Modulation of lysosomal Cl- mediates migration and apoptosis through the TRPML1 as a lysosomal Cl- sensor

Lysosomes are responsible for protein degradation and clearance in cellular recycling centers. It has been known that the lysosomal chloride level is enriched and involved in intrinsic lysosomal function. However, the mechanism by which chloride levels can be sensed and the chloride-mediated lysosomal function is unknown. In this study, we verified that reduced chloride levels acutely induced lysosomal calcium release through TRPML1 and lysosomal repositioning toward juxtanuclear region. Functionally, low chloride-induced lysosomal calcium release attenuated cellular migration. In addition, spontaneous exposure to low chloride levels dysregulated lysosomal biogenesis and subsequently induced the delayed migration and promoted apoptosis. Two chloride-sensing GXXXP motifs in the TRPML1 were identified. Mutations in the GXXXP motif of TRPML1 did not affect chloride levels and no changes in migratory ability. In this study, we demonstrated that the depleted chloride approach induces reformation of the lysosomal calcium pool, and subsequent dysregulated cancer progression will assist in improving therapeutic strategies for lysosomal accumulation-associated diseases or cancer cell apoptosis. * 340/380 : fura2 fluorescence ratio Baf : bafilomycin A1 BAPTA : 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid [Ca2+]i : intracellular Ca2+ concentration CBM : chloride binding motif [Cl—]i : intracellular Cl— concentration CPA : cyclopiazonic acid DAPI : 4’,6-diamidino-2-phenylindole DW : distilled water EGFR : epidermal growth factor receptor FBS : fetal bovine serum Fura : Fura-2-AM GPN : glycyl-Phenylalanine β-naphthylamide MQAE : ethoxycarbonylmethyl-6-methoxyquinolinium bromide NPPB : 5-Nitro-2-(3-phenylpropylamino) benzoic acid PCR : polymerase chain reaction PFA : paraformaldehyde pHi : intracellular pH PI : propidium iodide Reg : physiological salt solution RPMI : Roswell Park Memorial Institute 1640 RT : room temperature SDS : sodium dodecyl sulfate siRNA : small interfering RNA TEM : transmission electron microscopy TFEB : transcription factor EB TPC : two pore channel.