arl8b gene locus  (Benchling Inc)

Journal: The Journal of Cell Biology

Article Title: Arl8b inactivates the Rab11a recycling pathway to promote LAMP1 sorting and lysosome biogenesis

doi: 10.1083/jcb.202509040

Figure Lengend Snippet: Increase in surface LAMP1 levels upon Arl8b depletion is independent of active lysosome exocytosis. (A) Lysates of HeLa, HEK293T, RPE-1, and THP-1 cells treated with control or Arl8b siRNA were IB with the indicated antibodies. The values indicate densitometric analysis of Arl8b levels normalized to α-tubulin. The bar graph represents the relative fold change in densitometric values of Arl8b normalized to α-tubulin. Statistical significance was calculated using the one-sample t test ( n = 3; ***P = 0.0005 (HeLa); ***P = 0.0002 (THP-1); ****P < 0.0001). The values are represented as the mean ± SEM. (B) Wild-type or Arl8b KO HeLa cell lysates were IB for the indicated proteins. The values represent densitometric analysis of Arl8b levels normalized to α-tubulin. (C) Graph represents the MFI of surface LAMP2 and surface EGFR levels upon Arl8b siRNA treatment and normalized to control siRNA ( n = 3). Statistical significance was calculated using the one-sample t test (n.s., non-significant). The values are represented as the mean ± SEM. (D) Representative histogram showing MFI of surface LAMP1 and LAMP2 in control or Arl8b siRNA-treated RPE-1, THP-1, and HEK293T cells as analyzed by flow cytometry. (E and F) Graphs represent the MFI of surface LAMP1 (E) ( n = 3; each dot represents a single experiment, *P = 0.0488; n.s., non-significant) and LAMP2 (F) ( n = 3; each dot represents a single experiment, *P = 0.0392; n.s., non-significant) upon Arl8b siRNA treatment and normalized to control siRNA. Statistical significance was calculated using the one-sample t test. The values are represented as the mean ± SEM. (G) Percentage of β-hexosaminidase release was quantified from the culture supernatant of HeLa cells treated with control or Arl8b siRNA upon ionomycin addition ( n = 3; each dot represents a single experiment). Statistical significance was calculated using the one-sample t test (**P = 0.0092, n.s., non-significant). The values are represented as the mean ± SEM. (H) Cathepsin D levels in lysates and culture supernatants of HeLa cells treated with control or Arl8b siRNA upon ionomycin addition were measured by immunoblotting, and amido black staining was performed to visualize proteins. The values represent densitometric analysis of cathepsin D levels normalized to α-tubulin (lysate) or total protein (supernatant). (I) Representative confocal micrographs from live-cell imaging of HeLa cells expressing SBP-GFP-LAMP1 and lysosomes labeled with the SiR-Lysosome probe (magenta). Representative inset images at 30 and 90 min after biotin addition are shown. The line profiles indicate fluorescence intensity of SBP-GFP-LAMP1 (green) and SiR-Lysosome probe (magenta) along the yellow line. Scale bar: 2 µm. (J) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with the SiR-Lysosome probe at different time points is shown from three independent experiments ( n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated using unpaired Student’s t test (**P = 0.0012). The values are represented as the mean ± SEM. (K) Confocal micrographs from live-cell imaging of HeLa cells co-expressing SBP-GFP-LAMP1 and CD63-RFP at 30 and 90 min after biotin addition are shown. The line profiles indicate fluorescence intensity of SBP-GFP-LAMP1 (green) and CD63-RFP (magenta) along the yellow line. Scale bar: 2 µm. (L) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with CD63-RFP at different time points is shown from three independent experiments ( n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated using unpaired Student’s t test (*P = 0.0182). The values are represented as the mean ± SEM. (M) Confocal micrographs of HeLa cells expressing SBP-GFP-LAMP1 and immunostained for LAMP2 (magenta) at 30 and 90 min after biotin addition are shown. The line profiles indicate fluorescence intensity of SBP-GFP-LAMP1 (green) and LAMP2 (magenta) along the yellow line. Scale bar: 2 µm. (N) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with LAMP2 at different time points is shown from three independent experiments ( n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated using unpaired Student’s t test (***P = 0.0003). The values are represented as the mean ± SEM. (O) HeLa cells expressing SBP-GFP-LAMP1 (Y404A) are labeled with LTR (magenta). Confocal micrographs of live-cell imaging are shown at different time points after biotin addition. Scale bar: 10 µm. (P) Graph indicates Manders’ colocalization coefficient quantification of SBP-GFP-LAMP1 (Y404A) with the LTR from three independent experiments ( n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated by one-way ANOVA with Dunnett’s multiple comparisons test (n.s., non-significant). The values are represented as the mean ± SEM. Source data are available for this figure: .

Article Snippet: Briefly, the guide RNA (gRNA) targeting the Arl8b gene locus was designed using the Benchling platform ( https://www.benchling.com ) and cloned into the SpCas9 pX330 (plasmid #42230; Addgene) vector ( ).

