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    Addgene inc n52a egfp mutants
    LRBA is endogenously expressed in HeLa cells and recruited to endosomes by Arfs. (A) Immunoblot analysis of LRBA presence in HeLa cells of two control and four LRBA KO clones using polyclonal LRBA antibody and α -tubulin as a loading control. (B) LRBA is localized at the perinuclear region and on vesicular structures in HeLa cells. Immunofluorescence analysis of endogenous LRBA in fixed HeLa cells. Scale bar, 10 μm, inlay 2 μm. (C) LRBA partly colocalizes with the cis-Golgi in HeLa cells. Immunofluorescence staining of endogenous LRBA and the cis-Golgi marker giantin in fixed HeLa cells. Squares show magnification of the perinuclear area. The labeling of the single channels represents the color of the channel on the merged image. Scale bar, 10 μm, inlays 2 μm. (D) LRBA does not colocalize with M6PR in HeLa cells. Immunofluorescence analysis of endogenous LRBA and endogenous M6PR colocalization in fixed HeLa cells. Squares show magnification of the perinuclear area. The labeling of the single channels represents the color of the channel on the merged image. Scale bar, 10 μm, inlays 2 μm. (E) Colocalization measurement of LRBA with giantin and M6PR. To measure LRBA colocalization with giantin one ROI at the perinuclear region was analyzed. To measure colocalization with M6PR, two ROIs per cell at the cell periphery were analyzed and the Pearson’s coefficient was measured using the JACoP plugin in Fiji. Mean and minimum to maximum are shown, box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. All data points are shown. Giantin = 60 cells, M6PR = 35 cells. (F) LRBA puncta disperse upon treatment with ArfGEF inhibitors. Live-cell imaging of 3xFlagEGFP-LRBA upon BFA (top panels) and GCA (lower panels) treatment for indicated timepoints. Scale bar, 10 μm. (G) Arf1 is recruited onto Rab4 + endosomes in the absence of LRBA. Control KO and LRBA KO HeLa cells were transfected with mCherry-Rab4 and <t>Arf1-EGFP</t> and cells were imaged live using a wide-field microscope at 37°C, 5% CO 2 atmosphere. Deconvolved images of single stacks are shown. Squares show magnification of the perinuclear area. The labeling of the single channels represents the color of the channel on the merged image. Scale bar, 10 μm, inlays 2 μm. (H and I) Colocalization measurement of Arf1-EGFP and mCherry-Rab4 in control and LRBA KO HeLa cells. Two ROIs per cell were analyzed and Mander’s coefficients were measured using the JACoP plugin in Fiji. Arf1 overlap with Rab4 (M1) is shown in H, Rab4 overlap with Arf1 (M2) is shown in I. All data points are shown. Ctr. KO clone1 = 32 cells, Ctr. KO clone2 = 39 cells, LRBA KO clone1 = 36 cells, LRBA KO clone 4 = 36 cells from n = 3 biological replicates; (H) one-way ANOVA using Tukey’s multiple comparison. (I) Kruskal–Wallis test using Dunn’s multiple comparisons test, **P = 0.0071, *P = 0.0201. (J) The amino acids isoleucine 46 and 49 of Arf1 and Arf3 were predicted to interact with LRBA. Both amino acids are conserved across species. The amino acid sequences of the yeast, C. elegans (CAEEL), Drosophila melanogaster (DROME), mouse and human Arf1 and human Arf3 were aligned. Labels: (*) conserved sequence; (:) conservative mutation; (.) semiconservative mutation; (−) gap. Sequence alignments were performed using Clustal Omega. (K) The amino acids leucine 861, arginine 910, and isoleucine 918 of LRBA were predicted to interact with Arf1 and Arf3. All three amino acids are conserved across species. The amino acid sequences of the C. elegans SEL-2 (SEL2-CAEEL), the mouse and the human LRBA, and the human neurobeachin (NBEA) were aligned. Labels: (*) conserved sequence; (:) conservative mutation; (.) semi-conservative mutation; (-) gap. Sequence alignments were performed using Clustal Omega. Source data are available for this figure: .
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    1) Product Images from "Arf1-dependent LRBA recruitment to Rab4 endosomes is required for endolysosome homeostasis"

    Article Title: Arf1-dependent LRBA recruitment to Rab4 endosomes is required for endolysosome homeostasis

