lamtor1 shrna aav  (Vector Biolabs)

Journal: Frontiers in Cellular Neuroscience

Article Title: LAMTOR1 regulates dendritic lysosomal positioning in hippocampal neurons through TRPML1 inhibition

doi: 10.3389/fncel.2024.1495546

Figure Lengend Snippet: Antibodies, chemicals, and plasmids used in this study.

Article Snippet: Cultured hippocampal neurons were infected with LAMTOR1 shRNA AAV, LAMTOR2 shRNA AAV, or scrambled shRNA AAV (Vector Biolabs) with GFP lentiviral vector (Santa Cruz Biotechnology) at DIV 7, and 24 h after infection, 2/3 medium was replaced with fresh medium.

Techniques: Virus, Control, shRNA, Plasmid Preparation, Recombinant

Journal: Frontiers in Cellular Neuroscience

Article Title: LAMTOR1 regulates dendritic lysosomal positioning in hippocampal neurons through TRPML1 inhibition

doi: 10.3389/fncel.2024.1495546

Figure Lengend Snippet: LAMTOR1 regulates lysosomal positioning in dendrites of cultured hippocampal neurons. See also – . (A) Images of cultured hippocampal neurons stained for LAMP2 (red). Neurons were infected with an shRNA AAV directed against LAMTOR1 (shLT1) or a scrambled shRNA control (shSc) with GFP co-expression before being processed for immunofluorescence assay and imaging. Insets: enlarged dendrites. Scale bar: 20 μm, and 5 μm in insets. (B) Quantification of lysosome distribution along the dendrites, as shown in (A) . Results are Means ± SEM of 17–18 neurons from 6 independent experiments, * p < 0.05, *** p < 0.001, as compared with shSc, two-way ANOVA with Sidak’s post-test. (C) Images of cultured hippocampal neurons stained for cathepsin B (red). Neurons were infected as described in (A) before being processed for immunofluorescence assay and imaging. Insets: enlarged dendrites. Scale bar: 20 μm, and 5 μm in insets. (D) Quantification of lysosome distribution along the dendrites, as shown in (C) . Results are Means ± SEM of 14–17 neurons from 3 independent experiments, * p < 0.05, *** p < 0.001, as compared with shSc, two-way ANOVA with Sidak’s post-test.

Article Snippet: Cultured hippocampal neurons were infected with LAMTOR1 shRNA AAV, LAMTOR2 shRNA AAV, or scrambled shRNA AAV (Vector Biolabs) with GFP lentiviral vector (Santa Cruz Biotechnology) at DIV 7, and 24 h after infection, 2/3 medium was replaced with fresh medium.

Techniques: Cell Culture, Staining, Infection, shRNA, Control, Expressing, Immunofluorescence, Imaging

Journal: Frontiers in Cellular Neuroscience

Article Title: LAMTOR1 regulates dendritic lysosomal positioning in hippocampal neurons through TRPML1 inhibition

doi: 10.3389/fncel.2024.1495546

Figure Lengend Snippet: The effects of LAMTOR1 KD on lysosomal positioning depend on TRMPL1-mediated Ca 2+ release. (A) The TRPML1 inhibitor, ML-SI1, blocked the effects of LAMTOR1 KD on dendritic lysosomal positioning. Cultured hippocampal neurons were infected with AAV expressing either a LAMTOR1 shRNA (shLT1) or a scrambled shRNA (shSc); they were then treated with vehicle control or ML-SI1 before being processed for LAMP2 staining. Results are Means ± SEM of 6–18 neurons from 3 to 6 independent experiments, ** p < 0.01, *** p < 0.001, as compared with shSc (green line compared to red line), ## p < 0.01, ### p < 0.001, as compared with shLAMTOR1 (purple line compared to green line), two-way ANOVA with Tukey’s post-test. (B) TRPML1 KD blocked the effects of LAMTOR1 KD on dendritic lysosomal positioning. Cultured hippocampal neurons were infected with shLAMTOR1 (shLT1) or shSc AAV and Accell TRPML1 siRNA or control siRNA; they were then being processed for LAMP2 staining. Results are Means ± SEM of 8–9 neurons from 3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, as compared with shSc/Accell siControl (green line compared to red line), # p < 0.05, ### p < 0.001, as compared with shLAMTOR1/Accell siControl (purple line compared to green line), two-way ANOVA with Tukey’s post-test. Note that the data for shSc and shLT1 in (A) are the same as those shown in . (C) Representative images of with shLT1 or shSc AAV and Accell TRPML1 siRNA or control siRNA and stained with LAMP2 and GFP. Scale bar: 20 μm.

Article Snippet: Cultured hippocampal neurons were infected with LAMTOR1 shRNA AAV, LAMTOR2 shRNA AAV, or scrambled shRNA AAV (Vector Biolabs) with GFP lentiviral vector (Santa Cruz Biotechnology) at DIV 7, and 24 h after infection, 2/3 medium was replaced with fresh medium.