Techniques: Control, Flow Cytometry, Western Blot, Staining, Live Cell Imaging, Expressing, Labeling, Fluorescence, Single Cell

Journal: The Journal of Cell Biology

Article Title: Arl8b inactivates the Rab11a recycling pathway to promote LAMP1 sorting and lysosome biogenesis

doi: 10.1083/jcb.202509040

Figure Lengend Snippet: Arl8b depletion leads to an increase in surface LAMP1 levels and characterization of newly synthesized LAMP1 trafficking to active lysosomes. (A) Representative histogram showing MFI of surface LAMP1 in control or Arl8b siRNA-treated HeLa cells as analyzed by flow cytometry. (B) Bar graph represents the percent MFI of surface LAMP1 in HeLa cells treated with indicated siRNAs normalized to control siRNA from three independent experiments ( n = 3). Statistical significance was calculated using one-sample t test (*P = 0.0122). The values are represented as the mean ± SEM. (C) Representative histogram of wild-type (WT) or Arl8b KO HeLa cells showing MFI of surface LAMP1 and LAMP2 as analyzed by flow cytometry. (D) Bar graph represents the percent MFI of surface LAMP1 and LAMP2 in Arl8b KO cells normalized to WT cells from four independent experiments ( n = 4). Statistical significance was calculated using one-sample t test (**P = 0.0064 [LAMP1]; **P = 0.0095 [LAMP2]). The values are represented as the mean ± SEM. (E) Schematic depiction of the RUSH assay used to investigate the trafficking of newly synthesized LAMP1. ER hook streptavidin-KDEL, along with a reporter (LAMP1) fused with SBP-GFP, is expressed in HeLa cells. The KDEL hook enables retention of the reporter in the ER through interaction between streptavidin and SBP. Biotin addition allows the reporter to be released and subsequently trafficked via the secretory pathway. The schematic is created using BioRender. (F) Representative confocal micrographs from a live-cell imaging experiment of HeLa cells expressing the RUSH reporter (SBP-GFP-LAMP1) and labeled with LTR. The line profiles indicate fluorescence intensity along the yellow lines for both channels: SBP-GFP-LAMP1 (green) and LTR (magenta). Scale bars: 10 μm (main); 2 μm (inset). (G) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with LTR is shown from three independent experiments ( n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated using unpaired Student’s t test (**P = 0.0022). The values are represented as the mean ± SEM. (H) Representative live-cell time-lapse images of HeLa cells expressing SBP-GFP LAMP1 (green) and LTR (magenta) after 80 min of biotin addition. The yellow arrowheads indicate SBP-GFP-LAMP1– and LTR-positive vesicles undergoing kiss-and-run events. Scale bar: 2 µm. (I) Representative confocal micrographs of HeLa cells expressing SBP-GFP-LAMP1. Cells were incubated with mCherry-tagged anti-GFP nanobody at the time of biotin addition, followed by fixation at the indicated time points. Scale bars: 10 μm (main); 2 μm (inset). (J) PCC of SBP-GFP-LAMP1 with mCherry-tagged anti-GFP nanobody at different time points of biotin addition from three independent experiments ( n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated by one-way ANOVA with Dunnett’s multiple comparisons test (****P < 0.0001). The values are represented as the mean ± SEM. MFI, mean fluorescence intensity; PCC, pearson's correlation coefficient.

Article Snippet: Briefly, the guide RNA (gRNA) targeting the Arl8b gene locus was designed using the Benchling platform ( https://www.benchling.com ) and cloned into the SpCas9 pX330 (plasmid #42230; Addgene) vector ( ).

Techniques: Synthesized, Control, Flow Cytometry, Live Cell Imaging, Expressing, Labeling, Fluorescence, Single Cell, Incubation

Journal: The Journal of Cell Biology

Article Title: Arl8b inactivates the Rab11a recycling pathway to promote LAMP1 sorting and lysosome biogenesis

