Many lysosome functions are determined by a lumenal pH of ∼5.0, including the activity of resident acid-activated hydrolases. Lysosome pH (pHlys) is often increased in neurodegenerative disorders and predicted to be decreased in cancers, making it a potential target for therapeutics to limit the progression of these diseases. Accurately measuring pHlys, however, is limited by currently used dyes that accumulate in multiple intracellular compartments and cannot be propagated in clonal cells for longitudinal studies or used for in vivo determinations. To resolve this limitation, we developed a genetically encoded ratiometric pHlys biosensor, pHLARE (pHLysosomal Activity REporter), which localizes predominantly in lysosomes, has a dynamic range of pH 4.0 to 6.5, and can be stably expressed in cells. Using pHLARE we show decreased pHlys with inhibiting activity of the mammalian target of rapamycin complex 1 (mTORC1), in breast, pancreatic, colon, and glioblastoma cancer cells compared with untransformed cells, and with the activated oncogenes H-RasV12 and R-RasV12. pHLARE is a new tool to accurately measure pHlys, for improved understanding of lysosome dynamics that could be a promising therapeutic target.

许多溶酶体功能由~5.0 的腔内 pH 值决定，包括常驻酸激活水解酶的活性。溶酶体 pH 值 (pHlys) 在神经退行性疾病中通常会升高，而在癌症中预计会降低，这使其成为限制这些疾病进展的治疗方法的潜在目标。准确地测量pHlys，但是，由积累在多个细胞内区室，并且不能在克隆细胞中繁殖为纵向研究或体内测定用于当前使用的染料的限制。要解决这个限制，我们开发了一种基因编码的比例pHlys生物传感器，pHLARE（PHL ysosomal一个ctivity RE搬运工），其主要是在局部化溶酶体，具有pH为4.0的6.5的动态范围，并能在细胞中稳定表达。使用pHLARE我们表明降低pHlys与抑制雷帕霉素络合物1（mTORC1的）的哺乳动物靶的活性，乳腺癌，胰腺癌，结肠癌和胶质母细胞瘤肿瘤细胞与未转化的细胞相比，并与活化的癌基因H-RasV12和R-RasV12。pHLARE是精确测量pHlys一个新的工具，以提高溶酶体的动态，可能是有希望的治疗目标的认识。