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: 7954565
• 负责人: KATHLEEN W. KINNALLY
• 金额: $ 1.12万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7954565
• 关键词: Antibodies; Apoptosis; Apoptotic; Appearance; Atomic Force Microscopy; BAX gene; BH4 Domain; Bax protein; Biological; Blood capillaries; Caliber; Carbon; Cell Death; Cell Line; Cells; Cellulose; Cessation of life; Computer Retrieval of Information on Scientific Projects Database; Cytolysis; Cytosol; Data; Defect; Development; Electrons; Family member; Film; Freezing; Functional disorder; Funding; Gold Colloid; Grant; Hour; Ice; Imagery; Immunoglobulin G; In Situ; Induction of Apoptosis; Institution; Interleukin-3; Label; Link; Lipids; Liposomes; Mammalian Cell; Membrane; Mitochondria; Mitochondrial Crista; Morphologic artifacts; Morphology; Mus; Outer Mitochondrial Membrane; Pathway interactions; Penetration; Permeability; Play; Procedures; Process; Proteins; Research; Research Personnel; Resolution; Resources; Series; Source; Specific qualifier value; Staging; Structure; Surface; Suspension substance; Suspensions; Techniques; Thick; Time; Tomogram; Tube; Ultramicrotomy; United States National Institutes of Health; Voltage-Dependent Anion Channel; Yeasts; abstracting; base; capillary; cytochrome c; deprivation; electron tomography; mitochondrial membrane; novel; pressure; pro-apoptotic protein; reconstruction; research study; respiratory

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 抽象的： 在细胞凋亡的早期阶段，促凋亡蛋白 Bax 从细胞质移动到线粒体外膜 (OM)，在那里寡聚化并启动一个过程，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)。 该过程由截短形式的蛋白质 Bid (tBid) 启动，并被 Bcl-2 等抗凋亡蛋白抑制。 关于细胞色素 c 的释放机制，存在多种假设，包括（1）Bax 和可能的其他蛋白质（例如 VDAC 通道）在 OM 中形成大孔； (2) OM 中结构缺陷的形成，可能涉及神经酰胺等脂质； (3) 内膜扩张，导致 OM 裂解，可能发生在 Bax 削弱的区域（例如，Korsmeyer 等人，2000；Shimizu 等人，2000；Mootha 等人，2001；Degterev 等人，2001；Waterhouse 等人，2002）。 电生理学研究有力地支持了假设 (1)，表明在小鼠 FL5.12 细胞中 IL-3 剥夺诱导的细胞凋亡早期阶段，线粒体外膜中出现了新的通道活性，同时释放了细胞色素 c (Pavlov 等，2001)。 两个观察结果表明，新通道（称为 MAC，线粒体凋亡诱导通道）可能是细胞色素 c 释放途径。 首先，其最大电导幅度与直径为 5 nm 或更大的蛋白质（细胞色素 c 的直径约为 3 nm）的渗透性一致。 其次，细胞色素 c 和其他碱性蛋白会导致通道电导短暂阻断，这与渗透和可能的易位一致（尽管后者尚未得到直接证明）。 我们将对处于细胞凋亡进展阶段（超过 24 小时）的哺乳动物细胞中的线粒体进行冷冻电子断层扫描研究。 