Techniques: Cell Culture, Infection, Expressing, shRNA, Control, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: LAMTOR1 regulates dendritic lysosomal positioning in hippocampal neurons through TRPML1 inhibition

doi: 10.3389/fncel.2024.1495546

Figure Lengend Snippet: TRPML1 activation by ML-SA1 or blockade of LAMTOR1/TRPML1 inhibitory interaction reproduced the effects of LAMTOR1 KD on lysosome positioning in a dynein-dependent manner. (A) The TRPML1 activator ML-SA1 induced lysosomal accumulation in proximal dendrites. Neurons were infected with shLAMTOR1 (shLT1) or shSc AAV and treated with either a vehicle control or ML-SA1 before being processed for LAMP2 staining. Results are Means ± SEM of 12–18 neurons from 3 to 6 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, as compared with shSc. (B) Disruption of LAMTOR1-TRPML1 interaction induced lysosomal accumulation in proximal dendrites. Neurons were infected with shLAMTOR1 (shLT1) or shSc AAV, treated with TAT or TAT-2031 (10 μM) for 3 h, before being processed for LAMP2 staining. Results are Means ± SEM of 14–17 neurons from 3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, as compared with shSc+TAT. (C) Inhibition of dynein blocked LAMTOR KD induced lysosomal accumulation. Neurons were infected with shLAMTOR1 (shLT1) or shSc AAV, treated with vehicle control or Ciliobrevin D (CilioD), before being processed for LAMP2 staining. Results are Means ± SEM of 10–18 neurons from 3 independent experiments, ** p < 0.01, *** p < 0.001, as compared with shSc, ### p < 0.001 compared with shLAMTOR1. P -values were derived by two-way ANOVA with Tukey’s post-test. Note that the data for shSc and shLAMTOR1 in (A,C) are the same as those shown in . (D) Diagram showing that deletion of LAMTOR1 or disruption of LAMTOR1/TRPML1 interaction (red X) enhances dynein-mediated retrograde lysosomal trafficking and proximal dendrite positioning.

Article Snippet: Cultured hippocampal neurons were infected with LAMTOR1 shRNA AAV, LAMTOR2 shRNA AAV, or scrambled shRNA AAV (Vector Biolabs) with GFP lentiviral vector (Santa Cruz Biotechnology) at DIV 7, and 24 h after infection, 2/3 medium was replaced with fresh medium.

Techniques: Activation Assay, Infection, Control, Staining, Disruption, Inhibition, Derivative Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: LAMTOR1 regulates dendritic lysosomal positioning in hippocampal neurons through TRPML1 inhibition

doi: 10.3389/fncel.2024.1495546

Figure Lengend Snippet: Antibodies, chemicals, and plasmids used in this study.

Article Snippet: For LAMTOR1 rescue experiments, LAMTOR1 shRNA AAV-infected neurons were infected with RNAi-resistant LAMTOR1 AAV (VectorBuilder) at DIV14, and neurons were analyzed at DIV21.

Techniques: Virus, Control, shRNA, Plasmid Preparation, Recombinant

Journal: Frontiers in Cellular Neuroscience

Article Title: LAMTOR1 regulates dendritic lysosomal positioning in hippocampal neurons through TRPML1 inhibition

doi: 10.3389/fncel.2024.1495546

Figure Lengend Snippet: LAMTOR1 regulates lysosomal positioning in dendrites of cultured hippocampal neurons. See also – . (A) Images of cultured hippocampal neurons stained for LAMP2 (red). Neurons were infected with an shRNA AAV directed against LAMTOR1 (shLT1) or a scrambled shRNA control (shSc) with GFP co-expression before being processed for immunofluorescence assay and imaging. Insets: enlarged dendrites. Scale bar: 20 μm, and 5 μm in insets. (B) Quantification of lysosome distribution along the dendrites, as shown in (A) . Results are Means ± SEM of 17–18 neurons from 6 independent experiments, * p < 0.05, *** p < 0.001, as compared with shSc, two-way ANOVA with Sidak’s post-test. (C) Images of cultured hippocampal neurons stained for cathepsin B (red). Neurons were infected as described in (A) before being processed for immunofluorescence assay and imaging. Insets: enlarged dendrites. Scale bar: 20 μm, and 5 μm in insets. (D) Quantification of lysosome distribution along the dendrites, as shown in (C) . Results are Means ± SEM of 14–17 neurons from 3 independent experiments, * p < 0.05, *** p < 0.001, as compared with shSc, two-way ANOVA with Sidak’s post-test.

Article Snippet: For LAMTOR1 rescue experiments, LAMTOR1 shRNA AAV-infected neurons were infected with RNAi-resistant LAMTOR1 AAV (VectorBuilder) at DIV14, and neurons were analyzed at DIV21.

Techniques: Cell Culture, Staining, Infection, shRNA, Control, Expressing, Immunofluorescence, Imaging

