REGULATION OF SUBCELLULAR ORGANIZATION OF EXCITABLE CELLS

调节转运和靶向膜蛋白到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组织的一个重要方面。该项目的目标是了解这些细胞在分化过程中是如何组织亚细胞结构域的，以及它们随后是如何被细胞活动塑造的。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性细胞中心。在肌肉分化和成熟过程中，高尔基复合体经历了显著的变化。它们的机制和调控都不为人所知。小鼠肌肉细胞系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池堆，它们沿着肌管核的外核膜排列，在细胞质中排列。用荧光蛋白GFP标记的高尔基复合体酶-甘露糖苷酶II永久转染的细胞系可以在活细胞上测量光漂白后的荧光恢复。我们已经证明肌管的高尔基复合体是由独立的元件组成的，这些元件位于内质网（ER）出口部位。我们现在已经证明，高尔基复合体的蛋白质通过内质网不断循环，更重要的是，这种逆行循环是高尔基复合体在分化过程中发生变化所必需的。这些结果很重要，因为它们表明有丝分裂和分化等不同的事件通过相似的途径影响高尔基复合体。我们在这项工作中发现，我们可以通过释放由MAP激酶p38抑制剂诱导的分化阻滞来同步C2培养物的分化。我们将继续开展这项工作。以大鼠肌肉单根肌纤维为模型，研究了体内肌肉成熟过程中高尔基复合体的变化。在成熟的肌纤维中，小堆的池池遍布纤维，既靠近表面，也在肌原纤维核心，确保了蛋白质的运输可以在大纤维的所有区域得到局部控制。我们已经观察到高尔基复合体和微管的分布与纤维类型有关。为了确定神经来源的营养因子或电活动是否对这种影响负责，我们检查了在去神经和慢性刺激2周的大鼠肌肉中高尔基复合物、内质网出口位点、中心体蛋白和微管的分布。我们发现，用快速刺激模式刺激快肌保留了所有标记的原始分布，用缓慢刺激模式刺激慢肌也是如此。然而，快速肌肉与缓慢刺激频率的交叉刺激或缓慢肌肉与快速刺激频率的交叉刺激会导致不同程度的标记分布变化。这些结果表明，模式电活动负责高尔基体复合体的组织，并在成年动物中保持可塑性。在未来，我们将尝试了解负责这种可塑性的分子途径。

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. Permanently transfected cell lines expressing the Golgi complex enzyme alpha-mannosidase II tagged with the fluorescent protein GFP have allowed measurements of FRAP (fluorescence recovery after photobleaching) on live cells. We have demonstrated that the Golgi complex of myotubes is made of independent elements, which are localized at the endoplasmic reticulum (ER) exit sites. We have now shown that there is constant recycling of the proteins of the Golgi complex through the ER and, more importantly, that this retrograde cycling is necessary for the changes in Golgi complex during differentiation to occur. These results are important because they show that events as different as mitosis and differentiation affect the Golgi complex by similar pathways. We have been helped in this work by the finding that we could synchronize the differentiation of the C2 cultures by releasing them from a differentiation block induced by inhibitors of the MAP kinase p38. We will pursue this work.Single muscle fibers prepared from rat muscles are used as a model to study the changes in the Golgi complex during muscle maturation in vivo. In mature muscle fibers, small stacks of cisternae are found throughout the fibers, both near the surface and in the myofibrillar core, ensuring that protein trafficking can be locally controlled in all areas of the large fibers. We have observed that the distribution of the Golgi complex and of the microtubules is fiber type dependent. To determine whether nerve-derived trophic factors or electrical activity are responsible for this effect, we have examined the distribution of the Golgi complex, of the ER exit sites, centrosomal proteins and microtubules in rat muscles that have been denervated and chronically stimulated for 2 weeks. We found that stimulation of a fast mucle with a fast stimulation pattern preserved the original distribution of all the markers, as did stimulation of a slow muscle with a slow stimulation pattern. However, cross-stimulation of a fast muscle with a slow stimulation frequency or of a slow muscle with a fast frequency led to changes in the distribution of the markers to different degrees. These results demonstrate that patterned electrical activity is responsible for the organization of the Golgi complex and that it remains plastic in the adult animal. In the future we will attempt to understand the molecular pathways responsible for this plasticity.