EMBO/soda-nel huggingface dataset:
Aster-C Coordinates with COP I Vesicles to Regulate Lysosomal Trafficking and Activation of mTORC1 (2020)
Figure 2

Figure 2 Aster-C regulates mTORC1 activity by selectively interacting with the GATOR2 complex

A. Confocal image analysis depicting the co-localization of Aster-C with the ER in C2C12 cells transiently expressing GFP-Aster-C and DsRed-ER under starvation and in response to AA stimulation. Scale bar, 5 μm.

B. Subcellular fractionation analysis of mTOR, GATOR2 complex and Aster-C localization in HEK293T cells transiently expressing FLAG-Aster-C under starvation and in response to AA stimulation. LAMP1, Sec63 and GAPDH were used as lysosomal (Lyso), rough ER (RER) and cytosol (Cyto) protein markers, respectively. Microsomal fraction (Micro) was also used in the blot.

C. Subcellular fractionation analysis of mTOR and GATOR2 complex in C2C12-Vector and Aster-C KO cells under starvation and in response to AA stimulation. LAMP1, Sec63 and GAPDH were used as lysosomal (Lyso), rough ER (RER) and cytosol (Cyto) protein markers, respectively.

D. Co-IP analysis of the interaction of Aster-C with exogenous mTOR in response to starvation and AA or insulin stimulation in HEK293T cells transiently expressing the indicated proteins. Anti-Myc antibody was used for immunoprecipitation.

E. Co-IP analysis of the interaction of Aster-C with endogenous mTOR and the GATOR2 complex using FLAG-Sestrin2 (SESN2) as a positive control in HEK293T cells transiently expressing the indicated proteins. Anti-FLAG antibody was used for immunoprecipitation.

F. Co-IP analysis of the interaction of Aster-C with mTOR, the GATOR2 complex, GATOR1 complex and the Ragulator-Rag complex using FLAG-DEPDC5 as a positive control in HEK293T cells transiently expressing the indicated proteins. Anti-FLAG antibody was used for immunoprecipitation. Black arrow indicates the RagC protein.

Data information: Data are representative of at least three independent experiments.
Aster-C Coordinates with COP I Vesicles to Regulate Lysosomal Trafficking and Activation of mTORC1 (2020)
Figure 3

Figure 3 COP I vesicle assembly is required for the trafficking, activation and lysosomal association of mTORC1

A. Co-IP analysis of the interaction of Aster-C with COPA and COPG in response to starvation and AA stimulation using FLAG-DEPDC5 as a negative control in HEK293T cells transiently expressing the indicated proteins. Anti-FLAG antibody was used for immunoprecipitation. Black arrow indicates the COPG protein.

B. Co-IP analysis of the interaction of COPA with mTOR in HEK293T cells transiently expressing GFP-COPA in response to starvation and AA stimulation. Anti-GFP antibody was used for immunoprecipitation. Black arrow indicates the COPG protein.

C. Co-IP analysis of the interaction of COPA with mTOR in C2C12-Vector and Aster-C KO cells in response to starvation and AA stimulation. Anti-COPA antibody was used for immunoprecipitation.

D. Confocal imaging analysis depicting the co-localization of YFP-mTOR (yellow) with GFP-COPA (green) and lysosomes (red) in live C2C12-Vector and Aster-C KO cells in response to nutrient starvation and re-stimulation by AA. Arrows highlight co-localization of mTOR, COPA and lysosomes. Scale bar, 20 μm.

E and F. Statistical analysis of the Pearson's correlation coefficient of mTOR/Lysosome (E) and COPA/Lysosome (F) in Figure 3D (n=10-12 cells per group). Data are represented as mean ± SD. ***p<0.001 by one-way ANOVA.

Data information: Data are representative of at least three independent experiments.
Aster-C Coordinates with COP I Vesicles to Regulate Lysosomal Trafficking and Activation of mTORC1 (2020)
Figure 4

Figure 4 COP I vesicle moves with mTORC1 to the lysosome surface upon amino acids stimulation.

Time-lapse confocal imaging analysis of the co-localization of YFP-mTOR (red) with GFP-COPA (green) and lysosomes (blue) in live C2C12 cells under nutrient starvation (0 min), and in response to AA re-stimulation for indicated time (0.5 min, 2 min, 6 min and 8 min). Arrows highlight COPA puncta that were co-localized with mTOR or COPA puncta alone prior to their association with mTOR and lysosomes.. Scale bar, 5 μm.

Data information: Data are representative of two independent experiments
Aster-C Coordinates with COP I Vesicles to Regulate Lysosomal Trafficking and Activation of mTORC1 (2020)
Figure 5

Figure 5 Inhibition of ARF1 attenuates the activation of mTORC1 by amino acids stimulation

A. Confocal imaging analysis depicting the lysosomal association of mTOR in response to AA stimulation in the presence or absence of BFA (10 μM) or Exo-2 (10 μM) in live C2C12 cells transiently expressing YFP-mTOR and stained with Lysotracker Red. Scale bar, 20 μm.

