Skip to main content
. Author manuscript; available in PMC: 2011 Jan 1.
Published in final edited form as: Nat Commun. 2010 Jul 13;1(4):38. doi: 10.1038/ncomms1037

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

Figure 7

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. Non-vacuolated cells are indicated with 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) 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%, while the 75% of EGFP-ML1-KK or EGFP-ML1-7Q-transfected cells contained enlarged vacuoles. d) Histogram analysis of the vacuole size/number in Vac14−/− fibroblasts transfected with indicated constructs. e) 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.