Regulation of Subcellular Organization in Skeletal Muscle

我们目前的工作主要集中在了解正常小鼠(WT)和Duchenne肌营养不良症(DMD)模型MDX小鼠之间微管组织的差异。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。在光成像科开发的软件TeDT用于分析微管的方向性，是定量评估微管组织的这种差异的重要工具(Liu等人，2014年)。 前些年的研究结果表明，一旦微管从成核的高尔基体元素开始生长，就可以观察到微管取向的差异。因此，肌肉微管的生长就好像高尔基体本身，或者锚定在高尔基体上的成核分子有特定的方向，在MDX肌肉中受到干扰。用常规显微镜和超分辨显微镜研究了高尔基体元素的取向。用两种抗体标记高尔基体元素，一个针对顺式高尔基体蛋白GM130，另一个针对反式高尔基体蛋白TGN38，使我们能够确定每个高尔基体元素的方向。用TeDT软件绘制高尔基体方向性图确实显示了WT和MDX小鼠肌肉之间的差异，证实了微管组织的差异在它们形成的早期阶段就被封存了。 我们还对WT和MDX小鼠2个月和5个月龄的三种不同的小鼠肌肉(FDB、EDL和比目鱼肌)进行了RNA-Seq分析。目的是比较MDX小鼠肌肉和人类DMD肌肉中RNA的变化(Kairallah等人。2012，Sci Signal，5,236)，特别关注微管蛋白、微管的成分和微管相关蛋白。这两个年龄的选择是基于同一篇论文中的报告，即微管在5岁时与MDX病理有关，但在2个月龄时不涉及。结果证实，与WT肌肉相比，几个微管蛋白mRNAs在MDX肌肉中有差异表达。然而，他们也指出了人类和小鼠微管蛋白亚型分布的差异。虽然在2个月和5个月大的小鼠中有几个不同的mRNAs表达，但微管蛋白mRNAs的情况并非如此。因此，这些年龄之间的反应差异必须在受影响的通路的其他部分进行研究。在结果分析完成后，我们将进一步调查假定在MDX网络解体中发挥作用的特定分子的作用。 在过去的一年里，我们的研究主要集中在由tubb6基因编码的β-微管蛋白亚型β6 V类。为简单起见，我们将其称为TUBB6。我们对这种微管蛋白的兴趣有两个动机：第一，当比较DMD和正常的人类肌肉转录组时，它的mRNA表达增加最多；第二，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微管网络，并对增殖细胞具有毒性(Bhattacharya等人)。2011年，Mol Biol Cell，22,1025-34)。我们决定追求两个目标：一方面研究TUBB6在正常小鼠肌肉中过表达的影响；另一方面探索当TUBB6在MDX肌肉中过表达时会发生什么。这两种方法都是成功的，并表明TUBB6是MDX小鼠和潜在的DMD的疾病修饰剂。过表达GFP构建的TUBB6导致WT小鼠肌肉中扭曲、致密的微管网络。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--但不是扰乱的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，而无论是否存在dystrophin，DMD和MDX中缺乏的蛋白质以前被认为是引导和正确组织微管所必需的。 如果TUBB6对肌肉有毒性，为什么MDX和DMD中TUBB6会增加？我们通过分析TUBB6在肌肉发育过程中的表达变化发现了一些线索：我们发现在小鼠肌肉细胞系C2分化过程中，TUBB6的表达增加了2倍以上。因此，TUBB6可能在肌肉再生过程中发挥有用的作用，即在肌肉疾病如DMD和MDX小鼠中发生的新肌肉纤维的形成。为了支持这一假说，我们发现TUBB6在MDX肌肉中并不是均匀分布的，而是高度集中在离散区域的肌管和肌纤维中，这些区域也显示出免疫球蛋白积聚和非肌肉细胞的渗透，表明它们是再生区。 因此，在MDX中(可能在DMD中)TUBB6的增加似乎是对肌肉炎症和退化的反应的一部分。然而，MDX/DMD的肌肉再生成为一种永久性的情况，因为这种疾病的根本原因--肌营养不良蛋白的缺乏--并没有消失。因此，TUBB6继续升级，消防员成为纵火犯。

Our current work focuses primarily on understanding the differences in microtubule organization between muscles of normal mice (WT) and of mdx mice, a model for Duchenne muscular dystrophy (DMD). Normal mouse muscles have a periodic grid-like microtubule network, whereas mdx mouse muscles have a disordered, denser network. The software TeDT, developed in the Light Imaging Section for the analysis of microtubule directionality, is an essential tool in the quantitative assessment of such differences in microtubule organization (Liu et al., 2014). Results obtained in previous years demonstrated that the differences in microtubule orientation can be observed as soon as microtubules start growing from the nucleating Golgi elements. Thus muscle microtubules grow as if Golgi elements themselves, or the nucleating molecules anchored to the Golgi elements, had a specific orientation, disturbed in mdx muscles. Conventional and super-resolution microscopy have been used to investigate the orientation of the Golgi elements. Labeling of the Golgi elements with two antibodies, one for the cis-Golgi protein GM130 and the other for the trans-Golgi protein TGN38 allowed us to determine the orientation of each Golgi element. Plotting Golgi directionality with the software TeDT indeed shows differences between WT and mdx mouse muscles, confirming that differences in microtubule organization are sealed at an early stage of their formation. We have also carried out RNA-Seq analysis of three different mouse muscles (FDB, EDL, and soleus) at two