor subunit (TOM20), and sterol carrier protein 2 (SCP2), representative of at least three independent experiments. Tubulin (TUB) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as loading controls. A densitometric quantification of the immunoblots is presented in Figure S1. (B) Cerotic acid (C26:0) peroxisomal β-oxidation activity in PNT1A, 22Rv1, and PC3 cells. Data represent means of three independent experiments and the bars represent standard error of the mean (SEM) of the mean. * p < 0.05 *** p < 0.001.
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Collected and cropped from Cancers (Basel) by CiteAb, provided under a CC-BY license
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In Nat Commun on 21 May 2020 by Yu, B., Ma, J., et al.
Fig.6.C
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Rescue of PGAM5−/− accelerated senescence phenotype by Drp-S637A overexpression.a Mitochondrial morphology outlined by Tom20 antibodies in WT and PGAM5−/− ARPE-19 cells overexpressing GFP or DRP1 S637A, respectively. Scale bar = 20 µm. n = 5. b Quantification of mitochondrial branch in a. n = 5, ...
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Rescue of PGAM5−/− accelerated senescence phenotype by Drp-S637A overexpression.a Mitochondrial morphology outlined by Tom20 antibodies in WT and PGAM5−/− ARPE-19 cells overexpressing GFP or DRP1 S637A, respectively. Scale bar = 20 µm. n = 5. b Quantification of mitochondrial branch in a. n = 5, ***p = 0.0007, ****p < 0.0001, n.s. no significant. two-way ANOVA Tukey’s multiple comparisons test. Error bars, mean ± s.d. c Upregulation of mitochondrial proteins (Tom20, CYTC, CYPD) were rescued by Drp1 S637A overexpression in PGAM5−/− ARPE-19 cells. α-Tubulin was used as loading control. n = 4. d Expression of phosphor-AMPK, phosphor-TSC2, total-TSC2, phosphor-S6, total S6 and Drp1 in WT and PGAM5−/− ARPE-19 cells transduced with GFP or Drp1 S637A expressing lentivirus. β-Actin was used as loading control. n = 4. e ATP level in short-term (1 week) culture of WT and PGAM5−/− ARPE-19 cells transduced with GFP or Drp1 S637A expressing lentivirus. n = 9 biologically independent samples. ***p = 0.0005, ****p < 0.0001, two-way ANOVA Tukey’s multiple comparisons test. Error bars, mean ± s.d. f ATP level in long-term (8 week) culture of WT and PGAM5−/− ARPE-19 cells transduced with GFP or Drp1 S637A expressing lentivirus. n = 9 biologically independent samples. ****p < 0.0001, two-way ANOVA Tukey’s multiple comparisons test. Error bars, mean ± s.d. g Partial rescue of senescence phenotype in PGAM5−/− ARPE-19 cells by Drp1 S637A overexpression as shown by SA-β-Gal assay. Scale bar = 100 µm. n = 4. h Quantification β-gal-positive cells in g. n = 4. ****p < 0.0001, two-way ANOVA Tukey’s multiple comparisons test. Error bars, mean ± s.d. For assays in the figure, n represents the number of biologically independent experiments unless otherwise specified. Images were captured under same settings, and representative images were shown. Source data are available as a Source Data file.
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
Image 1 of 42
In Sci Rep on 6 November 2019 by Santos, M., Morais, S., et al.
Fig.5.B
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Homo sapiens (Human)
Only WT parkin is able to redistribute to depolarized mitochondria. (A) Immunofluorescence localization of EGFP-tagged parkin clones in HEK293T cells treated with FCCP or control vehicle DMSO. EGFP was directly visualized. TOM20 labelled mitochondria. Photographs were acquired using a Zeiss Axio ...
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Only WT parkin is able to redistribute to depolarized mitochondria. (A) Immunofluorescence localization of EGFP-tagged parkin clones in HEK293T cells treated with FCCP or control vehicle DMSO. EGFP was directly visualized. TOM20 labelled mitochondria. Photographs were acquired using a Zeiss Axio Imager Z1 microscope. Bars, 10 μm. (B) Mitochondria isolation from HEK293T cells expressing parkin clones. Presence of parkin in cytosol (C) or mitochondria (Mt) fractions was analyzed by immunoblotting with anti-EGFP antibody and normalized to caspase 3 or TOM20, respectively. Original blots are presented in Supplementary Fig. S6. Quantification data (graph) are presented as the mean ± SD of three independent experiments; *p < 0.05, compared with control DMSO (two-tailed Student’s t-test).