Journal: Frontiers in Cellular Neuroscience

Article Title: LAMTOR1 regulates dendritic lysosomal positioning in hippocampal neurons through TRPML1 inhibition

doi: 10.3389/fncel.2024.1495546

Figure Lengend Snippet: The effects of LAMTOR1 KD on lysosomal positioning depend on TRMPL1-mediated Ca 2+ release. (A) The TRPML1 inhibitor, ML-SI1, blocked the effects of LAMTOR1 KD on dendritic lysosomal positioning. Cultured hippocampal neurons were infected with AAV expressing either a LAMTOR1 shRNA (shLT1) or a scrambled shRNA (shSc); they were then treated with vehicle control or ML-SI1 before being processed for LAMP2 staining. Results are Means ± SEM of 6–18 neurons from 3 to 6 independent experiments, ** p < 0.01, *** p < 0.001, as compared with shSc (green line compared to red line), ## p < 0.01, ### p < 0.001, as compared with shLAMTOR1 (purple line compared to green line), two-way ANOVA with Tukey’s post-test. (B) TRPML1 KD blocked the effects of LAMTOR1 KD on dendritic lysosomal positioning. Cultured hippocampal neurons were infected with shLAMTOR1 (shLT1) or shSc AAV and Accell TRPML1 siRNA or control siRNA; they were then being processed for LAMP2 staining. Results are Means ± SEM of 8–9 neurons from 3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, as compared with shSc/Accell siControl (green line compared to red line), # p < 0.05, ### p < 0.001, as compared with shLAMTOR1/Accell siControl (purple line compared to green line), two-way ANOVA with Tukey’s post-test. Note that the data for shSc and shLT1 in (A) are the same as those shown in . (C) Representative images of with shLT1 or shSc AAV and Accell TRPML1 siRNA or control siRNA and stained with LAMP2 and GFP. Scale bar: 20 μm.

Article Snippet: For LAMTOR1 rescue experiments, LAMTOR1 shRNA AAV-infected neurons were infected with RNAi-resistant LAMTOR1 AAV (VectorBuilder) at DIV14, and neurons were analyzed at DIV21.

Techniques: Cell Culture, Infection, Expressing, shRNA, Control, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: LAMTOR1 regulates dendritic lysosomal positioning in hippocampal neurons through TRPML1 inhibition

doi: 10.3389/fncel.2024.1495546

Figure Lengend Snippet: TRPML1 activation by ML-SA1 or blockade of LAMTOR1/TRPML1 inhibitory interaction reproduced the effects of LAMTOR1 KD on lysosome positioning in a dynein-dependent manner. (A) The TRPML1 activator ML-SA1 induced lysosomal accumulation in proximal dendrites. Neurons were infected with shLAMTOR1 (shLT1) or shSc AAV and treated with either a vehicle control or ML-SA1 before being processed for LAMP2 staining. Results are Means ± SEM of 12–18 neurons from 3 to 6 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, as compared with shSc. (B) Disruption of LAMTOR1-TRPML1 interaction induced lysosomal accumulation in proximal dendrites. Neurons were infected with shLAMTOR1 (shLT1) or shSc AAV, treated with TAT or TAT-2031 (10 μM) for 3 h, before being processed for LAMP2 staining. Results are Means ± SEM of 14–17 neurons from 3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, as compared with shSc+TAT. (C) Inhibition of dynein blocked LAMTOR KD induced lysosomal accumulation. Neurons were infected with shLAMTOR1 (shLT1) or shSc AAV, treated with vehicle control or Ciliobrevin D (CilioD), before being processed for LAMP2 staining. Results are Means ± SEM of 10–18 neurons from 3 independent experiments, ** p < 0.01, *** p < 0.001, as compared with shSc, ### p < 0.001 compared with shLAMTOR1. P -values were derived by two-way ANOVA with Tukey’s post-test. Note that the data for shSc and shLAMTOR1 in (A,C) are the same as those shown in . (D) Diagram showing that deletion of LAMTOR1 or disruption of LAMTOR1/TRPML1 interaction (red X) enhances dynein-mediated retrograde lysosomal trafficking and proximal dendrite positioning.

Article Snippet: For LAMTOR1 rescue experiments, LAMTOR1 shRNA AAV-infected neurons were infected with RNAi-resistant LAMTOR1 AAV (VectorBuilder) at DIV14, and neurons were analyzed at DIV21.

Techniques: Activation Assay, Infection, Control, Staining, Disruption, Inhibition, Derivative Assay

Journal: The EMBO Journal

Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

doi: 10.15252/embj.2021108119

Figure Lengend Snippet: A Disruption of lysosomes with GPN abolished LysoTracker staining (red) in GFP‐expressing hippocampal neurons. Scale bar, 20 µm. B Upper panels: Kymographs of LysoTracker‐labeled lysosomes in the proximal dendrites of neurons infected with scrambled shRNA (shSc), LAMTOR1 shRNA (shLAMTOR1), or LAMTOR1 shRNA and RNAi‐resistant LAMTOR1 (rLAMTOR1). Stationary, anterograde, and retrograde traces generated using the KymoGraphClear plugin for ImageJ were coded blue, red, and green, respectively. Lower panels: Tracks of mobile lysosomes. Scale bar, 5 µm. C Quantitative analysis of lysosomal movement from kymographs. Results were expressed as percent of total lysosomes ( N = 44, 49, and 16 neurons for shSc, shLAMTOR1, and shLAMTOR+rLAMTOR1 respectively from 3 to 10 independent experiments). D, E Quantification of track speed (D) and displacement length (E) of lysosomes with low (0–1,000 arbitrary unit) and high (> 1,000) intensity of LysoTracker (dashed line, median; thin dashed line, quartiles; same length dendrites for each group from 3 independent experiments were analyzed). F, G FRAP analysis of dendritic lysosome movement. (F) Representative images of neurons at 0 and 4 min after photobleaching. Photobleached regions are indicated by the cyan and red lines, and lysosomes traveling anterogradely (crossing cyan line first) or retrogradely (crossing red line first) are labeled cyan and red, respectively. (G) Quantitative analysis for (F) ( N = 7, 7, and 9 neurons for shSc, shLAMTOR1, and shLAMTOR+rLAMTOR1, respectively, from 3 independent experiments). H Left: Kymographs of Magic Red cathepsin B substrate‐labeled degradative lysosomes in the proximal dendrites of neurons infected with shSc and shLAMTOR1. Scale bar, 5 µm. Right: Quantitative analysis of vesicular movement from kymographs ( N = 16 neurons from 3 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two‐way ANOVA with Tukey’s (C), Dunnett’s post‐test (D, E, G), or Mann–Whitney U test (H). * P < 0.05, ** P < 0.01, *** P < 0.001, as compared to shSc; # P < 0.05, ### P < 0.001, as compared to shLAMTOR1 (C‐E, G); n.s., not significant. See also Fig and Appendix Fig . Source data are available online for this figure.

