Live imaging of intra-lysosome pH in cell lines and primary neuronal culture using a novel genetically encoded biosensor.
Live imaging of intra-lysosome pH in cell lines and primary neuronal culture using a novel genetically encoded biosensor.
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使用新型遗传编码生物传感器对细胞系和原代神经元培养物中溶酶体内pH进行实时成像。
DOI：
10.1080/15548627.2020.1771858
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发表时间：
2021-06
期刊：
影响因子：
13.3
通讯作者：
Stagi M
中科院分区：
文献类型：
--
作者：
Ponsford AH;Ryan TA;Raimondi A;Cocucci E;Wycislo SA;Fröhlich F;Swan LE;Stagi M
越来越多的研究发现，溶酶体生理学障碍是神经发育障碍和散发性衰老疾病快速增长的病理基础。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传导和蛋白水解能力的缺陷。我们已经开发了一种遗传编码的比率探针，以测量溶酶体的pH值加上纯化标签，以有效地纯化溶酶体的蛋白质组学和体外评价其功能。使用我们的探针，我们表明，溶酶体pH值是非常稳定的一段时间内，在各种类型的细胞。此外，该探针可用于确定通过TFEB的溶酶体应激信号传导与溶酶体pH的总体变化解偶联。最后，我们证明了虽然ARL 8B GTdR的过表达导致HEK 293 T细胞中外周溶酶体的显著碱化，但外周溶酶体本身的酸性不低于我们细胞系中的单核溶酶体。缩略语：ARL8B：ADP核糖基化因子，如GT3 β 8 B; ATP：三磷酸腺苷; ATP 5 F1 B/ATPB：ATP合成酶F1亚基β; ATP 6 V1 A：ATP酶H+转运V1亚基A; Baf：巴弗洛霉素A1; 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; Cos 7：非洲绿色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; EEE 8：胚胎第8天; EEA 1：早期内体抗原1; EGTA：乙二醇双（β-氨乙基醚）-N，N，N′，N′-四乙酸; ER：内质网; FBS：胎牛血清; FITC：异硫氰酸荧光素; GABARAPL 2：GABA A型受体相关蛋白样2; GAPDH：甘油醛-3-磷酸脱氢酶; GOLGA 2/GM 130：golgin A2; GTP：三磷酸鸟苷; HEK 293 T：人胚肾293细胞，表达SV 40大T抗原的突变体; HeLa：亨里埃塔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; IGF 2 R/ciM 6PR：胰岛素样生长因子2受体; LAMP 1/2：溶酶体相关膜蛋白1/2; LMAN 2/VIP 36：凝集素，甘露糖结合2; MAP 1 LC 3/LC 3：微管相关蛋白1轻链3; MTORC 1：雷帕霉素激酶复合物1的机制靶标; PCR：聚合酶链反应; PDL：聚-d-赖氨酸; PGK1p：来自人磷酸甘油酸激酶1的启动子; PIKFYVE：磷酸肌醇激酶，含FYVE型锌指; PPT 1/CLN 1：棕榈酰蛋白硫酯酶1; RPS 6 KB 1/p70：核糖体蛋白S6激酶B1; STAT 3：信号转导和转录激活因子3; TAX 1BP 1：Tax 1结合蛋白1; TFEB：转录因子EB; TGN：trans-Golgi网络; TGOLN 2/TGN 46：trans-Golgi网络蛋白2; TIRF：全内反射荧光; TMEM 106 B：跨膜蛋白106 B; TOR：雷帕霉素靶标; TRPM 2：瞬时受体电位阳离子通道亚家族M成员2; V-ATP酶：空泡型质子易位ATP酶; VPS 35：VPS 35逆转录复合物组分。
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.
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DOI：
10.1085/jgp.201110596
发表时间：
2011-04
期刊：
The Journal of general physiology
影响因子：
--
作者：
DiCiccio JE;Steinberg BE
通讯作者：
Steinberg BE
DOI：
10.1126/science.aag1417
发表时间：
2017-03-24
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Castellano BM;Thelen AM;Moldavski O;Feltes M;van der Welle RE;Mydock-McGrane L;Jiang X;van Eijkeren RJ;Davis OB;Louie SM;Perera RM;Covey DF;Nomura DK;Ory DS;Zoncu R
通讯作者：
Zoncu R
影响因子：
4.4
作者：
Cox, Juergen;Neuhauser, Nadin;Mann, Matthias
通讯作者：
Mann, Matthias
影响因子：
48
作者：
Ai, Hui-wang;Hazelwood, Kristin L.;Campbell, Robert E.
通讯作者：
Campbell, Robert E.
影响因子：
13.3
作者：
Bourdenx, Mathieu;Daniel, Jonathan;Dehay, Benjamin
通讯作者：
Dehay, Benjamin