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
Image 1 of 42
In Cell Death Differ on 1 May 2018 by Goiran, T., Duplan, E., et al.
Fig.6.A
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Homo sapiens (Human)
TP53 knockout triggers a pro-mitophagic response.PINK1 (a,b, N = 8), LC3-2/LC3-1 (a,c, N = 8), p62 (a,d, N = 12), TIM23 (a,e, N = 10), TOM20 (a,f, N = 9), HSP60 (a,g, N = 16), optineurin (a,h, N = 9) and NDP52 (a,i, N = 8) protein levels were analyzed in control (p53+/+) or TP53-deficient (p53−/−...
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TP53 knockout triggers a pro-mitophagic response.PINK1 (a,b, N = 8), LC3-2/LC3-1 (a,c, N = 8), p62 (a,d, N = 12), TIM23 (a,e, N = 10), TOM20 (a,f, N = 9), HSP60 (a,g, N = 16), optineurin (a,h, N = 9) and NDP52 (a,i, N = 8) protein levels were analyzed in control (p53+/+) or TP53-deficient (p53−/−) HAP1 cells as described in the Methods. Bars represent the means ± SEM of 3-4 independent experiments performed in triplicate and are expressed as percent of control (p53+/+) cells. Actin expression is provided as a representative gel loading control in a. Statistical analyses were performed with GraphPad Prism software by using unpaired Student’s t-test. Significant differences are: ** p < 0.01, ***p < 0.001 and ****p < 0.0001.
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Collected and cropped from Cell Death Differ by CiteAb, provided under a CC-BY license
Image 1 of 42
In Cell Death Differ on 1 May 2018 by Goiran, T., Duplan, E., et al.
Fig.8.A
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p53 repression of autophagy is PINK1-dependent.LC3-2/LC3-1 (a,b, N = 12), p62 (a,c, N = 12), TIM23 (a,d, N = 12), TOM20 (a,e, N = 12) and HSP60 (a,f, N = 12) protein levels were analyzed in control (PINK+/+) or Pink1-deficient (PINK−/−) HAP cells treated with vehicle (Ct), pifithrin-α (PFT, 30 µM...
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p53 repression of autophagy is PINK1-dependent.LC3-2/LC3-1 (a,b, N = 12), p62 (a,c, N = 12), TIM23 (a,d, N = 12), TOM20 (a,e, N = 12) and HSP60 (a,f, N = 12) protein levels were analyzed in control (PINK+/+) or Pink1-deficient (PINK−/−) HAP cells treated with vehicle (Ct), pifithrin-α (PFT, 30 µM, 4 h) or CCCP (CP, 10 µM, 6 h) as described in the Methods. Bars represent the means ± SEM of 4 independent experiments performed in triplicates and are expressed as percent of control HAP1 (HAP+/+, Ct) cells. Actin expression is provided as a representative gel loading control in a. Statistical analyses were performed with GraphPad Prism software by using unpaired Student’s t-test. Significant differences are: ns not significant, *p < 0.05, ** p < 0.01 and ***p < 0.001.
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Collected and cropped from Cell Death Differ by CiteAb, provided under a CC-BY license
Image 1 of 42
In EMBO J on 17 January 2018 by Jimenez-Orgaz, A., Kvainickas, A., et al.
Fig.1.H
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IF
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Homo sapiens (Human)
RAB7 localizes to mitochondria and the ERAll images show formaldehyde‐fixed HeLa cells.HeLa cells stained for endogenous RAB7a (green) and endogenous LAMP2 (red). White arrows indicate sites of vesicular RAB7 that appears to be budding from a network of RAB7.Immunofluorescent co‐staining of endog...
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RAB7 localizes to mitochondria and the ERAll images show formaldehyde‐fixed HeLa cells.HeLa cells stained for endogenous RAB7a (green) and endogenous LAMP2 (red). White arrows indicate sites of vesicular RAB7 that appears to be budding from a network of RAB7.Immunofluorescent co‐staining of endogenous RAB7a (green) and the mitochondria marker TOM20 (red).Co‐staining of endogenous RAB7 (green) and the trans‐Golgi network marker TGN46 (red).Co‐staining of endogenous RAB7 (green) and the ER marker protein disulfide isomerase (PDI, red).Co‐staining of lentivirally expressed GFP‐RAB7a and endogenous TOM20 (red).Co‐staining of endogenous RAB7 (green) and TOM20 (red) in cells treated with Cas9 and a mixture of three gRNAs targeting the RAB7a locus. The Western blot of this cell population shows an almost complete loss of RAB7 in the treated cells compared to parental HeLa cells.GFP‐RAB7 was expressed in RAB7a KO HeLa cells and imaged in live cells using a spinning disk confocal microscope. Mitochondria were visualized with MitoTracker Red.Parental HeLa cells and clonal VPS29 KO cells were co‐stained for endogenous RAB7a (green) and endogenous TOM20 (red), and co‐localization was analyzed across two independent experiments.Data information: All scale bars = 10 μm, all error bars = SD, and *P < 0.05 in a t‐test of the respective condition compared to the control cells.Source data are available online for this figure.