doi: 10.1083/jcb.202509040

Figure Lengend Snippet: Arl8b localizes to the newly synthesized post-endocytic LAMP1 vesicles. (A) Lysates of WT and Arl8b-KI HeLa cells were IB with indicated antibodies. (B) Representative confocal micrographs from a live-cell imaging experiment of HeLa cells expressing the RUSH reporter (SBP-mCherry-LAMP1) and labeled with LTR. Scale bars: 10 μm (main); 2 μm (inset). (C) Quantification of Manders’ colocalization coefficient of SBP-mCherry-LAMP1 with LTR is shown from three independent experiments ( n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated using unpaired Student’s t test (****P < 0.0001). The values are represented as the mean ± SEM. (D) Representative images of HeLa cells co-expressing SBP-GFP-LAMP1 (green) and Arl8b-Halo (magenta), followed by live-cell imaging. Cells were incubated with biotin, and image insets for indicated time points are shown. The box with vertical lines represents blank space. Scale bars: 10 μm (main); 2 μm (inset). (E) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with Arl8b-Halo is shown from three independent experiments ( n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated by one-way ANOVA with Dunnett’s multiple comparisons test (*P = 0.0114; ***P = 0.0007; n.s., non-significant). The values are represented as the mean ± SEM. (F) Representative images of HeLa cells co-expressing SBP-GFP-LAMP1 (Y404A) (green) and Arl8b-Halo (magenta), followed by live-cell imaging. Cells were incubated with biotin, and image insets for indicated time points are shown. The black box with vertical lines represents blank space. Scale bars: 10 μm (main); 2 μm (inset). (G) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 (Y404A) with Arl8b-Halo at the indicated time points is shown from three independent experiments ( n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated by one-way ANOVA with Dunnett’s multiple comparisons test (n.s., non-significant). The values are represented as the mean ± SEM. (H) HeLa cell lysates treated with control or Vps33a siRNAs were IB for the indicated proteins. (I) HeLa cells treated with the control or Arl8b siRNA and expressing SBP-GFP-LAMP1 were imaged after 30 min of biotin addition along with pitstop2 (30 µM). The trafficking of newly synthesized RUSH-LAMP1 from the TGN to the plasma membrane was visualized by surface staining with an anti-GFP antibody, followed by labeling with Alexa Fluor 568–conjugated secondary antibody. Scale bar: 10 µm. (J) Graph represents the relative ratio of surface signal (measured by an anti-GFP antibody) to total GFP signal of SBP-GFP-LAMP1 in HeLa cells treated with indicated siRNAs ( n = 3; each dot represents a single experiment). Statistical significance was calculated using the one-sample t test (n.s., non-significant). The values are represented as the mean ± SEM. Source data are available for this figure: .

Article Snippet: Briefly, the guide RNA (gRNA) targeting the Arl8b gene locus was designed using the Benchling platform ( https://www.benchling.com ) and cloned into the SpCas9 pX330 (plasmid #42230; Addgene) vector ( ).

Techniques: Synthesized, Live Cell Imaging, Expressing, Labeling, Single Cell, Incubation, Control, Clinical Proteomics, Membrane, Staining

Journal: The Journal of Cell Biology

Article Title: Arl8b inactivates the Rab11a recycling pathway to promote LAMP1 sorting and lysosome biogenesis

doi: 10.1083/jcb.202509040

Figure Lengend Snippet: Arl8b localizes to the newly synthesized post-endocytic RUSH-LAMP1 vesicles prior to fusion with active lysosomes. (A) Representative confocal images of Arl8b EN -mStayGold KI HeLa cells expressing SBP-mCherry-LAMP1. Live-cell imaging was performed after biotin addition, and representative images for indicated time points are shown. Images are maximum-intensity projections of z-stack micrographs. The line profiles indicate fluorescence intensity along the white lines for both channels: SBP-mCherry-LAMP1 (red) and Arl8b EN -mStayGold (green). Scale bars: 10 μm (main); 2 μm (inset). (B) Quantification of Manders’ colocalization coefficient of SBP-mCherry-LAMP1 with Arl8b EN -mStayGold at the indicated time points is shown from three independent experiments ( n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated by one-way ANOVA with Dunnett’s multiple comparisons test (***P = 0.0006; ****P < 0.0001). The values are represented as the mean ± SEM. (C) Representative confocal micrographs of HeLa cells treated with control or Vps33a siRNA, followed by co-expression of SBP-GFP-LAMP1 (green) and Arl8b-Halo (red). Live-cell imaging was performed after cells were incubated with LTR (blue) dye to label acidic compartments, followed by biotin addition. Blue arrowheads indicate SBP-GFP-LAMP1– and Arl8b-Halo–positive vesicles. The line profiles indicate fluorescence intensity along the blue lines for all channels: SBP-GFP-LAMP1 (green), Arl8b-Halo (red), and LTR (blue). Scale bars: 10 μm (main); 2 μm (inset). (D) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with LTR in HeLa cells treated with the indicated siRNAs is shown from three independent experiments ( n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated using unpaired Student’s t test (***P = 0.0003). The values are represented as the mean ± SEM. (E) Quantification of Manders’ colocalization coefficient for SBP-GFP-LAMP1 with Arl8b-Halo in HeLa cells treated with the control or Vps33a siRNA is shown from three independent experiments ( n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated using unpaired Student’s t test (n.s., non-significant). The values are represented as the mean ± SEM.

Article Snippet: Briefly, the guide RNA (gRNA) targeting the Arl8b gene locus was designed using the Benchling platform ( https://www.benchling.com ) and cloned into the SpCas9 pX330 (plasmid #42230; Addgene) vector ( ).

