Regulation of Subcellular Organization in Skeletal Muscle

骨骼肌亚细胞组织的调节

• 批准号: 8939419
• 负责人: Evelyn Ralston
• 金额: $ 36.08万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 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/8939419
• 关键词: Blood Vessels; Brain; Cells; Characteristics; Complementary DNA; Cytoplasm; Cytoskeleton; Defect; Detection; Disease; Duchenne muscular dystrophy; Elements; Fiber; Filament; Fluorescence Microscopy; Gel; Glycogen storage disease type II; Goals; Golgi Apparatus; Growth; Image; Image Analysis; Immunofluorescence Immunologic; Life; Light; Link; Lysosomes; Mammalian Cell; Manuscripts; Mechanics; Membrane Lipids; Microscopy; Microtubules; Mus; Muscle; Muscle Cells; Muscle Fibers; Myopathy; Nerve; Organelles; Pathology; Pathway interactions; Play; Positioning Attribute; Process; Proliferating; Protein Dynamics; Proteins; RNA Sequences; Regulation; Resistance; Resolution; Role; Seminal; Skeletal Muscle; Staining method; Stains; Structure; Techniques; Tissue Embedding; Tissues; Wild Type Mouse; Work; human disease; in vivo; insight; interest; intravital microscopy; mdx mouse; mechanical pressure; mouse model; software development; tool

Our main project deals with understanding the organization of the unique grid-like network of skeletal muscle fiber microtubules and its implication in muscle diseases, particularly Duchenne muscular dystrophy (DMD). Results obtained in previous years by Sarah Oddoux, Kristien Zaal and others using fluorescent protein constructs expressed in muscle fibers ex vivo and in vivo showed that this microtubule network, apparently static, is in fact composed of highly dynamic microtubules moving along each other as along tracks (Oddoux et al., 2013). We now work on understanding how microtubule organization in the mdx mouse, the mouse model for DMD, differs from that in wild-type (WT) mice. Mdx muscle fibers have an abnormal microtubule network; instead of a regular grid, microtubules are disordered, dense, and mostly oblique. Software developed in the Light Imaging Section for the analysis of microtubule directionality is essential in the automated assessment of muscle microtubules (Liu et al., 2014). Microscopy on live fibers has been carried out to compare mdx and WT muscle microtubule dynamics. We hypothesized that mdx microtubules would mostly differ by their orientation/directionality, not by their growth characteristics. The results obtained, so far, confirm our predictions. Furthermore, the results demonstrate 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. Super-resolution microscopy will be used to investigate the orientation of the Golgi elements. A secondary project has investigated the CLARITY technique for making whole muscle transparent in fluorescence microscopy. The initial technique (Chung & Deisseroth, Nat. Meth. 2013, 10 (6) 508-13) has been applied to the brain. The goal is to make the tissue transparent to allow the analysis of large structures such as nerve tracts in the brain. Lipid membranes are removed by a process that embeds the tissue in a gel, while simultaneously fixing proteinaceous structures. We thought that it would be advantageous to apply this technique to skeletal muscle, in order to follow nerve and vascular organization, muscle fiber distribution, and their changes in muscle diseases. Andy Milgroom succeeded in importing the technique to LIS and obtained the first example of CLARITIzed skeletal muscle. Manuscripts are currently prepared from both projects.

我们的主要项目涉及了解骨骼肌纤维微管独特的网格状网络的组织及其在肌肉疾病，特别是杜氏肌营养不良症（DMD）中的意义。 Sarah Oddoux、Kristien Zaal和其他人在前几年使用在离体和体内肌纤维中表达的荧光蛋白构建体获得的结果表明，这种微管网络，表面上是静态的，实际上是由高度动态的微管组成的，这些微管沿着彼此移动，就像沿着轨道一样（Oddoux et al.，2013年）。 我们现在致力于了解mdx小鼠（DMD的小鼠模型）中的微管组织与野生型（WT）小鼠中的微管组织有何不同。Mdx肌纤维有一个异常的微管网络;而不是一个规则的网格，微管是无序的，密集的，大部分是倾斜的。在光成像部分开发的用于微管方向性分析的软件在肌肉微管的自动评估中是必不可少的（Liu等人，2014年）。 对活纤维进行显微镜检查以比较mdx和WT肌肉微管动力学。我们假设mdx微管的差异主要在于它们的取向/方向性，而不是它们的生长特性。到目前为止，所获得的结果证实了我们的预测。此外，结果表明，微管方向的差异，可以观察到，一旦微管开始从成核高尔基体元件生长。因此，肌肉微管的生长就好像高尔基体元件本身或锚定在高尔基体元件上的成核分子具有特定的方向，在mdx肌肉中受到干扰。超分辨率显微镜将被用来调查的高尔基体元件的方向。 第二个项目研究了在荧光显微镜下使整个肌肉透明的透明性技术。初始技术（Chung & Deisseroth，Nat. Meth. 2013，10（6）508-13）已经应用于大脑。目标是使组织透明，以便分析大脑中的神经束等大型结构。脂质膜通过将组织包埋在凝胶中的过程去除，同时固定蛋白质结构。我们认为，这将是有利的，将这项技术应用于骨骼肌，以跟踪神经和血管的组织，肌纤维分布，以及它们在肌肉疾病的变化。Andy Milgroom成功地将该技术引入LIS，并获得了第一个骨骼肌钙化的例子。 目前正在编写这两个项目的清单。