ABSTRACT

Disorders of lysosomal physiology have increasingly been found to underlie the pathology of a rapidly growing cast of neurodevelopmental disorders and sporadic diseases of aging. One cardinal aspect of lysosomal (dys)function is lysosomal acidification in which defects trigger lysosomal stress signaling and defects in proteolytic capacity. We have developed a genetically encoded ratiometric probe to measure lysosomal pH coupled with a purification tag to efficiently purify lysosomes for both proteomic and in vitro evaluation of their function. Using our probe, we showed that lysosomal pH is remarkably stable over a period of days in a variety of cell types. Additionally, this probe can be used to determine that lysosomal stress signaling via TFEB is uncoupled from gross changes in lysosomal pH. Finally, we demonstrated that while overexpression of ARL8B GTPase causes striking alkalinization of peripheral lysosomes in HEK293 T cells, peripheral lysosomes per se are no less acidic than juxtanuclear lysosomes in our cell lines.

Abbreviations: ARL8B: ADP ribosylation factor like GTPase 8B; ATP: adenosine triphosphate; ATP5F1B/ATPB: ATP synthase F1 subunit beta; ATP6V1A: ATPase H+ transporting V1 subunit A; Baf: bafilomycin A₁; BLOC-1: biogenesis of lysosome-related organelles complex 1; BSA: bovine serum albumin; Cos7: African green monkey kidney fibroblast-like cell line; CQ: chloroquine; CTSB: cathepsin B; CYCS: cytochrome c, somatic; DAPI: 4′,6-diamidino −2- phenylindole; DIC: differential interference contrast; DIV: days in vitro; DMEM: Dulbecco′s modified Eagle′s medium;‎ E8: embryonic day 8; EEA1: early endosome antigen 1; EGTA: ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; ER: endoplasmic reticulum; FBS: fetal bovine serum; FITC: fluorescein isothiocyanate; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GOLGA2/GM130: golgin A2; GTP: guanosine triphosphate; HEK293T: human embryonic kidney 293 cells, that expresses a mutant version of the SV40 large T antigen; HeLa: Henrietta Lacks-derived cell; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HRP: horseradish peroxidase; IGF2R/ciM6PR: insulin like growth factor 2 receptor; LAMP1/2: lysosomal associated membrane protein 1/2; LMAN2/VIP36: lectin, mannose binding 2; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; PCR: polymerase chain reaction; PDL: poly-d-lysine; PGK1p: promotor from human phosphoglycerate kinase 1; PIKFYVE: phosphoinositide kinase, FYVE-type zinc finger containing; PPT1/CLN1: palmitoyl-protein thioesterase 1; RPS6KB1/p70: ribosomal protein S6 kinase B1; STAT3: signal transducer and activator of transcription 3; TAX1BP1: Tax1 binding protein 1; TFEB: transcription factor EB; TGN: trans-Golgi network; TGOLN2/TGN46: trans-Golgi network protein 2; TIRF: total internal reflection fluorescence; TMEM106B: transmembrane protein 106B; TOR: target of rapamycin; TRPM2: transient receptor potential cation channel subfamily M member 2; V-ATPase: vacuolar-type proton-translocating ATPase; VPS35: VPS35 retromer complex component.

摘要

越来越多的人发现，溶酶体生理障碍是迅速增长的神经发育障碍和散发性衰老疾病的病理基础。溶酶体 (dys) 功能的一个主要方面是溶酶体酸化，其中缺陷触发溶酶体应激信号和蛋白水解能力缺陷。我们开发了一种基因编码的比率探针来测量溶酶体的 pH 值，并结合一个纯化标签来有效地纯化溶酶体，以便对其功能进行蛋白质组学和体外评估。使用我们的探针，我们发现溶酶体 pH 值在几天内在各种细胞类型中非常稳定。此外，该探针可用于确定溶酶体应激信号通过TFEB 与溶酶体 pH 值的总体变化无关。最后，我们证明了虽然 ARL8B GTPase 的过表达导致 HEK293 T 细胞中外周溶酶体显着碱化，但外周溶酶体本身的酸性不低于我们细胞系中的近核溶酶体。

缩写： ARL8B：ADP 核糖基化因子，如 GTPase 8B；ATP：三磷酸腺苷；ATP5F1B/ATPB：ATP合酶F1亚基β；ATP6V1A：ATPase H+ 转运 V1 亚基 A；Baf：巴弗洛霉素A ₁；BLOC-1：溶酶体相关细胞器复合物 1 的生物发生；BSA：牛血清白蛋白；Cos7：非洲绿猴肾成纤维细胞样细胞系；CQ：氯喹；CTSB：组织蛋白酶 B；CYCS：细胞色素 c，体细胞；DAPI：4'，6-二脒基-2-苯基吲哚；DIC：微分干涉对比；DIV：体外天数; DMEM：Dulbecco 改良 Eagle 培养基；E8：胚胎第 8 天；EEA1：早期内体抗原 1；EGTA：乙二醇-双(β-氨基乙基醚)-N,N,N',N'-四乙酸；ER：内质网；FBS：胎牛血清；FITC：异硫氰酸荧光素；GABARAPL2：GABA A 型受体相关蛋白样 2；GAPDH：3-磷酸甘油醛脱氢酶；GOLGA2/GM130：高尔金A2；GTP：鸟苷三磷酸；HEK293T：人胚肾 293 细胞，表达 SV40 大 T 抗原的突变版本；HeLa：Henrietta Lacks 衍生细胞；HEPES：4-(2-羟乙基)-1-哌嗪乙磺酸；HRP：辣根过氧化物酶；IGF2R/ciM6PR：胰岛素样生长因子 2 受体；LAMP1/2：溶酶体相关膜蛋白 1/2；LMAN2/VIP36：凝集素，甘露糖结合 2；MAP1LC3/LC3：微管相关蛋白 1 轻链 3；MTORC1：雷帕霉素激酶复合物 1 的机械靶点；PCR：聚合酶链式反应；PDL：聚-d-赖氨酸；PGK1p：人磷酸甘油酸激酶 1 的启动子；PIKFYVE：磷酸肌醇激酶，含有FYVE型锌指；PPT1/CLN1：棕榈酰蛋白硫酯酶 1；RPS6KB1/p70：核糖体蛋白 S6 激酶 B1；STAT3：信号转导和转录激活因子 3；TAX1BP1：Tax1 结合蛋白 1；TFEB：转录因子EB；TGN：跨高尔基网络；TGOLN2/TGN46：反式高尔基网络蛋白 2；TIRF：全内反射荧光；TMEM106B：跨膜蛋白106B；TOR：雷帕霉素靶点；TRPM2：瞬时受体电位阳离子通道亚家族 M 成员 2；V-ATP酶：液泡型质子转运ATP酶；VPS35：VPS35 逆转录酶复合物。