Article Snippet: For the rescue experiment, LAMTOR1 shRNA AAV‐infected neurons were infected with RNAi‐resistant LAMTOR1 AAV (VectorBuilder) at DIV14.

Techniques: Staining, Expressing, Labeling, Infection, shRNA, Generated, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

doi: 10.15252/embj.2021108119

Figure Lengend Snippet: A LysoTracker (red) live imaging in GFP‐positive hippocampal neurons. Insets: enlarged dendrites. Arrowheads indicate LysoTracker‐labeled puncta in spines. Scale bar: left, 20 µm; inset, 10 µm. B Western blot analysis using anti‐LAMTOR1 and GAPDH antibodies of lysates from MEFs transfected with scrambled shRNA (shSc) or LAMTOR1 shRNA (shLAMTOR1) with or without shRNA‐resistant LAMTOR1 (rLAMTOR1). C Images of cultured hippocampal neurons immunostained for LAMTOR1 (red). Neurons were infected with shRNA AAV directed against LAMTOR1 with GFP co‐expression or scrambled shRNA control with or without shRNA‐resistant LAMTOR1 before processing for immunofluorescence assay and imaging. Scale bar, 20 µm. D, E Shown is the scatter plot of the track speed (D) and track displacement length (E) of individual mobile lysosomes versus its mean fluorescent intensity of LysoTracker. F Cumulative number of LysoTracker‐labeled mobile vesicles exhibiting the binned intensity values for different experimental groups (note that the stationary vesicles are not included). G Effects of LAMTOR1 KD on the movement of LAMP1‐YFP‐positive vesicles. Left: Images of LAMP1‐YFP in dendrites (upper) and their kymographs (lower) from neurons transfected with either control siRNA (siControl) or LAMTOR1 siRNA (siLAMTOR1). Scale bar, 5 µm. Right: Quantitative analysis of vesicular movement from kymographs ( N = 16 neurons from 3 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Kolmogorov‐Smirnov test (F) and Mann‐Whitney U test (G). * P < 0.05 as compared to siControl, ** P < 0.01 as compared to shSc, # P < 0.05, as compared to shLAMTOR1. Source data are available online for this figure.

Article Snippet: For the rescue experiment, LAMTOR1 shRNA AAV‐infected neurons were infected with RNAi‐resistant LAMTOR1 AAV (VectorBuilder) at DIV14.

Techniques: Imaging, Labeling, Western Blot, Transfection, shRNA, Cell Culture, Infection, Expressing, Immunofluorescence, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

doi: 10.15252/embj.2021108119

Figure Lengend Snippet: A Representative kymographs of LysoTracker‐labeled lysosomes in proximal dendrites of hippocampal neurons infected with scrambled shRNA (shSc), LAMTOR2 shRNA (shLAMTOR2), or Raptor shRNA (shRaptor). Scale bar, 5 µm. B Quantitative analysis of lysosomal movement from kymographs ( N = 44, 24, and 19 neurons for shSc, shLAMTOR2, and shRaptor, respectively, from 3 to 10 independent experiments). C, D FRAP analysis of dendritic lysosomal movement in Raptor KD neurons. (C) Representative images of a neuron at 0 and 4 min after photobleaching. (D) Quantification of the lysosomes in (C) undergoing retrograde (red) or anterograde (cyan) transport ( N = 7 and 8 neurons for shSc and shRaptor, respectively, from 3 independent experiments). E LAMTOR1 KD did not affect trafficking of vesicles labeled with Alexa 594‐conjugated transferrin (red, Tf 594) in dendrites. Shown are dendritic segments (upper) and kymographs (lower). Scale bar, 5 µm. F Quantitative analysis of vesicular movement from kymographs ( N = 10 neurons from 3 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two‐way ANOVA with Sidak’s post‐test (B) and Mann–Whitney U test (D, F). *** P < 0.001 as compared to shSc; n.s., not significant. See also Appendix Fig . Source data are available online for this figure.

Article Snippet: For the rescue experiment, LAMTOR1 shRNA AAV‐infected neurons were infected with RNAi‐resistant LAMTOR1 AAV (VectorBuilder) at DIV14.

