In Vivo Molecular Probes for the Membrane Repair Pathway in Muscle

 描述(申请人提供)：肌肉营养不良，尤其是那些与肌营养不良蛋白糖蛋白复合体功能紊乱相关的肌肉，其特征是肌肉对机械损伤敏感。肌肉损伤机制的一个重要特征是失去肌肉纤维质膜的完整性，最终导致严重的肌肉纤维变性、肌肉纤维丢失，从而导致肌肉质量的丧失和进行性无力。像许多细胞一样，有丝分裂后的肌肉纤维具有显著的修复膜损伤的能力，但对肌肉纤维中的膜修复机制知之甚少。基因突变与人类LGMD2B和Myoshi肌病有关。由于异铁蛋白与其他铁蛋白和突触素的相似性，异铁蛋白被认为与膜转运有关。在小鼠体内，去铁蛋白的丢失似乎扰乱了正常的膜修复途径。膜修复研究的主要局限性之一是，大多数膜修复的研究方法都是间接的，只显示了膜能有效排除膜损伤探针的程度，但不一定区分膜损伤的大小和修复效率的差异。这项提议的总体目标是开发一套新颖的 活细胞分子探针可以特异性地标记活体肌肉纤维和肌肉中的膜修复途径，并研究膜在肌肉纤维中直接转运的机制，以响应实验和生理损伤。我们使用这些新颖的记者之一的初步数据挑战了当前的模型，即含有脱铁蛋白的预先存在的囊泡或细胞器隔室负责修复质膜损伤。因此，本论文的工作主要集中在两个方面：1)建立一套活体小鼠细胞分子探针，用于研究实验性和生理性肌肉损伤下的膜修复途径。2)剖析膜下肌动蛋白细胞骨架是如何通过在膜损伤部位募集异位素来促进膜修复的。长期的目标是确定膜修复的机制，希望能被利用或增强，以修复几种形式的遗传性肌营养不良的肌肉损伤。

 DESCRIPTION (provided by applicant): Muscular dystrophies, particularly those associated with disruption of the function of the dystrophin glycoprotein complex, are characterized by muscles that are sensitive to mechanical damage. A key feature in the mechanism of muscle damage is the loss of muscle fiber plasma membrane integrity which ultimately results in severe muscle fiber degeneration, loss of muscle fibers, and consequently loss of muscle mass and progressive weakness. Post-mitotic muscle fibers, like many cells, have a remarkable capacity to repair membrane lesions, but little is known about the mechanisms of membrane repair in muscle fibers. Mutations in the protein dysferlin, are associated with LGMD 2B and Myoshi Myopathy in humans. Due to the similarity of dysferlin to other ferlins and synaptotagmin, dysferlin is believed to be involved in membrane trafficking. Loss of dysferlin in mice appears to disrupt the normal membrane repair pathway. One of the major limitations in studying membrane repair is that most of the approaches to study membrane repair are indirect, only showing how well membranes can effectively exclude membrane impermeant probes but doesn't necessarily differentiate differences in the magnitude of membrane wounding versus differences in efficiency of repair. The overall goal of this proposal is to develop a novel set of live cell molecular probes to specifically label the membrane repair pathway in live muscle fibers and muscles, and study the mechanisms of membrane trafficking directly in muscle fibers in response to experimental and physiological injury. Our preliminary data using one of these novel reporters challenges the current model that pre-existing vesicle or organelle compartments containing dysferlin are responsible for repairing plasma membrane lesions. Therefore, the proposed work will focus on two major aims: 1) Develop a set of live cell molecular probes in living mice to study the membrane repair pathway under experimental and physiological muscle injury. 2) Dissect how the submembrane actin cytoskeleton contributes to membrane repair by recruiting dysferlin to the site of membrane injury. The long term goal is to identify the mechanisms of membrane repair that hopefully can be exploited or enhanced in order to repair muscle damage in several forms of inherited muscular dystrophy.