STRUCTURAL CHANGES IN MITOCHONDRIAL MEMBRANES DURING APOPTOSIS

细胞凋亡过程中线粒体膜的结构变化

• 批准号: 7357266
• 负责人: KATHLEEN W. KINNALLY
• 金额: $ 0.11万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7357266
• 关键词: STRUCTURAL; CHANGES; MITOCHONDRIAL; MEMBRANES; DURING

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. ABSTRACT: During the early stages of apoptosis, the pro-apoptotic protein Bax moves from the cytosol to the mitochondrial outer membrane (OM) where it oligomerizes and initiates a process that results in release of cytochrome c and other proteins from the intermembrane space (IMS) into the cytosol (Wei et al., 2000). This process is initiated by the truncated form of the protein Bid (tBid) and is inhibited by anti-apoptotic proteins like Bcl-2. There are several hypotheses in play regarding the mechanism of release of cytochrome c, including (1) the formation of large pores in the OM by Bax and possibly other proteins, such as the channel VDAC; (2) the formation of structural defects in the OM, perhaps involving lipids like ceremides; and (3) inner membrane expansion, causing OM lysis, perhaps at regions weakened by Bax (e.g., Korsmeyer et al., 2000; Shimizu et al., 2000; Mootha et al., 2001; Degterev et al., 2001; Waterhouse et al., 2002). Strong support for hypothesis (1) has been provided by electrophysiological studies indicating the appearance of a new channel activity in mitochondrial outer membranes concurrent with release of cytochrome c during early stages of apoptosis induced by IL-3 deprivation in murine FL5.12 cells (Pavlov et al., 2001). That the new channel (called MAC for Mitochondrial Apoptosis-induced Channel) might be the cytochrome c release pathway is suggested by two observations. First, its maximum conductance amplitudes are consistent with permeability to proteins having diameters of 5 nm or larger (the diameter of cytochrome c is around 3 nm). Second, cytochrome c and other basic proteins cause a transient blockade of the channel conductance, consistent with penetration and possible translocation (although the latter has not been directly demonstrated). We will undertake cryo-electron tomographic studies of mitochondria in mammalian cells at progressive stages of apoptosis (over 24 hours). For in-situ studies, the cells will be deprived of IL-3 and high-pressure frozen (in our Bal-Tec HPM 010) at times (4, 8 and 16 hours) characterized in terms of MAC activity and cytochrome c release in parallel experiments. Freezing will be done in 200-¿m-diameter cellulose capillary tubes, which will then be sectioned by either cryo-ultramicrotomy or FIB-milling. However, while these techniques are under development, first experiments will be done with mitochondria isolated from the murine cell line at the specified times following induction of apoptosis. The mitochondria would be embedded in ca. 400-nm-thick films of vitreous ice by plunge-freezing suspensions on EM grids (coated with a holey carbon film) using the FEI Vitrobot. Cryo-electron tomography (using the JEOL 4000 with GIF) will be undertaken initially on randomly selected sets of 6-8 mitochondria at each time point. Control reconstructions will be done on equal numbers of randomly selected mitochondria from untreated cells at the same time points. Double-axis tilt series would be needed for these studies to reduce artifacts and information loss due to the missing wedge in single-axis data (Penczek et al, 1995). We estimate 3-4 nm resolution should be attainable in our best tomograms. At