超分辨率径向波动(SRRF)作为一种无需特殊硬件的计算成像方法,为活细胞超分辨率成像提供了有力工具,然而该技术的成像效果高度依赖参数设置。为建立针对活细胞线粒体的SRRF成像优化方案,该研究以HeLa细胞为模型,利用Abberior Live Orang...超分辨率径向波动(SRRF)作为一种无需特殊硬件的计算成像方法,为活细胞超分辨率成像提供了有力工具,然而该技术的成像效果高度依赖参数设置。为建立针对活细胞线粒体的SRRF成像优化方案,该研究以HeLa细胞为模型,利用Abberior Live Orange Mito染料标记线粒体,通过转盘共聚焦显微镜获取时间序列图像,并使用NanoJ-SRRF系统评估了环半径、径向放大倍数、时序分析模式等关键参数对成像质量的影响。重建图像的分辨率与质量分别采用半高全宽和NanoJ-SQUIRREL的分辨率尺度皮尔逊相关系数与分辨率尺度误差进行定量评估。结果表明,环半径是最关键参数,较小取值(0.1~1.0)可实现超越衍射极限的分辨率;径向放大倍数与环内轴数对结构质量影响较小;在时序分析模式中,时序径向性平均法能提供最稳定且无伪影的重建结果;而梯度加权会引入明显伪影并显著降低图像质量。该研究针对转盘共聚焦成像、Abberior Live Orange Mito标记、108 nm像素尺寸、20 Hz采集、100帧时间序列的实验条件,提供了经量化评估的NanoJ-SRRF参数优化方案,为该条件下的活细胞线粒体纳米级成像提供了经量化评估的参数优化起点。展开更多
目的:探讨敲除溶酶体跨膜蛋白175(transmembrane protein 175,TMEM175)对HeLa细胞线粒体功能的影响。方法:传代培养HeLa细胞系,分为野生型(wild-type,WT)组和TMEM175敲除(knockout,KO)组。采用Western blot技术检测TMEM175、溶酶体关联...目的:探讨敲除溶酶体跨膜蛋白175(transmembrane protein 175,TMEM175)对HeLa细胞线粒体功能的影响。方法:传代培养HeLa细胞系,分为野生型(wild-type,WT)组和TMEM175敲除(knockout,KO)组。采用Western blot技术检测TMEM175、溶酶体关联膜蛋白1(lysosomal-associated membrane protein 1,LAMP1)、LAMP2和转录因子EB(transcription factor EB,TFEB)等蛋白表达;LysoSensor染色测定溶酶体酸度变化;分别采用JC-1和Mito-SOX探针测定线粒体膜电位及活性氧(reactive oxygen species,ROS)水平;应用O2K高分辨率线粒体呼吸测定系统和ATP试剂盒测定线粒体耗氧率和ATP含量;采用Western blot技术测定线粒体动力学及能量代谢关键分子包括线粒体融合蛋白2(mitofusin 2,Mfn2)、视神经萎缩蛋白1(optic atrophy 1,OPA1)、发动蛋白相关蛋白1(dynaminrelated protein 1,Drp1)、腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)及电子呼吸链复合体等的蛋白表达。结果:与WT组相比,KO组细胞中TMEM175蛋白表达水平显著降低(P<0.01),溶酶体酸度增加(P<0.05),LAMP1表达水平降低(P<0.05),LAMP2和磷酸化TFEB蛋白水平升高(P<0.05)。缺失TMEM175后细胞线粒体膜电位降低(P<0.01),线粒体ROS水平升高(P<0.01),线粒体基础呼吸和漏呼吸能力减弱(P<0.01),ATP生成相关呼吸及ATP含量增加(P<0.05或P<0.01)。WT组和KO组细胞中Mfn2和OPA1蛋白表达无显著差异(P>0.05),KO组细胞磷酸化Drp1增加(P<0.05),AMPK蛋白表达及活性均显著增加(P<0.05),线粒体复合体I、IV蛋白表达水平降低(P<0.05或P<0.01)。结论:敲除HeLa细胞TMEM175引起溶酶体酸化,溶酶体相关蛋白表达改变,并抑制线粒体功能,激活Drp1和AMPK信号。展开更多
Golgi apparatus(GA)-associated secretome runs through the endomembrane system and is critical for inter-and intracellular communication networks.However,achieving in situ dissection of the GA-associated secretome rema...Golgi apparatus(GA)-associated secretome runs through the endomembrane system and is critical for inter-and intracellular communication networks.However,achieving in situ dissection of the GA-associated secretome remains challenging owing to the scarcity of specific labeling methods.This work develops an aggregation-induced emission(AIE)luminogen-mediated photocatalytic proximity labeling strategy that allows profiling of the GA-associated secretome with high spatiotemporal precision.Leveraging an AIE luminogen-derived GA-targeting photocatalyst,this strategy achieves efficient labeling of proteins in minutes within the Golgi lumen upon light activation,which enables spatiotemporally resolved modification of histidine and tyrosine residues.We succeed in profiling secretome in both living HeLa cells and hard-to-transfect macrophage HMC3 cells,and a significant subset of GA-associated secretome with 80%specificity is determined,linking the distinct GA-associated secretory profiles to cellular characteristics.The method is further applied to proteome mapping of brain and bone metastatic lung cancer cells,which reveals the underlying roles the GA-associated secretome plays in extracellular matrix organization during metastasis.This work delivers a robust tool to break the dilemma of chemical labeling of GA-associated secretome in living cells,and provides mechanistic insights into secretion regulation at the subcellular level.展开更多