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.202401167

    LRBA is endogenously expressed in HeLa cells and recruited to endosomes by Arfs. (A) Immunoblot analysis of LRBA presence in HeLa cells of two control and four LRBA KO clones using polyclonal LRBA antibody and α -tubulin as a loading control. (B) LRBA is localized at the perinuclear region and on vesicular structures in HeLa cells. Immunofluorescence analysis of endogenous LRBA in fixed HeLa cells. Scale bar, 10 μm, inlay 2 μm. (C) LRBA partly colocalizes with the cis-Golgi in HeLa cells. Immunofluorescence staining of endogenous LRBA and the cis-Golgi marker giantin in fixed HeLa cells. Squares show magnification of the perinuclear area. The labeling of the single channels represents the color of the channel on the merged image. Scale bar, 10 μm, inlays 2 μm. (D) LRBA does not colocalize with M6PR in HeLa cells. Immunofluorescence analysis of endogenous LRBA and endogenous M6PR colocalization in fixed HeLa cells. Squares show magnification of the perinuclear area. The labeling of the single channels represents the color of the channel on the merged image. Scale bar, 10 μm, inlays 2 μm. (E) Colocalization measurement of LRBA with giantin and M6PR. To measure LRBA colocalization with giantin one ROI at the perinuclear region was analyzed. To measure colocalization with M6PR, two ROIs per cell at the cell periphery were analyzed and the Pearson’s coefficient was measured using the JACoP plugin in Fiji. Mean and minimum to maximum are shown, box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. All data points are shown. Giantin = 60 cells, M6PR = 35 cells. (F) LRBA puncta disperse upon treatment with ArfGEF inhibitors. Live-cell imaging of 3xFlagEGFP-LRBA upon BFA (top panels) and GCA (lower panels) treatment for indicated timepoints. Scale bar, 10 μm. (G) Arf1 is recruited onto Rab4 + endosomes in the absence of LRBA. Control KO and LRBA KO HeLa cells were transfected with mCherry-Rab4 and Arf1-EGFP and cells were imaged live using a wide-field microscope at 37°C, 5% CO 2 atmosphere. Deconvolved images of single stacks are shown. Squares show magnification of the perinuclear area. The labeling of the single channels represents the color of the channel on the merged image. Scale bar, 10 μm, inlays 2 μm. (H and I) Colocalization measurement of Arf1-EGFP and mCherry-Rab4 in control and LRBA KO HeLa cells. Two ROIs per cell were analyzed and Mander’s coefficients were measured using the JACoP plugin in Fiji. Arf1 overlap with Rab4 (M1) is shown in H, Rab4 overlap with Arf1 (M2) is shown in I. All data points are shown. Ctr. KO clone1 = 32 cells, Ctr. KO clone2 = 39 cells, LRBA KO clone1 = 36 cells, LRBA KO clone 4 = 36 cells from n = 3 biological replicates; (H) one-way ANOVA using Tukey’s multiple comparison. (I) Kruskal–Wallis test using Dunn’s multiple comparisons test, **P = 0.0071, *P = 0.0201. (J) The amino acids isoleucine 46 and 49 of Arf1 and Arf3 were predicted to interact with LRBA. Both amino acids are conserved across species. The amino acid sequences of the yeast, C. elegans (CAEEL), Drosophila melanogaster (DROME), mouse and human Arf1 and human Arf3 were aligned. Labels: (*) conserved sequence; (:) conservative mutation; (.) semiconservative mutation; (−) gap. Sequence alignments were performed using Clustal Omega. (K) The amino acids leucine 861, arginine 910, and isoleucine 918 of LRBA were predicted to interact with Arf1 and Arf3. All three amino acids are conserved across species. The amino acid sequences of the C. elegans SEL-2 (SEL2-CAEEL), the mouse and the human LRBA, and the human neurobeachin (NBEA) were aligned. Labels: (*) conserved sequence; (:) conservative mutation; (.) semi-conservative mutation; (-) gap. Sequence alignments were performed using Clustal Omega. Source data are available for this figure: .
    Figure Legend Snippet: LRBA is endogenously expressed in HeLa cells and recruited to endosomes by Arfs. (A) Immunoblot analysis of LRBA presence in HeLa cells of two control and four LRBA KO clones using polyclonal LRBA antibody and α -tubulin as a loading control. (B) LRBA is localized at the perinuclear region and on vesicular structures in HeLa cells. Immunofluorescence analysis of endogenous LRBA in fixed HeLa cells. Scale bar, 10 μm, inlay 2 μm. (C) LRBA partly colocalizes with the cis-Golgi in HeLa cells. Immunofluorescence staining of endogenous LRBA and the cis-Golgi marker giantin in fixed HeLa cells. Squares show magnification of the perinuclear area. The labeling of the single channels represents the color of the channel on the merged image. Scale bar, 10 μm, inlays 2 μm. (D) LRBA does not colocalize with M6PR in HeLa cells. Immunofluorescence analysis of endogenous LRBA and endogenous M6PR colocalization in fixed HeLa cells. Squares show magnification of the perinuclear area. The labeling of the single channels represents the color of the channel on the merged image. Scale bar, 10 μm, inlays 2 μm. (E) Colocalization measurement of LRBA with giantin and M6PR. To measure LRBA colocalization with giantin one ROI at the perinuclear region was analyzed. To measure colocalization with M6PR, two ROIs per cell at the cell periphery were analyzed and the Pearson’s coefficient was measured using the JACoP plugin in Fiji. Mean and minimum to maximum are shown, box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. All data points are shown. Giantin = 60 cells, M6PR = 35 cells. (F) LRBA puncta disperse upon treatment with ArfGEF inhibitors. Live-cell imaging of 3xFlagEGFP-LRBA upon BFA (top panels) and GCA (lower panels) treatment for indicated timepoints. Scale bar, 10 μm. (G) Arf1 is recruited onto Rab4 + endosomes in the absence of LRBA. Control KO and LRBA KO HeLa cells were transfected with mCherry-Rab4 and Arf1-EGFP and cells were imaged live using a wide-field microscope at 37°C, 5% CO 2 atmosphere. Deconvolved images of single stacks are shown. Squares show magnification of the perinuclear area. The labeling of the single channels represents the color of the channel on the merged image. Scale bar, 10 μm, inlays 2 μm. (H and I) Colocalization measurement of Arf1-EGFP and mCherry-Rab4 in control and LRBA KO HeLa cells. Two ROIs per cell were analyzed and Mander’s coefficients were measured using the JACoP plugin in Fiji. Arf1 overlap with Rab4 (M1) is shown in H, Rab4 overlap with Arf1 (M2) is shown in I. All data points are shown. Ctr. KO clone1 = 32 cells, Ctr. KO clone2 = 39 cells, LRBA KO clone1 = 36 cells, LRBA KO clone 4 = 36 cells from n = 3 biological replicates; (H) one-way ANOVA using Tukey’s multiple comparison. (I) Kruskal–Wallis test using Dunn’s multiple comparisons test, **P = 0.0071, *P = 0.0201. (J) The amino acids isoleucine 46 and 49 of Arf1 and Arf3 were predicted to interact with LRBA. Both amino acids are conserved across species. The amino acid sequences of the yeast, C. elegans (CAEEL), Drosophila melanogaster (DROME), mouse and human Arf1 and human Arf3 were aligned. Labels: (*) conserved sequence; (:) conservative mutation; (.) semiconservative mutation; (−) gap. Sequence alignments were performed using Clustal Omega. (K) The amino acids leucine 861, arginine 910, and isoleucine 918 of LRBA were predicted to interact with Arf1 and Arf3. All three amino acids are conserved across species. The amino acid sequences of the C. elegans SEL-2 (SEL2-CAEEL), the mouse and the human LRBA, and the human neurobeachin (NBEA) were aligned. Labels: (*) conserved sequence; (:) conservative mutation; (.) semi-conservative mutation; (-) gap. Sequence alignments were performed using Clustal Omega. Source data are available for this figure: .

    Techniques Used: Western Blot, Control, Clone Assay, Immunofluorescence, Staining, Marker, Labeling, Live Cell Imaging, Transfection, Microscopy, Comparison, Sequencing, Mutagenesis

