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

项目摘要/摘要 钙是一种关键的第二信使，在许多细胞过程中都是必需的。胞浆钙(cCa~(2+)) 由于高度负的膜电位，瞬间由线粒体塑造，并通过 线粒体钙单转运体(MCU)。基质脱氢酶利用线粒体钙离子(mCa2+) 维持细胞生物能量学。反过来，中风条件下钙离子的异常升高， 缺血/再灌注损伤导致线粒体钙超载，进而导致线粒体通透性转换孔 打开后会引发坏死细胞死亡。因此，人们认为，防止钙超载可以通过 在钙离子升高条件下具有保护作用。与此相反，小鼠被淘汰为MCU，这表明 没有线粒体钙摄取，因此没有线粒体肿胀，令人惊讶的是，没有对IR提供任何保护 介导细胞死亡，表明MCU介导的钙超载的丧失不足以保护细胞 由钙离子引起的坏死。为了了解升高的钙离子诱导细胞死亡的分子机制，我们 对肝脏特异性Mcu-/-(McuHEP)和McUf1/fl动物的肝脏进行了超微结构分析。 电子显微镜研究显示线粒体的形状形成鲜明对比：MCUFL/FL肝脏切片 线粒体长而细丝状(意大利面条样)，而MCUHEP线粒体短而圆 (像甜甜圈一样)。我们假设了这种线粒体形状转变现象，我们在下文中称之为 雾，是由钙离子诱导的，不依赖于线粒体肿胀或DRp1介导的线粒体分裂。 根据我们的初步结果，我们假设钙离子的病理生理升高导致雾和 这是Miro-1驱动的。因为细胞线粒体网络允许代谢物、蛋白质、 线粒体DNA和势能分布，存在局部线粒体失效的广泛风险 在整个网络中迅速传播，并影响蜂窝能量转换。比如电力网络， 用断路器在物理上分割元素，我们假设薄雾保护线粒体网络 防止传播本地故障。我们最近完成的全基因组CRISPR/Cas9文库筛选 MEFS鉴定出一个保守的蛋白质S100z是MIST的胞浆组分。我们预计雾将成为一种 顺序步骤，主要决定因素是钙离子瞬变和要共享的分子成分 由胞浆(S100Z)和线粒体(Miro1)组成。我们还假设薄雾很可能是守恒的 并通过自噬/有丝分裂吞噬来促进溶酶体的去除，这取决于不同的钙离子 瞬变，从而保持线粒体网络的质量。这种钙离子诱导的启示 现象和分子组分的识别将解决时空分子的问题 雾化的机理。使用我们的蜂窝， 生化和成像技术将真实地证明薄雾是维持 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.