Regulation Of Subcellular Organization Of Excitable Cell

跨膜蛋白的正确靶向和定位是细胞组织的重要方面，特别是在包含不同膜结构域的大细胞如肌肉中。高尔基复合体是负责这一任务的亚细胞器。在肌肉中，高尔基复合体的组织在分化和再生期间发生显著变化。人们对这些变化的机制知之甚少。肌肉的高尔基复合体的组织也取决于收缩活动的模式，这种调节根本不清楚。我们的目标是了解高尔基复合体在肌肉中的分布是如何调节的，并与肌肉的功能需求有关。 在过去，我们已经揭示了分化过程中发生的变化的基本方面。我们已经确定，每个成肌细胞的高尔基复合体碎片成数百个较小的独立的高尔基复合体，位于细胞核周围和多核肌管和肌纤维的整个细胞质中。这些小的高尔基体元件不是随机分布的，而是保留在专门将蛋白质输出到高尔基体复合体的内质网位点旁边。这种特定的定位表明，肌肉分化过程中高尔基复合体的碎片类似于微管解聚时高尔基复合体经历的碎片。我们确实证明了这些过程之间的相似性，因此确定微管细胞骨架的变化是分化过程中发生变化的关键因素。 模式化活动是肌肉代谢和收缩的重要调节因子，但它从未与蛋白质分泌途径的组织联系起来。我们已经发现，高尔基复合体，内质网出口网站和微管的分布是塑料在成熟的肌纤维，并响应收缩活动的模式，导致纤维类型依赖的组织。我们假设这种可塑性是重要的，并允许肌肉满足不同的代谢需求取决于模式的活动。 我们过去的工作给出了，第一次，肌肉中的高尔基复合体的组织的描述。为了了解它是如何被诸如模式化活动等因素控制的，我们需要确定所涉及的几种囊泡和细胞骨架元件中的哪一种是组织其他元件的触发器。例如，确定微管变化是否导致分化期间内质网出口位点的重组或两者是否独立发生是至关重要的。 一个大的努力已经面向观察内质网出口网站在活细胞作为微管状态的函数。这是通过同时观察C2小鼠肌肉细胞系中两种不同颜色的荧光构建体以及用破坏微管的药理学试剂处理来完成的。结果是一致的，到目前为止，与模型中，微管组织的ER出口网站上的ER本身的作用。 到目前为止，我们所有的结果，因此，微管的首要重要性，作为调节器的高尔基体在肌肉中的分布。因此，了解微管本身是如何组织的变得很重要。在分化过程中微管成核的变化的调查显示，成核，在分化的肌肉，发生在三种类型的网站，不同的成肌细胞中心体，虽然似乎都涉及蛋白g-微管蛋白。因此，从细胞质库中募集g-微管蛋白的新模式可能是分化过程中微管组织变化的原因。 微管是亚细胞结构的重要整合者和调节者。我们的研究结果表明，他们可能发挥另一个重要的作用，作为传感器的模式收缩活动。

The correct targeting and localization of transmembrane proteins is an essential aspect of cellular organization, particularly in large cells such as muscle which contains distinct membrane domains. The Golgi complex is the subcellular organelle responsible for this task. In muscle, the organization of the Golgi complex changes dramatically during differentiation and regeneration. Very little is known of the mechanism of these changes. The organization of the Golgi complex of muscle also depends on the pattern of contractile activity and this regulation is not understood at all. Our goal is to understand how the distribution of the Golgi complex in muscle is regulated and is linked to the functional needs of muscle. In the past we have uncoverered basic aspects of the changes that take place during differentiation. We have established that the Golgi complex of each myoblast fragments into hundreds of smaller independent Golgi complexes placed around the nuclei and throughout the cytoplasm of the multinucleated myotubes and muscle fibers. These small Golgi elements are not distributed randomly but are retained next to endoplasmic reticulum sites specialized in the export of proteins to the Golgi complex. This specific localization suggested that the fragmentation of the Golgi complex during muscle differentiation resembles the fragmentation that the Golgi complex undergoes when microtubules are depolymerized. We have indeed demonstrated the similarity between these processes, therefore identifying changes in the microtubule cytoskeleton as a key factor in the changes that take place during differentiation. Patterned activity is an important regulator of muscle metabolism and contraction but it had never been linked to the organization of the protein secretory pathway. We have discovered that the distribution of Golgi complex, endoplasmic reticulum exit sites and microtubules is plastic in mature muscle fibers and responds to the pattern of contractile activity, causing a fiber type-dependent organization. We hypothesize that this plasticity is important and allows muscle to fulfill different metabolic demands depending on patterned activity. Our past work gives, for the first time, a description of the organization of the Golgi complex in muscle. In order to understand how it is controlled by factors such as patterned activity, we need to determine which of the several vesicular and cytoskeletal elements involved is the trigger that organizes the others. For example, it is fundamental to determine whether microtubule changes cause the reorganization of the endoplasmic reticulum exit sites during differentiation or whether the two take place independently. A large effort has been geared towards the observation of endoplasmic reticulum exit sites in live cells as a function of microtubule status. This has been done by the simultaneous observation of fluorescent constructs of two different colors in the C2 mouse muscle cell line, together with treatment with pharmacological agents that disrupt microtubules. The results are consistent, so far, with a model in which microtubules organize the ER exit sites by acting on the ER itself. All our results, so far, thus point to the primary importance of microtubules as regulators of the Golgi complex distribution in muscle. It becomes then important to understand how microtubules themselves are organized. An investigation of the changes in microtubule nucleation during differentiation is showing that nucleation, in differentiated muscle, takes place at three types of sites which differ from the myoblast centrosomes, although all seem to involve the protein g-tubulin. Therefore, new modes of recruitment of g-tubulin from a cytoplasmic pool may be responsible for the changes in microtubule organization during differentiation. Microtubules are essential integrators and regulators of subcellular architecture. Our results suggest that they may be play another important role as sensors of patterned contractile activity.