Techniques: Labeling, Infection, shRNA, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

doi: 10.15252/embj.2021108119

Figure Lengend Snippet: A Quantification of the percent of lysosomes moving in the anterograde or retrograde direction in neurons infected with AAV expressing either LAMTOR1 shRNA (shLAMTOR1) or scrambled shRNA (shSc) and Accell lyspersin siRNA or control siRNA. Neurons were imaged with LysoTracker to visualize lysosomal trafficking in dendrites. N = 17–31 neurons from 3 to 6 independent experiments. B Targets of pharmacological reagents and genetic manipulation used in this study. Blue text box, TRPML1 inhibition including TRPML1 inhibitor ML‐SI1, TRPML1 siRNA, and dynein inhibitor ciliobrevin D (CilioD); red text box, TRPML1 activation induced by TRPML1 activator ML‐SA1. Dash line separates the lysosome into lysosomes under control and LAMTOR1 (LT1) KD conditions. C–F Quantification of the percent of lysosomes moving in the anterograde or retrograde direction. (C, E, F) Cultured hippocampal neurons were infected with AAV expressing either shLAMTOR1 or shSc; they were treated with vehicle control or ML‐SI1 (20 µM, C, n = 16–49 neurons from 3 to 10 independent experiments), or ML‐SA1 (20 µM, E, n = 13–49 neurons from 3 to 10 independent experiments), or CilioD (20 µM, F, n = 12–49 neurons from 3 to 10 independent experiments), and imaged with LysoTracker to visualize lysosomal trafficking in dendrites. Note that the data of shSc and shLAMTOR1 in (C, E, and F) are the same as shown in Fig . (D) Neurons were infected with shLAMTOR1 or shSc AAV and Accell TRPML1 siRNA or control siRNA; they were imaged as described above. N = 7–31 neurons from 3 to 6 independent experiments. Note that the data of shSc/Accell siControl and shLAMTOR1/Accell siControl in (D) are the same as shown in (A). Data information: Data with error bars are represented as means ± SEM. *** P < 0.001 compared with shSc or shSc/Accell siControl; ## P < 0.01, ### P < 0.001 compared with shLAMTOR1 or shLAMTOR1/Accell siControl; && P < 0.01, &&& P < 0.001 compared with shSc/Accell siLyspersin; n.s., not significant; two‐way ANOVA with Tukey’s post‐test. See also Appendix Fig . Source data are available online for this figure.

Article Snippet: For the rescue experiment, LAMTOR1 shRNA AAV‐infected neurons were infected with RNAi‐resistant LAMTOR1 AAV (VectorBuilder) at DIV14.

Techniques: Infection, Expressing, shRNA, Inhibition, Activation Assay, Cell Culture

Journal: The EMBO Journal

Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

doi: 10.15252/embj.2021108119

Figure Lengend Snippet: A Schematic structure of LAMTOR1 mutants. GCC are lipidation sites. Blue cans indicate α‐helices. Dashed lines indicate the locations of amino acid deletions. Arrowheads indicate the locations of amino acid substitutions. WT, wild‐type; ∆N, N‐terminal deletion; ∆C, C‐terminal deletion; ∆K1, K20R; ∆K2, K31R. B–E Lysates from Hela cells cotransfected with LAMTOR1‐Flag or its mutants (∆N and ∆C in B, ∆N1, and ∆N2 in D, ∆K1, and ∆K2 in E) and TRPML1‐YFP were immunoprecipitated with anti‐GFP or control IgG antibodies and probed with the indicated antibodies. Left, input proteins; right, immunoprecipitated (IP) proteins. Note one group (Starved) of Hela cells transfected with LAMTOR1‐Flag and TRPML1‐YFP in (E) were incubated in medium without amino acids and serum for 2 h. (C) Lysates from Hela cells transfected as described in (B) were immunoprecipitated with anti‐Flag or control IgG antibodies and probed with Flag and GFP antibodies. See Appendix Fig for negative controls. F Quantification of co‐IP results. N = 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one‐way ANOVA with Dunnett’s post‐test (F). * P < 0.05, *** P < 0.001. See also Fig and Appendix Fig . Source data are available online for this figure.

Article Snippet: For the rescue experiment, LAMTOR1 shRNA AAV‐infected neurons were infected with RNAi‐resistant LAMTOR1 AAV (VectorBuilder) at DIV14.

Techniques: Immunoprecipitation, Transfection, Incubation, Co-Immunoprecipitation Assay

Journal: The EMBO Journal

Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

doi: 10.15252/embj.2021108119

Figure Lengend Snippet: A, B Colocalization of LAMTOR1‐Flag with LAMP2 in Hela cells. Hela cells were transfected with LAMTOR1‐Flag or its mutants before being processed for LAMP2 (red) and Flag (green) immunofluorescence assay and imaging. Scale bar, 10 µm. (B) Quantification of LAMTOR1‐Flag and LAMP2 colocalization shown in (A). N = 8–17 cells from 3 independent experiments. C, D Co‐localization of LAMTOR1‐Flag with TRPML1‐YFP in Hela cells. Hela cells were transfected with LAMTOR1‐Flag or its mutants and TRPML1‐YFP (green) before processing for Flag (red) immunofluorescence assay and imaging. Scale bar, 10 µm. (D) Quantification of LAMTOR1‐Flag and LAMP2 colocalization shown in (C). N = 8–20 cells from 3 independent experiments. E, F Effects of TAT‐2031 on the interaction between LAMTOR1 and other members of the Ragulator (E) and mTORC1 signaling (F) in mouse hippocampus. E Lysates from fresh hippocampal tissue were immunoprecipitated with anti‐LAMTOR1 antibodies or negative control anti‐HA antibodies and probed with the indicated antibodies. Top, representative Western blot images; bottom, quantification of the relative abundance of LAMTOR2‐5 pulled down by LAMTOR1 in naïve, TAT or TAT‐2031‐treated mice. F Protein lysates of hippocampal tissue from TAT or TAT‐2031‐treated mice were prepared for Western blot analysis. Top, representative Western blot images; bottom, quantitative analysis. N = 3 mice. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one‐way ANOVA with Dunnett’s post‐test (B, D) and Student’s t ‐test (E, F). n.s., not significant. Source data are available online for this figure.