B and C. Western blot analysis of mTORC1 activity in C2C12 cells in CM, in response to nutrient starvation and AA stimulation in the presence or absence of BFA (10 μM) (B) or Exo-2 (10 μM) (C).

D. Western blot analysis of mTORC1 activity in C2C12-Vector and ARF1 KO cells in CM, in response to nutrient starvation and AA stimulation.

E. Confocal imaging analysis depicting COPA puncta (green) formation and co-localization with lysosomes (red) in live C2C12 cells transiently expressing GFP-COPA and stained with Lysotracker Red in response to nutrient starvation and AA stimulation in the presence or absence of BFA (10 μM) or Exo-2 (10 μM). Arrows highlight co-localization of COPA with lysosomes. Scale bar, 5 μm.

F. Statistical analysis of the Pearson's correlation coefficient of COPA and lysosomes in Figure 5E (n=10-15 cells per group). Data are represented as mean ± SD. ***p<0.001 by one-way ANOVA.

Data information: Data are representative of at least three independent experiments.
Aster-C Coordinates with COP I Vesicles to Regulate Lysosomal Trafficking and Activation of mTORC1 (2020)
Figure 6

Figure 6 Aster-C coordinates with non-muscle myosin to regulate mTORC1 trafficking and activation

A. Co-IP analysis of the interaction of Aster-C with COPA, MYH10, WDR24 and Mios in HEK293T cells transiently expressing the indicated proteins in response to treatment with brefeldin A (BFA, 10 μM) and/or blebbistatin (BBS, 10 μM), inhibitors of ARFs and MYH10, respectively. Anti-FLAG antibody was used for immunoprecipitation.

B. Western blot analysis of mTORC1 activity in wild type (WT) and MYH10 KO MEF cells cultured in CM, or in response to starvation and AA stimulation.

C. Western blot analysis of mTORC1 activity in MYH10 KO MEF cells with stably re-expressed exogenous mCherry-MYH10 in response to starvation and AA stimulation. MYH10 KO MEF cells were transfected with mCherry-MYH10 and selected with G418 for 2 weeks.

D. Confocal imaging analysis depicting COPA puncta (green) formation and co-localization with mTOR (red) in live C2C12 cells transiently expression GFP-COPA and YFP-mTOR in response to nutrient starvation in the presence or absence of BBS (10 µM). Arrows highlight the co-localization of mTOR with COPA. Scale bar, 20 μm.

E. Statistical analysis of the Pearson's correlation coefficient of mTOR and COPA in Figure 6D (n=10-12 cells per group). Data are represented as mean ± SD. ***p<0.001 by student's t test.

Data information: Data are representative of at least three independent experiments.
Aster-C Coordinates with COP I Vesicles to Regulate Lysosomal Trafficking and Activation of mTORC1 (2020)
Figure 7

Figure 7 Actin filament is required for the trafficking and activation of mTORC1

A. Confocal imaging analysis depicting mTOR puncta (green) formation on actin filaments (red) in COS-7 cells transiently expressing YFP-mTOR in response to AA stimulation in the presence or absence of latrunculin B (LA-B, 100 nM). Actin filaments were stained using BODIPY™ 558/568-Phalloidin. Arrows indicated the association of mTOR with actin filaments. Scale bar, 5 μm.

B. Immunostaining of endogenous mTOR and LAMP1 in C2C12 cells under amino acids starvation (-AA), or in response to AA stimulation (+AA) in the presence or absence of LA-B (100 nM). Scale bar, 20 μm.

C. Western blot analysis of mTORC1 activity in C2C12 cells in CM, in response to nutrient starvation and AA stimulation in the presence or absence of LA-B (100 nM).

D. Co-IP analysis of the interaction of Aster-C with COPA and MYH10 in the presence or absence of LA-B (100 nM) or cytochalasin D (Cyto-D, 250 nM) in HEK293T cells transiently expressing FLAG-Aster-C. Anti-FLAG antibody was used for immunoprecipitation.

Data information: Data are representative of at least three independent experiments.

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LAMTOR1 inhibition of TRPML1-dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function (2021)
Figure 1

Figure 1. LAMTOR1 regulates lysosomal trafficking in dendrites of hippocampal neurons.

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 3-10 independent experiments).

D, E Quantification of track speed (D) and displacement length (E) of lysosomes with low (0-1000 arbitrary unit) and high (>1000) 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 labelled 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 EV1 and Appendix Fig S1. Source data are available online for this figure.
LAMTOR1 inhibition of TRPML1-dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function (2021)
Figure 2

Figure 2. LAMTOR1 regulates dendritic lysosome trafficking independently of mTORC1.

