REGULATION OF SUBCELLULAR ORGANIZATION OF EXCITABLE CELLS

兴奋细胞亚细胞组织的调节

• 批准号: 6290649
• 负责人: Evelyn Ralston
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6290649
• 关键词: Golgi apparatus; axon; cell line; cellular polarity; dendrites; developmental neurobiology; genetic translation; glucose transporter; immunofluorescence technique; intracellular transport; laboratory rat; messenger RNA; muscle cells; nerve /myelin protein; neuromuscular junction; neurons; organelles; protein transport; tissue /cell culture; transfection; transferrin receptor; vesicle /vacuole

Regulated trafficking and targeting of membrane proteins to specific cellular domains is especially crucial for the very large excitable cells of the nervous system, 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. In mitotic cells, its organization is dictated by the needs of cell division. In post-mitotic differentiated cells, in contrast, its organization changes according to cell morphology and needs. The changes in the organization of the Golgi complex during muscle differentiation and maturation are striking. 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 been cloned and characterized. Measurements of FRAP (fluorescence recovery after photobleaching) on myoblasts and myotubes have demonstrated that the Golgi complex of myotubes is made of independent elements, which are not in rapid dynamic exchange with the ER, as has been suggested in mitotic cells. It has also shown that these elements are relatively immobile and are localized at the ER exit sites. These results suggest a model in which the changes that occur during differentiation of muscle cells resemble the events taking place in other mammalian cells when microtubules are severed. In the near future, we will continue to investigate this model.Single muscle fibers prepared from several 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 is fiber type dependent. A quantitative study by confocal light microscopy has shown that about 75% of all Golgi elements are found in a thin layer of cytoplasm at the surface of the slow fibers, whereas Golgi elements are more uniformly distributed throughout fast fibers. At the neuromuscular junction, however, the organization is independent of fiber type. The differential distribution of the Golgi complex is explained by a fiber type dependent distribution of microtubules. This differential distribution may have important physiological consequences for muscle fiber metabolism. Future work will examine whether muscle activity or nerve-derived factors are responsible for the fiber type differences. - skeletal muscle, differentiation,subcellular organelles, Golgi complex, C2C12, rat soleus, fiber type, confocal microscopy.

对于神经系统中非常大的可兴奋细胞，如神经元和肌肉纤维，调节膜蛋白的运输和靶向到特定的细胞结构域尤为重要。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。在有丝分裂细胞中，它的组织是由细胞分裂的需要决定的。相比之下，在有丝分裂后分化的细胞中，其组织根据细胞形态和需要而变化。在肌肉分化和成熟过程中，高尔基复合体组织的变化是惊人的。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标记表达高尔基复合物酶-甘露糖苷酶II的永久转染细胞系已被克隆和鉴定。对肌母细胞和肌管的FRAP（光漂白后荧光恢复）测量表明，肌管的高尔基复合体是由独立的元素组成的，而不是像有丝分裂细胞那样与内质网进行快速动态交换。它还表明，这些元素是相对不动的，并且定位在内质网出口位点。这些结果表明，在肌肉细胞分化过程中发生的变化类似于其他哺乳动物细胞在微管切断时发生的事件。在不久的将来，我们将继续研究这个模型。以大鼠单根肌肉纤维为模型，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的分布与纤维类型有关。共聚焦光学显微镜的定量研究表明，大约75%的高尔基元存在于慢纤维表面的细胞质薄层中，而高尔基元则更均匀地分布在快纤维中。然而，在神经肌肉连接处，组织与纤维类型无关。高尔基复合体的差异分布可以用微管的纤维类型依赖性分布来解释。这种差异分布可能对肌纤维代谢有重要的生理影响。未来的工作将研究是肌肉活动还是神经源性因素导致了纤维类型的差异。-骨骼肌，分化，亚细胞细胞器，高尔基复合体，C2C12，大鼠比目鱼，纤维类型，共聚焦显微镜。