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

Ca2+诱导后生动物线粒体形态转变的分子机制

基本信息

批准号：
10331786
负责人：
MADESH MUNISWAMY
金额：
$ 39.97万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-15 至 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10331786
关键词：
Animals Autophagocytosis Binding Biochemical Bioenergetics CRISPR library CRISPR/Cas technology Calcium Calcium Oscillations Cell Death Cell Survival Cell physiology Cells Cessation of life Complex Cytosol Data Disease Dissociation EF-Hand Domain Electron Microscopy Elements Embryo Excision Exhibits Failure Future Genes Glutamates Harvest Hepatocyte Imaging Techniques In Vitro Infarction Investigation Ischemia Kinesin Knock-in Knock-out Knockout Mice Liver Mediating Membrane Potentials Mitochondria Mitochondrial DNA Mitochondrial Matrix Mitochondrial Swelling Modeling Molecular Mus Mutation Necrosis Neurologic Neurons Outer Mitochondrial Membrane Oxidants Oxidation-Reduction Oxidoreductase Permeability Physiology Play Potential Energy Proteins Quality Control Regulation Reperfusion Injury Reperfusion Therapy Resistance Risk Role Second Messenger Systems Shapes Signal Transduction Stress Stroke Testing Toxic effect Tubulin base calcium uniporter cell type cellular pathology deep sequencing experimental study genome wide screen genome-wide indexing loss of function mitochondrial dysfunction mitochondrial permeability transition pore next generation preservation prevent response rho GTP-Binding Proteins sensor spatiotemporal therapeutic development therapeutic target treatment strategy uptake whole genome

项目摘要

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.

项目概要/摘要 Ca2+ 是多种细胞过程所需的重要第二信使。由于高负膜电位，瞬态由线粒体形成，并通过线粒体钙单向转运蛋白 (MCU) 被基质脱氢酶利用。反过来，在中风的情况下维持细胞生物能失调，缺血/再灌注损伤导致 mCa2+ 过载，进而导致线粒体通透性转换孔因此，人们认为可以防止 mCa2+ 过载。与此相反，MCU 敲除的小鼠在 cCa2+ 升高的条件下具有保护作用。没有 mCa2+ 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 Ca2+ 超载的丧失不足以保护细胞为了了解 Ca2+ 升高诱导细胞死亡的分子机制，我们对从肝脏特异性 MCU-/- (MCUHEP) 和 MCUfl/fl 动物中采集的肝脏进行超微结构分析。电子显微镜研究显示线粒体形状形成鲜明对比：MCUfl/fl 肝脏切片显示出长且丝状的线粒体（意大利面条状），而 MCUHEP 线粒体则短且呈圆形（类似甜甜圈）。 MiST，由 cCa2+ 诱导，且不依赖于线粒体肿胀或 Drp1 介导的线粒体裂变。根据我们的初步结果，我们认为 cCa2+ 的病理生理学升高会诱发 MiST 和这是由 Miro-1 驱动的，因为细胞线粒体网络允许共享代谢物、蛋白质、蛋白质。线粒体 DNA 和势能分布，存在局部线粒体故障的广泛风险迅速蔓延到整个网络并损害细胞能量转换，就像电力网络一样。带有断路器的物理段元件，我们发现 MiST 保护线粒体网络我们最近完成了全基因组 CRISPR/Cas9 文库筛选。 MEF 鉴定出一种保守蛋白 S100z 是 MiST 的胞质成分，我们预计 MiST 是一种保守蛋白。连续步骤，主要决定因素是 cCa2+ 瞬态和要共享的分子成分我们还发现 MiST 可能是保守的。在后生动物中，根据不同的 cCa2+，可以通过自噬/线粒体自噬促进溶酶体去除瞬变，从而保持线粒体网络的质量。Ca2+ 诱导的揭示。现象和分子成分的识别将解决时空分子问题 MiST 的机制使用我们的细胞成功完成了我们提出的实验，生化和成像技术将真实地证明 MiST 是维持健康的关键调节剂病理生理条件下的线粒体质量控制。