MITOCHONDRIA FROZEN WITH TREHALOSE RETAIN BIOLOGICAL FUNCTIONS

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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.