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Collected and cropped from EMBO J by CiteAb, provided under a CC-BY license
Image 1 of 42
In EMBO J on 17 January 2018 by Jimenez-Orgaz, A., Kvainickas, A., et al.
Fig.1.C
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IF
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Homo sapiens (Human)
Loss of retromer leads to a pronounced shift in RAB7 distributionParental HeLa cells, two clonal VPS35 knockout cell lines, and one clonal VPS29 KO cell line were fixed in PFA and co‐stained for endogenous RAB7a (green) and endogenous LAMP2 (red). Co‐localization was analyzed across three indepen...
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Loss of retromer leads to a pronounced shift in RAB7 distributionParental HeLa cells, two clonal VPS35 knockout cell lines, and one clonal VPS29 KO cell line were fixed in PFA and co‐stained for endogenous RAB7a (green) and endogenous LAMP2 (red). Co‐localization was analyzed across three independent experiments.A clonal RAB7a knockout cell line was mixed 1:1 with parental HeLa cells and seeded onto coverslips. Following PFA fixation, the mixed cells were stained for endogenous RAB7a (green) and endogenous LAMP2 (red). Note that the RAB7a signal completely disappears in the cells not expressing RAB7a.Parental HeLa cells and clonal VPS35 KO cells were co‐stained for endogenous RAB7a (green) and endogenous TOM20 (red, upper panel) or for endogenous RAB7a and a mCherry‐tagged ER marker (red, lower panel), and co‐localization between RAB7a and the respective marker was analyzed across two independent experiments. To show that RAB7a localizes to the ER and mitochondria, endogenous TOM20 (blue) was co‐stained in the lower panel.Parental HeLa cells and clonal VPS35 and VPS29 KO cells were transduced with a lentivirus expressing GFP‐FIS1TM (eGFP with a C‐terminal mitochondrial targeting sequence and transmembrane domain of the mitochondrial protein FIS1) and disrupted through a fine needle in detergent‐free sucrose buffer followed by isolation of the mitochondria from postnuclear supernatants with GFP‐trap agarose beads. The amount of RAB7 precipitating with the mitochondria was quantified over four independent experiments.Data information: All scale bars = 10 μm, all error bars = SD, and *P < 0.05 in a t‐test of the respective condition compared to the control cells. Source data are available online for this figure.
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Collected and cropped from EMBO J by CiteAb, provided under a CC-BY license
Image 1 of 42
In EMBO J on 17 January 2018 by Jimenez-Orgaz, A., Kvainickas, A., et al.
Fig.1.E
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IF
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Homo sapiens (Human)
RAB7 localizes to mitochondria and the ERAll images show formaldehyde‐fixed HeLa cells.HeLa cells stained for endogenous RAB7a (green) and endogenous LAMP2 (red). White arrows indicate sites of vesicular RAB7 that appears to be budding from a network of RAB7.Immunofluorescent co‐staining of endog...
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RAB7 localizes to mitochondria and the ERAll images show formaldehyde‐fixed HeLa cells.HeLa cells stained for endogenous RAB7a (green) and endogenous LAMP2 (red). White arrows indicate sites of vesicular RAB7 that appears to be budding from a network of RAB7.Immunofluorescent co‐staining of endogenous RAB7a (green) and the mitochondria marker TOM20 (red).Co‐staining of endogenous RAB7 (green) and the trans‐Golgi network marker TGN46 (red).Co‐staining of endogenous RAB7 (green) and the ER marker protein disulfide isomerase (PDI, red).Co‐staining of lentivirally expressed GFP‐RAB7a and endogenous TOM20 (red).Co‐staining of endogenous RAB7 (green) and TOM20 (red) in cells treated with Cas9 and a mixture of three gRNAs targeting the RAB7a locus. The Western blot of this cell population shows an almost complete loss of RAB7 in the treated cells compared to parental HeLa cells.GFP‐RAB7 was expressed in RAB7a KO HeLa cells and imaged in live cells using a spinning disk confocal microscope. Mitochondria were visualized with MitoTracker Red.Parental HeLa cells and clonal VPS29 KO cells were co‐stained for endogenous RAB7a (green) and endogenous TOM20 (red), and co‐localization was analyzed across two independent experiments.Data information: All scale bars = 10 μm, all error bars = SD, and *P < 0.05 in a t‐test of the respective condition compared to the control cells.Source data are available online for this figure.
