ULTRASTRUCTURE OF APOPTOTIC MITOCHONDRIA

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。列出的机构是 中心，不一定是研究者的机构。 我们正在研究一种在含海藻糖的缓冲液中长期储存线粒体的方法。 在细胞凋亡中，Bcl-2家族蛋白调节线粒体外膜（mtDNA）的屏障功能，控制促凋亡蛋白从膜间隙（IMS）释放到细胞质中。这个过程可以在体外研究新鲜分离的小鼠肝线粒体。不幸的是，在标准蔗糖-甘露醇缓冲液中冷冻/解冻的线粒体变得渗漏并且对于凋亡研究无用。我们所知道的关于线粒体凋亡途径的大部分已经从使用分离的小鼠肝脏线粒体的实验中确定。这些孤立的细胞器的外膜往往会随着时间的推移而恶化，并在冻结后立即破裂解冻。因此，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）的功能分析目前需要新鲜制备的细胞器。为了减少对昂贵和耗时的制备的需要，我们试图确定一种保存线粒体功能，特别是外膜功能的储存介质。为此，我们分析了快速冷冻后MOMs的完整性在含有各种碳水化合物的缓冲液中解冻。该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NCMR与Perkins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.