文摘超分辨率径向波动(SRRF)作为一种无需特殊硬件的计算成像方法,为活细胞超分辨率成像提供了有力工具,然而该技术的成像效果高度依赖参数设置。为建立针对活细胞线粒体的SRRF成像优化方案,该研究以HeLa细胞为模型,利用Abberior Live Orange Mito染料标记线粒体,通过转盘共聚焦显微镜获取时间序列图像,并使用NanoJ-SRRF系统评估了环半径、径向放大倍数、时序分析模式等关键参数对成像质量的影响。重建图像的分辨率与质量分别采用半高全宽和NanoJ-SQUIRREL的分辨率尺度皮尔逊相关系数与分辨率尺度误差进行定量评估。结果表明,环半径是最关键参数,较小取值(0.1~1.0)可实现超越衍射极限的分辨率;径向放大倍数与环内轴数对结构质量影响较小;在时序分析模式中,时序径向性平均法能提供最稳定且无伪影的重建结果;而梯度加权会引入明显伪影并显著降低图像质量。该研究针对转盘共聚焦成像、Abberior Live Orange Mito标记、108 nm像素尺寸、20 Hz采集、100帧时间序列的实验条件,提供了经量化评估的NanoJ-SRRF参数优化方案,为该条件下的活细胞线粒体纳米级成像提供了经量化评估的参数优化起点。
文摘目的:探讨敲除溶酶体跨膜蛋白175(transmembrane protein 175,TMEM175)对HeLa细胞线粒体功能的影响。方法:传代培养HeLa细胞系,分为野生型(wild-type,WT)组和TMEM175敲除(knockout,KO)组。采用Western blot技术检测TMEM175、溶酶体关联膜蛋白1(lysosomal-associated membrane protein 1,LAMP1)、LAMP2和转录因子EB(transcription factor EB,TFEB)等蛋白表达;LysoSensor染色测定溶酶体酸度变化;分别采用JC-1和Mito-SOX探针测定线粒体膜电位及活性氧(reactive oxygen species,ROS)水平;应用O2K高分辨率线粒体呼吸测定系统和ATP试剂盒测定线粒体耗氧率和ATP含量;采用Western blot技术测定线粒体动力学及能量代谢关键分子包括线粒体融合蛋白2(mitofusin 2,Mfn2)、视神经萎缩蛋白1(optic atrophy 1,OPA1)、发动蛋白相关蛋白1(dynaminrelated protein 1,Drp1)、腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)及电子呼吸链复合体等的蛋白表达。结果:与WT组相比,KO组细胞中TMEM175蛋白表达水平显著降低(P<0.01),溶酶体酸度增加(P<0.05),LAMP1表达水平降低(P<0.05),LAMP2和磷酸化TFEB蛋白水平升高(P<0.05)。缺失TMEM175后细胞线粒体膜电位降低(P<0.01),线粒体ROS水平升高(P<0.01),线粒体基础呼吸和漏呼吸能力减弱(P<0.01),ATP生成相关呼吸及ATP含量增加(P<0.05或P<0.01)。WT组和KO组细胞中Mfn2和OPA1蛋白表达无显著差异(P>0.05),KO组细胞磷酸化Drp1增加(P<0.05),AMPK蛋白表达及活性均显著增加(P<0.05),线粒体复合体I、IV蛋白表达水平降低(P<0.05或P<0.01)。结论:敲除HeLa细胞TMEM175引起溶酶体酸化,溶酶体相关蛋白表达改变,并抑制线粒体功能,激活Drp1和AMPK信号。
基金support from the National Key R&D Program of China(2023YFA0913902)the National Natural Science Foundation of China(32088101,22074140,and 22474136)the Dalian Institute of Chemical Physics,Chinese Academy of Sciences(DICP-I202316).
文摘Golgi apparatus(GA)-associated secretome runs through the endomembrane system and is critical for inter-and intracellular communication networks.However,achieving in situ dissection of the GA-associated secretome remains challenging owing to the scarcity of specific labeling methods.This work develops an aggregation-induced emission(AIE)luminogen-mediated photocatalytic proximity labeling strategy that allows profiling of the GA-associated secretome with high spatiotemporal precision.Leveraging an AIE luminogen-derived GA-targeting photocatalyst,this strategy achieves efficient labeling of proteins in minutes within the Golgi lumen upon light activation,which enables spatiotemporally resolved modification of histidine and tyrosine residues.We succeed in profiling secretome in both living HeLa cells and hard-to-transfect macrophage HMC3 cells,and a significant subset of GA-associated secretome with 80%specificity is determined,linking the distinct GA-associated secretory profiles to cellular characteristics.The method is further applied to proteome mapping of brain and bone metastatic lung cancer cells,which reveals the underlying roles the GA-associated secretome plays in extracellular matrix organization during metastasis.This work delivers a robust tool to break the dilemma of chemical labeling of GA-associated secretome in living cells,and provides mechanistic insights into secretion regulation at the subcellular level.
