Regulation Of Subcellular Organization Of Excitable Cell

调节膜蛋白的运输和靶向特定的亚细胞结构域是非常大的细胞，如神经元和肌肉纤维的组织的一个重要方面。该项目的目标是了解亚细胞结构域在分化过程中如何在这些细胞中组织，以及它们随后如何通过细胞活动形成。我们认为，这些结构域的形成取决于高尔基复合体组织的变化，高尔基复合体是膜蛋白分选和靶向的战略细胞中心。在肌肉分化和成熟过程中，高尔基复合体发生了显著的变化。它们的机制和规则都不清楚。小鼠肌细胞系C2是我们研究分化的模型。在分化过程中，高尔基复合体似乎分裂成小的池堆，其沿着肌管核的外核膜定位，并在细胞质中成行。我们已经证明，高尔基复合体的肌管是由独立的元素，这是本地化的内质网（ER）出口网站。分化过程中高尔基复合体变化的基础过程类似于所有哺乳动物细胞中微管被破坏时发生的过程。我们已经提出，微管的成核及其极性相对于核膜的变化解释了肌肉中高尔基复合体的破碎。这些结果很重要，因为它们表明简单的药物治疗，例如微管解聚药物诺考达唑，模仿并可以作为肌肉分化期间发生的生理事件的模型。在过去的一年里，我们专注于ER退出网站。这些被认为是静态的，并且可能沿ER膜沿着均匀分布。然而，我们的研究结果表明，ER出口位点在分化过程中重组，并且在药物如诺考达唑和布雷菲德菌素A治疗后也重组。有趣的是，这似乎不仅反映了整个ER的重组，而且还反映了ER出口位点的定位受到直接控制。目前正在进行几项实验，以研究这种控制是如何发生的。我们正在分离C2细胞以获得两种荧光蛋白的同时表达，例如一种标记ER，另一种标记ER出口位点。通过组合绿色荧光蛋白GFP的两种变体，我们能够在活细胞中同时观察两种标记物，这是一种强大的工具。我们正在通过EM免疫金评估各种药物对ER出口位点的影响，而抗体和显性阴性构建体的显微注射将使我们能够更多地了解ER出口位点定位的机制以及它在分化过程中如何变化。我们还继续研究高尔基复合体在体内肌纤维中的组织结构。为了检验中间丝可能有助于高尔基复合体定位的假设，我们开始研究结蛋白-/-敲除的肌肉中各种标记物的分布。初步检查显示，在高尔基复合体的本地化没有重大缺陷。有趣的是，微管出现上调。转基因小鼠的检查应有助于确定特定分子在健康和疾病中肌纤维细胞组织中的作用。

Regulated trafficking and targeting of membrane proteins to specific subcellular domains is an essential aspect of the organization of very large cells such as neurons and muscle fibers. The goal of this project is to understand how subcellular domains are organized in these cells during differentiation, and how they are subsequently shaped by cellular activity. We believe that the formation of such domains depends on changes in the organization of the Golgi complex, the strategic cellular center for membrane protein sorting and targeting. During muscle differentiation and maturation, the Golgi complex undergoes striking changes. Neither their mechanism nor their regulation is understood. The mouse muscle cell line C2 is our model to study differentiation. During differentiation, the Golgi complex appears to fragment into small stacks of cisternae which are positioned along the outer nuclear membrane of the myotube nuclei and in rows in the cytoplasm. We have demonstrated that the Golgi complex of myotubes is made of independent elements, which are localized at the endoplasmic reticulum (ER) exit sites. The process which underlies the changes of the Golgi complex during differentiation resembles that taking place when microtubules are disrupted in all mammalian cells. We have proposed that changes in the nucleation of microtubules and in their polarity relative to the nuclear membrane explain the Golgi complex fragmentation in muscle. These results are important because they show that simple drug treatments, for example with the microtubule-depolymerizing drug nocodazole, mimick and can serve as a model for the physiological events taking place during muscle differentiation. In the past year we have focused on the ER exit sites. These have been thought to be static and probably uniformly distributed along the ER membranes. Our results however show that ER exit sites are reorganized during differentiation and are also reorganized following treatments by drugs such as nocodazole and brefeldin A. Interestingly, it appears that this is not merely reflecting a reorganization of the whole ER but that the ER exit site localization is directly controlled. Several experiments are in progress to investigate how such control takes place. We are transfecting C2 cells to obtain simultaneous expression of two fluorescent proteins, for example one labeling the ER and the other one the ER exit sites. With a combination of two variants of the green fluorescent protein GFP we are then able to visualize both markers simultaneously in live cells, a powerful tool. We are assessing the effect of various drugs on the ER exit sites by EM immunogold, while micro-injection of antibodies and dominant negative constructs will allow us to learn more about the mechanism of ER exit site localization and how it changes during differentiation. We have also continued to investigate how the Golgi complex is organized in muscle fibers in vivo. In order to test the hypothesis that intermediate filaments may contribute to the localization of the Golgi complex, we started to examine the distribution of various markers in muscles from a desmin -/- knockout. A preliminary examination revealed no gross defects in the localization of the Golgi complex. Interestingly, microtubules appeared upregulated. The examination of transgenic mice should be useful to determine the role of specific molecules in the cellular organization of muscle fibers in health and disease.