Techniques: Synthesized, Expressing, Live Cell Imaging, Fluorescence, Single Cell, Control, Incubation

Journal: The Journal of Cell Biology

Article Title: Arl8b inactivates the Rab11a recycling pathway to promote LAMP1 sorting and lysosome biogenesis

doi: 10.1083/jcb.202509040

Figure Lengend Snippet: Time-lapse imaging of HeLa cells co-expressing SBP-GFP-LAMP1 and Arl8b-Halo. The cells were incubated with biotin, and the route of SBP-GFP-LAMP1 trafficking was followed from 40 to 103 min after biotin addition. The video is captured at 2.22 frame/sec with a 3-min time interval between the frames. The movie is shown at 2 frame/sec, and the total number of frames displayed is 22.

Article Snippet: Briefly, the guide RNA (gRNA) targeting the Arl8b gene locus was designed using the Benchling platform ( https://www.benchling.com ) and cloned into the SpCas9 pX330 (plasmid #42230; Addgene) vector ( ).

Techniques: Imaging, Expressing, Incubation

Journal: The Journal of Cell Biology

Article Title: Arl8b inactivates the Rab11a recycling pathway to promote LAMP1 sorting and lysosome biogenesis

doi: 10.1083/jcb.202509040

Figure Lengend Snippet: Arl8b depletion enhances recycling of the newly synthesized LAMP1 to the plasma membrane, impairing its lysosomal delivery. (A) Representative confocal micrographs of HeLa cells treated with control or Arl8b siRNA, transfected with SBP-GFP-LAMP1, followed by labeling with LTR (magenta). Images are taken at indicated time points after biotin addition. Scale bars: 10 µm (main); 2 μm (inset). (B) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with LTR in HeLa cells treated with indicated siRNAs is shown from three independent experiments ( n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated using unpaired Student’s t test (***P = 0.0006). The values are represented as the mean ± SEM. (C) Schematic representation of the recycling assay showing endocytosis of anti-GFP antibody–labeled SBP-GFP-LAMP1 after 30 min of biotin addition, followed by acid wash to remove non-internalized anti-GFP antibody from the cell surface. Following this, pitstop2 (30 µM) was added to block endocytosis, and cells were incubated for 30 min to allow recycling of internalized anti-GFP antibody–bound SBP-GFP-LAMP1 back to the cell surface. Cells were fixed, and the surface staining was performed using an Alexa Fluor 568–conjugated secondary antibody without permeabilization. The schematic is created using BioRender. (D) Recycling assay was performed in HeLa cells treated with control or Arl8b siRNA, and representative micrographs depicting staining of surface-recycled SBP-GFP-LAMP1 are shown. Scale bar: 10 µm. (E) Graph represents the relative ratio of surface-recycled signal intensity (measured by anti-GFP antibody) to the total intensity of SBP-GFP-LAMP1 (total GFP signal) ( n = 3; each dot represents a single experiment). Statistical significance was calculated using the one-sample t test (**P = 0.0048). The values are represented as the mean ± SEM.

Article Snippet: Briefly, the guide RNA (gRNA) targeting the Arl8b gene locus was designed using the Benchling platform ( https://www.benchling.com ) and cloned into the SpCas9 pX330 (plasmid #42230; Addgene) vector ( ).

Techniques: Synthesized, Clinical Proteomics, Membrane, Control, Transfection, Labeling, Single Cell, Blocking Assay, Incubation, Staining

Journal: The Journal of Cell Biology

Article Title: Arl8b inactivates the Rab11a recycling pathway to promote LAMP1 sorting and lysosome biogenesis

doi: 10.1083/jcb.202509040

Figure Lengend Snippet: Time-lapse imaging of control and Arl8b siRNA-treated HeLa cells expressing SBP-GFP-LAMP1 and incubated with LTR to label active lysosomes. The cells were incubated with biotin, and the route of SBP-GFP-LAMP1 trafficking was followed after 90 min of biotin addition. The control and Arl8b siRNA videos are captured at 2.44 frames/sec with no time interval between the frames. The movies are shown at 2 frames/sec, and the total number of frames displayed is 25.

Article Snippet: Briefly, the guide RNA (gRNA) targeting the Arl8b gene locus was designed using the Benchling platform ( https://www.benchling.com ) and cloned into the SpCas9 pX330 (plasmid #42230; Addgene) vector ( ).

Techniques: Imaging, Control, Expressing, Incubation

Journal: The Journal of Cell Biology

Article Title: Arl8b inactivates the Rab11a recycling pathway to promote LAMP1 sorting and lysosome biogenesis

