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首页> 外文期刊>Protoplasma: An International Journal of Cell Biology >Single-cell, real-time measurements of extracellular oxygen and proton fluxes from Spirogyra grevilleana
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Single-cell, real-time measurements of extracellular oxygen and proton fluxes from Spirogyra grevilleana

机译：单细胞实时测量螺旋藻的细胞外氧气和质子通量

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We have adapted the self-referencing microelectrode technique to allow sensitive and noninvasive measurement of oxygen fluxes around single cells. The self-referencing technique is based on the translational movement of a selective microelectrode through the gradient next to the cell wall or membrane. The electrode is moved at a known frequency and between known points. The differential electrode output values are converted into a directional measurement of flux by the Fick equation. By coupling the newly developed oxygen-selective self-referencing electrochemical microelectrode (SREM-O-2) system with self-referencing ion-selective proton measurements (SRIS-H+) we have characterized oxygen and proton fluxes from a single cell of the filamentous green alga Spirogyra gre illeana (Hass.). Oxygen showed a net efflux and protons showed a net influx when the cell was illuminated. These photosynthesis-dependent fluxes were found to be spatially associated with the chloroplasts and were sensitive to treatment with dichlorophenyldimethylurea. In the dark the directions of oxygen and proton fluxes were reversed. This oxygen influx was associated with mitochondrial respiration and was reduced by 78% when the cells was treated with 0.5 mM KCN. The residual cyanide-resistant respiration was inhibited by the application of 5 mM salicylhydroxamic acid, an inhibitor of the alternative oxidase. Similarly the cytochrome pathway was also inhibited by the presence of 20 mu M NO, while the cyanide-resistant alternative oxidase was not. These results demonstrate the use of the newly developed SREM-O-2 system to measure and characterize metabolic fluxes at a level of sensitivity that allows for subcellular resolution. These measurements, in conjunction with SERIS-H+ measurements, have led to new insights in our understanding of basic cellular physiology in plant cells. [References: 46]
机译：我们采用了自参考微电极技术，可以对单细胞周围的氧气通量进行灵敏且无创的测量。自参考技术基于选择性微电极通过细胞壁或细胞膜旁的梯度的平移运动。电极以已知频率在已知点之间移动。差分电极输出值通过Fick方程转换为通量的方向测量值。通过将新开发的氧选择自参考电化学微电极（SREM-O-2）系统与自参考离子选择质子测量（SRIS-H +）耦合，我们表征了来自单丝绿色电极的氧和质子通量海藻Spirogyra gre illeana（Hass。）。当细胞被照亮时，氧气显示净流出，质子显示净流入。发现这些依赖光合作用的通量在空间上与叶绿体相关，并且对二氯苯基二甲基脲的处理敏感。在黑暗中，氧气和质子通量的方向相反。这种氧气流入与线粒体呼吸有关，当用0.5 mM KCN处理细胞时，这种氧气流入减少了78％。通过使用5 mM水杨基异羟肟酸（另一种氧化酶的抑制剂）可抑制残留的抗氰化物呼吸作用。同样，细胞色素途径也被20μM NO抑制，而抗氰化物的替代氧化酶则不受抑制。这些结果证明了使用新开发的SREM-O-2系统以允许亚细胞分辨率的敏感性水平测量和表征代谢通量。这些测量结果与SERIS-H +测量结果一起，为我们对植物细胞基本细胞生理学的理解带来了新的见解。 [参考：46]

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