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 3-10 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 S2. Source data are available online for this figure.
LAMTOR1 inhibition of TRPML1-dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function (2021)
Figure 3

Figure 3. LAMTOR1 KD-induced changes in lysosomal trafficking require TRMPL1-mediated Ca2+ release and dynein activation.

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 3-6 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 3-10 independent experiments), or ML-SA1 (20 µM, E, n = 13-49 neurons from 3-10 independent experiments), or CilioD (20 µM, F, n = 12-49 neurons from 3-10 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 1C. 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 3-6 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 S3. Source data are available online for this figure.
LAMTOR1 inhibition of TRPML1-dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function (2021)
Figure 5

Figure 5. Effects of TAT-2031 on the interaction between LAMTOR1 and TRPML1 in vivo and lysosomal trafficking in dendrites of hippocampal neurons.

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 S5E 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 EV2 and Appendix Figs S3 and S5. Source data are available online for this figure.
LAMTOR1 inhibition of TRPML1-dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function (2021)
Figure 6

Figure 6. Detection of TRPML1-mediated lysosomal Ca2+ release and its regulation by LAMTOR1.

A Structure of the TRPML1-GCaMP6m-based Ca2+ sensor.

B Colocalization of TRPML1-GCaMP6m (green) with LysoTracker (red) in Hela cells. Scale bar, 5 µm.

C ML-SA1 (20 µM)-induced peak GCaMP6m responses (ΔF/F0) were increased in CRISPR-Cas9-mediated LAMTOR1 KD cells. N = 9-10 cells from 4 independent experiments.

D TAT-2031 treatment (10 µM) increased ML-SA1-induced TRPML1 Ca2+ release in both control and LAMTOR1 KD cells.

E Quantification of peak responses as shown in D. N = 11-19 cells from 5 independent experiments.

F Colocalization of TRPML1-GCaMP6m (green) with LysoTracker (red) in hippocampal neurons. Scale bar, 10 µm.

G Treatment with GPN (200 µM), BAPTA-AM (20 µM), or ML-SI1 (20 µM) blocked ML-SA1-induced TRPML1-GCaMP6m responses in neurons.

H Quantification of responses in G. N = 5-8 cells from 3 independent experiments.

I Both LAMTOR1 KD and TAT-2031 treatment (10 µM) increased ML-SA1-induced TRPML1-GCaMP6m responses in neurons.

J Quantification of peak responses as shown in I. N = 6-13 cells from 3 independent experiments.

K LAMTOR1 KD increased PI(3,5)P2 (0.5 µM)-induced TRPML1-GCaMP6m responses and the blocking effect of ML-SI1 treatment (20 µM).

L 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 EV3. Source data are available online for this figure.
LAMTOR1 inhibition of TRPML1-dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function (2021)
Figure 7

Figure 7. LAMTOR1 KD reduces LTP and enhances LTD in field CA1 of hippocampus by increasing TRPML1-mediated Ca2+ release and calcineurin activation.

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 4-5 mice) or LFS (D, n = 4-8 slices from 4-8 mice) in different groups.

E Effects of ML-SI1 treatment on LFS-induced LTD in CA1 from 2-3 weeks old mice.

F Means ± SEM of fEPSPs measured 45 min after LFS in different groups. N = 6-8 slices from 6-8 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 3-4 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 3-4 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 EV4 and 5. Source data are available online for this figure.
LAMTOR1 inhibition of TRPML1-dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function (2021)
Figure 8

Figure 8. Effects of LAMTOR1 KD and ML-SI1 treatment on GluA1 phosphorylation and levels in hippocampal neurons.

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 4-6 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 co-localized 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 S6. Source data are available online for this figure.
LAMTOR1 inhibition of TRPML1-dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function (2021)
Figure 9

Figure 9. LAMTOR1 KD-induced learning and memory impairment in mice is ameliorated by TRPML1 inhibition.

A 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).

B % freezing for different experimental groups in context memory (N = 7-19 mice).

C Model illustrating the proposed role of LAMTOR1-mediated inhibition of lysosomal Ca2+ 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 Ca2+ release and LAMTOR1 KD increased its Ca2+ release and dynein-dependent dendritic lysosome trafficking (①). LAMTOR1 KD-induced TRPML1 Ca2+ release also activates CaN, efficiently dephosphorylating GluA1 and targeting internalized AMPARs to lysosomes for degradation, thereby regulating synaptic plasticity (②). Figs 3B, 5A, and 8C 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 S6G. Source data are available online for this figure.

Table 1 Antibodies, chemicals, and plasmids used in this study

Further information and requests for resources and reagents should be directed to and will be fulfilled by Corresponding author, Xiaoning Bi (xbi@westernu.edu).

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