Article Snippet: For the rescue experiment, LAMTOR1 shRNA AAV‐infected neurons were infected with RNAi‐resistant LAMTOR1 AAV (VectorBuilder) at DIV14.

Techniques: Transfection, Immunofluorescence, Imaging, Immunoprecipitation, Negative Control, Western Blot

Journal: The EMBO Journal

Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

doi: 10.15252/embj.2021108119

Figure Lengend Snippet: A Interactions between LAMTOR1 and TRPML1 in mouse hippocampus. Binding of LAMTOR1 to TRPML1 in vivo was disrupted by systemic administration of the TAT‐2031 peptide. Wes protein analysis with anti‐LAMTOR1 and ‐TRPML1 antibodies of immunoprecipitation performed with anti‐LAMTOR1 antibodies or negative control anti‐HA antibodies using whole hippocampal homogenates from naïve, TAT or TAT‐2031‐treated mice. B Quantification of the relative abundance of TPRML1 pulled down by LAMTOR1 in naïve, TAT or TAT‐2031‐treated mice. N = 3 mice for each group. C Representative images from proximity ligation assay (PLA) performed on brain slices from naïve, TAT or TAT‐2031‐treated mice. Evidence of proximity between LAMTOR1 and TRPML1 is indicated by the appearance of red puncta. Nuclei are counterstained with DAPI (blue). Scale bar, 10 μm. See Appendix Fig for negative controls. D Quantification of the number of PLA signals in CA1 from naïve, TAT or TAT‐2031‐treated mice. N = 6 mice for each group. E TAT‐2031 treatment increased lysosomal mobility. Neurons were infected with shLAMTOR1 or shSc AAV; they were treated with TAT or TAT‐2031 (10 µM) and imaged with LysoTracker to visualize lysosomal trafficking in dendrites. N = 17–22 neurons from 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one‐way ANOVA with Dunnett's post‐test (B, D), or two‐way ANOVA with Tukey’s post‐test (E). ** P < 0.01, *** P < 0.001. See also Fig and Appendix Figs . Source data are available online for this figure.

Article Snippet: For the rescue experiment, LAMTOR1 shRNA AAV‐infected neurons were infected with RNAi‐resistant LAMTOR1 AAV (VectorBuilder) at DIV14.

Techniques: Binding Assay, In Vivo, Immunoprecipitation, Negative Control, Proximity Ligation Assay, Infection

Journal: The EMBO Journal

Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

doi: 10.15252/embj.2021108119

Figure Lengend Snippet: Structure of the TRPML1‐GCaMP6m‐based Ca 2+ sensor. Colocalization of TRPML1‐GCaMP6m (green) with LysoTracker (red) in Hela cells. Scale bar, 5 µm. ML‐SA1 (20 µM)‐induced peak GCaMP6m responses (ΔF/F 0 ) were increased in CRISPR‐Cas9‐mediated LAMTOR1 KD cells. N = 9–10 cells from 4 independent experiments. TAT‐2031 treatment (10 µM) increased ML‐SA1‐induced TRPML1 Ca 2+ release in both control and LAMTOR1 KD cells. Quantification of peak responses as shown in (D). N = 11–19 cells from 5 independent experiments. Colocalization of TRPML1‐GCaMP6m (green) with LysoTracker (red) in hippocampal neurons. Scale bar, 10 µm. Treatment with GPN (200 µM), BAPTA‐AM (20 µM), or ML‐SI1 (20 µM) blocked ML‐SA1‐induced TRPML1‐GCaMP6m responses in neurons. Quantification of responses in (G). N = 5–8 cells from 3 independent experiments. Both LAMTOR1 KD and TAT‐2031 treatment (10 µM) increased ML‐SA1‐induced TRPML1‐GCaMP6m responses in neurons. Quantification of peak responses as shown in I. N = 6–13 cells from 3 independent experiments. LAMTOR1 KD increased PI(3,5)P2 (0.5 µM)‐induced TRPML1‐GCaMP6m responses and the blocking effect of ML‐SI1 treatment (20 µM). Quantification of peak TRPML1‐GCaMP6m responses as shown in (K). N = 6–16 cells from 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Student’s t ‐test (C), two‐way ANOVA with Tukey’s post‐hoc analysis (E, J, L), and one‐way ANOVA with Dunnett’s post‐test (H). * P < 0.05, ** P < 0.01, *** P < 0.001, ### P < 0.001, n.s., not significant. Note that the traces in D, G, I, and K represent the mean values of each group. See also Fig . Source data are available online for this figure.

