DISRUPTION OF MITOCHONDRIAL FUNCTION DURING APOPTOSIS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在细胞凋亡过程中，促凋亡的Bcl2家族成员诱导线粒体外膜通透性和细胞色素c的释放，从而激活caspase。激活的caspase的第一个靶点是通透性的线粒体本身，导致电子传递中断，线粒体跨膜电位丧失，ATP水平下降，产生活性氧(ROS)，失去线粒体结构的完整性。2003年，我们发现呼吸复合体I的75 kDa亚基NDUFS1是线粒体中主要的caspase底物。表达p75(D255A)裂解位点突变的细胞在凋亡过程中维持？M和ATP水平，并在凋亡刺激下产生减少的ROS。不能切割的p75突变体不影响细胞色素c的释放和DNA片段化，但死亡细胞中线粒体的形态保持不变，质膜完整性的丧失延迟。因此，NDUFS1的caspase裂解促进了线粒体的凋亡改变。这部作品已提交出版。 2003年，我们开始研究NCMIR正在开发的新标记技术。ReAsH特别有用，因为它可以用于荧光显微镜和电子显微镜。当在固定样品中强烈照射时，ReAsH产生单线态氧，进而可以将二氨基联苯胺氧化成高度定域的聚合物，这可以与四氧化三铯染色(Gaietta等人，2002年)。我们已经开始使用ReAsH来定位线粒体中的细胞色素c-4C，并确定它在内膜池中的隔离程度。此外，使用从表达细胞色素c-4C的细胞中分离的线粒体，我们正在通过重组截短形式的促凋亡的Bcl-2家族成员BID来诱导线粒体外膜的通透性，就像我们之前所做的那样(Kuwana等人，2002年)。这导致快速的线粒体外膜通透性(MOMP)，我们应该能够看到，至少在细胞色素c是如何释放的方面(即，是否有至少暂时被困在这个过程中)。类似地，我们也在检测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经历MOMP的分离线粒体的超微结构。 一旦这些方法完善，我们将从半厚切片中完全重建线粒体，以产生MOMP前后线粒体的三维重建，并完成定位的细胞色素c-4C。我们将与Guy Perkins和Tom Deerinck合作，最好地确定如何使用电子断层扫描来处理我们标记的样本，其优势首先由Perkins等人描述。(1997年b)。在确定了线粒体凋亡效应蛋白释放的动力学后，我们需要快速修复我们的细胞，以捕捉MOMP期间这些蛋白释放的事件。需要对标记的线粒体进行三维分析，以确定释放的区域。我们需要电子断层扫描的高分辨率能力来将释放点与线粒体的结构元素相关联，例如接触位点和克里斯塔连接(Perkins等人，1997a)。首先，我们将使用JEOL 4000型显微镜(工作电压为400kV)，按经验优化方法，以0.5m或更薄的截面进行断层三维重建。在新的JEOL 300千伏能量过滤显微镜投入使用后，我们将使用它，因为它将允许以与老式400千伏显微镜相同的分辨率进行更厚的切片(几米厚)。

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.