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Collected and cropped from EMBO J by CiteAb, provided under a CC-BY license
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In EMBO J on 17 January 2018 by Jimenez-Orgaz, A., Kvainickas, A., et al.
Fig.8.B
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IF
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Homo sapiens (Human)
Control of RAB7 activity is required for efficient removal of mitochondria through mitophagyAll image panels show PFA‐fixed HeLa cells. mCherry‐Parkin‐transduced parental HeLa cells, VPS35 KO cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs cells wer...
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Control of RAB7 activity is required for efficient removal of mitochondria through mitophagyAll image panels show PFA‐fixed HeLa cells. mCherry‐Parkin‐transduced parental HeLa cells, VPS35 KO cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs cells were incubated with CCCP over 4 h, followed by staining of endogenous RAB7a (green) and TOM20 (blue). Co‐localization between RAB7 and TOM20 as well as RAB7 and mCherry‐Parkin was quantified over two independent experiments.RAB7a KO cells transduced with mCherry‐Parkin and GFP‐RAB7 or GFP‐RAB7‐Q67L were treated with CCCP for 4 h, followed by co‐staining with endogenous TOM20 (blue). Co‐localization between the GFP‐RAB7 variants and TOM20 was quantified over two independent experiments.Parental HeLa cells, RAB7 KO cells, and RAB7 KO cells transduced with the indicated GFP‐RAB7 rescue constructs were treated with CCCP for 16 h, followed by staining of endogenous TOM20 (blue). The residual TOM20 signal after CCCP treatment was quantified over two independent experiments.Data information: All scale bars = 10 μm, all error bars = SD, and *P < 0.05 in a t‐test of the respective condition compared to the control cells.
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Collected and cropped from EMBO J by CiteAb, provided under a CC-BY license
Image 1 of 42
In EMBO J on 17 January 2018 by Jimenez-Orgaz, A., Kvainickas, A., et al.
Fig.8.A
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IF
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Homo sapiens (Human)
Control of RAB7 activity is required for efficient removal of mitochondria through mitophagyAll image panels show PFA‐fixed HeLa cells. mCherry‐Parkin‐transduced parental HeLa cells, VPS35 KO cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs cells wer...
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Control of RAB7 activity is required for efficient removal of mitochondria through mitophagyAll image panels show PFA‐fixed HeLa cells. mCherry‐Parkin‐transduced parental HeLa cells, VPS35 KO cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs cells were incubated with CCCP over 4 h, followed by staining of endogenous RAB7a (green) and TOM20 (blue). Co‐localization between RAB7 and TOM20 as well as RAB7 and mCherry‐Parkin was quantified over two independent experiments.RAB7a KO cells transduced with mCherry‐Parkin and GFP‐RAB7 or GFP‐RAB7‐Q67L were treated with CCCP for 4 h, followed by co‐staining with endogenous TOM20 (blue). Co‐localization between the GFP‐RAB7 variants and TOM20 was quantified over two independent experiments.Parental HeLa cells, RAB7 KO cells, and RAB7 KO cells transduced with the indicated GFP‐RAB7 rescue constructs were treated with CCCP for 16 h, followed by staining of endogenous TOM20 (blue). The residual TOM20 signal after CCCP treatment was quantified over two independent experiments.Data information: All scale bars = 10 μm, all error bars = SD, and *P < 0.05 in a t‐test of the respective condition compared to the control cells.
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Collected and cropped from EMBO J by CiteAb, provided under a CC-BY license
Image 1 of 42
In EMBO J on 17 January 2018 by Jimenez-Orgaz, A., Kvainickas, A., et al.
Fig.9.C
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IF
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Homo sapiens (Human)
Hyperactive RAB7 results in ATG9a trafficking and autophagosome formation defectsmCherry‐Parkin‐expressing parental HeLa cells, VPS35 KO cells, and RAB7 KO cells transduced with untagged RAB7‐Q67L were treated with CCCP for 5 h, fixed in PFA, and co‐stained for endogenous ATG9a (green) and the TG...
