Figure 7: Overexpression of TRPML1 rescues the enlarged endolysosome phenotype of PI(3,5)P₂-deficient mouse fibroblasts.

(a, b) The effects of overexpression of WT TRPML1 and pore (ML1-KK) or PI(3,5)P₂-insensitive (ML1-7Q) mutant TRPML1 on the number and size of the vacuoles in Vac14^−/− fibroblasts. Cultured Vac14^−/− mouse fibroblast cells exhibited variable numbers (1–20) of large (>3 μm) vacuoles/endolysosomes. Left, differential interference contrast image, right epifluorescence. Green fluorescence, mCit (vector only), mCit-Vac14, GFP-ML1, GFP-ML1-KK, GFP-ML1-7Q. Non-vacuolated cells are indicated by asterisk. Scale bar, 20 μm. (b) TRPML1, ML1-KK and ML1-7Q proteins were co-localized in Lamp1-positive compartments of Vac14^−/− fibroblast cells. (c) Histogram analysis of the vacuole size/number in Vac14^−/− fibroblasts transfected with indicated constructs. (d) Large vacuoles in 75% of vector (mCit)-transfected Vac14^−/− fibroblast cells. Overexpression of Vac14-mCit or EGFP-ML1 reduced the percentage (of enlarged vacuoles) to approximately 15%, whereas the 75% of EGFP-ML1-KK or EGFP-ML1-7Q-transfected cells contained enlarged vacuoles. Data are presented as the mean ± s.e.m.; the n numbers are in parentheses. Statistical comparisons were made using analysis of variance: ***P<0.001. (e) Cellular organelle fractionation analysis reveals co-localization of TRPML1 and TRPML1-7Q with Lamp-1. Gradient cellular fractionations were obtained using ultracentrifugation. Both TRPML1 and TRPML1-7Q proteins were concentrated in Lamp1-rich fractionations. Arrow shows full-length TRPML1-EGFP.