    LRBA is recruited onto endosomes by Arf1 and Arf3. (A) Colocalization analysis of LRBA and TGN46 or Arf1 on endosomes in HeLa cells. For the colocalization analysis with the TGN, HeLa cells were fixed with 4% PFA and stained for endogenous TGN46 and LRBA. For colocalization analysis with Arf1, HeLa cells were transfected with ARF1-mCherry, fixed with 4% PFA and stained for endogenous LRBA. Squares show magnification of the perinuclear area. The labeling of the single channels represents the color of the channel on the merged image. Scale bar, 10 μm, inlays 2 μm. (B) Colocalization measurements of LRBA with TGN46 and Arf1 at the cell periphery. To measure LRBA colocalization with TGN46 one ROI per image, with Arf1 two ROIs per image were analyzed and the Pearson’s coefficient was measured using the JACoP plugin in Fiji. Mean and minimum to maximum are shown, the box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. All data points are shown. TGN46 (periphery) = 40 cells, Arf1 (periphery) = 35 cells from n = 3 biological replicates. (C) Immunoblot analysis of Arf1 and Arf3 expression in parental, ARF1 KO, ARF3 KO, and ARF1+3 dKO HeLa cells. Actin was used as a loading control. (D) LRBA is absent from endosomes in ARF1 and ARF3 dKO HeLa cells. Note that LRBA is still present on the Golgi. Parental, ARF1 KO, ARF3 KO, and ARF1+3 dKO HeLa cells were seeded on coverslips, fixed, and stained for endogenous LRBA. Maximum intensity projections of confocal images are shown. (E) The number of LRBA + endosomes in parental, ARF1 KO, ARF3 KO, and ARF1+3 dKO cells was measured using two ROIs per cell. Mean and minimum to maximum are shown, box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. All data points are shown. Parental = 95 cells, ARF1 KO = 49 cells, ARF3 KO = 32 cells, and Arf1+3 dKO = 34 cells were analyzed from n = 3 biological replicates; one-way ANOVA using Dunnett’s multiple comparison, **P = 0.0010 (parental versus ARF1 KO), **P = 0.0097 (parental versus ARF3 KO), ****P < 0.0001. (F) Arf1-EGFP re-expression rescues LRBA + endosomes absent in ARF1+3 dKO cells. Parental HeLa cells were transfected with Arf1-EGFP, and ARF1+3 dKO cells were transfected either with EGFP as a control or with Arf1-EGFP. Cells were then fixed and stained for endogenous LRBA and with Hoechst. Maximum intensity projection of confocal Z-stacks is shown. Rectangles show the magnified area in the upper right corner. Scale bar on inlays 2 µm. (G) The number of LRBA + puncta are counted based on data in F. Two ROIs at the cell periphery per cell are analyzed. Mean and minimum to maximum are shown, box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. All data points are shown. Parental = 49 cells, EGFP rescue = 42 cells, Arf1-EGFP rescue = 39 cells were analyzed from n = 3 biological replicates; Kruskal–Wallis test using Dunn’s multiple comparison, ****P < 0.0001. (H) (Endo)lysosomal structures are enlarged in ARF1+3 dKO cells. Parental and ARF1+3 dKO HeLa cells were seeded on coverslips, fixed, and stained for LAMP1 and with Hoechst. Maximum intensity projections of confocal Z-stacks. Squares show magnification of the (endo)lysosomes. Scale bar, 10 μm, inlays 2 μm. (I) Quantification of lysosome diameter based on images shown in H. The diameter of round lysosomes was measured manually in Fiji. Mean and minimum to maximum are shown, box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. Parental = 60 cells, ARF1+3 dKO = 64 cells were analyzed from n = 3 biological replicates. All data points are shown. Mann–Whitney test, ****P < 0.0001. Source data are available for this figure: .
    Figure Legend Snippet: LRBA is recruited onto endosomes by Arf1 and Arf3. (A) Colocalization analysis of LRBA and TGN46 or Arf1 on endosomes in HeLa cells. For the colocalization analysis with the TGN, HeLa cells were fixed with 4% PFA and stained for endogenous TGN46 and LRBA. For colocalization analysis with Arf1, HeLa cells were transfected with ARF1-mCherry, fixed with 4% PFA and stained for endogenous LRBA. Squares show magnification of the perinuclear area. The labeling of the single channels represents the color of the channel on the merged image. Scale bar, 10 μm, inlays 2 μm. (B) Colocalization measurements of LRBA with TGN46 and Arf1 at the cell periphery. To measure LRBA colocalization with TGN46 one ROI per image, with Arf1 two ROIs per image were analyzed and the Pearson’s coefficient was measured using the JACoP plugin in Fiji. Mean and minimum to maximum are shown, the box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. All data points are shown. TGN46 (periphery) = 40 cells, Arf1 (periphery) = 35 cells from n = 3 biological replicates. (C) Immunoblot analysis of Arf1 and Arf3 expression in parental, ARF1 KO, ARF3 KO, and ARF1+3 dKO HeLa cells. Actin was used as a loading control. (D) LRBA is absent from endosomes in ARF1 and ARF3 dKO HeLa cells. Note that LRBA is still present on the Golgi. Parental, ARF1 KO, ARF3 KO, and ARF1+3 dKO HeLa cells were seeded on coverslips, fixed, and stained for endogenous LRBA. Maximum intensity projections of confocal images are shown. (E) The number of LRBA + endosomes in parental, ARF1 KO, ARF3 KO, and ARF1+3 dKO cells was measured using two ROIs per cell. Mean and minimum to maximum are shown, box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. All data points are shown. Parental = 95 cells, ARF1 KO = 49 cells, ARF3 KO = 32 cells, and Arf1+3 dKO = 34 cells were analyzed from n = 3 biological replicates; one-way ANOVA using Dunnett’s multiple comparison, **P = 0.0010 (parental versus ARF1 KO), **P = 0.0097 (parental versus ARF3 KO), ****P < 0.0001. (F) Arf1-EGFP re-expression rescues LRBA + endosomes absent in ARF1+3 dKO cells. Parental HeLa cells were transfected with Arf1-EGFP, and ARF1+3 dKO cells were transfected either with EGFP as a control or with Arf1-EGFP. Cells were then fixed and stained for endogenous LRBA and with Hoechst. Maximum intensity projection of confocal Z-stacks is shown. Rectangles show the magnified area in the upper right corner. Scale bar on inlays 2 µm. (G) The number of LRBA + puncta are counted based on data in F. Two ROIs at the cell periphery per cell are analyzed. Mean and minimum to maximum are shown, box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. All data points are shown. Parental = 49 cells, EGFP rescue = 42 cells, Arf1-EGFP rescue = 39 cells were analyzed from n = 3 biological replicates; Kruskal–Wallis test using Dunn’s multiple comparison, ****P < 0.0001. (H) (Endo)lysosomal structures are enlarged in ARF1+3 dKO cells. Parental and ARF1+3 dKO HeLa cells were seeded on coverslips, fixed, and stained for LAMP1 and with Hoechst. Maximum intensity projections of confocal Z-stacks. Squares show magnification of the (endo)lysosomes. Scale bar, 10 μm, inlays 2 μm. (I) Quantification of lysosome diameter based on images shown in H. The diameter of round lysosomes was measured manually in Fiji. Mean and minimum to maximum are shown, box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. Parental = 60 cells, ARF1+3 dKO = 64 cells were analyzed from n = 3 biological replicates. All data points are shown. Mann–Whitney test, ****P < 0.0001. Source data are available for this figure: .

    Techniques Used: Staining, Transfection, Labeling, Western Blot, Expressing, Control, Comparison, MANN-WHITNEY

