MITOCHONDRIA FROZEN WITH TREHALOSE RETAIN BIOLOGICAL FUNCTIONS

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。在细胞凋亡中，Bcl-2家族蛋白Bax和Bak破坏线粒体外膜（MOM）的正常屏障功能，导致细胞色素c、Omi和Smac等促凋亡蛋白从膜间隙（IMS）释放到细胞质中。这一过程可以在体外用新鲜分离的小鼠肝脏线粒体维持生理平衡的缓冲液进行研究。这些细胞器的mom保持完整，除非Bak被激活，例如通过与bh3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mom立即泄漏，使其无法用于细胞凋亡研究。然而，我们发现，在含有海藻糖的缓冲液中冷冻的线粒体保持了它们的MOM完整性和对BH3-only蛋白的反应，就像新鲜的线粒体一样。海藻糖缓冲液中冷冻还可以保存ATP合成、钙诱导肿胀、跨膜电位和线粒体蛋白输入等生物功能。储存活性线粒体的能力将有助于研究细胞凋亡和其他依赖于完整MOM的线粒体功能。

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.