In Vivo Molecular Probes for the Membrane Repair Pathway in Muscle

肌肉膜修复途径的体内分子探针

• 批准号: 9060873
• 负责人: Daniel E Michele
• 金额: $ 17.11万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9060873
• 关键词: Actins; Animals; Binding; Biological Assay; Calcium; Cell membrane; Cells; Cessation of life; Communities; Complex; Confocal Microscopy; Cytoskeleton; DYSF gene; Data; Deterioration; Disease Outcome; Dyes; Dystrophin; Early Ambulation; Engineering; Event; Face; Family; Fiber; Future; Genes; Glycoproteins; Goals; Gold; Health; Hereditary Disease; Human; Inherited; Injury; Knockout Mice; Label; Lasers; Lesion; Life; Limb-Girdle Muscular Dystrophies; Lipid Binding; Measures; Mechanics; Membrane; Membrane Protein Traffic; Methodology; Methods; Mitotic; Modeling; Molecular; Molecular Probes; Monitor; Mus; Muscle; Muscle Cells; Muscle Fibers; Muscle Weakness; Muscular Dystrophies; Mutation; Myopathy; Natural regeneration; Organelles; Outcome; Pathway interactions; Patients; Peptides; Phospholipids; Physiological; Process; Proteins; Recruitment Activity; Recycling; Reporter; Research; Respiratory physiology; Role; Sarcolemma; Series; Site; Striated Muscles; Testing; Therapeutic; Time; Total Internal Reflection Fluorescent; Transgenic Organisms; Vesicle; Work; base; cell injury; gamma Actin; improved; in vivo; injury and repair; link protein; loss of function; loss of function mutation; member; mouse model; muscle degeneration; muscle form; novel; prevent; repaired; research study; response; sensor; synaptotagmin; targeted treatment; tool; trafficking; two-photon; uptake; wound

 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.

 描述（由申请人提供）：肌营养不良，特别是与肌营养不良蛋白糖蛋白复合物功能破坏相关的肌营养不良，其特征在于肌肉对机械损伤敏感。肌肉损伤机制中的一个关键特征是肌纤维质膜完整性的丧失，其最终导致严重的肌纤维变性、肌纤维损失，并因此导致肌肉质量损失和进行性虚弱。有丝分裂后的肌纤维与许多细胞一样，具有显着的修复膜损伤的能力，但人们对肌纤维膜修复的机制知之甚少。蛋白dysferlin的突变与人类LGMD 2B和Myoshi肌病有关。由于dysferlin与其他ferlin和synaptotagmin的相似性，dysferlin被认为参与膜运输。在小鼠中dysferlin的丢失似乎破坏了正常的膜修复途径。研究膜修复的主要限制之一是，大多数研究膜修复的方法是间接的，仅显示膜可以有效地排除膜不渗透探针，但不一定区分膜损伤的程度与修复效率的差异。该提案的总体目标是开发一套新颖的 活细胞分子探针，特异性标记活肌纤维和肌肉中的膜修复途径，并研究肌纤维中直接响应实验和生理损伤的膜运输机制。我们的初步数据，使用这些新的记者挑战目前的模型，即预先存在的囊泡或细胞器隔间含有dysferlin负责修复质膜病变。因此，本论文的主要工作将集中在两个方面：1）开发一套活体小鼠的活细胞分子探针，用于研究实验性和生理性肌肉损伤下的膜修复途径。2)解剖膜下肌动蛋白细胞骨架如何通过将dysferlin募集到膜损伤部位来促进膜修复。长期目标是确定膜修复的机制，希望可以利用或增强，以修复几种形式的遗传性肌营养不良症的肌肉损伤。