ULTRASTRUCTURE OF APOPTOTIC MITOCHONDRIA

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。 我们正在研究一种在含有海藻糖的缓冲液中长期保存线粒体的方法。在细胞凋亡中，Bcl2家族蛋白调节线粒体外膜(MOM)的屏障功能，控制促凋亡蛋白从膜间隙(IMS)释放到细胞质中。这一过程可以用新鲜分离的小鼠肝脏线粒体在体外进行研究。不幸的是，在标准的蔗糖-甘露醇缓冲液中冷冻/解冻的线粒体变得漏水，对细胞凋亡研究毫无用处。我们对线粒体凋亡途径的许多了解都是通过使用分离的小鼠肝脏线粒体进行的实验确定的。这些孤立细胞器的外膜往往会随着时间的推移而恶化，并在冰冻后立即破裂。因此，目前对小鼠肝脏线粒体(以及可能来自其他来源的哺乳动物线粒体)的功能分析需要新鲜准备的细胞器。为了减少昂贵和耗时的准备工作，我们试图寻找一种保存线粒体功能，特别是外膜功能的存储介质。为此，我们分析了妈妈们在含有各种碳水化合物的缓冲液中快速冷冻后的完整性。这项分析的一部分是确定分离的线粒体的结构保存。这项工作是在NCMIR与珀金斯博士合作完成的。

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. We are investigating a method for the long-term storage of mitochondria in trehalose-containing buffer. In apoptosis, Bcl-2-family proteins regulate the barrier function of the mitochondrial outer membrane (MOM), controlling the release of proapoptotic proteins from the intermembrane space (IMS) into the cytoplasm. This process can be studied in vitro with freshly isolated mouse liver mitochondria. Unfortunately, mitochondria frozen/thawed in standard sucrose-mannitol buffers become leaky and useless for apoptosis research. Much of what we know about the mitochondrial apoptotic pathway has been determined from experiments using isolated mouse liver mitochondria. The outer membranes of these isolated organelles tend to deteriorate over time and rupture immediately upon freezethaw. Thus, the functional analysis of mouse liver mitochondria (and presumably mammalian mitochondria from other sources) currently requires freshly prepared organelles. To reduce the need for costly and timeconsuming preparations, we sought to identify a storage medium that preserves mitochondrial function, particularly of the outer membrane. To that end, we analyzed the integrity of MOMs after rapid freezethaw in buffers containing various carbohydrates. Part of this analysis was to determine the structural preservation of isolated mitochondria. This work was done at the NCMIR in collaboration with Dr. Perkins.