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dr。海德堡德国癌症研究所的赫伯特·斯普林和迈克尔·特伦登堡说。他们计划测试目前的概念，即钠选择性电离层莫能菌素如何在植物和动物细胞中特异性地阻断高尔基体功能。人们普遍认为，莫能菌素处理细胞的电子显微照片显示扩大，紊乱的高尔基池。然而，有证据表明，这些影响可能取决于细胞在电子显微镜下的固定方式。通过合作，他们将结合最先进的光学和电子显微镜方法来观察活细胞中莫能菌素诱导的高尔基体肿胀。高压冷冻替代将被用来比较这种技术产生的图像与传统冷冻替代电子显微镜观察到的图像。莫尔博士是最早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视频增强观看方法和计划实验所需的样品室设计。此外，他们还希望与海德堡大学的E.施奈尔夫博士（Dr. E. Schnepf）合作，探索激光扫描共聚焦显微镜的使用。施奈尔夫博士是激光扫描共聚焦显微镜的研发人员之一。莫能菌素是一种钠离子载体，能够使钠和氢的梯度坍塌。作为研究高尔基体功能和确定亚细胞囊泡交通的分子途径的生化和生物学研究工具，它已被广泛接受。莫能菌素如何在活细胞中起作用的整个假设是基于反高尔基体池在莫能菌素治疗后肿胀的假设。用莫能菌素处理过的各种植物和动物细胞，以及用乙醛固定剂制备的电子显微镜观察到的细胞肿胀。然而，其他固定剂较少或不存在肿胀的形态学证据。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作出反应，批判性地评估莫能菌素肿胀假说的总体有效性。该合作计划还提供了一个测试德国激光扫描共聚焦显微镜使用的机会，这是细胞生物学领域一种非常新的、概念上令人兴奋的、昂贵得令人望而却步的工具。共聚焦扫描仪很可能能够达到解决当前项目中提出的问题所必需的分辨率。如果是这样，这将是细胞生物学中一项重要的开拓性成就，对涉及细胞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.