MITOCHONDRIA FROZEN WITH TREHALOSE RETAIN BIOLOGICAL FUNCTIONS

用海藻糖冷冻线粒体保留生物功能

• 批准号: 7601060
• 负责人: DONALD DAVID NEWMEYER
• 金额: $ 1.09万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7601060
• 关键词: ATP Synthesis Pathway; Apoptosis; Bax protein; Biological Process; Buffers; Calcium; Computer Retrieval of Information on Scientific Projects Database; Cytoplasm; Equilibrium; Freezing; Funding; Grant; In Vitro; Institution; Liver Mitochondria; Mannitol; Membrane Potentials; Mitochondria; Mitochondrial Proteins; Mus; Organelles; Outer Mitochondrial Membrane; Process; Protein Family; Protein Import; Proteins; Research; Research Personnel; Resources; Source; Standards of Weights and Measures; Sucrose; Swelling; Trehalose; United States National Institutes of Health; cytochrome c; pro-apoptotic protein; sugar

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它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。可以使用维持在生理平衡缓冲液中的新鲜分离的小鼠肝线粒体在体外研究该过程。这些细胞器的 MOM 保持完整，除非 Bak 被激活，例如通过与仅 BH3 的蛋白质（例如 tBid）一起孵育。相比之下，在标准蔗糖-甘露醇缓冲液中冷冻/解冻的线粒体 MOM 立即变得渗漏，使它们无法用于细胞凋亡研究。然而，我们在这里表明，在含有糖、海藻糖的缓冲液中冷冻的线粒体保持了其 MOM 完整性和对仅 BH3 蛋白的反应性，就像新鲜线粒体一样。在海藻糖缓冲液中冷冻还保留了生物学功能，例如 ATP 合成、钙诱导的肿胀、跨膜电位和线粒体蛋白输入。储存活性线粒体的能力将促进细胞凋亡和其他依赖于完整 MOM 的线粒体功能的研究。