McKenzie Matthew, Lim Sze C, Duchen Michael R
Centre for Genetic Diseases, Hudson Institute of Medical Research; The Department of Molecular and Translational Sciences, Monash University;
Centre for Genetic Diseases, Hudson Institute of Medical Research.
J Vis Exp. 2017 Jan 24(119):55166. doi: 10.3791/55166.
Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca) buffers to tightly regulate intracellular Ca concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (ΔΨm) to sequester Ca. The influx of Ca into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains ΔΨm, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix. We describe here a method for simultaneously measuring mitochondria Ca uptake and ΔΨm in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca can be measured using the fluorescent Ca indicator Fluo-4, AM, with measurement of ΔΨm using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca and ΔΨm simultaneously. Using the sequential addition of Ca aliquots, mitochondrial Ca uptake can be monitored, and the concentration at which Ca induces mitochondrial membrane permeability transition and the loss of ΔΨm determined.
除了在产生三磷酸腺苷（ATP）中发挥关键作用外，线粒体还作为局部钙（Ca）缓冲剂，严格调节细胞内钙浓度。为此，线粒体利用其内膜上的电化学势（ΔΨm）来隔离钙。钙流入线粒体刺激柠檬酸循环的三种限速脱氢酶，增加通过氧化磷酸化（OXPHOS）复合物的电子传递。这种刺激维持了ΔΨm，当正钙离子穿过线粒体内膜进入线粒体基质时，ΔΨm会暂时消散。我们在此描述一种使用共聚焦显微镜同时测量活细胞中线粒体钙摄取和ΔΨm的方法。通过使细胞通透，可使用荧光钙指示剂Fluo-4，AM测量线粒体钙，并使用荧光染料四甲基罗丹明甲酯高氯酸盐（TMRM）测量ΔΨm。该系统的优点是荧光染料之间的光谱重叠非常小，从而能够同时准确测量线粒体钙和ΔΨm。通过逐次添加钙等分试样，可以监测线粒体钙摄取，并确定钙诱导线粒体膜通透性转变和ΔΨm丧失的浓度。
