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Cited by 28Autophagy in Disease and TherapyCalcium signaling and nucleotide metabolismCellular transport and secretion

The DOI matches its Crossref record; 46 of the 46 references it cites were found in a registry or an index; 34 of the 39 sources its claims cite were checked for support.

TMEM55B links autophagy flux, lysosomal repair, and TFE3 activation in response to oxidative stress

Open access
Jeong, Eutteum, Willett, Rose, Rissone, Alberto, La Spina, Martina, Puertollano, Rosa·2024·Nature Communications
DOI 10.1038/s41467-023-44316-6PMC PMC10761734
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Abstract

Lysosomes have emerged as critical regulators of cellular homeostasis. Here we show that the lysosomal protein TMEM55B contributes to restore cellular homeostasis in response to oxidative stress by three different mechanisms: (1) TMEM55B mediates NEDD4-dependent PLEKHM1 ubiquitination, causing PLEKHM1 proteasomal degradation and halting autophagosome/lysosome fusion; (2) TMEM55B promotes recruitment of components of the ESCRT machinery to lysosomal membranes to stimulate lysosomal repair; and (3) TMEM55B sequesters the FLCN/FNIP complex to facilitate translocation of the transcription factor TFE3 to the nucleus, allowing expression of transcriptional programs that enable cellular adaptation to stress. Knockout of tmem55 genes in zebrafish embryos increases their susceptibility to oxidative stress, causing early death of tmem55-KO animals in response to arsenite toxicity. Altogether, our work identifies a role for TMEM55B as a molecular sensor that coordinates autophagosome degradation, lysosomal repair, and activation of stress responses.

Overview

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Written from the paper’s full text.

TL;DR

TMEM55B acts as a lysosomal sensor that, under acute oxidative stress, coordinates three responses: halting autophagosome–lysosome fusion via NEDD4-dependent PLEKHM1 degradation, promoting ESCRT-mediated lysosomal repair, and activating TFE3 by sequestering FLCN/FNIP. Zebrafish tmem55-knockout embryos show increased susceptibility to arsenite, supporting an in vivo protective role.

Key findings

  • Under NaAsO2 (300 μM, 2 h), TMEM55B recruits NEDD4-like E3 ligases via a PPXY motif; TMEM55B is ubiquitinated at seven CD-domain sites (K96, K103, K114, K120, K121, K134, K148), and phosphorylation at S162 increases from 6.98% to almost 30%.
  • TMEM55B binds PLEKHM1 via its PH1 domain under oxidative stress, causing NEDD4-dependent PLEKHM1 ubiquitination and proteasomal degradation (blocked by MG132, not bafilomycin A1), which reduces autophagosome/lysosome fusion.
  • TMEM55B promotes ESCRT recruitment to lysosomes through both PPXY and P(S/T)AP motifs; TMEM55B-KO cells show higher galectin3 accumulation after 6 h NaAsO2 and significantly more necrosis after 10 h NaAsO2.
  • TMEM55B sequesters FLCN/FNIP under NaAsO2, promoting TFE3 nuclear translocation and TFE3-S321 dephosphorylation; tmem55-KO zebrafish embryos have lower survival under continuous 2 mM NaAsO2 (logrank test, P < 0.0001).

Methods

  • U2OS and HeLa cells with CRISPR/Cas9 TMEM55B knockout, siRNA depletion, plasmid/adenovirus expression, drug treatments (including NaAsO2 300 μM), immunoprecipitation, immunoblotting, immunofluorescence, flow cytometry, and mass spectrometry.
  • Zebrafish: multiplex CRISPR/Cas9 targeting tmem55ba and tmem55bb, plus gRNAs against tmem55a; embryos treated with 2 mM NaAsO2 from 1 dpf, with survival analyzed by Kaplan-Meier curves.
  • Statistics: Student’s t test, one-way or two-way ANOVA, and logrank tests; data presented as mean ± SEM, with experiments performed at least three times independently unless otherwise noted.