this resolution, the largest MAC pores expected on the basis of conductance amplitude (approximately 8-10 nm) should be detectable in the tomograms, especially if MAC forms clusters. Immuno-labelling of mitochondria with anti-Bax antibodies suggests that Bax does cluster on the OM. Likewise, unusual surface topology, including defects such as linear cracks or tears, on the order of 10-20 nm or larger, should be visible in OM profiles. Finally, unusual inner membrane morphologies (Scorrano et al., 2002) or herniation of the OM by local bulging of the IM (Mootha et al, 2001) would be noted. In parallel experiments, we will employ immuno-labeling of intact mitochondria with anti-Bax and anti-cytochrome c antibodies (commercially available). Decoration of OM regions by the distinctive IgG molecules (in the absence of colloidal gold-labeled secondary antibody) may help to localize the structural features we hope to characterize. It should be noted that an earlier study failed to detect unusual structures in the mitochondrial outer membrane during apoptosis (Kuwana et al., 2002). That study employed conventional TEM and SEM preparative procedures, which may have limited resolution and/or obscured fine detail. It should be noted in this regard that pores 5-200 nm in diameter have been detected in flattened Bax-treated liposomes by atomic force microscopy (Epand et al. 2002). While there is no guarantee that we will detect the structural basis for cytochrome c release from mitochondria in these studies, we will ? at the very least ? establish a lower limit for the physical size of the pore or other membrane structural feature underlying this very important phenomenon. References. 1. Degterev, A., Boyce, M., and Yuan, J. (2001). The channel of death. J Cell Biol 155:695-697. 2. Epand, R. F., Martinou, J. C., Montessuit, S., Epand, R. M., and Yip, C. M. (2002). Direct evidence for membrane pore formation by the apoptotic protein Bax. Biochem Biophys Res Comm 298:744-749. 3. Korsmeyer, S. J., Wei, M. C., Saito, M., Weiler, S., Oh, K. J., and Schlesinger, P. H. (2000). Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c. Cell Death Differ 7:1166-1173. 4. Kuwana, T., Mackey, M., Perkins, G., Ellisman, M., Latterich, M., Schneiter, R., Green, D. R., and Newmeyer, D. D. (2002). Bid, Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane. Cell 111:331-342. 5. Mootha, V. K., Wei, M. C., Buttle, K., Scorrano, L., Panoutsakopoulou, V., Mannella, C. A., and Korsmeyer, S. J. (2001). A reversible component of mitochondrial respiratory dysfunction in apoptosis can be rescued by exogenous cytochrome c. EMBO J 20:661-671. 6. Pavlov, E. V., Priault, M., Pietkiewicz, D., Cheng, E. H. Y., Antonsson, B., Manon, S., Korsmeyer, S. J., Mannella, C. A., and Kinnally, K. W. (2001). A novel, high conductance channel of mitochondria linked to apoptosis in mammalian cells and Bax expression in yeast. J Cell Biol 155:719-724. 7. Penczek, P., Marko, M., Buttle, K., and Frank, J. (1995). Double-tilt electron tomography. Ultramicroscopy 60:393-410. 8. Scorrano, L., Ashiya, M., Buttle, K., Oakes, S. A., Mannella, C. A., and Korsmeyer, S. J. (2002). A distinct pathway remodels mitochondrial cristae and mobilizes cytochrome c during apoptosis. Devel Cell 2:55-67. 9. Shimizu, S., Konishi, A., Kodama, T., and Tsujimoto, Y. (2000). BH4 domain of antiapoptotic Bcl-2 family members closes voltage-dependent anion channel and inhibits apoptotic mitochondrial changes and cell death. Proc Natl Acad Sci USA 97:3100-3105. 10. Waterhouse, N. J., Ricci, J.-E., and Green, D. R. (2002). All of a sudden it's over: mitochondrial outer-membrane permeabilization in apoptosis. Biochimie 84:113-121. In previous work at the RVBC, mitochondria in four cultured neuronal cells were laser-ablated. The cells were processed for same-cell correlative LM/EM, and serially-sectioned. Tomographic reconstructions were made, using the HVEM, from semi-thick sections of ablated and control mitochondria. Work on this project will continue shortly.

