REGULATION OF SUBCELLULAR ORGANIZATION OF EXCITABLE CELLS

膜蛋白被调控的运输和靶向特定的亚细胞结构域是超大型细胞如神经元和肌肉纤维组织的一个重要方面。这个项目的目标是了解这些细胞在分化过程中亚细胞结构域是如何组织的，以及随后它们是如何由细胞活动塑造的。我们认为，这些结构域的形成取决于高尔基复合体的组织结构的变化，高尔基复合体是膜蛋白分类和靶向的战略细胞中心。在肌肉分化和成熟的过程中，高尔基复合体发生了显著的变化。既不了解它们的机制，也不了解它们的监管。小鼠肌肉细胞系C2是我们研究分化的模型。在分化过程中，高尔基复合体似乎碎裂成小池，沿着肌管核的外核膜排列在细胞质中。用荧光蛋白GFP标记的表达高尔基体复合酶α-甘露糖苷酶II的永久性转基因细胞株可以在活细胞上测量FRAP(光漂白后荧光恢复)。我们已经证明，肌管高尔基复合体是由独立的元件组成的，这些元件定位于内质网(ER)出口部位。我们现在已经证明，高尔基复合体中的蛋白质通过内质网不断地循环，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，像有丝分裂和分化这样不同的事件通过相似的途径影响高尔基复合体。我们在这项工作中得到的帮助是，我们可以通过将C2培养物从MAP激酶p38抑制剂诱导的分化阻滞剂中释放出来，使C2培养物的分化同步。我们将继续这项工作。以大鼠肌肉制备的单一肌肉纤维为模型，研究肌肉在体内成熟过程中高尔基复合体的变化。在成熟的肌肉纤维中，在靠近表面和肌原纤维核心的整个纤维中都发现了小池，这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。为了确定神经源性营养因子或电活动是否对这种效应负责，我们研究了去神经和慢性刺激2周的大鼠肌肉中高尔基复合体、内质网出口部位、中心体蛋白和微管的分布。我们发现，快速粘液和快速刺激模式的刺激保留了所有标志物的原始分布，慢速肌肉的刺激模式也是如此。然而，对慢刺激频率的快肌肉或快频率的慢肌肉的交叉刺激会导致标志物分布的不同程度的变化。这些结果表明，图案化的电活动负责高尔基复合体的组织，并且在成年动物中它仍然是可塑性的。在未来，我们将试图了解导致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.