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: 161
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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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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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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PPTC7 antagonizes mitophagy by promoting BNIP3 and NIX degradation via SCFFBXL4.
In EMBO Reports on 1 August 2024 by Nguyen, G. D. T., Townsend, B., et al.
Mitophagy must be carefully regulated to ensure that cells maintain appropriate numbers of functional mitochondria. The SCFFBXL4 ubiquitin ligase complex suppresses mitophagy by controlling the degradation of BNIP3 and NIX mitophagy receptors, and FBXL4 mutations result in mitochondrial disease as a consequence of elevated mitophagy. Here, we reveal that the mitochondrial phosphatase PPTC7 is an essential cofactor for SCFFBXL4-mediated destruction of BNIP3 and NIX, suppressing both steady-state and induced mitophagy. Disruption of the phosphatase activity of PPTC7 does not influence BNIP3 and NIX turnover. Rather, a pool of PPTC7 on the mitochondrial outer membrane acts as an adaptor linking BNIP3 and NIX to FBXL4, facilitating the turnover of these mitophagy receptors. PPTC7 accumulates on the outer mitochondrial membrane in response to mitophagy induction or the absence of FBXL4, suggesting a homoeostatic feedback mechanism that attenuates high levels of mitophagy. We mapped critical residues required for PPTC7-BNIP3/NIX and PPTC7-FBXL4 interactions and their disruption interferes with both BNIP3/NIX degradation and mitophagy suppression. Collectively, these findings delineate a complex regulatory mechanism that restricts BNIP3/NIX-induced mitophagy.
© 2024. The Author(s).
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In Acta Neuropathologica Communications on 8 June 2024 by Eysert, F., Kinoshita, P. F., et al.
Mitochondrial dysfunctions are key features of Alzheimer's disease (AD). The occurrence of these disturbances in the peripheral cells of AD patients and their potential correlation with disease progression are underinvestigated. We studied mitochondrial structure, function and mitophagy in fibroblasts from healthy volunteers and AD patients at the prodromal (AD-MCI) or demented (AD-D) stages. We carried out correlation studies with clinical cognitive scores, namely, (i) Mini-Mental State Examination (MMSE) and (ii) Dementia Rating-Scale Sum of Boxes (CDR-SOB), and with (iii) amyloid beta (Aβ) plaque burden (PiB-PET imaging) and (iv) the accumulation of peripheral amyloid precursor protein C-terminal fragments (APP-CTFs). We revealed alterations in mitochondrial structure as well as specific mitochondrial dysfunction signatures in AD-MCI and AD-D fibroblasts and revealed that defective mitophagy and autophagy are linked to impaired lysosomal activity in AD-D fibroblasts. We reported significant correlations of a subset of these dysfunctions with cognitive decline, AD-related clinical hallmarks and peripheral APP-CTFs accumulation. This study emphasizes the potential use of peripheral cells for investigating AD pathophysiology.
© 2024. The Author(s).
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Cell Biology
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Neuroscience
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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.
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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.
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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.
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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.
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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 35
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 35
In Cell Death Differ on 1 May 2018 by Goiran, T., Duplan, E., et al.
Fig.6.A
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WB
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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 35
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 35
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 35
In EMBO J on 17 January 2018 by Jimenez-Orgaz, A., Kvainickas, A., et al.
Fig.1.B
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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 35
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 35
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 35
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 35
In EMBO J on 17 January 2018 by Jimenez-Orgaz, A., Kvainickas, A., et al.
Fig.6.D
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IF
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Homo sapiens (Human)
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 35
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
Image 1 of 35
In EMBO J on 17 January 2018 by Jimenez-Orgaz, A., Kvainickas, A., et al.
Fig.7.B
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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
Image 1 of 35
In EMBO J on 17 January 2018 by Jimenez-Orgaz, A., Kvainickas, A., et al.
Fig.7.A
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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
Image 1 of 35
In EMBO J on 17 January 2018 by Jimenez-Orgaz, A., Kvainickas, A., et al.
Fig.6.A
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IF
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Homo sapiens (Human)
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 35
In EMBO J on 17 January 2018 by Jimenez-Orgaz, A., Kvainickas, A., et al.
Fig.2.A
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IF
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Homo sapiens (Human)
Retromer controls RAB7 activity levels and mobility/membrane turnoverGDP‐locked (inactive) GFP‐RAB7‐T22N and GTP‐locked (constitutively active) GFP‐RAB7‐Q67L were lentivirally expressed in RAB7 KO cells and co‐stained with the mitochondrial marker TOM20 (red). Co‐localization was analyzed over tw...
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Retromer controls RAB7 activity levels and mobility/membrane turnoverGDP‐locked (inactive) GFP‐RAB7‐T22N and GTP‐locked (constitutively active) GFP‐RAB7‐Q67L were lentivirally expressed in RAB7 KO cells and co‐stained with the mitochondrial marker TOM20 (red). Co‐localization was analyzed over two independent experiments with 10 images each.Lysates from parental HeLa cells and clonal VPS35 KO cells were probed with immobilized GST‐RILP protein, and GST‐RILP‐bound (active) RAB7a was detected and quantified by fluorescent Western blotting across three independent experiments.Lysates from parental HeLa cells and clonal VPS29 KO cells were probed with immobilized GST‐RILP protein, and GST‐RILP‐bound (active) RAB7a was detected and quantified by fluorescent Western blotting across three independent experiments.Lysates from parental HeLa cells and clonal VPS29 KO cells and VPS29 KO cells with lentivirally re‐expressed VPS29‐myc were probed with immobilized GST‐RILP protein, and GST‐RILP‐bound (active) RAB7a was detected and quantified by fluorescent Western blotting across three independent experiments.Lentivirally expressed GFP‐RAB7 was precipitated from parental and from VPS35 KO cells, and the precipitates were analyzed for the presence of the endogenous RAB‐chaperone GDI2.Lentivirally expressed GFP‐GDI1 was precipitated from parental and from VPS35 KO cells, and the precipitates were analyzed for the presence of endogenous RAB14 and endogenous RAB7a.GFP‐RAB7 was transduced into parental HeLa cells and VPS35 KO cells and analyzed for its mobility/membrane turnover using FRAP imaging in live cells. The recovery kinetics were obtained by averaging 15 FRAP recoveries acquired in two independent experiments.GFP‐RAB7 was transduced into parental HeLa cells and VPS35 KO cells and analyzed for its mobility/membrane turnover using FLIP imaging in live cells. The depletion kinetics (in area A, as indicated) were obtained by averaging 18 FLIP depletions 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 35
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.
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Collected and cropped from EMBO J by CiteAb, provided under a CC-BY license
Image 1 of 35
In EMBO J on 17 January 2018 by Jimenez-Orgaz, A., Kvainickas, A., et al.
Fig.2.A
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WB
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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 35