Limitations

  • Stated in the paper: no statistical method was used to predetermine sample size; no data were excluded; experiments were not randomized; investigators were not blinded to allocation during experiments and outcome assessment.
  • Evident from the text: mechanistic dissection relies mainly on U2OS/HeLa cell lines and zebrafish, with no mammalian in vivo model because TMEM55B depletion in mice was previously shown to be embryonic lethal.
  • Evident from the text: several key interactions were specific to NaAsO2 or high-ROS inducers, so the generality of the proposed TMEM55B stress-response axis across other oxidative stresses remains to be established.

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Crossref · 10.1038/s41467-023-44316-6

Cites

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Figures

Fig. 1 TMEM55B interacts with members of the NEDD4-like family of E3 ligases. a Schematic of the predicted TMEM55B membrane topology. Illustration created with BioRender.com. b Multi-sequence alignment of TMEM55B orthologs in different species. The PPXY motif is indicated in red. c U2OS cells were transfected with plasmids encoding TMEM55B-GFP-WT full length (FL), TMEM55B-GFP-WT cytosolic domain (CD), TMEM55B-GFP-P66A FL, or P66A CD. Cells were lysed and pulled down with GFP beads. The results are representative of three independent experiments. WT (Wild-type), P66A (P66A mutant). d U2OS TMEM55B KO cells stably expressing mCherry-NEDD4 (red) were transfected with plasmids encoding GFP, TMEM55B-GFP-WT or P66A (green). Cells were fixed and permeabilized for immunofluorescence. Scale bars, 20 μm, n = 3. e U2OS cells were infected with adenovirus expressing TMEM55B-GFP-WT or P66A and pulled down with GFP beads. The results are representative of three independent experiments. f U2OS cells infected with adenovirus expressing TMEM55B-GFP-WT were treated with various drugs and pulled down with GFP beads. EBSS for 4 h, NaAsO2 (300 μM) for 2 h, H2O2 (500 μM) for 4 h, LLOMe (1 mM) for 2 h, CCCP (25 μM) for 4 h, Tunicamycin (10 μg/ml) for 4 h, Thapsigargin (10 μM) for 4 h. The results are representative of three independent experiments. g U2OS cells infected with adenovirus expressing TMEM55B-GFP-WT or P66A (green) were treated with or without NaAsO2 (300 μM) for 2 h. Cells were fixed and immunostained with antibodies against ubiquitin (red) and LAMTOR1 (blue). Scale bars, 20 μm, n = 3. h U2OS cells were transfected with plasmids encoding TMEM55B-GFP-WT or P66A and treated with or without NaAsO2 (300 μM) for 2 h. Cells were lysed and immunoprecipitated with GFP beads under denaturing condition. The results are representative of three independent experiments. Source data are provided as a Source Data file.
Fig. 1 TMEM55B interacts with members of the NEDD4-like family of E3 ligases. a Schematic of the predicted TMEM55B membrane topology. Illustration created with BioRender.com. b Multi-sequence alignment of TMEM55B orthologs in different species. The PPXY motif is indicated in red. c U2OS cells were transfected with plasmids encoding TMEM55B-GFP-WT full length (FL), TMEM55B-GFP-WT cytosolic domain (CD), TMEM55B-GFP-P66A FL, or P66A CD. Cells were lysed and pulled down with GFP beads. The results are representative of three independent experiments. WT (Wild-type), P66A (P66A mutant). d U2OS TMEM55B KO cells stably expressing mCherry-NEDD4 (red) were transfected with plasmids encoding GFP, TMEM55B-GFP-WT or P66A (green). Cells were fixed and permeabilized for immunofluorescence. Scale bars, 20 μm, n = 3. e U2OS cells were infected with adenovirus expressing TMEM55B-GFP-WT or P66A and pulled down with GFP beads. The results are representative of three independent experiments. f U2OS cells infected with adenovirus expressing TMEM55B-GFP-WT were treated with various drugs and pulled down with GFP beads. EBSS for 4 h, NaAsO2 (300 μM) for 2 h, H2O2 (500 μM) for 4 h, LLOMe (1 mM) for 2 h, CCCP (25 μM) for 4 h, Tunicamycin (10 μg/ml) for 4 h, Thapsigargin (10 μM) for 4 h. The results are representative of three independent experiments. g U2OS cells infected with adenovirus expressing TMEM55B-GFP-WT or P66A (green) were treated with or without NaAsO2 (300 μM) for 2 h. Cells were fixed and immunostained with antibodies against ubiquitin (red) and LAMTOR1 (blue). Scale bars, 20 μm, n = 3. h U2OS cells were transfected with plasmids encoding TMEM55B-GFP-WT or P66A and treated with or without NaAsO2 (300 μM) for 2 h. Cells were lysed and immunoprecipitated with GFP beads under denaturing condition. The results are representative of three independent experiments. