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Regulation of Subcellular Organization in Skeletal Muscle

骨骼肌亚细胞组织的调节

基本信息

批准号：
10006383
负责人：
Evelyn Ralston
金额：
$ 27.43万
依托单位：
NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
依托单位国家：
美国
项目类别：
财政年份：
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/10006383
关键词：
Affect Animal Model Binding Sites Cell Culture Techniques Cells Cellular biology Characteristics Complement Complementary DNA Cytoplasm Cytoskeleton Defect Detection Developmental Biology Disease Duchenne muscular dystrophy Dystrophin Elements Fiber Filament Genetic Diseases Golgi Apparatus Human Image Analysis Immunofluorescence Immunologic Link Lysosomes Mammalian Cell Microscopy Microtubule Alteration Microtubules Minor Modification Mus Muscle Muscle Cells Muscle Development Muscle Fibers Muscular Atrophy Mutant Strains Mice Natural regeneration Organelles Pathology Pathway interactions Periodicity Pharmacology Physiological Play Positioning Attribute Proliferating Protein Dynamics Protein Isoforms Proteins Publications Publishing RNA Regulation Reporting Research Resistance Role Sampling Seminal Site Skeletal Muscle Stains Structure Techniques Tissues Transgenic Organisms Uncertainty Wild Type Mouse Work alpha Tubulin beta Tubulin density dimer frontier human disease in vivo insight interest loss of function mature animal mdx mouse mechanical force mechanical pressure microscopic imaging mouse model muscle degeneration muscle regeneration overexpression small hairpin RNA tool transcriptome sequencing

项目摘要

Skeletal muscle degeneration and loss of function is at the basis of Duchenne muscular dystrophy (DMD) pathology. Our work focuses on understanding differences between normal, wild-type mouse muscle and the same muscle in the mdx mouse, a spontaneous mutant mouse line that is an animal model for DMD. The differences we study affect muscle microtubule organization. Normal mouse muscles have a periodic grid-like microtubule network, whereas mdx mouse muscles have a disordered, denser network. We still understand little of muscle microtubule organization despite the essential and diverse roles played by microtubules in all mammalian cells and their dramatic reorganization at every stage of muscle development. Furthermore, physiological and pharmacological research on mdx muscle, complemented by RNAseq of human DMD samples, has led others to claim that microtubules underlie mdx and DMD pathology. Previously we showed that microtubules are dynamic, constantly growing, and that their path is aligned with that of the protein dystrophin, which is lacking in mdx and DMD and directly interacts with microtubules. However, a dystrophin construct lacking the microtubule binding site was able to rescue microtubules in a transgenic mdx mouse line, casting doubt on the overall role of dystrophin in microtubule organization. We decided to search for other molecules potentially responsible for the differences in microtubule organization. Microtubules are assembled from linear chains of alpha- and beta-tubulin dimers. Both alpha- and beta-tubulins have 8 to 10 isotypes, allowing for structural and possibly functional diversity. We noticed that the RNA for one of the beta-tubulin isotypes, tubb6 (beta 6 class V), is most increased in DMD compared to normal muscle; furthermore, overexpression of this ubiquitous, minor beta-tubulin isoform modifies the microtubule network of proliferating cells (Bhattacharya et al. 2011, Mol Biol Cell, 22, 1025-34). Tubb6 thus appeared worth investigating. Last year we reported that tubb6 is remarkably increased in mdx compared to wild-type mice (from 3- to 20- fold depending on specific muscle). Additionally, overexpression of a GFP construct of tubb6 causes distortion and density increase of the microtubule network in WT mouse muscle. In comparison, overexpression of the same GFP construct of another beta tubulin, TUBB5, caused no modification of the microtubule network. When tubb6 was decreased or suppressed in mdx muscle by shRNA treatment, the fibers expressing the shRNA --but not a non-specific shRNA or an RNA against TUBB5-- showed a nearly normal microtubule network. Thus, manipulating the level of tubb6 allows us to modify the organization of muscle microtubules, regardless of the presence or absence of dystrophin, suggesting that tubb6 plays a role in muscle microtubule organization and in the microtubule alterations of mdx. But why would tubb6 be increased in mdx and DMD, if it is toxic to muscle? We found that tubb6 is incrreased in regenerating fibers. Its increase in mdx muscle is proportional to the degree of regeneration.The change in microtubule organization observed in mdx muscle fibers is therefore not a simple direct consequence of the lack of dystrophin, but is linked to muscle regeneration. This work has been published recently (Randazzo et al., 2019). Some of our past work showed that in skeletal muscle, dynamic microtubules are nucleated on static Golgi elements. It is therefore possible that the microtubule disorder characteristic of mdx muscle fibers results from mispositioning of the Golgi elements. We have therefore investigated Golgi complex organizaton in live muscle fibers expressing fluorescently tagged microtubule markers. We have concluded that Golgi elements are anchored by an association with ER exit sites that is maintained in mdx muscle and thus does not require dystrophin. In contrast the positioning of the Golgi-ER exit site assemblies is perturbed in mdx muscle and thus may be linked to dystrophin. This work has been accepted for publication in Frontiers in Cell and Developmental Biology.