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Hyperactive RAB7 results in ATG9a trafficking and autophagosome formation defectsmCherry‐Parkin‐expressing parental HeLa cells, VPS35 KO cells, and RAB7 KO cells transduced with untagged RAB7‐Q67L were treated with CCCP for 5 h, fixed in PFA, and co‐stained for endogenous ATG9a (green) and the TGN marker TGN46 (blue). Co‐localization between ATG9a and TGN46 was analyzed across 14 images acquired in two independent experiments.Parental HeLa cells and VPS35 KO cells were transduced with GFP‐RAB7 and mCherry‐Parkin and treated with CCCP for 5 h. The cells were then fixed in PFA and stained for endogenous ATG9a (blue). Co‐localization between GFP‐RAB7 and ATG9a was quantified across 14 images acquired in two independent experiments.mCherry‐Parkin‐expressing parental HeLa cells and RAB7 KO cells transduced with untagged RAB7‐Q67L were treated with CCCP for 5 h (upper panel) and 8 h (lower panel), fixed in methanol, and stained for endogenous TOM20 (blue) and endogenous LC3b (green). Co‐localization between LC3b and TOM20 was quantified across 14 images acquired in two independent experiments.Data information: All scale bars = 10 μm, all error bars = SD, and *P < 0.05 in a t‐test of the respective condition compared to the control cells.
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Collected and cropped from EMBO J by CiteAb, provided under a CC-BY license
Image 1 of 42
In EMBO J on 17 January 2018 by Jimenez-Orgaz, A., Kvainickas, A., et al.
Fig.7.D
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IF
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Homo sapiens (Human)
Autophagosome formation and ATG9a trafficking are impaired upon loss of retromerParental HeLa cells and VPS35 KO cells were transduced with mCherry‐Parkin and GFP‐LC3b and incubated with CCCP for 2 and 4 h. Co‐localization of GFP‐LC3b and endogenous TOM20 (blue) was quantified over two independen...
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Autophagosome formation and ATG9a trafficking are impaired upon loss of retromerParental HeLa cells and VPS35 KO cells were transduced with mCherry‐Parkin and GFP‐LC3b and incubated with CCCP for 2 and 4 h. Co‐localization of GFP‐LC3b and endogenous TOM20 (blue) was quantified over two independent experiments.Parental HeLa cells and VPS29 and VPS35 KO cells transduced with mCherry‐Parkin were treated with CCCP for the indicated time points, followed by staining of endogenous ATG16L1 (green) and endogenous TOM20 (blue).HeLa cells were transfected with humanized Cas9 and a mix of three distinct gRNAs targeting the ATG9a gene. Note that the vesicular ATG9a antibody signal (green) completely disappears in the CRISPR‐treated cells, indicating a high degree of specificity.Parental HeLa cells and VPS29 and VPS35 KO cells transduced with mCherry‐Parkin were treated with CCCP for the indicated time points, followed by staining of endogenous ATG9a (green) and endogenous TOM20 (blue).Data information: All scale bars = 10 μm, all error bars = SD, and *P < 0.05 in a t‐test of the respective condition compared to the control cells.
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Collected and cropped from EMBO J by CiteAb, provided under a CC-BY license
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In EMBO J on 17 January 2018 by Jimenez-Orgaz, A., Kvainickas, A., et al.
Fig.6.B
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Mitophagy is defective upon loss of retromermCherry‐Parkin‐transduced parental HeLa cells, VPS35 KO cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs were treated with CCCP over 16 h, followed by PFA fixation and staining of endogenous TOM20 (green). ...
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Mitophagy is defective upon loss of retromermCherry‐Parkin‐transduced parental HeLa cells, VPS35 KO cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs were treated with CCCP over 16 h, followed by PFA fixation and staining of endogenous TOM20 (green). Residual TOM20 after CCCP treatment was quantified over two independent experiments.The cells described for panel (A) were lysed after 16‐h CCCP treatment, and residual TOM20 was detected by Western blotting (left) and quantified over three independent experiments (right).SHSY‐5Y cells that had been transduced with a construct expressing tandem mCherry‐eGFP‐FIS1TM (mitochondria‐anchored mCherry‐GFP tandem) were transfected with control and VPS35‐specific siRNA and treated with the iron chelator deferiprone for 24 h. The cells were then assessed for red‐shifted dots representing mitophagy events by confocal microscopy. Efficiency of the siRNA treatment was confirmed by Western blotting against endogenous VPS35. Mitophagy events were counted in 20 images per condition, acquired in two independent experiments.Parental HeLa cells and VPS29 and VPS35 KO cells were transduced with mCherry–Parkin, and mitophagy was induced for the indicated time points with the proton uncoupler CCCP. The cells were then fixed in methanol and co‐stained for endogenous LC3b (green) and TOM20 (blue). (D′) Co‐localization between LC3b and TOM20 was analyzed over 12 images per condition acquired in two independent experiments.Data information: All scale bars = 10 μm, all error bars = SD, and *P < 0.05 in a t‐test of the respective condition compared to the control cells.Source data are available online for this figure.