doi: 10.1083/jcb.202509040

Figure Lengend Snippet: Arl8b depletion results in LAMP1 missorting to Rab11a-positive REs. (A) Representative confocal images of HeLa cells expressing SBP-GFP-LAMP1 (green) and stained for endogenous AP-3 (magenta) at the indicated time points after biotin addition. The line profiles indicate fluorescence intensity along the yellow lines for both channels: SBP-GFP-LAMP1 (green) and AP-3 (magenta). Scale bars: 10 μm (main); 2 μm (inset). (B) Manders’ colocalization coefficient quantification of SBP-GFP-LAMP1 with AP-3 at the indicated time points after biotin addition is shown from three independent experiments ( n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated by one-way ANOVA with Dunnett’s multiple comparisons test (*P = 0.0146; n.s., non-significant). The values are represented as the mean ± SEM. (C) Representative confocal micrographs of HeLa cells treated with control or Arl8b siRNA, followed by the expression of SBP-GFP-LAMP1 (green). Cells were fixed at 60 min after biotin addition and immunostained for endogenous AP-3 (magenta). The line profiles indicate fluorescence intensity along the yellow lines for both channels: SBP-GFP-LAMP1 (green) and AP-3 (magenta). Scale bars: 10 μm (main); 2 μm (inset). (D) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with AP-3 in HeLa cells treated with the indicated siRNAs is shown from three independent experiments ( n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated using unpaired Student’s t test (**P = 0.0088). The values are represented as the mean ± SEM. (E) Representative confocal images of HeLa cells treated with control or Arl8b siRNA, followed by the expression of SBP-GFP-LAMP1 (green). Cells were fixed at 60 min after biotin addition and immunostained for endogenous Rab11a (magenta). The line profiles indicate fluorescence intensity along the yellow lines for both channels: SBP-GFP-LAMP1 (green) and Rab11a (magenta). Scale bars: 10 μm (main); 2 μm (inset). (F) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with Rab11a or Rab14 in HeLa cells treated with the indicated siRNAs is shown from three independent experiments ( n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated using unpaired Student’s t test (*P = 0.0237; **P = 0.0028). The values are represented as the mean ± SEM. (G) HeLa cells stably expressing 3x-FLAG-TFR1 were treated with control or Arl8b siRNA, and the lysates were subjected to anti-FLAG IP. The precipitates were IB with the indicated antibodies. (H) Graph represents the relative fold change in densitometric values of the indicated proteins normalized to input and direct IP of 3x-FLAG-TFR1. Statistical significance was calculated using the one-sample t test ( n = 4; *P = 0.0108; n.s., non-significant). The values are represented as the mean ± SEM. (I) Bar graph represents the percent MFI of surface LAMP1 in HeLa cells treated with the indicated siRNAs and normalized to control siRNA ( n = 3; each dot represents a single experiment). Statistical significance was calculated using the one-sample t test (**P = 0.0061; n.s., non-significant). The values are represented as the mean ± SEM. IP, immunoprecipitation; IB, immunoblotted. Source data are available for this figure: .

Article Snippet: Briefly, the guide RNA (gRNA) targeting the Arl8b gene locus was designed using the Benchling platform ( https://www.benchling.com ) and cloned into the SpCas9 pX330 (plasmid #42230; Addgene) vector ( ).

Techniques: Expressing, Staining, Fluorescence, Single Cell, Control, Stable Transfection, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: Arl8b inactivates the Rab11a recycling pathway to promote LAMP1 sorting and lysosome biogenesis

doi: 10.1083/jcb.202509040

Figure Lengend Snippet: Arl8b-positive vesicles transiently interact with AP-3–positive endosomes, which represent the sorting station for the newly synthesized LAMP1 to active lysosomes. (A) HeLa cell lysates treated with control or AP-3 siRNA were IB for the indicated proteins. The values represent densitometric analysis of AP-3 levels normalized to α-tubulin. (B) Representative confocal micrographs of HeLa cells treated with control or AP-3 siRNA, followed by the expression of SBP-GFP-LAMP1 (green). Live-cell imaging was performed after cells were incubated with LTR (magenta) dye to label acidic lysosomes, followed by biotin addition. Yellow arrowheads in the insets denote the colocalized pixels. Scale bars: 10 μm (main); 2 μm (inset). (C) Manders’ colocalization coefficient quantification of SBP-GFP-LAMP1 with LTR in HeLa cells treated with control or AP-3 siRNA is shown from three independent experiments ( n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated using unpaired Student’s t test (****P < 0.0001). The values are represented as the mean ± SEM. (D) Bar graph represents the percent MFI of surface LAMP1 in HeLa cells treated with the indicated siRNAs and normalized to control siRNA ( n = 3; each dot represents a single experiment). Statistical significance was calculated using the one-sample t test (**P = 0.0050). The values are represented as the mean ± SEM. (E) Representative confocal images of HeLa cells treated with control or AP-3 siRNA, followed by co-expression of SBP-GFP-LAMP1 (green) and Arl8b-Halo (magenta). Cells were incubated with biotin, and live-cell imaging was performed. Scale bars: 10 μm (main); 2 μm (inset). (F) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with Arl8b-Halo in HeLa cells treated with the indicated siRNAs is shown from three independent experiments ( n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated using unpaired Student’s t test (n.s., non-significant). The values are represented as the mean ± SEM. ( G) Representative confocal micrographs of Arl8b EN -mStayGold KI (green) HeLa cells immunostained for endogenous AP-3 (magenta). Yellow arrowheads in the insets denote the colocalized pixels. The line profiles indicate fluorescence intensity along the yellow lines for both channels: Arl8b EN -mStayGold (green) and AP-3 (magenta). Scale bars: 10 μm (main); 2 μm (inset). (H) Quantification of Manders’ colocalization coefficient (M1 and M2) of Arl8b EN -mStayGold with AP-3 in HeLa cells is shown from three independent experiments ( n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. The values are represented as the mean ± SEM. (I) Representative confocal micrograph from a live-cell imaging experiment of HeLa cells co-expressing Arl8b-GFP (green) and AP-3 (M1)-mScarlet (magenta); insets show time-lapse images. Micrographs are maximum-intensity projections of z-stack images from a live-cell video. Yellow arrowheads in the insets denote the colocalized pixels. The line profiles indicate fluorescence intensity along the yellow lines for both channels: Arl8b-GFP (green) and AP-3 (M1)-mScarlet (magenta). Scale bars: 10 μm (main); 2 μm (inset). (J) Representative confocal micrographs of HeLa cells immunostained for endogenous AP-3 (magenta) and Rab14 (green) (left panel). Representative confocal images of HeLa cells expressing RUFY1-FLAG were immunostained with anti-FLAG (green) and anti-AP-3 antibodies (magenta) (right panel). Yellow arrowheads in the insets denote the colocalized pixels. Scale bars: 10 μm (main); 2 μm (inset). (K) Quantification of Manders’ colocalization coefficient of AP-3 with Rab14 and RUFY1-FLAG, respectively, from three independent experiments ( n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. The values are represented as the mean ± SEM. (L) Representative confocal images of HeLa cells treated with control or Arl8b siRNA, followed by the expression of SBP-GFP-LAMP1 (green). Cells were fixed at 60 min after biotin addition and immunostained for endogenous Rab14 (magenta). Scale bars: 10 μm (main); 2 μm (inset). (M) HeLa cell lysates treated with the control or Rab11a siRNAs were IB for the indicated proteins. The values represent densitometric analysis of Rab11a levels normalized to α-tubulin. Source data are available for this figure: .

