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.
