Regulation of Subcellular Organization in Skeletal Muscle

为了向目标1迈进，我们一直在研究小鼠的指屈肌短肌，从那里可以可靠地、可重复地制备胶原酶解离纤维，用药物处理，用抗体染色，并用免疫荧光观察。在微管生长的实验条件下，我们观察到典型的短微管“紫菀”。在大多数细胞类型中，这种细胞起源于中心体，尽管一群特殊的微管似乎集中在高尔基复合体上。在肌肉发育过程中中心体被分解。在肌纤维中，我们观察到的紫苑以高尔基复合体元素为中心。我们已经证明了高尔基复合体元素是成核机制的一部分，通过证明用Brefeldin A（一种使高尔基复合体塌陷到内质网的药物）处理过的纤维中的微管紫晶在数量和大小上都严重减少。我们现在正在研究特定高尔基复合体蛋白在成核过程中的作用。第一个候选是高尔基复合体蛋白GM130，一种中心高尔基基质蛋白。在初步实验中，我们已经测试了shrna，并成功地在C2肌肉细胞培养中敲除GM130。我们还优化了将cdna注入小鼠FDB肌肉的技术。因此，我们应该能够在FDB肌纤维中敲除GM130，并评估其对微管成核和体内高尔基分布的影响。

To advance towards objective 1, we have been working with the flexor digitorum brevis muscle of the mouse, from which collagenase-dissociated fibers can be reliably and reproducibly prepared, treated with drugs, stained with antibodies and observed in immunofluorescence. In experimental conditions of microtubule growth, we observe typical "asters" of short microtubules. In most cell types such asters originate from the centrosome, although a population of specialized microtubules appears to be centered on the Golgi complex. During muscle development the centrosomes are disassembled. In muscle fibers, the asters we observe are centered on Golgi complex elements. We have shown that the Golgi complex elements are part of the nucleating mechanism by demonstrating that microtubule asters are severely reduced in number and in size in fibers treated with Brefeldin A, a drug that collapses the Golgi complex into the endoplasmic reticulum. We are now investigating the role of specific Golgi complex proteins in this nucleation. The first candidate is the Golgi complex protein GM130, a central Golgi matrix protein. In preliminary experiments we have tested shRNAs and succeded in knocking GM130 down in C2 muscle cell cultures. We have also optimized techniques for injecting cDNAs into mouse FDB muscle. We should therefore be able to knock down GM130 in FDB muscle fibers and assess the effects on microtubule nucleation and Golgi distribution in vivo. In order to observe microtubule dynamics in muscle in vivo (objective 2), we have constructed and tested DNA plasmids encoding fragments or the whole of two microtubule-associated proteins: MAP4, and the ensconsin microtubule-binding domain (EMTB),linked to fluorescent proteins. This preliminary work is necessary because the most obvious microtubule marker, tubulin-GFP, does not fully incorporate in existing microtubules, and does not track microtubule dynamics faithfully in C2 cultures. At low degrees of overexpression, MAP4 and EMTB show dynamic behavior in myoblasts and myotubes and, satisfy all our criteria for usefulness for in vivo experiments. The next stage will be to introduce them into muscle fibers in vivo. To evaluate the mechanism and consequences of the microtubule defects in disease models (objective 3) we have observed microtubule nucleation in mdx and Pompe disease mice and have found profound defects which we are currently analyzing. In the past year we have finalized a study showing that the initial reorganization of centrosomal proteins during myogenesis in muscle cell cultures drives all the other steps and takes place independently of microtubule reorganization. We have also continued our collaboration with Drs. Plotz and Raben on the study of Pompe Disease. Our results on microtubule nucleation in muscle in vivo are turning around the way we understand microtubule-organelle relationships. They will help us understand muscle phenotype in several diseases as well as the difficulties encountered by enzyme replacement therapy in Pompe disease.