Article Snippet: Briefly, the guide RNA (gRNA) targeting the Arl8b gene locus was designed using the Benchling platform ( https://www.benchling.com ) and cloned into the SpCas9 pX330 (plasmid #42230; Addgene) vector ( ).

Techniques: Synthesized, Control, Expressing, Live Cell Imaging, Incubation, Single Cell, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Arl8b inactivates the Rab11a recycling pathway to promote LAMP1 sorting and lysosome biogenesis

doi: 10.1083/jcb.202509040

Figure Lengend Snippet: Time-lapse imaging of HeLa cells co-expressing AP-3 (M1)-mScarlet (magenta) and Arl8b-GFP (green). The video is captured at 1.34 frames/sec with no time interval between the frames. The video is the maximum-intensity projection of three z-stacks captured. The movies (whole cell and marked inset) are shown at 4 frames/sec, and the total number of frames displayed is 50 for each. The yellow arrows in the inset movie depict the Arl8b-positive vesicles docking and interacting with AP-3–positive endosomes.

Article Snippet: Briefly, the guide RNA (gRNA) targeting the Arl8b gene locus was designed using the Benchling platform ( https://www.benchling.com ) and cloned into the SpCas9 pX330 (plasmid #42230; Addgene) vector ( ).

Techniques: Imaging, Expressing

Journal: The Journal of Cell Biology

Article Title: Arl8b inactivates the Rab11a recycling pathway to promote LAMP1 sorting and lysosome biogenesis

doi: 10.1083/jcb.202509040

Figure Lengend Snippet: TBC1D9A is a Rab11a GAP and interacts with Arl8b via its N-terminal region encompassing the GRAM1 domain. (A) Yeast two-hybrid assay to detect the interaction between TBC1D9B (WT and RYQ →A mutant) and different forms of Arl8b. Co-transformants were spotted on -Leu/-Trp and -Leu/-Trp/-His media to confirm viability and interactions, respectively. (B) Percentage of cells showing relocalization of GFP or GFP-TBC1D9B (WT and its mutants) to mitochondria with HA-tagged Arl8b (Q75L)-MitoID ( n = 3; each dot represents a single experiment). The values are represented as the mean ± SEM. (C) Structural model of interactions between Arl8b and the N-terminal fragment of TBC1D9B (1–410 residues) was generated using the AlphaFold3 tool and visualized using Chimera software. The cyan chain denotes Arl8b, and the orange chain indicates TBC1D9B (1–410 residues). (D) Yeast two-hybrid assay to detect interaction of TBC1D9B (WT or indicated mutants) with Arl8b (Q75L). Co-transformants were spotted on -Leu/-Trp and -Leu/-Trp/-His media to confirm viability and interactions, respectively. In the assay, LC3B was used as a positive control for binding to TBC1D9B. (E) ConSurf server tool was used to predict the evolutionary conservation score (represented by a color-coded scale) of Arl8b binding–defective mutants of TBC1D9B (highlighted in blue) predicted by the AlphaFold3 server. (F) Kinetic analysis of GTP hydrolysis activity of Rab11a in the presence of TBC1D9A (WT or RYQ→A) or TBC1D9B (WT or RYQ→A) was performed by measuring the release of free inorganic phosphate (P i ) using malachite green reagent. Absorbance was recorded at 650 nm, and plotted values were calculated by subtracting the signal of GDP-loaded samples from GTP-loaded samples. (G) Yeast two-hybrid assay was performed to check the interaction of Arl8b (Q75L) and LC3B with TBC1D9A (WT or E95A). The co-transformants were spotted on -Leu/-Trp and -Leu/-Trp/-His media to confirm viability and interactions, respectively. (H) Endogenous immunoprecipitation was performed by incubating HEK293T cell lysates with an anti-Arl8 antibody, followed by immunoblotting with the indicated antibodies. Source data are available for this figure: .