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 2 PLEKHM1 interacts with TMEM55B upon NaAsO2 treatment. a Volcano plot of hits identified in immunoprecipitation and mass spectrometry analysis from U2OS cells infected with adenovirus expressing TMEM55B-GFP-WT treated with NaAsO2 (300 μM), compared to untreated cells. The data were analyzed with two-tailed t test. b, c U2OS cells were treated with or without NaAsO2 (300 μM) for 2 h and immunoprecipitated with anti-TMEM55B antibody. The results are representative of three independent experiments. d U2OS cells infected with adenovirus expressing TMEM55B-GFP-WT were treated with various drugs and pulled down with GFP beads. EBSS for 4 h, NaAsO2 (300 μM) for 2 h, H2O2 (500 μM) for 4 h, LLOMe (1 mM) for 2 h, CCCP (25 μM) for 4 h, Tunicamycin (10 μg/ml) for 4 h, Thapsigargin (10 μM) for 4 h. The results are representative of three independent experiments. e Hela cells infected with adenovirus expressing TMEM55B-GFP were treated with NaAsO2 (300 μM) or LLOMe (1 mM) for 2 h and pulled down with GFP beads. The results are representative of three independent experiments. f Schematic representation of the PLEKHM1 and truncated mutants. g U2OS cells were transfected with Flag-tagged PLEKHM1 plasmid variants and treated with or without NaAsO2 (300 μM) for 2 h. Cells were lysed and immunoprecipitated with Flag beads. The results are representative of two independent experiments. Source data are provided as a Source Data file.
Fig. 2 PLEKHM1 interacts with TMEM55B upon NaAsO2 treatment. a Volcano plot of hits identified in immunoprecipitation and mass spectrometry analysis from U2OS cells infected with adenovirus expressing TMEM55B-GFP-WT treated with NaAsO2 (300 μM), compared to untreated cells. The data were analyzed with two-tailed t test. b, c U2OS cells were treated with or without NaAsO2 (300 μM) for 2 h and immunoprecipitated with anti-TMEM55B antibody. The results are representative of three independent experiments. d U2OS cells infected with adenovirus expressing TMEM55B-GFP-WT were treated with various drugs and pulled down with GFP beads. EBSS for 4 h, NaAsO2 (300 μM) for 2 h, H2O2 (500 μM) for 4 h, LLOMe (1 mM) for 2 h, CCCP (25 μM) for 4 h, Tunicamycin (10 μg/ml) for 4 h, Thapsigargin (10 μM) for 4 h. The results are representative of three independent experiments. e Hela cells infected with adenovirus expressing TMEM55B-GFP were treated with NaAsO2 (300 μM) or LLOMe (1 mM) for 2 h and pulled down with GFP beads. The results are representative of three independent experiments. f Schematic representation of the PLEKHM1 and truncated mutants. g U2OS cells were transfected with Flag-tagged PLEKHM1 plasmid variants and treated with or without NaAsO2 (300 μM) for 2 h. Cells were lysed and immunoprecipitated with Flag beads. The results are representative of two independent experiments. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 3 NaAsO2 induces TMEM55B phosphorylation and PLEKHM1 ubiquitination. a U2OS cells were treated with or without NaAsO2 (300 μM) for 2 h. Cell lysates and samples immunoprecipitated with anti-TMEM55B antibody were incubated with or without Lambda phosphatase. The results are representative of three independent experiments. b Quantification of immunoblots shown in (a). The data represent means ± SEM, n = 3 independent experiments. Statistical significance was determined by using two-tailed unpaired t test. c U2OS cells were treated with NaAsO2 (300 μM), H2O2 (500 μM), Acrolein (200 μM) or Spermidine (300 μM) as indicated times. The results are representative of two independent experiments. d U2OS