Live imaging of intra-lysosome pH in cell lines and primary neuronal culture using a novel genetically encoded biosensor

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. 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.

越来越多的研究表明，溶酶体生理功能异常是一类快速增多的神经发育疾病以及散发性衰老相关疾病的病理基础。溶酶体功能异常的核心特征之一是溶酶体酸化缺陷，此类缺陷会触发溶酶体应激信号通路，并导致蛋白水解能力受损。我们开发了一种基因编码的比率型探针，可用于检测溶酶体pH，并结合纯化标签以高效分离纯化溶酶体，用于蛋白质组学分析以及溶酶体功能的体外评估。利用该探针，我们证实多种细胞类型中的溶酶体pH在数日时间内均保持显著稳定。此外，该探针可用于验证：经由TFEB（转录因子EB，transcription factor EB）介导的溶酶体应激信号通路，与溶酶体整体pH变化并无关联。最后，我们证明：尽管过表达ARL8B GTP酶（ADP核糖基化因子样GTP酶8B，ADP ribosylation factor like GTPase 8B）会导致HEK293T细胞（表达SV40大T抗原突变体的人胚肾293细胞，human embryonic kidney 293 cells, that expresses a mutant version of the SV40 large T antigen）中外周溶酶体出现显著碱化，但在本研究使用的细胞系中，外周溶酶体的酸性程度并不亚于近核溶酶体。
以下为术语对照表：
ARL8B: ADP核糖基化因子样GTP酶8B（ADP ribosylation factor like GTPase 8B）
ATP: 三磷酸腺苷（adenosine triphosphate）
ATP5F1B/ATPB: ATP合酶F1亚基β（ATP synthase F1 subunit beta）
ATP6V1A: ATP酶H+转运V1亚基A（ATPase H+ transporting V1 subunit A）
Baf: 巴弗洛霉素A1（bafilomycin A1）
BLOC-1: 溶酶体相关细胞器生物发生复合物1（biogenesis of lysosome-related organelles complex 1）
BSA: 牛血清白蛋白（bovine serum albumin）
Cos7: 非洲绿猴肾成纤维样细胞系（African green monkey kidney fibroblast-like cell line）
CQ: 氯喹（chloroquine）
CTSB: 组织蛋白酶B（cathepsin B）
CYCS: 体细胞型细胞色素c（cytochrome c, somatic）
DAPI: 4′,6-二脒基-2-苯基吲哚（4′,6-diamidino −2- phenylindole）
DIC: 微分干涉对比（differential interference contrast）
DIV: 体外培养天数（days in vitro）
DMEM: 杜尔贝科改良伊格尔培养基（Dulbecco′s modified Eagle′s medium）
E8: 胚胎第8天（embryonic day 8）
EEA1: 早期内体抗原1（early endosome antigen 1）
EGTA: 乙二醇双(β-氨基乙基醚)-N,N,N′,N′-四乙酸（ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid）
ER: 内质网（endoplasmic reticulum）
FBS: 胎牛血清（fetal bovine serum）
FITC: 异硫氰酸荧光素（fluorescein isothiocyanate）
GABARAPL2: γ-氨基丁酸A型受体相关蛋白样2（GABA type A receptor associated protein like 2）
GAPDH: 甘油醛-3-磷酸脱氢酶（glyceraldehyde-3-phosphate dehydrogenase）
GOLGA2/GM130: 高尔基体蛋白A2（golgin A2）
GTP: 三磷酸鸟苷（guanosine triphosphate）
HEK293T: 表达SV40大T抗原突变体的人胚肾293细胞（human embryonic kidney 293 cells, that expresses a mutant version of the SV40 large T antigen）
HeLa: 海拉细胞（源自Henrietta Lacks的细胞系，Henrietta Lacks-derived cell）
HEPES: 4-(2-羟乙基)-1-哌嗪乙磺酸（4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid）
HRP: 辣根过氧化物酶（horseradish peroxidase）
IGF2R/ciM6PR: 胰岛素样生长因子2受体（insulin like growth factor 2 receptor）
LAMP1/2: 溶酶体相关膜蛋白1/2（lysosomal associated membrane protein 1/2）
LMAN2/VIP36: 甘露糖结合凝集素2（lectin, mannose binding 2）
MAP1LC3/LC3: 微管相关蛋白1轻链3（microtubule-associated protein 1 light chain 3）
MTORC1: 机械靶向雷帕霉素激酶复合物1（mechanistic target of rapamycin kinase complex 1）
PCR: 聚合酶链式反应（polymerase chain reaction）
PDL: 聚-D-赖氨酸（poly-d-lysine）
PGK1p: 人磷酸甘油酸激酶1启动子（promotor from human phosphoglycerate kinase 1）
PIKFYVE: 含FYVE型锌指结构的磷酸肌醇激酶（phosphoinositide kinase, FYVE-type zinc finger containing）
PPT1/CLN1: 棕榈酰蛋白硫酯酶1（palmitoyl-protein thioesterase 1）
RPS6KB1/p70: 核糖体蛋白S6激酶B1（ribosomal protein S6 kinase B1）
STAT3: 信号转导与转录激活因子3（signal transducer and activator of transcription 3）
TAX1BP1: Tax1结合蛋白1（Tax1 binding protein 1）
TFEB: 转录因子EB（transcription factor EB）
TGN: 反式高尔基体网络（trans-Golgi network）
TGOLN2/TGN46: 反式高尔基体网络蛋白2（trans-Golgi network protein 2）
TIRF: 全内反射荧光（total internal reflection fluorescence）
TMEM106B: 跨膜蛋白106B（transmembrane protein 106B）
TOR: 雷帕霉素靶标（target of rapamycin）
TRPM2: 瞬时受体电位阳离子通道M亚家族成员2（transient receptor potential cation channel subfamily M member 2）
V-ATPase: 囊泡型质子转运ATP酶（vacuolar-type proton-translocating ATPase）
VPS35: VPS35 retromer复合物组分（VPS35 retromer complex component）