    Structure of LRBA and potential interaction site with Arfs as predicted by Alphafold. (A and B) Alphafold monomer prediction of human LRBA structure. Model confidence values (A) and domains (B) are indicated with colors. (C) Predicted interaction sites between LRBA (amino acids 1–1373) and Arf1 (amino acids 1–181) using Alphafold multimer. PAE plot shows the confidence scores of the interaction. The LRBA–ARF1 model has PTM and iPTM scores of 0.583 and 0.504, respectively. (D) Schematic of Arf1/3 structure and its domains. The conserved amino acid isoleucine 49 of Arf1/3 is shown since most of our models indicated that it could potentially interact with LRBA. (E) The predicted LRBA interaction site of Arf1, isoleucine 46 and 49 are conserved among human Arf1, Arf3, Arf4, and Arf5. Sequence alignments were performed using Clustal Omega. Labels: (*) conserved sequence; (:) conservative mutation; (.) semi-conservative mutation; (-) gap. (F) Colocalization analysis of mutated Arf1-EGFP constructs with Rab4 + endosomes. ARF1+3dKO HeLa cells were transfected with mCherry-Rab4a and with Arf1-EGFP constructs. Confocal images of single focal planes. Scale bar, 10 μm, inlays 2 μm. (G and H) Colocalization measurement of Rab4 and Arf1 mutants determined by Mander’s coefficients. Arf1 I46A, I49A -EGFP and Arf1 I46S, I49S- EGFP are still efficiently recruited to Rab4 + endosomes. Arf1 I46A, I49A, N52A -EGFP recruitment to Rab4 endosomes is strongly reduced compared with wild-type Arf1-EGFP. Arf1-EGFP = 42 cells, Arf1 I46A, I49A -EGFP = 43 cells, Arf1 I46S, I49S- EGFP = 38 cells, and Arf1 I46A, I49A, N52A = 36 cells were analyzed from n = 3 biological replicates, one-way ANOVA using Tukey’s multiple comparison test; ****P < 0.0001. (I) Arf1 I46S, I49S- EGFP fails to rescue LRBA + puncta in ARF1+3 dKO cells. ARF1+3 dKO cells were transfected with EGFP, Arf1-EGFP, Arf1 I46A, I49A -EGFP, Arf1 I46S, I49S- EGFP or with Arf1 I46A, I49A, N52A -EGFP, fixed and stained for endogenous LRBA and with Hoechst. Scale bar, 10 μm, inlays 2 μm. (J) Quantification of the number of LRBA + puncta based on images as shown on panel I. Two ROIs at the cell periphery per cell were analyzed. Mean and minimum to maximum are shown, box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. All data points are shown. EGFP = 42 cells, Arf1-EGFP = 39 cells, Arf1 I46A, I49A -EGFP = 48 cells, Arf1 I46S, I49S- EGFP = 36 cells, Arf1 I46A, I49A, N52A = 49 cells were analyzed from n = 3 independent experiments; Kruskal–Wallis test using Dunn’s multiple comparison, ****P < 0.0001. EGFP and Arf1-EGFP measurements are identical to the one shown in as the data were obtained in the same experiment.
    Figure Legend Snippet: Structure of LRBA and potential interaction site with Arfs as predicted by Alphafold. (A and B) Alphafold monomer prediction of human LRBA structure. Model confidence values (A) and domains (B) are indicated with colors. (C) Predicted interaction sites between LRBA (amino acids 1–1373) and Arf1 (amino acids 1–181) using Alphafold multimer. PAE plot shows the confidence scores of the interaction. The LRBA–ARF1 model has PTM and iPTM scores of 0.583 and 0.504, respectively. (D) Schematic of Arf1/3 structure and its domains. The conserved amino acid isoleucine 49 of Arf1/3 is shown since most of our models indicated that it could potentially interact with LRBA. (E) The predicted LRBA interaction site of Arf1, isoleucine 46 and 49 are conserved among human Arf1, Arf3, Arf4, and Arf5. Sequence alignments were performed using Clustal Omega. Labels: (*) conserved sequence; (:) conservative mutation; (.) semi-conservative mutation; (-) gap. (F) Colocalization analysis of mutated Arf1-EGFP constructs with Rab4 + endosomes. ARF1+3dKO HeLa cells were transfected with mCherry-Rab4a and with Arf1-EGFP constructs. Confocal images of single focal planes. Scale bar, 10 μm, inlays 2 μm. (G and H) Colocalization measurement of Rab4 and Arf1 mutants determined by Mander’s coefficients. Arf1 I46A, I49A -EGFP and Arf1 I46S, I49S- EGFP are still efficiently recruited to Rab4 + endosomes. Arf1 I46A, I49A, N52A -EGFP recruitment to Rab4 endosomes is strongly reduced compared with wild-type Arf1-EGFP. Arf1-EGFP = 42 cells, Arf1 I46A, I49A -EGFP = 43 cells, Arf1 I46S, I49S- EGFP = 38 cells, and Arf1 I46A, I49A, N52A = 36 cells were analyzed from n = 3 biological replicates, one-way ANOVA using Tukey’s multiple comparison test; ****P < 0.0001. (I) Arf1 I46S, I49S- EGFP fails to rescue LRBA + puncta in ARF1+3 dKO cells. ARF1+3 dKO cells were transfected with EGFP, Arf1-EGFP, Arf1 I46A, I49A -EGFP, Arf1 I46S, I49S- EGFP or with Arf1 I46A, I49A, N52A -EGFP, fixed and stained for endogenous LRBA and with Hoechst. Scale bar, 10 μm, inlays 2 μm. (J) Quantification of the number of LRBA + puncta based on images as shown on panel I. Two ROIs at the cell periphery per cell were analyzed. Mean and minimum to maximum are shown, box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. All data points are shown. EGFP = 42 cells, Arf1-EGFP = 39 cells, Arf1 I46A, I49A -EGFP = 48 cells, Arf1 I46S, I49S- EGFP = 36 cells, Arf1 I46A, I49A, N52A = 49 cells were analyzed from n = 3 independent experiments; Kruskal–Wallis test using Dunn’s multiple comparison, ****P < 0.0001. EGFP and Arf1-EGFP measurements are identical to the one shown in as the data were obtained in the same experiment.

    Techniques Used: Sequencing, Mutagenesis, Construct, Transfection, Comparison, Staining

    Arf1 coimmunoprecipitates with LRBA. (A) Coimmunoprecipitation analysis of Arf1-EGFP, Arf1 Q71L -EGFP (constitutively active [CA]), Arf1 I46S I49S- EGFP binding mutant and Arf1 T31N -EGFP (dominant-negative [DN]). Arf1-EGFP constructs were pulled-down using GFP-trap magnetic beads. The eluted proteins were immunoblotted and Arf1 constructs were detected using polyclonal GFP antibodies. LRBA was detected by the polyclonal LRBA antibody. Wild-type and the constitutively active Arf1 interact with LRBA. This interaction is reduced with the Arf1 I46S I49S -EGFP and Arf1 T31N -EGFP DN mutant. (B) Quantification of immunoblots as shown on panel (A) from n = 6 biological replicates, one-way ANOVA using Tukey’s multiple comparison test, **P = 0.0040, *P = 0.0163. (C) Model of endosomal trafficking in the presence and in the absence of LRBA. Source data are available for this figure: .
    Figure Legend Snippet: Arf1 coimmunoprecipitates with LRBA. (A) Coimmunoprecipitation analysis of Arf1-EGFP, Arf1 Q71L -EGFP (constitutively active [CA]), Arf1 I46S I49S- EGFP binding mutant and Arf1 T31N -EGFP (dominant-negative [DN]). Arf1-EGFP constructs were pulled-down using GFP-trap magnetic beads. The eluted proteins were immunoblotted and Arf1 constructs were detected using polyclonal GFP antibodies. LRBA was detected by the polyclonal LRBA antibody. Wild-type and the constitutively active Arf1 interact with LRBA. This interaction is reduced with the Arf1 I46S I49S -EGFP and Arf1 T31N -EGFP DN mutant. (B) Quantification of immunoblots as shown on panel (A) from n = 6 biological replicates, one-way ANOVA using Tukey’s multiple comparison test, **P = 0.0040, *P = 0.0163. (C) Model of endosomal trafficking in the presence and in the absence of LRBA. Source data are available for this figure: .