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。摘要：在细胞凋亡的早期阶段，促凋亡蛋白Bax从细胞质转移到线粒体外膜（OM），在那里它寡聚并启动一个过程，导致细胞色素c和其他蛋白质从膜间隙（IMS）释放到细胞质中（Wei et al., 2000）。该过程由蛋白Bid （tBid）的截断形式启动，并被抗凋亡蛋白如Bcl-2抑制。关于细胞色素c的释放机制有几种假设，包括：(1)Bax和其他可能的蛋白质，如通道VDAC，在OM中形成大孔；(2) OM结构缺陷的形成，可能涉及类脂质缺陷；(3)内膜膨胀，可能在被Bax削弱的区域引起OM的溶解（例如，Korsmeyer等，2000；Shimizu等，2000；Mootha等，2001；Degterev等，2001；Waterhouse等，2002）。电生理学研究为假设(1)提供了强有力的支持，该研究表明，在小鼠FL5.12细胞IL-3剥夺诱导的凋亡的早期阶段，线粒体外膜中出现了一种新的通道活性，同时细胞色素c的释放（Pavlov et al., 2001）。两个观察结果表明，新的通道（称为线粒体凋亡诱导通道MAC）可能是细胞色素c释放途径。首先，它的最大电导振幅与对直径为5nm或更大的蛋白质的渗透性一致（细胞色素c的直径约为3nm）。其次，细胞色素c和其他碱性蛋白会导致通道电导的短暂阻断，这与渗透和可能的易位一致（尽管后者尚未被直接证明）。我们将在哺乳动物细胞凋亡的渐进阶段（超过24小时）进行线粒体的低温电子断层扫描研究。在原位研究中，细胞将被剥夺IL-3和高压冷冻（在我们的Bal-Tec HPM 010中），在平行实验中（4,8和16小时）以MAC活性和细胞色素c释放为特征。冷冻将在直径200- m的纤维素毛细管中进行，然后通过冷冻-超显微切开术或fib -铣削进行切片。然而，虽然这些技术正在开发中，但第一次实验将在诱导细胞凋亡后的特定时间从小鼠细胞系中分离出线粒体。使用FEI Vitrobot将线粒体嵌入到约400纳米厚的玻璃冰薄膜中，方法是在EM网格（涂有多孔碳膜）上进行俯冲冷冻悬浮液。在每个时间点随机选择6-8组线粒体进行低温电子断层扫描（使用JEOL 4000带GIF）。对照重建将在相同时间点从未处理的细胞中随机选择相同数量的线粒体进行。这些研究需要双轴倾斜系列，以减少由于单轴数据中缺失楔形而导致的伪影和信息损失（Penczek等人，1995）。我们估计在我们最好的层析成像中应该可以达到3-4纳米的分辨率。在这个分辨率下，根据电导幅度（大约8-10 nm）预期的最大MAC孔隙应该可以在层析图中检测到，特别是当MAC形成簇时。用抗Bax抗体对线粒体进行免疫标记表明Bax确实聚集在线粒体上。同样，不寻常的表面拓扑结构，包括10-20纳米或更大的线性裂纹或撕裂等缺陷，应该在OM剖面中可见。最后，会注意到不寻常的内膜形态（Scorrano et al., 2002）或由IM局部膨出引起的OM突出（Mootha et al., 2001）。在平行实验中，我们将使用抗bax和抗细胞色素c抗体（市售）对完整线粒体进行免疫标记。独特的IgG分子（在没有胶体金标记的二抗的情况下）修饰OM区域可能有助于定位我们希望表征的结构特征。值得注意的是，早期的研究未能在细胞凋亡过程中检测到线粒体外膜的异常结构（Kuwana et al., 2002）。该研究采用了传统的TEM和SEM制备程序，可能具有有限的分辨率和/或模糊的细节。在这方面应该注意的是，通过原子力显微镜可以在经过bax处理的扁平脂质体中检测到直径为5- 200nm的孔隙（Epand et al. 2002）。虽然不能保证在这些研究中我们会检测到线粒体中细胞色素c释放的结构基础，但我们会？至少？为这一重要现象背后的孔隙物理大小或其他膜结构特征建立一个下限。引用。1。Degterev， A., Boyce， M.和Yuan， J.（2001）。死亡通道。[J] .中国生物医学工程学报。2. Epand， R. F., Martinou， J. C., Montessuit， S., Epand， R. M., Yip， C. M.（2002）。凋亡蛋白Bax形成膜孔的直接证据。生物化学学报，28(2):744-749。3. Korsmeyer， S. J, Wei， M. C, Saito， M., Weiler， S., Oh， K. J，和Schlesinger， P. H.（2000）。促凋亡级联激活BID，它将BAK或BAX寡聚到毛孔中，导致细胞色素c的释放。4. Kuwana， T., Mackey， M., Perkins， G., Ellisman， M., Latterich， M., Schneiter， R., Green， D., and Newmeyer， D.（2002）。Bid， Bax和脂质协同形成线粒体外膜的超分子开口。细胞111:331 - 342。5. Mootha， V. K, Wei， M. C, Buttle， K, Scorrano， L., Panoutsakopoulou， V., Mannella， C. A.和Korsmeyer， S. J.（2001）。细胞凋亡中线粒体呼吸功能障碍的可逆组分可通过外源性细胞色素拯救[J] . EMBO, 20:661-671。6. Pavlov， E. V, Priault， M., Pietkiewicz， D., Cheng， E. H. Y., Antonsson， B., Manon， S., Korsmeyer， S. J., Mannella， C. A.和Kinnally， K. W.（2001）。一种与哺乳动物细胞凋亡和酵母Bax表达相关的新型高导线粒体通道。[J]中国生物医学工程学报（英文版）。7. 潘克，P.，马尔科，M.，巴特尔，K.和弗兰克，J.（1995）。双倾斜电子断层扫描。Ultramicroscopy 60:393 - 410。8. Scorrano， L., Ashiya， M., Buttle， K., Oakes， S. A., Mannella， C. A.和Korsmeyer， S. J.（2002）。在细胞凋亡过程中，一个独特的途径重塑线粒体嵴并动员细胞色素c。Devel Cell 2:55-67。9. Shimizu， S., Konishi， A., Kodama， T., Tsujimoto， Y.（2000）。抗凋亡Bcl-2家族成员的BH4结构域关闭电压依赖性阴离子通道，抑制线粒体凋亡改变和细胞死亡。美国科学进展97:3100-3105。10. 沃特豪斯，n.j.，里奇，j.e。Green, dr . R.（2002）。突然间一切都结束了：细胞凋亡中的线粒体外膜渗透。Biochimie 84:113 - 121。在RVBC之前的工作中，四个培养的神经元细胞中的线粒体被激光消融。对细胞进行同细胞相关LM/EM处理，并进行连续切片。利用HVEM对消融和对照线粒体的半厚切片进行层析重建。这个项目的工作不久将继续进行。