对于原位研究，细胞将被剥夺 IL-3 并在平行实验中不时（4、8 和 16 小时）高压冷冻（在我们的 Bal-Tec HPM 010 中），以 MAC 活性和细胞色素 c 释放为特征。 冷冻将在直径 200 米的纤维素毛细管中进行，然后通过冷冻超薄切片或 FIB 铣削进行切片。 然而，虽然这些技术正在开发中，但第一个实验将在诱导细胞凋亡后的特定时间用从鼠细胞系分离的线粒体进行。 线粒体将嵌入约。使用 FEI Vitrobot 在 EM 网格（涂有多孔碳膜）上冷冻悬浮液，形成 400 nm 厚的玻璃冰膜。 首先将在每个时间点对随机选择的 6-8 个线粒体组进行冷冻电子断层扫描（使用带有 GIF 的 JEOL 4000）。 将在相同时间点对来自未处理细胞的相同数量的随机选择的线粒体进行对照重建。 这些研究需要双轴倾斜系列，以减少由于单轴数据中缺少楔块而导致的伪影和信息丢失（Penczek 等，1995）。 我们估计在我们最好的断层扫描中应该可以达到 3-4 nm 的分辨率。 在此分辨率下，基于电导幅度（大约 8-10 nm）预期的最大 MAC 孔隙应该可以在断层图中检测到，特别是如果 MAC 形成簇。 用抗 Bax 抗体对线粒体进行免疫标记表明 Bax 确实聚集在 OM 上。 同样，不寻常的表面拓扑结构，包括 10-20 nm 或更大的线性裂纹或撕裂等缺陷，在 OM 剖面中应该是可见的。 最后，会注意到异常的内膜形态（Scorrano 等人，2002）或因 IM 局部膨胀而导致的 OM 突出（Mootha 等人，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。 用独特的 IgG 分子修饰 OM 区域（在没有胶体金标记二抗的情况下）可能有助于定位我们希望表征的结构特征。 应该指出的是，早期的研究未能检测到细胞凋亡期间线粒体外膜的异常结构（Kuwana 等，2002）。 该研究采用了传统的 TEM 和 SEM 制备程序，其分辨率可能有限和/或模糊了精细细节。 在这方面应该注意的是，通过原子力显微镜在扁平的 Bax 处理的脂质体中检测到直径为 5-200 nm 的孔（Epand 等人，2002）。 虽然不能保证在这些研究中我们能够检测到线粒体释放细胞色素 c 的结构基础，但我们至少会为这一非常重要现象背后的孔或其他膜结构特征的物理尺寸确定下限。 参考。 1. Degterev, A.、Boyce, M. 和 Yuan, J. (2001)。死亡通道。细胞生物学杂志 155：695-697。 2. Epand, R. F.、Martinou, J. C.、Montessuit, S.、Epand, R. M. 和 Yip, C. M. (2002)。凋亡蛋白 Bax 形成膜孔的直接证据。生物化学生物物理学研究通讯 298：744-749。 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，将 BAK 或 BAX 寡聚到孔中，导致细胞色素 c 的释放。细胞死亡差异 7：1166-1173。 4. Kuwana, T.、Mackey, M.、Perkins, G.、Ellisman, M.、Latterich, M.、Schneiter, R.、Green, D. R. 和 Newmeyer, D. D. (2002)。 Bid、Bax 和脂质协同作用，在线粒体外膜上形成超分子开口。单元格 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成分可以通过外源细胞色素 c 来挽救。 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. 和 Kinnally, K. W. (2001)。线粒体的一种新型高电导通道与哺乳动物细胞凋亡和酵母中 Bax 表达相关。细胞生物学杂志 155：719-724。 7. Penczek, P.、Marko, M.、Buttle, K. 和 Frank, J. (1995)。双倾斜电子断层扫描。超显微镜学 60：393-410。 8. Scorrano, L.、Ashiya, M.、Buttle, K.、Oakes, S. A.、Mannella, C. A. 和 Korsmeyer, S. J. (2002)。细胞凋亡过程中，一条独特的途径重塑线粒体嵴并动员细胞色素 c。开发细胞 2：55-67。 9. Shimizu, S.、Konishi, A.、Kodama, T. 和 Tsujimoto, Y. (2000)。抗凋亡 Bcl-2 家族成员的 BH4 结构域可关闭电压依赖性阴离子通道并抑制凋亡线粒体变化和细胞死亡。美国国家科学院院报 97：3100-3105。 10. Waterhouse, N. J.、Ricci, J.-E. 和 Green, D. R. (2002)。突然间一切都结束了：细胞凋亡中的线粒体外膜透化。生物化学 84：113-121。