    Techniques Used: Binding Assay, Mutagenesis, Dominant Negative Mutation, Construct, Magnetic Beads, Western Blot, Comparison



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    LRBA is endogenously expressed in HeLa cells and recruited to endosomes by Arfs. (A) Immunoblot analysis of LRBA presence in HeLa cells of two control and four LRBA KO clones using polyclonal LRBA antibody and α -tubulin as a loading control. (B) LRBA is localized at the perinuclear region and on vesicular structures in HeLa cells. Immunofluorescence analysis of endogenous LRBA in fixed HeLa cells. Scale bar, 10 μm, inlay 2 μm. (C) LRBA partly colocalizes with the cis-Golgi in HeLa cells. Immunofluorescence staining of endogenous LRBA and the cis-Golgi marker giantin in fixed HeLa cells. Squares show magnification of the perinuclear area. The labeling of the single channels represents the color of the channel on the merged image. Scale bar, 10 μm, inlays 2 μm. (D) LRBA does not colocalize with M6PR in HeLa cells. Immunofluorescence analysis of endogenous LRBA and endogenous M6PR colocalization in fixed HeLa cells. Squares show magnification of the perinuclear area. The labeling of the single channels represents the color of the channel on the merged image. Scale bar, 10 μm, inlays 2 μm. (E) Colocalization measurement of LRBA with giantin and M6PR. To measure LRBA colocalization with giantin one ROI at the perinuclear region was analyzed. To measure colocalization with M6PR, two ROIs per cell at the cell periphery were analyzed and the Pearson’s coefficient was measured using the JACoP plugin in Fiji. Mean and minimum to maximum are shown, box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. All data points are shown. Giantin = 60 cells, M6PR = 35 cells. (F) LRBA puncta disperse upon treatment with ArfGEF inhibitors. Live-cell imaging of 3xFlagEGFP-LRBA upon BFA (top panels) and GCA (lower panels) treatment for indicated timepoints. Scale bar, 10 μm. (G) Arf1 is recruited onto Rab4 + endosomes in the absence of LRBA. Control KO and LRBA KO HeLa cells were transfected with mCherry-Rab4 and <t>Arf1-EGFP</t> and cells were imaged live using a wide-field microscope at 37°C, 5% CO 2 atmosphere. Deconvolved images of single stacks are shown. Squares show magnification of the perinuclear area. The labeling of the single channels represents the color of the channel on the merged image. Scale bar, 10 μm, inlays 2 μm. (H and I) Colocalization measurement of Arf1-EGFP and mCherry-Rab4 in control and LRBA KO HeLa cells. Two ROIs per cell were analyzed and Mander’s coefficients were measured using the JACoP plugin in Fiji. Arf1 overlap with Rab4 (M1) is shown in H, Rab4 overlap with Arf1 (M2) is shown in I. All data points are shown. Ctr. KO clone1 = 32 cells, Ctr. KO clone2 = 39 cells, LRBA KO clone1 = 36 cells, LRBA KO clone 4 = 36 cells from n = 3 biological replicates; (H) one-way ANOVA using Tukey’s multiple comparison. (I) Kruskal–Wallis test using Dunn’s multiple comparisons test, **P = 0.0071, *P = 0.0201. (J) The amino acids isoleucine 46 and 49 of Arf1 and Arf3 were predicted to interact with LRBA. Both amino acids are conserved across species. The amino acid sequences of the yeast, C. elegans (CAEEL), Drosophila melanogaster (DROME), mouse and human Arf1 and human Arf3 were aligned. Labels: (*) conserved sequence; (:) conservative mutation; (.) semiconservative mutation; (−) gap. Sequence alignments were performed using Clustal Omega. (K) The amino acids leucine 861, arginine 910, and isoleucine 918 of LRBA were predicted to interact with Arf1 and Arf3. All three amino acids are conserved across species. The amino acid sequences of the C. elegans SEL-2 (SEL2-CAEEL), the mouse and the human LRBA, and the human neurobeachin (NBEA) were aligned. Labels: (*) conserved sequence; (:) conservative mutation; (.) semi-conservative mutation; (-) gap. Sequence alignments were performed using Clustal Omega. Source data are available for this figure: .
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    LRBA is endogenously expressed in HeLa cells and recruited to endosomes by Arfs. (A) Immunoblot analysis of LRBA presence in HeLa cells of two control and four LRBA KO clones using polyclonal LRBA antibody and α -tubulin as a loading control. (B) LRBA is localized at the perinuclear region and on vesicular structures in HeLa cells. Immunofluorescence analysis of endogenous LRBA in fixed HeLa cells. Scale bar, 10 μm, inlay 2 μm. (C) LRBA partly colocalizes with the cis-Golgi in HeLa cells. Immunofluorescence staining of endogenous LRBA and the cis-Golgi marker giantin in fixed HeLa cells. Squares show magnification of the perinuclear area. The labeling of the single channels represents the color of the channel on the merged image. Scale bar, 10 μm, inlays 2 μm. (D) LRBA does not colocalize with M6PR in HeLa cells. Immunofluorescence analysis of endogenous LRBA and endogenous M6PR colocalization in fixed HeLa cells. Squares show magnification of the perinuclear area. The labeling of the single channels represents the color of the channel on the merged image. Scale bar, 10 μm, inlays 2 μm. (E) Colocalization measurement of LRBA with giantin and M6PR. To measure LRBA colocalization with giantin one ROI at the perinuclear region was analyzed. To measure colocalization with M6PR, two ROIs per cell at the cell periphery were analyzed and the Pearson’s coefficient was measured using the JACoP plugin in Fiji. Mean and minimum to maximum are shown, box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. All data points are shown. Giantin = 60 cells, M6PR = 35 cells. (F) LRBA puncta disperse upon treatment with ArfGEF inhibitors. Live-cell imaging of 3xFlagEGFP-LRBA upon BFA (top panels) and GCA (lower panels) treatment for indicated timepoints. Scale bar, 10 μm. (G) Arf1 is recruited onto Rab4 + endosomes in the absence of LRBA. Control KO and LRBA KO HeLa cells were transfected with mCherry-Rab4 and Arf1-EGFP and cells were imaged live using a wide-field microscope at 37°C, 5% CO 2 atmosphere. Deconvolved images of single stacks are shown. Squares show magnification of the perinuclear area. The labeling of the single channels represents the color of the channel on the merged image. Scale bar, 10 μm, inlays 2 μm. (H and I) Colocalization measurement of Arf1-EGFP and mCherry-Rab4 in control and LRBA KO HeLa cells. Two ROIs per cell were analyzed and Mander’s coefficients were measured using the JACoP plugin in Fiji. Arf1 overlap with Rab4 (M1) is shown in H, Rab4 overlap with Arf1 (M2) is shown in I. All data points are shown. Ctr. KO clone1 = 32 cells, Ctr. KO clone2 = 39 cells, LRBA KO clone1 = 36 cells, LRBA KO clone 4 = 36 cells from n = 3 biological replicates; (H) one-way ANOVA using Tukey’s multiple comparison. (I) Kruskal–Wallis test using Dunn’s multiple comparisons test, **P = 0.0071, *P = 0.0201. (J) The amino acids isoleucine 46 and 49 of Arf1 and Arf3 were predicted to interact with LRBA. Both amino acids are conserved across species. The amino acid sequences of the yeast, C. elegans (CAEEL), Drosophila melanogaster (DROME), mouse and human Arf1 and human Arf3 were aligned. Labels: (*) conserved sequence; (:) conservative mutation; (.) semiconservative mutation; (−) gap. Sequence alignments were performed using Clustal Omega. (K) The amino acids leucine 861, arginine 910, and isoleucine 918 of LRBA were predicted to interact with Arf1 and Arf3. All three amino acids are conserved across species. The amino acid sequences of the C. elegans SEL-2 (SEL2-CAEEL), the mouse and the human LRBA, and the human neurobeachin (NBEA) were aligned. Labels: (*) conserved sequence; (:) conservative mutation; (.) semi-conservative mutation; (-) gap. Sequence alignments were performed using Clustal Omega. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: Arf1-dependent LRBA recruitment to Rab4 endosomes is required for endolysosome homeostasis

