REGULATION OF SUBCELLULAR ORGANIZATION IN SKELETAL MUSCL

1)背景 跨膜蛋白的正确靶向和定位是细胞组织的一个重要方面，特别是在像肌肉这样包含不同膜域的大细胞中。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过程中发生了巨大的变化。人们对这些变化的机制知之甚少。肌肉高尔基复合体的组织也依赖于收缩活动的模式，而这一规律根本不被理解。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，我们已经发现了分化过程中发生的变化的基本方面。我们已经证实，每个成肌细胞的高尔基复合体分裂成数百个较小的独立高尔基复合体，分布在细胞核周围以及多核肌管和肌纤维的细胞质中。这些小的高尔基体元素并不是随机分布的，而是被保留在内质网点旁边，专门将蛋白质输出到高尔基复合体。这种特殊的定位表明，在肌肉分化过程中高尔基复合体的碎裂类似于微管解聚时高尔基复合体所经历的碎裂。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。 模式活动是肌肉新陈代谢和收缩的重要调节因素，但它从未与蛋白质分泌途径的组织联系在一起。我们发现，高尔基复合体、内质网出口部位和微管在成熟肌肉纤维中的分布是可塑性的，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，并允许肌肉根据模式活动满足不同的新陈代谢需求。 2)本研究的目的 我们过去的工作第一次描述了肌肉中高尔基复合体的组织。为了了解它是如何被模式活动等因素控制的，我们需要确定所涉及的几个囊泡和细胞骨架元素中的哪一个是组织其他元素的触发因素。例如，确定在分化过程中微管的变化是否导致内质网出口部位的重组，或者这两者是否独立发生，这是至关重要的。同样重要的是，确定除了微管外，是否还有其他细胞骨架元件参与高尔基复合体的重新分布。例如，其他人的最新结果表明，结蛋白中间丝网络可能发挥了作用，这是我们需要探索的一种可能性。 3)过去一年的业绩 随着微管状态的变化，人们已经致力于观察活细胞中内质网的退出位置。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结构，以及用破坏微管的药物治疗来完成的。到目前为止，结果与微管通过作用于内质网本身来组织内质网退出部位的模型是一致的。 我们通过研究结蛋白缺失小鼠肌肉纤维中高尔基复合体蛋白和相关细胞元件的分布来探讨结蛋白中间丝的作用(由贝勒医学院Capetanaki博士提供)。结蛋白缺失纤维的高尔基复合体在纤维表面附近受到扰动，但在其核心似乎是正常的。有趣的是，我们发现微管在结蛋白缺失纤维的表面也受到了扰动，但在核心却没有。 到目前为止，我们的所有结果都指出微管作为肌肉中高尔基复合体分布的调节因素的首要重要性。因此，了解微管本身是如何组织的就变得很重要。一项对分化过程中微管成核变化的研究表明，在分化的肌肉中，成核发生在三种不同于成肌细胞中心体的位置，尽管似乎都涉及到蛋白质-微管蛋白。因此，G-微管蛋白从细胞质池中募集的新模式可能是微管组织在分化过程中发生变化的原因。 4)结论和意义 多年来，细胞生物学家一直在争论高尔基复合体和内质网之间的关系。我们的结果表明，在分化的肌肉中，高尔基复合体与内质网紧密相连。小但功能齐全的高尔基体确实可以被视为内质网的附属物，而不是独立的细胞器。这一发现可能有助于我们理解在某些病理条件下发生的情况，例如在神经退行性疾病中，高尔基复合体的碎裂被认为是导致疾病的原因之一。 微管是亚细胞结构的重要整合者和调节者。我们的结果表明，它们可能作为模式收缩活动的传感器发挥着另一种重要作用。在结蛋白缺失的小鼠中观察到的微管缺陷也增加了微管可能对这些小鼠的肌肉病理起作用的可能性。

1) Background 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. 2) Objective of present studies 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. It is also important to identify whether other cytoskeletal elements, besides microtubules, are involved in the redistribution of the Golgi complex. Recent results by others, for example, suggest that the desmin intermediate filament network might play a role and this is a possibility we need to explore. 3) Results during the past year 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. We have probed the role of the desmin intermediate filaments by studying the distribution of the Golgi complex proteins and associated cellular elements in muscle fibers of desmin-null mice (provided by Dr. Capetanaki, Baylor College of Medicine). The Golgi complex of desmin-null fibers is perturbed near the surface of the fibers but apparently normal in their core. Interestingly, we find that microtubules as well are perturbed at the surface but not in the core of the desmin-null fibers. 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 gamma-tubulin. Therefore, new modes of recruitment of g-tubulin from a cytoplasmic pool may be responsible for the changes in microtubule organization during differentiation. 4) Conclusions and significance Cell biologists have been debating the relationship between Golgi complex and endoplasmic reticulum for several years. Our results suggest that in differentiated muscle the Golgi complex is tightly linked to the ER. The small but fully functional Golgi elements may indeed be viewed as appendages to the ER rather than independent organelles. This discovery may help us to understand what is happening in some pathological conditions, such as in neurodegenerative diseases, in which fragmentation of the Golgi complex has been suggested to contribute to the disease. 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. The observation of microtubule defects in desmin-null mice also raises the possibility that microtubules may contribute to the muscle pathology of these mice.