different ages, 2 and 5 months, from both WT and mdx mice. The goal was a comparison of RNA changes in the mdx mouse muscles compared to human DMD muscles (Khairallah et al. 2012, Sci Signal, 5, 236) with special attention to tubulins, the constituents of microtubules, and microtubule-associated proteins. The two ages were selected based on the report in the same paper that microtubules are implicated in the mdx pathology at 5 but not at 2 months of age. The results confirm that several tubulin mRNAs are differentially expressed in mdx compared to WT muscles. However, they also point to differences between human and mouse tubulin isoform distribution. Although several mRNAs are differentially expressed in 2 and 5 month-old mice, this is not the case for tubulin mRNAs. Thus, the difference in response between these ages must be searched in other parts of the pathways affected. After analysis of the results is complete we will further investigate the role of specific molecules hypothesized to play a role in the disorganization of the mdx network. Our research over the past year has focused on the beta-tubulin isoform beta 6 class V which is encoded by the gene tubb6. For the sake of simplicity we refer to it as TUBB6. There are two motives for our interest for this tubulin: first, its mRNA is the most increased when comparing DMD to normal human muscle transcriptome; second, overexpression of this ubiquitous, minor beta-tubulin isoform modifies the microtubule network and is toxic to proliferating cells (Bhattacharya et al. 2011, Mol Biol Cell, 22, 1025-34). We decided to pursue two goals: on the one hand investigate the effects of overexpressing TUBB6 in normal mouse muscle; and on the other explore what happens when TUBB6 is decreased in mdx muscle. Both avenues have been successful and suggest TUBB6 as a disease modifier for the mdx mouse and potentially for DMD. Overexpression of a GFP construct of TUBB6 led to a distorted, dense 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 scrambled 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, the protein lacking in DMD and mdx that was previously thought to be necessary for the guidance and proper organization of microtubules. Why would TUBB6 be increased in mdx and DMD, if it is toxic to muscle? We found some hints by analyzing changes in TUBB6 expression during muscle development: we found out that TUBB6 increases more than 2-fold during differentiation of the mouse muscle cell line C2. It is therefore possible that TUBB6 plays a useful role during muscle regeneration, i.e. the formation of new muscle fibers that takes place in muscle diseases such as DMD and in the mdx mouse. In support of this hypothesis, we found that TUBB6 in mdx muscle is not uniformly distributed but is highly concentrated in myotubes and muscle fibers in discrete areas that also show IgG accumulation and non-muscle cell infiltration identifying them as regeneration areas. It therefore appears that the increase of TUBB6 in mdx (and probably in DMD) is part of the response to muscle inflammation and degeneration. However, muscle regeneration in mdx/DMD becomes a permanent situation because the root cause of the disease --the absence of dystrophin-- does not go away. Therefore TUBB6 continues to escalate, and the fireman becomes arsonist.