REGULATION OF SUBCELLULAR ORGANIZATION IN SKELETAL MUSCLE

在过去的一年里，我们完成了一项关于肌营养不良蛋白和微管组成蛋白微管蛋白之间相互作用的合作研究(Prins等人，2009年)。营养不良蛋白是一种很大的蛋白质，它的突变导致了最常见的肌肉疾病--Duchenne肌营养不良症(DMD)。已知dystrophin与肌动蛋白细胞骨架和中间丝蛋白相互作用，这表明它可能作为细胞连接蛋白，一种连接细胞细胞骨架所有成分的蛋白质。我们的合作者James Erasti(美国明尼苏达州大学)和他的实验室使用对照和DMD小鼠模型MDX小鼠的肌肉提取物，获得了支持dystrophin和微管蛋白之间直接相互作用的生化证据。Amisha Mehta和后来的Victoria Tate in Li对对照组和mdx小鼠以及utroin基因敲除小鼠和mdx-utroin双基因敲除小鼠的单个完整肌肉纤维进行了免疫荧光染色。在MDX和MDX-utroin双重敲除的肌肉中，微管失去了正常的组织，但在utroin敲除的肌肉中没有。此外，我们还发现，在尚未开始经历疾病特征的退化-再生周期的年轻(3周大)小鼠中，已经存在微管缺陷。这些实验支持体内营养不良蛋白和微管之间的特定联系。我们还显示了正常小鼠快速肌肉中的微管沿着dystrophin的横向和纵向条带，证实了dystrophin有助于组织微管。因此，这项工作将dystrophin归类为肌肉纤维中的一种细胞连接蛋白，并可能有助于理解在MDX小鼠和DMD中缺乏dystrophin的一些后果。 我们也朝着了解成熟肌肉纤维中微管的组织的目标前进。维多利亚·泰特一直在使用一种培养系统，该系统使用的是通过酶消化从老鼠短屈肌中分离出来的肌肉纤维。她已经确定了从活纤维中解聚动态微管的条件，然后观察它们的再聚合。这些实验很重要，因为微管通常在中心体有核，但一些细胞类型，如骨骼肌，缺乏典型的中心体。除中心体外，有些类型的细胞在高尔基复合体也可见微管成核。在骨骼肌中，中心体蛋白、微管和高尔基复合体都重新分布，首先是在分化过程中，然后是在肌肉纤维成熟过程中。分化的肌肉培养显示在核膜上形成微管，但在活体肌肉纤维中微管的动力学从未被研究过。有趣的是，肌肉纤维中微管的成核似乎起源于高尔基复合体元素。维多利亚还研究了微管在高尔基体与肌肉纤维中的溶酶体(LAMP-1阳性结构)之间紧密联系中的作用。大多数高尔基体(75%)在对照和诺可达唑处理的纤维中有并列的溶酶体，但寒冷和诺可达唑各自增加了与高尔基体无关的溶酶体的比例。这表明动态微管和稳定的、诺可达唑抗性的谷氨酰化微管都参与了高尔基体-溶酶体的结合。我们的发现为理解肌肉纤维中的微管和高尔基复合体组织提供了模型基础。他们还将基于高尔基复合体的微管成核扩展到高度分化的组织。这些机制可能并不完全相同。海报(泰特等人)将在2009年美国细胞生物学学会会议上公布。 在我们研究肌肉纤维的同时，我们还开展了肌肉培养方面的工作，使我们对肌肉细胞分化过程中发生的变化有了基本的了解。Tan Zhang和Kristien Zaal一直在研究微管相关蛋白EB1在肌肉分化中的作用。在其他哺乳动物细胞中，EB1是微管稳定所必需的。我们推测，EB1在肌肉发育过程中可能扮演着类似的角色。事实上，我们发现EB1的显性-负性结构影响成肌细胞的伸长。击倒EB1会永久性地阻止微管的稳定，并且出人意料地阻止肌肉细胞的分化(Zhang等人，2009)。此外，Tan Zhang现在已经证明，有一个与高尔基情结相关的EB1池，它可能在高尔基情结(手稿准备中)的完整性中发挥作用。 我们还继续与Plotz博士和Raben博士合作研究Pompe病，这是一种溶酶体储存障碍，心肌和骨骼肌是其病理来源(Raben等人，2008&2009)。

Over the past year we have completed a collaborative study of the interaction between the muscle protein dystrophin and the microtubule constituent protein tubulin (Prins et al., 2009). Dystrophin is a large protein whose mutations are responsible for the most prevalent muscle disease, Duchenne Muscular Dystrophy (DMD). Dystrophin was already known to interact with the actin cytoskeleton and with intermediate filament proteins, suggesting that it may act as a cytolinker, a protein that serves to link all constituents of the cell cytoskeleton. Using muscle extracts from control and from mdx mouse, the mouse model for DMD, our collaborator James Ervasti (U. Minnesota) and his laboratory obtained biochemical evidence supporting a direct interaction between dystrophin and tubulin. Amisha Mehta and, later, Victoria Tate in LI performed immunofluorescence staining of single whole muscle fibers from control and mdx mice, as well as from utrophin knockdown mice and mdx-utrophin double knockdown mice. Microtubules lost their normal organization in mdx and mdx-utrophin double knockdown muscles, but not in the utrophin knockdown muscles. In addition, we showed that microtubule defects are already present in young (3 week-old) mice which have not started undergoing the cycles of degeneration-regeneration characteristic of the disease. These experiments support a specific link between dystrophin and microtubules in vivo. We also showed that microtubules in fast muscles of a normal mouse course along transverse and longitudinal bands of dystrophin, confirming that dystrophin contributes to organizing microtubules. This work thus classifies dystrophin