cells transfected with plasmids encoding PLEKHM1-Flag were treated with or without NaAsO2 (300 μM) for 2 h and immunoprecipitated with Flag beads. The results are representative of three independent experiments. e U2OS cells were treated with or without NaAsO2 (300 μM) for 2 h and immunoprecipitated with anti-PLEKHM1 antibody. The results are representative of two independent experiments. f U2OS cells were treated with NaAsO2 (300 μM) for 2 h and immunoprecipitated with anti-PLEKHM1 antibody under denaturing conditions. The results are representative of three independent experiments. g U2OS cells were treated with NaAsO2 (300 μM) for indicated times and analyzes by immunoblot with the indicated antibodies. The results are representative of three independent experiments. h Quantification of immunoblots shown in (g). The data represent means ± SEM, n = 3 independent experiments. Statistical significance was determined by using one-way ANOVA with Dunnett’s multiple comparisons. Source data are provided as a Source Data file.
Fig. 3 NaAsO2 induces TMEM55B phosphorylation and PLEKHM1 ubiquitination. a U2OS cells were treated with or without NaAsO2 (300 μM) for 2 h. Cell lysates and samples immunoprecipitated with anti-TMEM55B antibody were incubated with or without Lambda phosphatase. The results are representative of three independent experiments. b Quantification of immunoblots shown in (a). The data represent means ± SEM, n = 3 independent experiments. Statistical significance was determined by using two-tailed unpaired t test. c U2OS cells were treated with NaAsO2 (300 μM), H2O2 (500 μM), Acrolein (200 μM) or Spermidine (300 μM) as indicated times. The results are representative of two independent experiments. d U2OS cells transfected with plasmids encoding PLEKHM1-Flag were treated with or without NaAsO2 (300 μM) for 2 h and immunoprecipitated with Flag beads. The results are representative of three independent experiments. e U2OS cells were treated with or without NaAsO2 (300 μM) for 2 h and immunoprecipitated with anti-PLEKHM1 antibody. The results are representative of two independent experiments. f U2OS cells were treated with NaAsO2 (300 μM) for 2 h and immunoprecipitated with anti-PLEKHM1 antibody under denaturing conditions. The results are representative of three independent experiments. g U2OS cells were treated with NaAsO2 (300 μM) for indicated times and analyzes by immunoblot with the indicated antibodies. The results are representative of three independent experiments. h Quantification of immunoblots shown in (g). The data represent means ± SEM, n = 3 independent experiments. Statistical significance was determined by using one-way ANOVA with Dunnett’s multiple comparisons. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 4 TMEM55B-mediated PLEKHM1 degradation decreases autophagy flux. a U2OS cells infected with adenovirus expressing TMEM55B-GFP-WT or P66A were treated with or without NaAsO2 (300 μM) for 2 h. Cells were lysed and pulled down with GFP beads. The results are representative of four independent experiments. Short expo. (Shorter exposure), long expo. (Longer exposure). b U2OS cells infected with adenovirus expressing TMEM55B-GFP-WT or P66A were treated with or without NaAsO2 (300 μM) for 2 h. Cells were lysed and immunoprecipitated with anti-PLEKHM1 antibody. The results are representative of three independent experiments. c U2OS cells infected with adenovirus expressing TMEM55B-GFP-WT or P66A were incubated with NaAsO2 (300 μM) in the presence of MG132 (50 μM) or Bafilomycin A1 (100 nM) for the indicated times. The results are representative of three independent experiments. d Quantification of immunoblots shown in (c). The data represent means ± SEM, n = 3 independent experiments. Statistical significance was determined by using two-way ANOVA with Sidak’s multiple comparisons. e siRNA transfected U2OS cells were treated with NaAsO2 (300 μM) for indicated times. The results are representative of three independent experiments. f Quantification of immunoblots shown in (e). The data represent means ± SEM, n = 3 independent experiments. Statistical significance was determined by using two-way ANOVA with Sidak’s multiple comparisons. g U2OS cells infected with adenovirus expressing TMEM55B-GFP-WT or P66A were incubated with NaAsO2 (300 μM) alone or NaAsO2 (300 μM) plus Bafilomycin A1 (200 nM) for indicated times. The results are representative of four independent experiments. h Quantification of immunoblots shown in (g). The data represent means ± SEM, n = 3 independent experiments. Statistical significance was determined by using two-way ANOVA with Sidak’s multiple comparisons. Source data are provided as a Source Data file.
