US-Federal Republic of Germany Cooperative Research: The Effect of Monensin on Golgi Apparatus (Cell Biology)

该奖项支持普渡大学的D.James Morre教授与海德堡德国癌症研究所的Herbert Spring博士和Michael Trendenlenburg博士合作进行研究。他们计划测试目前的概念，即钠选择性离子载体莫能菌素是如何发挥作用的，特别是在植物和动物细胞中阻断高尔基体功能。人们普遍认为，莫能菌素处理的细胞的电子显微镜显示出增大的、排列紊乱的高尔基池。然而，有证据表明，这些效应可能取决于细胞在电子显微镜下的固定方式。通过合作，他们将结合使用最先进的光学和电子显微镜方法来观察莫能菌素引起的活细胞中高尔基体的肿胀。将采用高压冷冻替代技术将该技术产生的图像与常规冷冻替代电子显微镜观察到的图像进行比较。莫雷博士是首批在高尔基体结构和功能研究中使用莫能菌素的人之一。德国的合作研究人员率先开发了计划中的实验所需的视频增强观看方法和样品室设计。此外，他们还希望与海德堡的E.Schnepf博士合作，探索激光扫描共聚焦显微镜的使用。莫能菌素是一种钠离子载体，能够分解钠和氢的梯度。作为研究高尔基体功能和识别亚细胞囊泡运输的分子途径的生化和生物学研究工具，它已被广泛接受。关于莫能菌素在活细胞中如何发挥作用的整个假说是基于莫能菌素治疗后的跨高尔基体池肿胀的假设。在用莫能菌素处理的植物和动物细胞中观察到了肿胀，并用乙醛固定剂准备了电子显微镜。然而，使用其他固定剂时，肿胀的形态证据很少或根本不存在。这项拟议的研究将通过明确确定活细胞中的高尔基体是否通过肿胀对莫能菌素做出反应，对莫能菌素肿胀假说的整体有效性进行批判性评估。这项合作计划还提供了测试德国激光扫描共聚焦显微镜使用的机会，这是一种在细胞生物学领域非常新的、概念上令人兴奋且昂贵得令人望而却步的工具。共焦扫描仪很可能能够达到回答当前项目中提出的问题所需的分辨率。如果是这样的话，这将是细胞生物学的一项重要的开创性成就，对涉及细胞内膜的广泛问题具有重要意义。

This award supports Professor D. James Morre of Purdue University to collaborate in research with Drs. Herbert Spring and Michael Trendenlenburg of the German Cancer Institute in Heidelberg. They plan to test the current concepts of how the sodium-selective ionophore monensin exerts its action in specifically blocking Golgi apparatus function in plant and animal cells. It is well accepted that electron micrographs of monensin-treated cells display enlarged, disarrayed Golgi cisternae. However, there is evidence that these effects may be dependent on how the cells were fixed for electron microscopy. Through collaboration, they will use a combination of state-of-the-art light and electron microscopic approaches to observe monensin-induced swelling of Golgi apparatus in living cells. High pressure freeze substitution will be employed to compare images generated by this technique for transmission electron microscopy with images observed with conventional freeze substitution electron microscopy. Dr. Morre was among the first to use monensin in studies of Golgi apparatus structure and function. The German co-investigators have pioneered in the development of the video-enhanced viewing methods and sample chamber design needed for the planned experiments. In addition, they expect to explore the use of a laser-scanning confocal microscope, in collaboration with Dr. E. Schnepf of Heidelberg, one of its developers. Monensin is a sodium ionophore capable of collapsing sodium and hydrogen gradients. It has gained wide acceptance as a biochemical and biological investigative tool to study Golgi apparatus function and to identify the molecular pathways of subcellular vesicular traffic. An entire hypothesis of how monensin works in the living cell is based upon the assumption that trans Golgi apparatus cisternae swell following monensin treatment. Swelling has been observed in a wide range of plant and animal cells treated with monensin and prepared for electron microscopy using aldehyde fixatives. However, morphological evidence of swelling is less or nonexistent with other fixatives. The proposed research will critically evaluate the overall validity of the monensin-swelling hypothesis by determining unequivocally whether Golgi apparatus in the living cell respond to monensin by swelling. The collaborative plan also offers an opportunity to test the uses of a German laser scan confocal microscope, a very new, conceptually exciting and prohibitively expensive tool in the field of cell biology. The confocal scanner may well be able to achieve the resolving power necessary to answer the questions posed in the current project. If so, it will be an important pioneering achievement in cell biology, with significance for a broad variety of questions involving intracellular membranes.