    doi: 10.1083/jcb.202401167

    Figure Lengend Snippet: LRBA is endogenously expressed in HeLa cells and recruited to endosomes by Arfs. (A) Immunoblot analysis of LRBA presence in HeLa cells of two control and four LRBA KO clones using polyclonal LRBA antibody and α -tubulin as a loading control. (B) LRBA is localized at the perinuclear region and on vesicular structures in HeLa cells. Immunofluorescence analysis of endogenous LRBA in fixed HeLa cells. Scale bar, 10 μm, inlay 2 μm. (C) LRBA partly colocalizes with the cis-Golgi in HeLa cells. Immunofluorescence staining of endogenous LRBA and the cis-Golgi marker giantin in fixed HeLa cells. Squares show magnification of the perinuclear area. The labeling of the single channels represents the color of the channel on the merged image. Scale bar, 10 μm, inlays 2 μm. (D) LRBA does not colocalize with M6PR in HeLa cells. Immunofluorescence analysis of endogenous LRBA and endogenous M6PR colocalization in fixed HeLa cells. Squares show magnification of the perinuclear area. The labeling of the single channels represents the color of the channel on the merged image. Scale bar, 10 μm, inlays 2 μm. (E) Colocalization measurement of LRBA with giantin and M6PR. To measure LRBA colocalization with giantin one ROI at the perinuclear region was analyzed. To measure colocalization with M6PR, two ROIs per cell at the cell periphery were analyzed and the Pearson’s coefficient was measured using the JACoP plugin in Fiji. Mean and minimum to maximum are shown, box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. All data points are shown. Giantin = 60 cells, M6PR = 35 cells. (F) LRBA puncta disperse upon treatment with ArfGEF inhibitors. Live-cell imaging of 3xFlagEGFP-LRBA upon BFA (top panels) and GCA (lower panels) treatment for indicated timepoints. Scale bar, 10 μm. (G) Arf1 is recruited onto Rab4 + endosomes in the absence of LRBA. Control KO and LRBA KO HeLa cells were transfected with mCherry-Rab4 and Arf1-EGFP and cells were imaged live using a wide-field microscope at 37°C, 5% CO 2 atmosphere. Deconvolved images of single stacks are shown. Squares show magnification of the perinuclear area. The labeling of the single channels represents the color of the channel on the merged image. Scale bar, 10 μm, inlays 2 μm. (H and I) Colocalization measurement of Arf1-EGFP and mCherry-Rab4 in control and LRBA KO HeLa cells. Two ROIs per cell were analyzed and Mander’s coefficients were measured using the JACoP plugin in Fiji. Arf1 overlap with Rab4 (M1) is shown in H, Rab4 overlap with Arf1 (M2) is shown in I. All data points are shown. Ctr. KO clone1 = 32 cells, Ctr. KO clone2 = 39 cells, LRBA KO clone1 = 36 cells, LRBA KO clone 4 = 36 cells from n = 3 biological replicates; (H) one-way ANOVA using Tukey’s multiple comparison. (I) Kruskal–Wallis test using Dunn’s multiple comparisons test, **P = 0.0071, *P = 0.0201. (J) The amino acids isoleucine 46 and 49 of Arf1 and Arf3 were predicted to interact with LRBA. Both amino acids are conserved across species. The amino acid sequences of the yeast, C. elegans (CAEEL), Drosophila melanogaster (DROME), mouse and human Arf1 and human Arf3 were aligned. Labels: (*) conserved sequence; (:) conservative mutation; (.) semiconservative mutation; (−) gap. Sequence alignments were performed using Clustal Omega. (K) The amino acids leucine 861, arginine 910, and isoleucine 918 of LRBA were predicted to interact with Arf1 and Arf3. All three amino acids are conserved across species. The amino acid sequences of the C. elegans SEL-2 (SEL2-CAEEL), the mouse and the human LRBA, and the human neurobeachin (NBEA) were aligned. Labels: (*) conserved sequence; (:) conservative mutation; (.) semi-conservative mutation; (-) gap. Sequence alignments were performed using Clustal Omega. Source data are available for this figure: .

    Article Snippet: Arf1 I46A, I49A -EGFP, Arf1 I46S, I49S -EGFP, and Arf1 I46A, I49A, N52A -EGFP mutants were generated in the pEGFP-N1-Arf1 (#39554; Addgene) using PCR KAPA HiFi DNA Polymerase and its HotStart ReadyMix.

    Techniques: Western Blot, Control, Clone Assay, Immunofluorescence, Staining, Marker, Labeling, Live Cell Imaging, Transfection, Microscopy, Comparison, Sequencing, Mutagenesis

    LRBA is recruited onto endosomes by Arf1 and Arf3. (A) Colocalization analysis of LRBA and TGN46 or Arf1 on endosomes in HeLa cells. For the colocalization analysis with the TGN, HeLa cells were fixed with 4% PFA and stained for endogenous TGN46 and LRBA. For colocalization analysis with Arf1, HeLa cells were transfected with ARF1-mCherry, fixed with 4% PFA and stained for endogenous LRBA. Squares show magnification of the perinuclear area. The labeling of the single channels represents the color of the channel on the merged image. Scale bar, 10 μm, inlays 2 μm. (B) Colocalization measurements of LRBA with TGN46 and Arf1 at the cell periphery. To measure LRBA colocalization with TGN46 one ROI per image, with Arf1 two ROIs per image were analyzed and the Pearson’s coefficient was measured using the JACoP plugin in Fiji. Mean and minimum to maximum are shown, the box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. All data points are shown. TGN46 (periphery) = 40 cells, Arf1 (periphery) = 35 cells from n = 3 biological replicates. (C) Immunoblot analysis of Arf1 and Arf3 expression in parental, ARF1 KO, ARF3 KO, and ARF1+3 dKO HeLa cells. Actin was used as a loading control. (D) LRBA is absent from endosomes in ARF1 and ARF3 dKO HeLa cells. Note that LRBA is still present on the Golgi. Parental, ARF1 KO, ARF3 KO, and ARF1+3 dKO HeLa cells were seeded on coverslips, fixed, and stained for endogenous LRBA. Maximum intensity projections of confocal images are shown. (E) The number of LRBA + endosomes in parental, ARF1 KO, ARF3 KO, and ARF1+3 dKO cells was measured using two ROIs per cell. Mean and minimum to maximum are shown, box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. All data points are shown. Parental = 95 cells, ARF1 KO = 49 cells, ARF3 KO = 32 cells, and Arf1+3 dKO = 34 cells were analyzed from n = 3 biological replicates; one-way ANOVA using Dunnett’s multiple comparison, **P = 0.0010 (parental versus ARF1 KO), **P = 0.0097 (parental versus ARF3 KO), ****P < 0.0001. (F) Arf1-EGFP re-expression rescues LRBA + endosomes absent in ARF1+3 dKO cells. Parental HeLa cells were transfected with Arf1-EGFP, and ARF1+3 dKO cells were transfected either with EGFP as a control or with Arf1-EGFP. Cells were then fixed and stained for endogenous LRBA and with Hoechst. Maximum intensity projection of confocal Z-stacks is shown. Rectangles show the magnified area in the upper right corner. Scale bar on inlays 2 µm. (G) The number of LRBA + puncta are counted based on data in F. Two ROIs at the cell periphery per cell are analyzed. Mean and minimum to maximum are shown, box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. All data points are shown. Parental = 49 cells, EGFP rescue = 42 cells, Arf1-EGFP rescue = 39 cells were analyzed from n = 3 biological replicates; Kruskal–Wallis test using Dunn’s multiple comparison, ****P < 0.0001. (H) (Endo)lysosomal structures are enlarged in ARF1+3 dKO cells. Parental and ARF1+3 dKO HeLa cells were seeded on coverslips, fixed, and stained for LAMP1 and with Hoechst. Maximum intensity projections of confocal Z-stacks. Squares show magnification of the (endo)lysosomes. Scale bar, 10 μm, inlays 2 μm. (I) Quantification of lysosome diameter based on images shown in H. The diameter of round lysosomes was measured manually in Fiji. Mean and minimum to maximum are shown, box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. Parental = 60 cells, ARF1+3 dKO = 64 cells were analyzed from n = 3 biological replicates. All data points are shown. Mann–Whitney test, ****P < 0.0001. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: Arf1-dependent LRBA recruitment to Rab4 endosomes is required for endolysosome homeostasis