Fig. 4 TMEM55B-mediated PLEKHM1 degradation decreases autophagy flux. a U2OS cells infected with adenovirus expressing TMEM55B-GFP-WT or P66A were treated with or without NaAsO2 (300 μM) for 2 h. Cells were lysed and pulled down with GFP beads. The results are representative of four independent experiments. Short expo. (Shorter exposure), long expo. (Longer exposure). b U2OS cells infected with adenovirus expressing TMEM55B-GFP-WT or P66A were treated with or without NaAsO2 (300 μM) for 2 h. Cells were lysed and immunoprecipitated with anti-PLEKHM1 antibody. The results are representative of three independent experiments. c U2OS cells infected with adenovirus expressing TMEM55B-GFP-WT or P66A were incubated with NaAsO2 (300 μM) in the presence of MG132 (50 μM) or Bafilomycin A1 (100 nM) for the indicated times. The results are representative of three independent experiments. d Quantification of immunoblots shown in (c). The data represent means ± SEM, n = 3 independent experiments. Statistical significance was determined by using two-way ANOVA with Sidak’s multiple comparisons. e siRNA transfected U2OS cells were treated with NaAsO2 (300 μM) for indicated times. The results are representative of three independent experiments. f Quantification of immunoblots shown in (e). The data represent means ± SEM, n = 3 independent experiments. Statistical significance was determined by using two-way ANOVA with Sidak’s multiple comparisons. g U2OS cells infected with adenovirus expressing TMEM55B-GFP-WT or P66A were incubated with NaAsO2 (300 μM) alone or NaAsO2 (300 μM) plus Bafilomycin A1 (200 nM) for indicated times. The results are representative of four independent experiments. h Quantification of immunoblots shown in (g). The data represent means ± SEM, n = 3 independent experiments. Statistical significance was determined by using two-way ANOVA with Sidak’s multiple comparisons. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 5 TMEM55B facilitates lysosomal repair by recruiting the ESCRT complex. a Volcano plot of hits identified by immunoprecipitation and mass spectrometry analysis of U2OS cells infected with adenovirus expressing TMEM55B-GFP-P66A compared to cells infected with adenovirus expressing TMEM55B-GFP-WT under NaAsO2 (300 μM) treatment. The data were analyzed with two-tailed t test. b Multi-sequence alignment of TMEM55B orthologs in different species. The P(S/T)AP motif is marked in red. c U2OS cells transfected with plasmid encoding TMEM55B-GFP-WT, S41A or P66A were treated with or without NaAsO2 (300 μM) for 2 h. Cells were lysed and pulled down with GFP beads. The results are representative of three independent experiments. d U2OS TMEM55B KO cells transfected with plasmid encoding TMEM55B-GFP WT, S41A, P66A (green) were treated with NaAsO2 (300 μM) for 2 h. Cells were fixed and immunostained with antibodies against CHMP2B (red) and LAMP1 (blue). Scale bars, 20 μm. Inset scale bars, 10 μm. n = 3. e Null and TMEM55B KO HeLa cells were treated with or without NaAsO2 (300 μM) for 6 h. Cells were fixed and immunostained with anti-Galectin3 antibody. Scale bars, 20 μm. f Quantification of immunofluorescence images shown in (e). The data represent means ± SEM. n = 200 cells examined over 3 independent experiments. Statistical significance was determined by using two-tailed unpaired t test. Source data are provided as a Source Data file.
