CYTOSKELETAL TARGETS OF NEURONAL SIGNALING

这些研究的总体目标是确定分子 突触后活动依赖的调节机制 细胞骨架组织。这些研究将集中在微管上， 肌动蛋白细丝和树突状微管相关蛋白 Map2.培养的神经元将被标记MAP2、F-肌动蛋白和/或 它们的相关蛋白质，并用双光子或双光子检测 荧光显微镜或高压电子显微镜。标注将使用以下工具完成 曙红偶联试剂光氧化电子 显微镜检查。肌动蛋白细丝的组织 树突棘将在无菌培养的情况下进行检查。 或谷氨酸激动剂的存在。进行的初步实验 在NCMIR上发现鬼臼草素-曙红的光氧化 提供美丽和选择性的树枝状刺的描绘 小脑片。我们将使用断层重建和 IVEM对相关光镜观察的影响 谷氨酸能刺激脊柱结构。我们也会用这个 检查肌动蛋白三维结构的技术 细胞骨架本身在不同的条件下。的相互作用 MAP2与肌动蛋白细丝或PKA的RII调节亚基原位结合 将使用FRET和高分辨率双光子相结合的方法进行检测 成像。这些数据将被集成到一个正在开发的模型中 细胞骨架元素之间的蛋白质-蛋白质相互作用 神经元突触后隔区及其神经调节 活动。确定最佳样品的初步实验 准备工作正在进行中。

The overall goal of these studies is to determine the molecular mechanisms for activity-dependent regulation of postsynaptic cytoskeletal organization. These studies will focus on microtubules, actin filaments, and the dendritic microtubule-associated protein MAP2. Cultured neurons will be labeled for MAP2, F-actin, and/or their associated proteins, and examined using either two-photon fluorescence microscopy or HVEM. Labeling will be accomplished using eosin conjugated reagents followed by photo-oxidation for electron microscopic examination. The organization of actin filaments in dendritic spines will be examined in cultures incubated in the absence or presence of glutamate agonists. Preliminary experiments performed at the NCMIR have shown that photo-oxidation of phalloidin-eosin provides beautiful and selective delineation of dendritic spines in slices of cerebellum. We will use tomographic reconstructions and IVEM to correlated light microscopic observations on the effects of glutamatergic stimulation on spine structure.. We will also use this technique to examine the 3-dimensional organization of the actin cytoskeleton itself under different conditions. The interaction of MAP2 in situ with actin filaments or the RII regulatory subunit of PKA will be examined using FRET combined with high resolution two-photon imaging. These data will be integrated into a developing model of the protein-protein interactions that occur among cytoskeletal elements in the postsynaptic compartment of neurons and their regulation by neural activity. Preliminary experiments to determine optimal specimen preparation procedures are underway.