    doi: 10.1083/jcb.202401167

    Figure Lengend Snippet: LRBA is recruited onto endosomes by Arf1 and Arf3. (A) Colocalization analysis of LRBA and TGN46 or Arf1 on endosomes in HeLa cells. For the colocalization analysis with the TGN, HeLa cells were fixed with 4% PFA and stained for endogenous TGN46 and LRBA. For colocalization analysis with Arf1, HeLa cells were transfected with ARF1-mCherry, fixed with 4% PFA and stained for endogenous LRBA. Squares show magnification of the perinuclear area. The labeling of the single channels represents the color of the channel on the merged image. Scale bar, 10 μm, inlays 2 μm. (B) Colocalization measurements of LRBA with TGN46 and Arf1 at the cell periphery. To measure LRBA colocalization with TGN46 one ROI per image, with Arf1 two ROIs per image were analyzed and the Pearson’s coefficient was measured using the JACoP plugin in Fiji. Mean and minimum to maximum are shown, the box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. All data points are shown. TGN46 (periphery) = 40 cells, Arf1 (periphery) = 35 cells from n = 3 biological replicates. (C) Immunoblot analysis of Arf1 and Arf3 expression in parental, ARF1 KO, ARF3 KO, and ARF1+3 dKO HeLa cells. Actin was used as a loading control. (D) LRBA is absent from endosomes in ARF1 and ARF3 dKO HeLa cells. Note that LRBA is still present on the Golgi. Parental, ARF1 KO, ARF3 KO, and ARF1+3 dKO HeLa cells were seeded on coverslips, fixed, and stained for endogenous LRBA. Maximum intensity projections of confocal images are shown. (E) The number of LRBA + endosomes in parental, ARF1 KO, ARF3 KO, and ARF1+3 dKO cells was measured using two ROIs per cell. Mean and minimum to maximum are shown, box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. All data points are shown. Parental = 95 cells, ARF1 KO = 49 cells, ARF3 KO = 32 cells, and Arf1+3 dKO = 34 cells were analyzed from n = 3 biological replicates; one-way ANOVA using Dunnett’s multiple comparison, **P = 0.0010 (parental versus ARF1 KO), **P = 0.0097 (parental versus ARF3 KO), ****P < 0.0001. (F) Arf1-EGFP re-expression rescues LRBA + endosomes absent in ARF1+3 dKO cells. Parental HeLa cells were transfected with Arf1-EGFP, and ARF1+3 dKO cells were transfected either with EGFP as a control or with Arf1-EGFP. Cells were then fixed and stained for endogenous LRBA and with Hoechst. Maximum intensity projection of confocal Z-stacks is shown. Rectangles show the magnified area in the upper right corner. Scale bar on inlays 2 µm. (G) The number of LRBA + puncta are counted based on data in F. Two ROIs at the cell periphery per cell are analyzed. Mean and minimum to maximum are shown, box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. All data points are shown. Parental = 49 cells, EGFP rescue = 42 cells, Arf1-EGFP rescue = 39 cells were analyzed from n = 3 biological replicates; Kruskal–Wallis test using Dunn’s multiple comparison, ****P < 0.0001. (H) (Endo)lysosomal structures are enlarged in ARF1+3 dKO cells. Parental and ARF1+3 dKO HeLa cells were seeded on coverslips, fixed, and stained for LAMP1 and with Hoechst. Maximum intensity projections of confocal Z-stacks. Squares show magnification of the (endo)lysosomes. Scale bar, 10 μm, inlays 2 μm. (I) Quantification of lysosome diameter based on images shown in H. The diameter of round lysosomes was measured manually in Fiji. Mean and minimum to maximum are shown, box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. Parental = 60 cells, ARF1+3 dKO = 64 cells were analyzed from n = 3 biological replicates. All data points are shown. Mann–Whitney test, ****P < 0.0001. Source data are available for this figure: .

    Article Snippet: Arf1 I46A, I49A -EGFP, Arf1 I46S, I49S -EGFP, and Arf1 I46A, I49A, N52A -EGFP mutants were generated in the pEGFP-N1-Arf1 (#39554; Addgene) using PCR KAPA HiFi DNA Polymerase and its HotStart ReadyMix.

    Techniques: Staining, Transfection, Labeling, Western Blot, Expressing, Control, Comparison, MANN-WHITNEY

    Structure of LRBA and potential interaction site with Arfs as predicted by Alphafold. (A and B) Alphafold monomer prediction of human LRBA structure. Model confidence values (A) and domains (B) are indicated with colors. (C) Predicted interaction sites between LRBA (amino acids 1–1373) and Arf1 (amino acids 1–181) using Alphafold multimer. PAE plot shows the confidence scores of the interaction. The LRBA–ARF1 model has PTM and iPTM scores of 0.583 and 0.504, respectively. (D) Schematic of Arf1/3 structure and its domains. The conserved amino acid isoleucine 49 of Arf1/3 is shown since most of our models indicated that it could potentially interact with LRBA. (E) The predicted LRBA interaction site of Arf1, isoleucine 46 and 49 are conserved among human Arf1, Arf3, Arf4, and Arf5. Sequence alignments were performed using Clustal Omega. Labels: (*) conserved sequence; (:) conservative mutation; (.) semi-conservative mutation; (-) gap. (F) Colocalization analysis of mutated Arf1-EGFP constructs with Rab4 + endosomes. ARF1+3dKO HeLa cells were transfected with mCherry-Rab4a and with Arf1-EGFP constructs. Confocal images of single focal planes. Scale bar, 10 μm, inlays 2 μm. (G and H) Colocalization measurement of Rab4 and Arf1 mutants determined by Mander’s coefficients. Arf1 I46A, I49A -EGFP and Arf1 I46S, I49S- EGFP are still efficiently recruited to Rab4 + endosomes. Arf1 I46A, I49A, N52A -EGFP recruitment to Rab4 endosomes is strongly reduced compared with wild-type Arf1-EGFP. Arf1-EGFP = 42 cells, Arf1 I46A, I49A -EGFP = 43 cells, Arf1 I46S, I49S- EGFP = 38 cells, and Arf1 I46A, I49A, N52A = 36 cells were analyzed from n = 3 biological replicates, one-way ANOVA using Tukey’s multiple comparison test; ****P < 0.0001. (I) Arf1 I46S, I49S- EGFP fails to rescue LRBA + puncta in ARF1+3 dKO cells. ARF1+3 dKO cells were transfected with EGFP, Arf1-EGFP, Arf1 I46A, I49A -EGFP, Arf1 I46S, I49S- EGFP or with Arf1 I46A, I49A, N52A -EGFP, fixed and stained for endogenous LRBA and with Hoechst. Scale bar, 10 μm, inlays 2 μm. (J) Quantification of the number of LRBA + puncta based on images as shown on panel I. Two ROIs at the cell periphery per cell were analyzed. Mean and minimum to maximum are shown, box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. All data points are shown. EGFP = 42 cells, Arf1-EGFP = 39 cells, Arf1 I46A, I49A -EGFP = 48 cells, Arf1 I46S, I49S- EGFP = 36 cells, Arf1 I46A, I49A, N52A = 49 cells were analyzed from n = 3 independent experiments; Kruskal–Wallis test using Dunn’s multiple comparison, ****P < 0.0001. EGFP and Arf1-EGFP measurements are identical to the one shown in as the data were obtained in the same experiment.