Article Snippet: Briefly, the guide RNA (gRNA) targeting the Arl8b gene locus was designed using the Benchling platform ( https://www.benchling.com ) and cloned into the SpCas9 pX330 (plasmid #42230; Addgene) vector ( ).

Techniques: Y2H Assay, Mutagenesis, Generated, Software, Positive Control, Binding Assay, Activity Assay, Immunoprecipitation, Western Blot

Journal: The Journal of Cell Biology

Article Title: Arl8b inactivates the Rab11a recycling pathway to promote LAMP1 sorting and lysosome biogenesis

doi: 10.1083/jcb.202509040

Figure Lengend Snippet: TBC1D9B interacts with Arl8b via its N-terminal region encompassing the GRAM1 domain. (A) GST and GST-Arl8b proteins were immobilized on glutathione resins and loaded with GTP or GDP, followed by incubation with HEK293T cell lysates. The precipitates were IB with anti-TBC1D9B antibody, and Ponceau S staining was done to visualize purified proteins. (B) WT and Arl8b KO HeLa cell lysates were subjected to endogenous IP using anti-Arl8b antibody, followed by IB with the indicated antibodies. (C) Domain architecture of TBC1D9B and its domain deletion mutants showing N-terminal GRAM1 and GRAM2 domains, TBC domain, and C-terminal EF hand. The image was created using IBS 2.0: Illustrator for Biological Sequences. (D) GST and GST-Arl8b (Q75L) proteins were immobilized on glutathione resins, followed by incubation with HEK293T cell lysates expressing GFP-tagged TBC1D9B or its domain deletion mutants. The precipitates were IB with anti-GFP antibody, and Ponceau S staining was done to visualize purified proteins. (E) HEK293T cell lysates expressing the indicated proteins were immunoprecipitated with anti-HA antibody and IB with the indicated antibodies. The values represent densitometric analysis of indicated proteins normalized to input and direct IP of Arl8b (Q75L)-HA. (F) HeLa cells were co-transfected with GFP alone or GFP-TBC1D9B (WT, 1–270 aa, or E91A) with Arl8b (Q75L or T34N)-MitoID (HA-tagged) and immunostained with an anti-HA (magenta) antibody. Scale bar: 10 µm. Source data are available for this figure: .

Article Snippet: Briefly, the guide RNA (gRNA) targeting the Arl8b gene locus was designed using the Benchling platform ( https://www.benchling.com ) and cloned into the SpCas9 pX330 (plasmid #42230; Addgene) vector ( ).

Techniques: Incubation, Staining, Purification, Expressing, Immunoprecipitation, Transfection

Journal: The Journal of Cell Biology

Article Title: Arl8b inactivates the Rab11a recycling pathway to promote LAMP1 sorting and lysosome biogenesis