Fig. 5 TMEM55B facilitates lysosomal repair by recruiting the ESCRT complex. a Volcano plot of hits identified by immunoprecipitation and mass spectrometry analysis of U2OS cells infected with adenovirus expressing TMEM55B-GFP-P66A compared to cells infected with adenovirus expressing TMEM55B-GFP-WT under NaAsO2 (300 μM) treatment. The data were analyzed with two-tailed t test. b Multi-sequence alignment of TMEM55B orthologs in different species. The P(S/T)AP motif is marked in red. c U2OS cells transfected with plasmid encoding TMEM55B-GFP-WT, S41A or P66A were treated with or without NaAsO2 (300 μM) for 2 h. Cells were lysed and pulled down with GFP beads. The results are representative of three independent experiments. d U2OS TMEM55B KO cells transfected with plasmid encoding TMEM55B-GFP WT, S41A, P66A (green) were treated with NaAsO2 (300 μM) for 2 h. Cells were fixed and immunostained with antibodies against CHMP2B (red) and LAMP1 (blue). Scale bars, 20 μm. Inset scale bars, 10 μm. n = 3. e Null and TMEM55B KO HeLa cells were treated with or without NaAsO2 (300 μM) for 6 h. Cells were fixed and immunostained with anti-Galectin3 antibody. Scale bars, 20 μm. f Quantification of immunofluorescence images shown in (e). The data represent means ± SEM. n = 200 cells examined over 3 independent experiments. Statistical significance was determined by using two-tailed unpaired t test. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 6 Depletion of TMEM55B reduces cell viability under oxidative stress conditions. a Null and TMEM55B KO U2OS cells were treated with or without NaAsO2 (300 μM) for 10 h. Cells were analyzed by Flow cytometry with Annexin V and 7AAD. 7AAD+ are necrotic, Annexin V+ are apoptotic and Annexin V + /7AAD+ are late apoptotic and necrotic cells. b Quantification of the population of live, necrotic (7AAD + ), apoptotic cells (Annexin V + ) and late apoptotic and necrotic cells (Annexin + /7AAD + ) from (a). The data represent means ± SEM, n = 3 independent experiments. Data taken from three independent experiments and statistical significance was determined by using two-way ANOVA with Sidak’s multiple comparisons. Source data are provided as a Source Data file.
Fig. 6 Depletion of TMEM55B reduces cell viability under oxidative stress conditions. a Null and TMEM55B KO U2OS cells were treated with or without NaAsO2 (300 μM) for 10 h. Cells were analyzed by Flow cytometry with Annexin V and 7AAD. 7AAD+ are necrotic, Annexin V+ are apoptotic and Annexin V + /7AAD+ are late apoptotic and necrotic cells. b Quantification of the population of live, necrotic (7AAD + ), apoptotic cells (Annexin V + ) and late apoptotic and necrotic cells (Annexin + /7AAD + ) from (a). The data represent means ± SEM, n = 3 independent experiments. Data taken from three independent experiments and statistical significance was determined by using two-way ANOVA with Sidak’s multiple comparisons. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 7 TMEM55B promotes TFE3 activation through FLCN sequestration. a U2OS cells treated with or without NaAsO2 (300 μM) for 2 h were lysed and immunoprecipitated with anti-TMEM55B antibody. The results are representative of three independent experiments. b U2OS cells treated with various drugs were lysed and immunoprecipitated with anti-TMEM55B antibody. EBSS for 4 h, NaAsO2 (300 μM) for 2 h, H2O2 (500 μM) for 4 h, LLOMe (1 mM) for 2 h, CCCP (25 μM) for 4 h, Tunicamycin (10 μg/ml) for 4 h, Thapsigargin (10 μM) for 4 h. The results are representative of three independent experiments. c Null and TMEM55B KO U2OS cells were treated with either EBSS for 4 h or NaAsO2 (300 μM) for 2 h. Cells were fixed and immunostained with antibodies against LAMP1 (red) and FLCN (green). Scale bars, 20 μm. n = 3. d Quantification of immunofluorescence images shown in (c). The data represent means ± SEM, n = 200 cells examined over 3 independent experiments. e Null and TMEM55B KO U2OS cells were treated with either NaAsO2 (300 μM) or Torin-1 (250 nM) for the indicated times. Samples were analyzed by immunoblot with the indicated antibodies. The results are representative of three independent experiments. f Quantification of immunoblots shown in (e). The data represent means ± SEM, n = 3 independent experiments. Statistical significance was determined by using two-way ANOVA with Sidak’s multiple comparisons. Source data are provided as a Source Data file.
