Molecular mechanism of Ca2+-induced mitochondrial shape transition in metazoans

项目总结/摘要 Ca 2+是一种重要的第二信使，是几种细胞过程所必需的。胞浆Ca 2+（cCa 2+） 由于高度负的膜电位和通过线粒体， 线粒体钙单向转运体（MCU）。线粒体Ca 2+（mCa 2+）被基质金属蛋白酶利用， 维持细胞的生物能量反过来，在中风条件下cCa 2+的失调升高， 缺血/再灌注损伤驱动mCa 2+过载，其进而导致线粒体通透性转换孔 导致坏死细胞死亡因此，认为防止mCa 2+过载可以 在升高的cCa 2+条件下具有保护作用。与此相反，小鼠敲除MCU，这表明 没有mCa 2+摄取，因此没有线粒体肿胀，令人惊讶的是，没有提供任何IR保护 介导的细胞死亡，表明MCU介导的Ca 2+过载的丧失不足以保护细胞 Ca 2+诱导的坏死。为了了解钙离子升高诱导细胞死亡的分子机制，我们 对从肝特异性MCU-/-（MCU β HEP）和MCUfl/fl动物收获的肝进行超微结构分析。 电子显微镜研究显示线粒体的形状形成鲜明对比： 显示长丝状线粒体（意大利面条状），而MCU HEP线粒体短圆形 （甜甜圈状）。我们假设这种线粒体形状转变现象，我们在下文中称为 MiST，是cCa 2+诱导的，独立于线粒体肿胀或Drp 1介导的线粒体分裂。 基于我们的初步结果，我们假设cCa 2+的病理生理升高诱导MiST， 是米罗一号驱动的因为细胞线粒体网络允许共享代谢物，蛋白质， 线粒体DNA和势能分布，存在局部线粒体失败的广泛风险， 快速地在整个网络上传播并损害蜂窝能量转换。比如电力网络， 我们假设MiST可以保护线粒体网络， 传播局部故障。我们最近完成的全基因组CRISPR/Cas9文库筛选， MEFs确定了一个保守的蛋白质，S100 z是MiST的胞质组分。我们希望MIST是一个 连续步骤，主要决定因素为cCa 2+瞬变和共享的分子组分 细胞质（S100 Z）和线粒体（Miro 1）。我们还假设MiST可能是保守的， 在后生动物中，并将促进溶酶体通过自噬/线粒体自噬去除，这取决于不同的cCa 2 + 因此，它可以抑制瞬时，从而保持线粒体网络的质量。这种Ca 2+诱导的 现象和分子组分的鉴定将解决时空分子 MiST的机制。成功完成我们提出的实验， 生物化学和成像技术将真实地证明MiST是维持免疫系统的关键调节因子。 病理生理条件下的线粒体质量控制。

PROJECT SUMMARY / ABSTRACT Ca2+ is a critical second messenger that is required for several cellular processes. Cytosolic Ca2+ (cCa2+) transients are shaped by the mitochondria due to the highly negative membrane potential and through the mitochondrial calcium uniporter (MCU). Mitochondrial Ca2+ (mCa2+) is utilized by the matrix dehydrogenases for maintaining cellular bioenergetics. Reciprocally, dysregulated elevation of cCa2+ under conditions of stroke, ischemia/reperfusion injury drives mCa2+ overload that in turn leads to mitochondrial permeability transition pore opening that triggers necrotic cell death. Hence, it was thought that preventing mCa2+ overload can be protective under conditions of elevated cCa2+. Contrary to this, mice knocked-out for MCU, which demonstrated no mCa2+ uptake and hence no mitochondrial swelling, surprisingly did not offer any protection from IR mediated cell death, suggesting that loss of MCU-mediated Ca2+ overload was not sufficient to protect cells from Ca2+-induced necrosis. To understand the molecular mechanisms of elevated Ca2+-induced cell death, we performed ultra-structural analysis of liver harvested from liver specific MCU-/- (MCUHEP) and MCUfl/fl animals. Electron microscopy studies revealed stark contrast in the shape of mitochondria: MCUfl/fl liver sections showed long and filamentous mitochondria (spaghetti-like) while MCUHEP mitochondria were short and circular (donut-like). We hypothesized this Mitochondrial Shape Transition phenomenon that we refer hereafter as MiST, to be cCa2+-induced and independent of mitochondrial swelling or Drp1-mediated mitochondrial fission. Based on our preliminary results, we hypothesize that pathophysiological elevation of cCa2+ induces MiST and that is Miro-1 driven. Because cellular mitochondrial networks allow for the sharing of metabolites, proteins, mitochondrial DNA and potential energy distribution, there is an extensive risk for local mitochondrial failures to quickly spread over the entire network and compromise cellular energy conversion. Like power networks that physically segment elements with circuit breakers, we hypothesize that MiST protects mitochondrial networks from propagating local failures. Our recently completed whole genome-wide CRISPR/Cas9 Library screen in MEFs identified a conserved protein, S100z to be the cytosolic component for MiST. We expect MiST to be a sequential step with a major determinant to be the cCa2+ transients and the molecular component to be shared by the cytosol (S100Z) and the mitochondria (Miro1). We also hypothesize that MiST is likely to be conserved in metazoans and would facilitate lysosomal removal by autophagy/mitophagy depending on the varying cCa2+ transients, thus preserving the quality of the mitochondrial network. The revelation of this Ca2+-induced phenomenon and the identification of the molecular components will resolve the spatio-temporal molecular mechanisms of MiST. Successful accomplishment of our proposed experiments using our cellular, biochemical, and imaging techniques will authentically demonstrate MiST to be key regulator in maintaining mitochondrial quality control under pathophysiological conditions.