as a cytolinker in muscle fibers and may help to understand some of the consequences of the absence of dystrophin, in the mdx mouse and in DMD. We have also progressed towards our goal of understanding the organization of microtubules in mature muscle fibers. Victoria Tate has been using a culture system using muscle fibers detached from the flexor digitorum brevis mouse muscle by enzymatic digestion. She has determined conditions to depolymerize the dynamic microtubules from the live fibers and to then observe their repolymerization. These experiments are important because microtubules are typically nucleated at the centrosome, but several cell types, such as skeletal muscle, lack a typical centrosome. Some cell types also show microtubule nucleation at the Golgi complex, apart from that seen at the centrosome. In skeletal muscle, centrosomal proteins, microtubules, and Golgi complex are all redistributed, first during differentiation, and then further during maturation of muscle fibers. Differentiated muscle cultures show microtubules forming at the nuclear membrane but microtubule dynamics have never been explored in muscle fibers in vivo. Interestingly, nucleation of microtubules in muscle fibers appears to originate at the Golgi complex elements. Victoria has also examined the role of microtubules in the close association between Golgi elements and lysosomes (LAMP-1 positive structures) in muscle fibers. Most Golgi elements (75%) have juxtaposed lysosomes in control and nocodazole-treated fibers but cold and nocodazole, each, increase the fraction of Golgi-unassociated lysosomes. This suggests that both dynamic microtubules and stable, nocodazole-resistant, glutamylated microtubules are involved in Golgi-lysosome association. Our findings provide the foundation of a model to understand microtubule and Golgi complex organization in muscle fibers. They also extend Golgi complex-based microtubule nucleation to a highly differentiated tissue. The mechanisms may not be identical. A poster (Tate et al.) will be presented at the 2009 meeting of the American Society for Cell biology. In parallel to our work on muscle fibers, we have pursued our work on muscle cultures which have provided us with a basic understanding of the changes taking place during differentiation of muscle cells. Tan Zhang together with Kristien Zaal has been investigating the role of the microtubule-associated protein EB1 in muscle differentiation. In other mammalian cells, EB1 is necessary for microtubule stabilization. We hypothesized that EB1 may play a similar role during muscle development. Indeed, we found that dominant-negative constructs of EB1 affect myoblast elongation. Knocking down EB1 permanently prevents microtubule stabilization and, unexpectedly, prevents differentiation of the muscle cells (Zhang et al., 2009). In addition, Tan Zhang has now demonstrated that there is an EB1 pool associated with the Golgi complex and that it may be play a role in the integrity of the Golgi complex (manuscript in preparation). We have also continued to collaborate with Drs. Plotz and Raben on the study of Pompe Disease, a lysosomal storage disorder in which cardiac and skeletal muscles are the source of the pathology (Raben et al., 2008 & 2009).