Fig. 7 TMEM55B promotes TFE3 activation through FLCN sequestration. a U2OS cells treated with or without NaAsO2 (300 μM) for 2 h were lysed and immunoprecipitated with anti-TMEM55B antibody. The results are representative of three independent experiments. b U2OS cells treated with various drugs were lysed and immunoprecipitated with anti-TMEM55B antibody. EBSS for 4 h, NaAsO2 (300 μM) for 2 h, H2O2 (500 μM) for 4 h, LLOMe (1 mM) for 2 h, CCCP (25 μM) for 4 h, Tunicamycin (10 μg/ml) for 4 h, Thapsigargin (10 μM) for 4 h. The results are representative of three independent experiments. c Null and TMEM55B KO U2OS cells were treated with either EBSS for 4 h or NaAsO2 (300 μM) for 2 h. Cells were fixed and immunostained with antibodies against LAMP1 (red) and FLCN (green). Scale bars, 20 μm. n = 3. d Quantification of immunofluorescence images shown in (c). The data represent means ± SEM, n = 200 cells examined over 3 independent experiments. e Null and TMEM55B KO U2OS cells were treated with either NaAsO2 (300 μM) or Torin-1 (250 nM) for the indicated times. Samples were analyzed by immunoblot with the indicated antibodies. The results are representative of three independent experiments. f Quantification of immunoblots shown in (e). The data represent means ± SEM, n = 3 independent experiments. Statistical significance was determined by using two-way ANOVA with Sidak’s multiple comparisons. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 8 Generation and characterization of tmem55-KO mutants. a (Top panel) Schematic representation of zebrafish tmem55ba, tmem55bb and tmem55a genes with exons shown as blue rectangles connected by introns. Red bars show the target region of the sgRNAs used for CRISPR/Cas9 knockout. Neither exons nor introns are drawn to scale. (Bottom panel) Nucleotide sequences of CRISPR target regions in exons 2 of tmem55ba and tmem55bb and exons 1 and 4 of tmem55a genes. sgRNAs are underlined, PAM sites are in bold and deleted nucleotides in the mutant alleles are marked by dashes. b Representative pictures of WT and tmem55-KO at 5 dpf. All embryos are depicted with anterior to the left. Scale bars = 500 μm. c Kaplan-Meier curves showing the survival rates in zebrafish embryos following continuous exposure to 2 mM NaAsO2 from 1 dpf stage. Untreated embryos are used as controls. The number of embryos per group is indicated in parenthesis. (Logrank test, P < 0.0001). Data in (b) and (c) show one representative experiment out of three independently performed. d Model depicting the proposed role of TMEM55B orchestrating cellular responses to oxidative stress. Illustration created with BioRender.com. Source data are provided as a Source Data file.
Fig. 8 Generation and characterization of tmem55-KO mutants. a (Top panel) Schematic representation of zebrafish tmem55ba, tmem55bb and tmem55a genes with exons shown as blue rectangles connected by introns. Red bars show the target region of the sgRNAs used for CRISPR/Cas9 knockout. Neither exons nor introns are drawn to scale. (Bottom panel) Nucleotide sequences of CRISPR target regions in exons 2 of tmem55ba and tmem55bb and exons 1 and 4 of tmem55a genes. sgRNAs are underlined, PAM sites are in bold and deleted nucleotides in the mutant alleles are marked by dashes. b Representative pictures of WT and tmem55-KO at 5 dpf. All embryos are depicted with anterior to the left. Scale bars = 500 μm. c Kaplan-Meier curves showing the survival rates in zebrafish embryos following continuous exposure to 2 mM NaAsO2 from 1 dpf stage. Untreated embryos are used as controls. The number of embryos per group is indicated in parenthesis. (Logrank test, P < 0.0001). Data in (b) and (c) show one representative experiment out of three independently performed. d Model depicting the proposed role of TMEM55B orchestrating cellular responses to oxidative stress. Illustration created with BioRender.com. Source data are provided as a Source Data file.CC BY · PubMed Central