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Collected and cropped from EMBO J by CiteAb, provided under a CC-BY license
Image 1 of 42
In EMBO J on 17 January 2018 by Jimenez-Orgaz, A., Kvainickas, A., et al.
Fig.2.D
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IF
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Homo sapiens (Human)
RAB7 activity state controls its localization to the ER, mitochondria, and lysosomesAMitochondria were purified from detergent‐free postnuclear supernatants using magnetic beads coated with a TOM22‐specific antibody from parental HeLa cells and from clonal VPS35 KO cells. The purified mitochondri...
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RAB7 activity state controls its localization to the ER, mitochondria, and lysosomesAMitochondria were purified from detergent‐free postnuclear supernatants using magnetic beads coated with a TOM22‐specific antibody from parental HeLa cells and from clonal VPS35 KO cells. The purified mitochondria were then subjected to a marker analysis using the indicated organelle markers. Note that only mitochondria (TOM20) and RAB7 are enriched over the inputs, with lower levels of RAB7 precipitating with the mitochondria of VPS35 KO cells. The quantification of RAB7 was done across three independent mitochondria isolations.BRAB7a KO cells transduced with the indicated GFP‐RAB7a variants were fixed and co‐stained for endogenous LAMP2. Note that the inactive RAB7 (T22N) does not localize to lysosomes, whereas the hyperactive variant (Q67L) fully localizes to lysosomes.C, DRAB7a KO cells transduced with the indicated GFP‐RAB7a variants and a mCherry‐ER marker were fixed and co‐stained for endogenous TOM20.ERAB7a KO cells were infected with the indicated GFP‐RAB7 variants, labeled with MitoTracker Red, and subjected to live cell imaging using a spinning disk confocal microscope.Data information: All scale bars = 10 μm, and all error bars = SD. Source data are available online for this figure.
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Collected and cropped from EMBO J by CiteAb, provided under a CC-BY license
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In EMBO J on 17 January 2018 by Jimenez-Orgaz, A., Kvainickas, A., et al.
Fig.2.C
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IF
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Homo sapiens (Human)
RAB7 activity state controls its localization to the ER, mitochondria, and lysosomesAMitochondria were purified from detergent‐free postnuclear supernatants using magnetic beads coated with a TOM22‐specific antibody from parental HeLa cells and from clonal VPS35 KO cells. The purified mitochondri...
more
RAB7 activity state controls its localization to the ER, mitochondria, and lysosomesAMitochondria were purified from detergent‐free postnuclear supernatants using magnetic beads coated with a TOM22‐specific antibody from parental HeLa cells and from clonal VPS35 KO cells. The purified mitochondria were then subjected to a marker analysis using the indicated organelle markers. Note that only mitochondria (TOM20) and RAB7 are enriched over the inputs, with lower levels of RAB7 precipitating with the mitochondria of VPS35 KO cells. The quantification of RAB7 was done across three independent mitochondria isolations.BRAB7a KO cells transduced with the indicated GFP‐RAB7a variants were fixed and co‐stained for endogenous LAMP2. Note that the inactive RAB7 (T22N) does not localize to lysosomes, whereas the hyperactive variant (Q67L) fully localizes to lysosomes.C, DRAB7a KO cells transduced with the indicated GFP‐RAB7a variants and a mCherry‐ER marker were fixed and co‐stained for endogenous TOM20.ERAB7a KO cells were infected with the indicated GFP‐RAB7 variants, labeled with MitoTracker Red, and subjected to live cell imaging using a spinning disk confocal microscope.Data information: All scale bars = 10 μm, and all error bars = SD. Source data are available online for this figure.
less
Collected and cropped from EMBO J by CiteAb, provided under a CC-BY license
Image 1 of 42