DISRUPTION OF MITOCHONDRIAL FUNCTION DURING APOPTOSIS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 在凋亡过程中，Bcl-2家族的促凋亡成员诱导线粒体外膜透化和细胞色素c释放，导致半胱天冬酶活化。 活化的半胱天冬酶的第一个靶点是透化的线粒体本身，导致电子传递中断，线粒体跨膜电位（？？m）、ATP水平下降、活性氧（ROS）产生和线粒体结构完整性丧失。 在2003年，我们确定了NDUFS 1，75 kDa的呼吸复合物I的亚基，作为一个主要的半胱天冬酶底物的线粒体。 表达p75裂解位点突变体（D255 A）的细胞持续？m和ATP水平，并产生减少的ROS响应于凋亡刺激。 虽然细胞色素c的释放和DNA片段不受不可裂解的p75突变体的影响，线粒体形态保持在垂死的细胞，质膜完整性的损失被推迟。因此，半胱天冬酶切割NDUFS 1促进细胞凋亡中的线粒体变化。 这项工作已提交出版。 在2003年，我们开始研究新的标签技术正在开发的NCMIR。ReAsH是特别有用的，因为它可以用于荧光和电子显微镜。 在固定样品中强烈照射时，ReAsH产生单线态氧，单线态氧又可以将二氨基联苯胺氧化成高度局部化的聚合物，并且这可以用四氧化锇染色（Gaietta等人，2002年）。 我们已经开始使用ReAsH定位线粒体中的细胞色素c-4C，并确定它可能被隔离在内膜池中的程度。 此外，使用从细胞色素c-4C表达细胞分离的线粒体，我们用促凋亡Bcl-2家族成员Bid的重组截短形式诱导线粒体外膜的透化，如我们先前所做的那样（库瓦纳等人，2002年）。 这导致了我们应该能够可视化的快速线粒体外膜通透性（MOMP），至少在细胞色素c如何释放方面（即，是否有任何人至少暂时被困在这个过程中。 同样，我们也在研究细胞凋亡过程中细胞色素c-4C的释放，以比较这些细胞中线粒体的超微结构与MOMP过程中分离的线粒体的超微结构。 一旦这些方法得到完善，我们将从半厚切片中完全重建线粒体，以在MOMP前后产生线粒体的三维重建，并完成局部细胞色素c-4C。我们将与Guy Perkins和Tom Deerinck合作，以确定如何最好地使用电子断层扫描与我们的标记样品，并具有Perkins等人（1997 b）首次描述的优点。在确定线粒体释放凋亡效应蛋白的动力学之后，我们将需要快速固定我们的细胞以捕获在MOMP期间释放这些蛋白的事件。 需要对标记的线粒体进行三维分析，以确定释放的区域。 我们需要电子断层摄影术的高分辨率能力来将释放点与线粒体的结构元件特征（例如接触位点和嵴连接）相关联（Perkins et al.，1997年a）。 最初，我们将进行断层三维重建使用JEOL 4000显微镜（在400千伏）与0.5？m或更薄的部分，根据经验优化。新的JEOL 300 kV能量过滤显微镜投入使用后，我们将使用它，因为它将允许更厚的切片（几个？m厚），分辨率与较旧的400 kV显微镜相同。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. During apoptosis, pro-apoptotic members of the Bcl-2 family induce mitochondrial outer membrane permeabilization and cytochrome c release resulting in caspase activation. Among the first targets of the activated caspases are the permeabilized mitochondria themselves, leading to disruption of electron transport, loss of mitochondrial transmembrane potential (??m), decline in ATP levels, production of reactive oxygen species (ROS) and loss of mitochondrial structural integrity. In 2003, we identified NDUFS1, the 75 kDa subunit of respiratory complex I, as a major caspase substrate in the mitochondria. Cells expressing a cleavage site mutant of p75 (D255A) sustained ??m and ATP levels during apoptosis and produced reduced ROS in response to apoptotic stimuli. While cytochrome c release and DNA fragmentation were unaffected by the uncleavable p75 mutant, mitochondrial morphology was maintained in the dying cells, and loss of plasma membrane integrity was delayed. Therefore, caspase cleavage of NDUFS1 promotes mitochondrial changes in apoptosis. This work has been submitted for publication. In 2003, we started studies with new labeling techniques being developed at NCMIR. ReAsH is particularly useful as it can be employed for both fluorescence and electron microscopy. Upon intense illumination in fixed samples, ReAsH generates singlet oxygen that in turn can oxidize diaminobenzidine into a highly localized polymer, and this can be stained with osmium tetroxide (Gaietta, et al., 2002). We have started to use ReAsH to localize the cytochrome c-4C in the mitochondria and determine the extent to which it may be sequestered in inner membrane cisternae. Further, using isolated mitochondria from cytochrome c-4C-expressing cells, we are inducing permeabilization of the mitochondrial outer membrane with a recombinant truncated form of the pro-apoptotic Bcl-2 family member Bid, as we have done previously (Kuwana, et al., 2002). This causes a rapid mitochondrial outer membrane permeability (MOMP) that we should be able to visualize, at least in terms of how the cytochrome c is released (i.e., whether any is at least temporarily trapped in the process. Similarly, we are also examining cytochrome c-4C release in cells undergoing apoptosis to compare the ultrastucture of the mitochondria in such cells to that of isolated mitochondria undergoing MOMP. Once these approaches are perfected, we will then fully reconstruct mitochodria from semi-thick sections to produce three-dimensional reconstructions of mitochondria before and after MOMP, complete with localized cytochrome c-4C. We will work with Guy Perkins and Tom Deerinck to best determine how to use electron tomography with our labeled samples with the advantages first described by Perkins et al. (1997b). After the kinetics of apoptotic effector protein release from mitochondria are determined, we will need to quickly fix our cells to capture the events of the release of these proteins during MOMP. A three-dimensional analysis of labeled mitochondria is needed to pinpoint the regions of release. We need the high resolution capabilities of electron tomography to correlate points of release with structural elements characteristic of mitochondria, such as contact sites and crista junctions (Perkins et al., 1997a). Initially, we will conduct tomographic three-dimensional reconstruction using the JEOL 4000 microscope (operated at 400 kV) with 0.5 ?m or thinner sections according to empirical optimization. After the new JEOL 300 kV energy-filtering microscope is operational, we will use it because it will allow for much thicker sections (several ?m thick) at the same resolution as the older 400 kV microscope.