Article Snippet: For the rescue experiment, LAMTOR1 shRNA AAV‐infected neurons were infected with RNAi‐resistant LAMTOR1 AAV (VectorBuilder) at DIV14.

Techniques: CRISPR, Blocking Assay

Journal: The EMBO Journal

Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

doi: 10.15252/embj.2021108119

Figure Lengend Snippet: A, B GPN (50 µM, A) and ML‐SA1 (20 µM, B)‐induced lysosomal Ca 2+ release in TRPML1‐GCaMP6m‐transfected Hela cells. C Increased cytosolic calcium triggered by thapsigargin (2 μM) was not detected by TRPML1‐GCaMP6m, but by BioTracker 609 Red Ca 2+ AM Dye in Hela cells. D Increased cytosolic calcium triggered by thapsigargin (2 μM) was not detected by TRPML1‐GCaMP6m in control and LAMTOR1 KD Hela cells. E Images of Hela cells immunostained for LAMTOR1 (green). Hela cells were transfected with CRISPR‐Cas9 plasmids (with mCherry reporter, indicated by asterisks) with (LAMTOR1 KD) or without (control) sgRNA targeting LAMTOR1 . Scale bar, 10 µm. F Representative images of Hela cells transfected with TRPML1‐GCaMP6m (green) and CRISPR‐Cas9 plasmids as described in E. Scale bar, 5 µm. G ML‐SA1 (20 µM)‐induced peak GCaMP6m responses (ΔF/F 0 ) were increased in Hela cells transfected with the LAMTOR1 mutant ΔN1 (lacking amino acids 20–31). Right, Quantification of peak responses as shown in the left panel. N = 14 cells from 3 independent experiments. H Increased cytosolic calcium triggered by thapsigargin (2 μM), under low (< 10 nM) external Ca 2+ was not detected by TRPML1‐GCaMP6m, but by BioTracker 609 Red Ca 2+ AM Dye in cultured neurons. Ionomycin (1 μM) following thapsigargin treatment under low external Ca 2+ increased cytosolic calcium but not TRPML1‐GCaMP6m response. The maximal GCaMP6m fluorescence signal was induced in Tyrode’s solution right after ionomycin treatment. I Representative images of cultured hippocampal neurons infected with shRNA AAV directed against LAMTOR1 (shLAMTOR1) with mCherry co‐expression or scrambled shRNA control (shSc) and transfected with TRPML1‐GCaMP6m (green). Scale bar, 10 µm. J Quantification of area under the curve (AUC) in Fig . N = 6–13 neurons from 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Student’s t ‐test (G) and two‐way ANOVA with Tukey’s post hoc analysis (J). * P < 0.05, *** P < 0.001, # P < 0.05. Note that the traces in (C, D, and G) represent the mean values of each group. Source data are available online for this figure.

Article Snippet: For the rescue experiment, LAMTOR1 shRNA AAV‐infected neurons were infected with RNAi‐resistant LAMTOR1 AAV (VectorBuilder) at DIV14.

Techniques: Transfection, CRISPR, Mutagenesis, Cell Culture, Fluorescence, Infection, shRNA, Expressing

Journal: The EMBO Journal

Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

doi: 10.15252/embj.2021108119

Figure Lengend Snippet: A Representative images of CA1 pyramidal neurons stained with anti‐LAMTOR1 (red) and ‐GFP (green) antibodies in scrambled shRNA (shSc) or LAMTOR1 shRNA (shLAMTOR1)‐injected mice. Scale bar, 100 µm. B, C Effects of LAMTOR1 knockdown in field CA1 of hippocampus on levels of LAMTOR1, LAMTOR2, TRPML1, LAMP2, cathepsin B, cathepsin D, and Rab7. (B) Representative Western blot images. (C) Quantitative analysis of blots in (B). N = 3 mice. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Student’s t ‐test. Source data are available online for this figure.

Article Snippet: For the rescue experiment, LAMTOR1 shRNA AAV‐infected neurons were infected with RNAi‐resistant LAMTOR1 AAV (VectorBuilder) at DIV14.

Techniques: Staining, shRNA, Injection, Western Blot

Journal: The EMBO Journal

Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

doi: 10.15252/embj.2021108119

Figure Lengend Snippet: A–D Effects of LAMTOR1 KD and ML‐SI1 treatment on TBS‐induced LTP (A) or LFS‐induced LTD (C) in CA1. (B, D) Means ± SEM of fEPSPs measured 40 min after TBS (B, n = 4–5 slices from 4 to 5 mice) or LFS (D, n = 4–8 slices from 4 to 8 mice) in different groups. E Effects of ML‐SI1 treatment on LFS‐induced LTD in CA1 from 2‐ to 3‐weeks‐old mice. F Means ± SEM of fEPSPs measured 45 min after LFS in different groups. N = 6–8 slices from 6 to 8 mice. G–J Effects of TAT‐2031 treatment on TBS‐induced LTP (G) or LFS‐induced LTD (I) in CA1. H, J Means ± SEM of fEPSPs measured 40 min after TBS (H, n = 6 slices from 6 mice) or LFS (J, n = 3–4 slices from 3 to 4 mice) in different groups. K Effects of LAMTOR1 KD and treatment with a calcineurin inhibitor, FK506, on LFS‐induced LTD in CA1. L Means ± SEM of fEPSPs measured 45 min after LFS in different groups ( n = 3–4 slices from 3 to 4 mice). Data information: Slopes of fEPSPs were normalized to the average values recorded during the first 10‐min baseline (A, C, E, G, I, K). Statistical significance was assessed by two‐way ANOVA with Tukey’s post‐test (B, D, L) and Student’s t ‐test (F, H, J). * P < 0.05, ** P < 0.01, *** P < 0.001 compared with shSc, Vehicle, or TAT, # P < 0.05, ## P < 0.01, ### P < 0.001 compared with shLAMTOR1. Insets show representative traces of evoked fEPSPs before and 40 min after TBS/LFS. Scale bar 0.5 mV/10 ms. See also Figs and . Source data are available online for this figure.

