Identification of genes responsible for sarcolemmal integrity in C. elegans

描述(申请人提供)：后生动物物种中的许多类型的细胞在机械应力条件下工作，并进化以应对质膜损伤。预防或修复质膜损伤的失败可导致疾病，或可影响疾病进展，如在几个肌肉营养不良症中观察到的那样。需要对膜修复有更详细的机制了解，才能找到各种疾病的治疗干预措施，在这些疾病中，膜损伤是病理生理的基础。对修复机制的详细了解的第一步是对组成分子的全面鉴定。我们开发了一种方法，利用微小的线虫线虫来鉴定介导受损肌膜(肌肉质膜)修复的分子和细胞成分。利用线虫体内可以检测到荧光蛋白的透明性，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膜损伤的程度。通过这项测试，我们发现线虫营养不良蛋白突变体(Duchenne肌营养不良症的模型)表现出肌膜渗漏和损伤，尽管很弱。基于这一发现，我们进行了基因筛选，以分离在肌膜修复方面存在缺陷的突变体，结果是加剧了肌营养不良蛋白突变体的肌膜损伤。在这个探索性的提案中，我们试图将筛选建立为识别负责肌膜修复的基因的有价值的工具。有了几个突出的突变体，我们特别建议通过遗传作图和全基因组测序相结合的方法来克隆肌膜修复缺陷的原因基因。一旦我们确定了负责的基因，我们将使用成熟的线虫基因技术来鉴定克隆的基因。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发挥保护和修复肌膜的功能。该项目的成功完成将有助于更好地了解肌膜修复的分子机制，更重要的是，将揭示潜在的药物靶点，用于阻断受膜修复影响的肌营养不良的进展。 与公共卫生相关：包括Duchenne肌营养不良症在内的许多形式的肌营养不良症都会导致肌膜完整性的破坏。因此，了解肌膜完整性是如何维持和修复的具有治疗意义。拟议中的线虫遗传学研究将识别和表征负责肌膜修复的基因。

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.