    Journal: The Journal of Cell Biology

    Article Title: Arf1-dependent LRBA recruitment to Rab4 endosomes is required for endolysosome homeostasis

    doi: 10.1083/jcb.202401167

    Figure Lengend Snippet: Structure of LRBA and potential interaction site with Arfs as predicted by Alphafold. (A and B) Alphafold monomer prediction of human LRBA structure. Model confidence values (A) and domains (B) are indicated with colors. (C) Predicted interaction sites between LRBA (amino acids 1–1373) and Arf1 (amino acids 1–181) using Alphafold multimer. PAE plot shows the confidence scores of the interaction. The LRBA–ARF1 model has PTM and iPTM scores of 0.583 and 0.504, respectively. (D) Schematic of Arf1/3 structure and its domains. The conserved amino acid isoleucine 49 of Arf1/3 is shown since most of our models indicated that it could potentially interact with LRBA. (E) The predicted LRBA interaction site of Arf1, isoleucine 46 and 49 are conserved among human Arf1, Arf3, Arf4, and Arf5. Sequence alignments were performed using Clustal Omega. Labels: (*) conserved sequence; (:) conservative mutation; (.) semi-conservative mutation; (-) gap. (F) Colocalization analysis of mutated Arf1-EGFP constructs with Rab4 + endosomes. ARF1+3dKO HeLa cells were transfected with mCherry-Rab4a and with Arf1-EGFP constructs. Confocal images of single focal planes. Scale bar, 10 μm, inlays 2 μm. (G and H) Colocalization measurement of Rab4 and Arf1 mutants determined by Mander’s coefficients. Arf1 I46A, I49A -EGFP and Arf1 I46S, I49S- EGFP are still efficiently recruited to Rab4 + endosomes. Arf1 I46A, I49A, N52A -EGFP recruitment to Rab4 endosomes is strongly reduced compared with wild-type Arf1-EGFP. Arf1-EGFP = 42 cells, Arf1 I46A, I49A -EGFP = 43 cells, Arf1 I46S, I49S- EGFP = 38 cells, and Arf1 I46A, I49A, N52A = 36 cells were analyzed from n = 3 biological replicates, one-way ANOVA using Tukey’s multiple comparison test; ****P < 0.0001. (I) Arf1 I46S, I49S- EGFP fails to rescue LRBA + puncta in ARF1+3 dKO cells. ARF1+3 dKO cells were transfected with EGFP, Arf1-EGFP, Arf1 I46A, I49A -EGFP, Arf1 I46S, I49S- EGFP or with Arf1 I46A, I49A, N52A -EGFP, fixed and stained for endogenous LRBA and with Hoechst. Scale bar, 10 μm, inlays 2 μm. (J) Quantification of the number of LRBA + puncta based on images as shown on panel I. Two ROIs at the cell periphery per cell were analyzed. Mean and minimum to maximum are shown, box ranges from the first (Q1–25th percentiles) to the third quartile (Q3–75th percentiles) of the distribution. All data points are shown. EGFP = 42 cells, Arf1-EGFP = 39 cells, Arf1 I46A, I49A -EGFP = 48 cells, Arf1 I46S, I49S- EGFP = 36 cells, Arf1 I46A, I49A, N52A = 49 cells were analyzed from n = 3 independent experiments; Kruskal–Wallis test using Dunn’s multiple comparison, ****P < 0.0001. EGFP and Arf1-EGFP measurements are identical to the one shown in as the data were obtained in the same experiment.

    Article Snippet: Arf1 I46A, I49A -EGFP, Arf1 I46S, I49S -EGFP, and Arf1 I46A, I49A, N52A -EGFP mutants were generated in the pEGFP-N1-Arf1 (#39554; Addgene) using PCR KAPA HiFi DNA Polymerase and its HotStart ReadyMix.

    Techniques: Sequencing, Mutagenesis, Construct, Transfection, Comparison, Staining

    Arf1 coimmunoprecipitates with LRBA. (A) Coimmunoprecipitation analysis of Arf1-EGFP, Arf1 Q71L -EGFP (constitutively active [CA]), Arf1 I46S I49S- EGFP binding mutant and Arf1 T31N -EGFP (dominant-negative [DN]). Arf1-EGFP constructs were pulled-down using GFP-trap magnetic beads. The eluted proteins were immunoblotted and Arf1 constructs were detected using polyclonal GFP antibodies. LRBA was detected by the polyclonal LRBA antibody. Wild-type and the constitutively active Arf1 interact with LRBA. This interaction is reduced with the Arf1 I46S I49S -EGFP and Arf1 T31N -EGFP DN mutant. (B) Quantification of immunoblots as shown on panel (A) from n = 6 biological replicates, one-way ANOVA using Tukey’s multiple comparison test, **P = 0.0040, *P = 0.0163. (C) Model of endosomal trafficking in the presence and in the absence of LRBA. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: Arf1-dependent LRBA recruitment to Rab4 endosomes is required for endolysosome homeostasis

    doi: 10.1083/jcb.202401167

    Figure Lengend Snippet: Arf1 coimmunoprecipitates with LRBA. (A) Coimmunoprecipitation analysis of Arf1-EGFP, Arf1 Q71L -EGFP (constitutively active [CA]), Arf1 I46S I49S- EGFP binding mutant and Arf1 T31N -EGFP (dominant-negative [DN]). Arf1-EGFP constructs were pulled-down using GFP-trap magnetic beads. The eluted proteins were immunoblotted and Arf1 constructs were detected using polyclonal GFP antibodies. LRBA was detected by the polyclonal LRBA antibody. Wild-type and the constitutively active Arf1 interact with LRBA. This interaction is reduced with the Arf1 I46S I49S -EGFP and Arf1 T31N -EGFP DN mutant. (B) Quantification of immunoblots as shown on panel (A) from n = 6 biological replicates, one-way ANOVA using Tukey’s multiple comparison test, **P = 0.0040, *P = 0.0163. (C) Model of endosomal trafficking in the presence and in the absence of LRBA. Source data are available for this figure: .

    Article Snippet: Arf1 I46A, I49A -EGFP, Arf1 I46S, I49S -EGFP, and Arf1 I46A, I49A, N52A -EGFP mutants were generated in the pEGFP-N1-Arf1 (#39554; Addgene) using PCR KAPA HiFi DNA Polymerase and its HotStart ReadyMix.

    Techniques: Binding Assay, Mutagenesis, Dominant Negative Mutation, Construct, Magnetic Beads, Western Blot, Comparison