骨骼肌变性和功能丧失是Duchenne肌营养不良症(DMD)的病理基础。我们的工作重点是了解正常的野生型小鼠肌肉与MDX小鼠的相同肌肉之间的差异，MDX小鼠是一种自发突变的小鼠系，是DMD的动物模型。我们研究的差异会影响肌肉微管组织。正常的小鼠肌肉有一个周期性的网格状微管网络，而MDX小鼠的肌肉有一个无序的、密度更高的网络。尽管微管在所有哺乳动物细胞中扮演着重要而多样的角色，并且在肌肉发育的每个阶段都有戏剧性的重组，但我们对肌肉微管组织的了解仍然很少。此外，对MDX肌肉的生理学和药理学研究，加上对人类DMD样本的RNAseq，导致其他人声称微管是MDX和DMD病理的基础。以前，我们发现微管是动态的，不断生长，它们的路径与Dstrophin蛋白的路径一致，Dstrophin蛋白缺乏MDX和DMD，并直接与微管相互作用。然而，缺乏微管结合位点的dystrophin结构能够挽救转基因mdx小鼠的微管，这让人们对dystrophin在微管组织中的整体作用产生了怀疑。我们决定寻找其他可能导致微管组织差异的分子。微管是由α-和β-微管蛋白二聚体的线性链组装而成的。α-微管蛋白和β-微管蛋白都有8到10种同工型，允许结构和可能的功能多样性。我们注意到，与正常肌肉相比，DMD中一种β-微管蛋白亚型Tubb6(β6类V)的RNA增加最多；此外，这种普遍存在的、少量的β-微管蛋白亚型的过度表达改变了增殖细胞的微管网络(Bhattacharya等人。2011年，Mol Biol Cell，22,1025-34)。因此，Tubb6似乎值得研究。去年，我们报道，与野生型小鼠相比，MDX小鼠的tubb6基因显著增加(根据特定肌肉的不同，从3倍增加到20倍)。此外，过度表达Tubb6的GFP结构会导致WT小鼠肌肉中微管网络的扭曲和密度增加。相比之下，另一种β微管蛋白TUBB5的相同GFP结构的过度表达并没有引起微管网络的改变。当通过shRNA处理减少或抑制MDX肌肉中的tubb6时，表达shRNA的纤维--但不是非特异性shRNA或针对TUBB5的RNA--显示出几乎正常的微管网络。因此，控制Tubb6的水平使我们能够改变肌肉微管的组织，而不考虑dystrophin的存在或不存在，这表明Tubb6在肌肉微管组织和MDX的微管改变中发挥作用。但是，如果Tubb6对肌肉有毒性，为什么在MDX和DMD中会增加呢？我们发现Tubb6在再生纤维中增加。它在MDX肌肉中的增加与再生程度成正比。因此，在MDX肌肉纤维中观察到的微管组织的变化不是缺乏dystrophin的简单直接结果，而是与肌肉再生有关。这项工作最近发表(Randazzo等人，2019年)。我们过去的一些工作表明，在骨骼肌中，动态微管是在静态高尔基体上成核的。因此，MDX肌纤维的微管紊乱特征可能是高尔基体错位所致。因此，我们研究了表达荧光标记微管标记的活体肌肉纤维中的高尔基复合体组织。我们得出结论，高尔基体通过与内质网出口部位的关联而被锚定，这种关联维持在MDX肌肉中，因此不需要抗肌营养不良蛋白。相反，高尔基体出口部位组件的定位在MDX肌肉中受到干扰，因此可能与Dstrophin有关。这项工作已被《细胞与发育生物学前沿》杂志接受发表。