Identification of genes responsible for sarcolemmal integrity in C. elegans

描述（由申请人提供）：在后生动物物种中，许多类型的细胞在机械应力条件下工作，并且进化到能够应对质膜损伤。在预防或修复质膜损伤方面的失败可导致疾病，或影响疾病进展，正如在几种肌营养不良症中所观察到的那样。我们需要对膜修复机制有更详细的了解，以找到各种疾病的治疗干预措施，其中膜损伤是病理生理学的基础。详细了解修复机制的第一步是对组成分子的全面鉴定。我们开发了一种方法，利用微观线虫秀丽隐杆线虫来鉴定介导受损肌膜（肌质膜）修复的分子和细胞成分。利用秀丽隐杆线虫体的透明性，可以在体内检测荧光蛋白，我们开发了一种新的、简单的检测方法，可以很容易地评估肌层损伤的程度。通过这个实验，我们发现秀丽隐杆线虫的肌营养不良蛋白突变体（杜氏肌营养不良的一种模型）表现出肌上皮渗漏和损伤，尽管很弱。基于这一发现，我们进行了基因筛选，以分离出在肌层修复中有缺陷的突变体，从而加重肌营养不良蛋白突变体的肌层损伤。在这个探索性的建议中，我们试图建立筛选作为鉴定负责肌上皮修复的基因的有价值的工具。在掌握了几个突出的突变体的情况下，我们特别建议通过遗传作图和全基因组测序相结合来克隆肌上皮修复缺陷的致病基因。一旦我们确定了负责基因，我们将利用成熟的秀丽隐杆线虫遗传技术对克隆基因进行表征。同时，我们将确定这些已识别基因之间的关系，以了解它们如何共同保护和修复肌膜。该项目的成功完成将有助于更好地理解肌上皮修复的分子机制，更重要的是，将揭示潜在的药物靶点，以阻断受膜修复影响的肌营养不良症的进展。

DESCRIPTION (provided by applicant): Many types of cells in metazoan species operate under conditions of mechanical stress, and are evolved to cope with plasma membrane damage. A failure in either the prevention or repair of plasma membrane damage can cause disease, or can influence disease progression, as observed in several muscular dystrophies. A more detailed mechanistic understanding of membrane repair is required to find therapeutic interventions for a variety of diseases where membrane damage underlies the pathophysiology. The first step towards the detailed understanding of the repair mechanism is a comprehensive identification of the component molecules. We developed a method that uses the microscopic nematode C. elegans for identifying molecular and cellular components that mediate the repair of damaged sarcolemma (muscle plasma membrane). By taking advantage of the transparency of the C. elegans body that allows detection of fluorescence proteins in vivo, we developed a novel, simple assay that can easily evaluate the degree of sarcolemmal damage. With this assay, we found that C. elegans dystrophin mutants (a model of Duchenne muscular dystrophy) exhibit sarcolemmal leakage and damage, albeit weak. Based on this finding, we performed a genetic screen to isolate mutants that have defects in sarcolemmal repair and, as a result, aggravate sarcolemmal damage of dystrophin mutants. In this exploratory proposal, we seek to establish the screen as a valuable tool for identifying genes responsible for sarcolemmal repair. With several prominent mutants in hands, we specifically propose to clone causal genes for defects in sarcolemmal repair by a combination of genetic mapping and whole genome sequencing. Once we identify the responsible genes, we will characterize the cloned genes using well-established C. elegans genetic techniques. In parallel, we will determine the relationship between these identified genes to understand how they function together to protect and repair the sarcolemma. The successful completion of this project will lead to a better understanding of the molecular mechanism of sarcolemmal repair and, more importantly, will reveal potential druggable targets for blocking the progression of muscular dystrophies that are influenced by membrane repair. PUBLIC HEALTH RELEVANCE: Many forms of muscular dystrophy, including Duchenne muscular dystrophy, cause a disruption of the integrity of the muscle membrane. Hence, understanding how muscle membrane integrity is maintained and repaired has a therapeutic implication. The proposed C. elegans genetic study will identify and characterize genes responsible for muscle membrane repair.