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  • The paper relies on this without support.

    NaAsO2 treatment induces oxidative stress as the relevant mechanism for the observed cellular effects.

    “generation of oxidative stress by treatment with NaAsO2”

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    ESCRT recruitment to lysosomes is an early event following lysosomal damage.

    “ESCRT recruitment to lysosomes is an early event following lysosomal damage.”

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    Extensive damage that cannot be repaired by the ESCRT complex leads to accumulation of galectins, triggering elimination of damaged lysosomes by lysophagy.

    “Extensive damage that cannot be repaired by the ESCRT complex leads to accumulation of galectins, triggering elimination of damaged lysosomes by lysophagy.”

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    TFEB and TFE3 orchestrate cellular response to stress by promoting expression of lysosomal and autophagic genes, contributing to elimination of waste material and restoration of energy homeostasis.

    “The transcription factors TFEB and TFE3 orchestrate cellular response to a variety of stress conditions. By promoting expression of lysosomal and autophagic genes, TFEB and TFE3 contribute to the elimination of waste material and the restoration of energy homeostasis.”

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    TMEM55B regulates lysosomal positioning by interacting with JIP4 to connect lysosomes to dynein for retrograde movement, relevant during starvation.

    “The role of TMEM55B in regulating lysosomal positioning is well established. Through its interaction with JIP4, TMEM55B connects lysosomes to the minus-end microtubule motor dynein, allowing retrograde movement of lysosomes from the periphery to the cell center. This function is particularly relevant during starvation conditions, when lysosomal retrograde movement facilitates contact and fusion of”
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Metadata

Authors
Jeong, E., Willett, R., Rissone, A., La Spina, M., & Puertollano, R.
Affiliations
National Institutes of Health (US)
Corresponding author
Puertollano, Rosa
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2024-01-02
Volume
15
Issue
1
Pages
93-93
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-023-44316-6
PMC
PMC10761734
OpenAlex
W4390499631
Cited by
25
References
46
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2026: 14 · 2025: 10 · 2024: 1
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3.95
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journal article
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en
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  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Padua Research Archive (University of Padova) (submittedVersion, cc-by)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
U.S. Department of Health & Human Services | National Institutes of Health (ZIA HL006075)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Autophagy in Disease and Therapy
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Physiology › Calcium signaling and nucleotide metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
Keywords
autophagic flux, oxidative stress, NEDD4, ESCRT machinery, lysosomal membrane repair, arsenite toxicity, zebrafish, lysosomes
MeSH terms
Animals, Autophagy, Lysosomes, Zebrafish, Oxidative Stress, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

A language model drew the claims from the paper on 2026-11-09, under version 3 of the claim definition (model deepseek-v4.1-flash, prompt extract-v3).

A second model labelled each claim on 2026-11-09 (model jev-1.13-20260917, definition revision gold-guideline-v0.2).

Cited sources are checked with a language model’s help (gpt-6-sol).

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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