doi: 10.1083/jcb.202509040

Figure Lengend Snippet: TBC1D9B localizes on peripheral non-acidic LAMP1-positive vesicles in an Arl8b-dependent manner. (A) Representative confocal micrographs of HeLa cells co-transfected with GFP-TBC1D9B (WT, E91A, or 1–270 aa) with Arl8b (WT, Q75L, or T34N)-Halo, followed by live-cell imaging. Micrographs are maximum-intensity projections of z-stack images from live-cell video. The line profiles indicate fluorescence intensity of GFP-TBC1D9B (green) and Arl8b-Halo (magenta) along the yellow line. Scale bars: 10 µm (main); 2 µm (inset). (B) Quantification of PCC of GFP-TBC1D9B with Arl8b-Halo is shown from three independent experiments ( n = 3). Colors in the SuperPlots indicate GFP-TBC1D9B (WT, E91A, or 1–270 aa), with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparisons test (****P < 0.0001). The values are represented as the mean ± SEM. (C) Quantification of Manders’ colocalization coefficient of GFP-TBC1D9B with Arl8b-Halo is shown from three independent experiments ( n = 3). Colors in the SuperPlots indicate GFP-TBC1D9B (WT, E91A, or 1–270 aa), with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparisons test (****P < 0.0001). The values are represented as the mean ± SEM. (D) Representative images of HeLa cells co-expressing GFP-TBC1D9B and LAMP1-RFP (upper image)/LTR (lower image) with Arl8b-HA (not shown). The line profiles indicate fluorescence intensity of GFP-TBC1D9B (green) and LAMP1-RFP or LTR (magenta) along the yellow line. Scale bars: 10 µm (main); 2 µm (inset). (E) Quantification of Manders’ colocalization coefficient of GFP-TBC1D9B with indicated markers is shown from three independent experiments ( n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. The values are represented as the mean ± SEM. (F) HEK293T cells stably expressing TMEM192-2x-FLAG were treated with control or Arl8b siRNA, and lysates were subjected to anti-FLAG IP. The precipitates were IB with the indicated antibodies. (G) Bar graph represents the relative fold change in densitometric values of the indicated protein normalized to input and direct IP of TMEM192-2x-FLAG. Statistical significance was calculated using the one-sample t test ( n = 3; **P = 0.0054; n.s., non-significant). The values are represented as the mean ± SEM. (H) Representative time-lapse confocal micrograph of a HeLa cell, co-expressing GFP-TBC1D9B (green), RUFY1-Halo (yellow), and Arl8b-DsRed (magenta). Micrographs are maximum-intensity projections of z-stack images from a live-cell video. The line profiles indicate fluorescence intensity along the blue lines for all channels: GFP-TBC1D9B (green), RUFY1-Halo (yellow), and Arl8b-DsRed (magenta). Scale bars: 10 µm (main); 2 µm (inset). Source data are available for this figure: .

Article Snippet: Briefly, the guide RNA (gRNA) targeting the Arl8b gene locus was designed using the Benchling platform ( https://www.benchling.com ) and cloned into the SpCas9 pX330 (plasmid #42230; Addgene) vector ( ).

Techniques: Transfection, Live Cell Imaging, Fluorescence, Single Cell, Expressing, Stable Transfection, Control

Journal: The Journal of Cell Biology

Article Title: Arl8b inactivates the Rab11a recycling pathway to promote LAMP1 sorting and lysosome biogenesis

doi: 10.1083/jcb.202509040

Figure Lengend Snippet: Time-lapse imaging of HeLa cells expressing Arl8b-HA (not stained) and GFP-TBC1D9B and either transfected with LAMP1-RFP or incubated with LTR to label active lysosomes. The videos are captured at 2.04 frames/sec with no time interval between the frames. The movies are shown at 4 frames/sec, and the total number of frames displayed is 25 each.

Article Snippet: Briefly, the guide RNA (gRNA) targeting the Arl8b gene locus was designed using the Benchling platform ( https://www.benchling.com ) and cloned into the SpCas9 pX330 (plasmid #42230; Addgene) vector ( ).

Techniques: Imaging, Expressing, Staining, Transfection, Incubation

Journal: The Journal of Cell Biology

Article Title: Arl8b inactivates the Rab11a recycling pathway to promote LAMP1 sorting and lysosome biogenesis

doi: 10.1083/jcb.202509040

Figure Lengend Snippet: Time-lapse imaging of HeLa cells expressing GFP-TBC1D9B (green), Arl8b-DsRed (magenta), and either RUFY1-Halo or Halo-Rab14 (yellow). The videos are captured at 1 frame/sec with no time interval between the frames. The movies are shown at 4 frames/sec, and the total number of frames displayed is 25 each.

Article Snippet: Briefly, the guide RNA (gRNA) targeting the Arl8b gene locus was designed using the Benchling platform ( https://www.benchling.com ) and cloned into the SpCas9 pX330 (plasmid #42230; Addgene) vector ( ).

Techniques: Imaging, Expressing

Journal: The Journal of Cell Biology

Article Title: Arl8b inactivates the Rab11a recycling pathway to promote LAMP1 sorting and lysosome biogenesis

doi: 10.1083/jcb.202509040

Figure Lengend Snippet: Proposed role of Arl8b and its effector, TBC1D9B, in trafficking of the newly synthesized LAMP1 to active lysosomes. Delivery of newly synthesized LAMP1 to active lysosomes follows an indirect trafficking pathway, wherein it exits the TGN in tubular carriers, followed by delivery to the plasma membrane. After endocytosis, LAMP1 is sorted from the AP-3–positive compartment in an Arl8b-dependent manner. Arl8b recruits the Rab11a GAP, TBC1D9B, which inactivates Rab11a to prevent recycling of LAMP1 to the cell surface and facilitates HOPS-mediated fusion of LAMP1-positive vesicles with pre-existing active lysosomes. Upon TBC1D9B depletion, Rab11a levels are increased on the newly synthesized LAMP1 vesicles, leading to recycling of LAMP1 to the cell surface.

Article Snippet: Briefly, the guide RNA (gRNA) targeting the Arl8b gene locus was designed using the Benchling platform ( https://www.benchling.com ) and cloned into the SpCas9 pX330 (plasmid #42230; Addgene) vector ( ).

Techniques: Synthesized, Clinical Proteomics, Membrane