MITOCHONDRIA FROZEN WITH TREHALOSE RETAIN BIOLOGICAL FUNCTIONS

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。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 在细胞凋亡中，Bcl-2家族蛋白Bax和巴克破坏线粒体外膜（mtDNA）的正常屏障功能，导致促细胞凋亡蛋白如细胞色素c、Omi和Smac从膜间隙（IMS）释放到细胞质中。这个过程可以在体外研究与新鲜分离的小鼠肝脏线粒体保持在生理平衡的缓冲液。这些细胞器的MOM保持完整，除非巴克被激活，例如通过与仅含BH 3的蛋白如tBid孵育。相比之下，在标准蔗糖-甘露醇缓冲液中冷冻/解冻的线粒体的M0 M立即变得渗漏，使得它们对于细胞凋亡研究无用。然而，在这里，我们表明，在含有糖，海藻糖的缓冲液中冷冻的线粒体，保持了它们的完整性和对BH 3-only蛋白质的反应性，就像新鲜的线粒体一样。在海藻糖缓冲液中冷冻也保留了生物学功能，如ATP合成，钙诱导的肿胀，跨膜电位和线粒体蛋白质输入。储存活性线粒体的能力将促进对细胞凋亡和依赖于完整线粒体的其他线粒体功能的研究。

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. In apoptosis, the Bcl-2 family proteins Bax and Bak disrupt the normal barrier function of the mitochondrial outer membrane (MOM), causing pro-apoptotic proteins such as cytochrome c, Omi and Smac to be released from the intermembrane space (IMS) into the cytoplasm. This process can be studied in vitro with freshly isolated mouse liver mitochondria maintained in a physiologically balanced buffer. The MOMs of these organelles remain intact, unless Bak is activated, e.g. by incubation with a BH3-only protein such as tBid. In contrast, the MOMs of mitochondria frozen/thawed in standard sucrose-mannitol buffers immediately become leaky, rendering them useless for apoptosis research. However, here we show that mitochondria frozen in buffer containing the sugar, trehalose, maintained their MOM integrity and responsiveness to BH3-only proteins, much like fresh mitochondria. Freezing in trehalose buffer also preserved biological functions such as ATP synthesis, calcium-induced swelling, transmembrane potential and mitochondrial protein import. The ability to store active mitochondria will facilitate research on apoptosis and other mitochondrial functions that rely on an intact MOM.