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: Shuttle PIP Kit, PI(3,5)P2 , Echelon Biosciences , Cat#P‐9035.
Techniques: CRISPR, Blocking Assay