Article Snippet: For the rescue experiment, LAMTOR1 shRNA AAV‐infected neurons were infected with RNAi‐resistant LAMTOR1 AAV (VectorBuilder) at DIV14.

Techniques:

Journal: The EMBO Journal

Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

doi: 10.15252/embj.2021108119

Figure Lengend Snippet: A–D LAMTOR1 KD significantly reduced levels of GluA1 phosphorylation and total GluA1 as well as the ratio of p‐GluA1 to GluA1 in hippocampal neurons, and this effect was reversed by ML‐SI1 treatment. (A) Representative images of CA1 pyramidal neurons stained with anti‐p‐GluA1 S845 (magenta), anti‐GluA1 (red), and anti‐GFP (green) antibodies. Scale bar = 20 µm. (B, C, D) Quantitative analysis of the mean fluorescence intensity (MFI) of p‐GluA1 S845 (B), GluA1 (C)‐immunoreactivity, and the ratio of p‐GluA1 to GluA1 (D) in hippocampal CA1 stratum radiatum (SR) 30 min after LFS. N = 4–6 slices from 4 to 6 mice. E Representative images of proximal dendrites of hippocampal neurons stained with anti‐GluA1 (green), anti‐LAMP2 (red), and anti‐GFP (gray) antibodies. Note that GluA1 labeled with Alexa Fluor 633 secondary antibodies was false‐colored green and GFP false‐colored gray to better show colocalization of GluA1 with LAMP2. Arrowheads indicate clearly colocalized puncta. Scale bar, 5 µm. F Quantitative analysis of the ratio of the number of GluA1/LAMP2 colocalized puncta to that of total lysosomes in (F) ( n = 17–29 neurons from 3 independent experiments). Data information: Data with error bars are represented as means ± SEM. * P < 0.05, ** P < 0.01 compared with shSc, # P < 0.05, ## P < 0.01 compared with shLAMTOR1; two‐way ANOVA with Tukey’s post‐test (B, C, D, F). A.U., Arbitrary unit. See also Appendix Fig . Source data are available online for this figure.

Article Snippet: For the rescue experiment, LAMTOR1 shRNA AAV‐infected neurons were infected with RNAi‐resistant LAMTOR1 AAV (VectorBuilder) at DIV14.

Techniques: Staining, Fluorescence, Labeling

Journal: The EMBO Journal

Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

doi: 10.15252/embj.2021108119

Figure Lengend Snippet: Mice received 10 min of training in an environment with two identical objects and received a retention test 24 h later in which one object was replaced with a novel one. LAMTOR1 shRNA‐injected mice exhibited a significant deficit 24 h after training, which was reversed by ML‐SI1 treatment ( N = 7–18 mice). % freezing for different experimental groups in context memory ( N = 7–19 mice). Model illustrating the proposed role of LAMTOR1‐mediated inhibition of lysosomal Ca 2+ release via TRPML1 in the regulation of lysosome motility in dendrites and synaptic plasticity. More lysosomes with lower pH move faster and longer distances in LAMTOR1 KD neurons, as compared to control neurons. LAMTOR1 interaction with TRPML1 inhibits its Ca 2+ release and LAMTOR1 KD increased its Ca 2+ release and dynein‐dependent dendritic lysosome trafficking (①). LAMTOR1 KD‐induced TRPML1 Ca 2+ release also activates CaN, efficiently dephosphorylating GluA1 and targeting internalized AMPARs to lysosomes for degradation, thereby regulating synaptic plasticity (②). Figs and , and were created with BioRender.com. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two‐way ANOVA with Tukey’s post‐test. * P < 0.05, *** P < 0.001, as compared to shSc; # P < 0.05, ### P < 0.001, as compared to shLAMTOR1. See also Appendix Fig . Source data are available online for this figure.

Article Snippet: For the rescue experiment, LAMTOR1 shRNA AAV‐infected neurons were infected with RNAi‐resistant LAMTOR1 AAV (VectorBuilder) at DIV14.

Techniques: shRNA, Injection, Inhibition

Journal: The EMBO Journal

Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

doi: 10.15252/embj.2021108119

Figure Lengend Snippet: Antibodies, chemicals, and plasmids used in this study.

Article Snippet: For the rescue experiment, LAMTOR1 shRNA AAV‐infected neurons were infected with RNAi‐resistant LAMTOR1 AAV (VectorBuilder) at DIV14.

Techniques: Recombinant, In Situ, CRISPR, Plasmid Preparation