Molecular mechanisms underlying repair of the plasma membrane at high spatial and temporal resolution

高空间和时间分辨率下质膜修复的分子机制

• 批准号: 260752559
• 负责人: Professor Dr. Gerd Ulrich Nienhaus
• 金额: --
• 依托单位: Institut für Angewandte Physik (APH)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/260752559?language=en
• 关键词: Molecular; mechanisms; underlying; repair; plasma

Cell membranes, especially the sarcolemma of muscle cells, suffer from small lesions due to shear stress. These membrane tears are rapidly sealed by a repair patch to prevent cell death. Although we have learnt a great deal about membrane repair in recent years, many key aspects of the mechanisms are still poorly understood. Almost all previous studies focused on membrane repair in tissue culture cells or large eggs and syncytial embryos as models.To investigate sarcolemmal repair in the natural tissue context, we took advantage of the transparent zebrafish embryo permitting the observation of repair of injured myofibers in the intact animal in real time. In order to visualize these processes down to the single molecule level, we implemented techniques that allow us to use super-resolution imaging. Moreover, we developed a new device that permits a correlative workflow combining fluorescence microscopy with electron tomography of the repair patch. We have previously shown that Dysferlin (Dysf) mediates phosphatidylserine (PS) enrichment in the repair patch. PS engages macrophages, which remove the patch as part of the repair process. Our preliminary data suggest Dysf and PS are recruited from the adjacent undamaged sarcolemma to the lesion site. Given the observation that Dysf can interact with caveolar proteins, we hypothesize that caveolae are reservoirs from which Dysf and PS are recruited to the repair patch. The overall objective of the proposal is to understand the molecular mechanisms that lead to repair patch formation. Specifically, key questions are: 1. Do PS and Dysf move to the repair patch from reservoirs in the adjacent, uninjured plasma membrane? Do they move as newly generated vesicles in parallel to the plasma membrane or do they travel in the plane of the sarcolemma? 2. Are caveolae reservoirs of Dysf and PS that release these molecules in response to injury? 3. Is Dysf processed prior to or in response to injury in zebrafish as in mammals? Is this part of the regulation or the correct spatial storage of Dysf in membrane reservoirs?We will approach these questions by an interdisciplinary combination of molecular and cellular studies tightly linked to development and adaptation of physical methods to image and quantify the underlying processes at highest resolution in real time in cells and intact animals. The results will advance our understanding of basic membrane repair processes. Biological membranes with their extraordinary functionalization with proteins have a high biotechnological potential. Understanding how the delicate structure of biological membranes is maintained is a prerequisite for technological exploitation. Moreover, this work will shed light on the function of a number of proteins involved in certain muscular dystrophies and may thus indirectly contribute to therapies.

细胞膜，特别是肌肉细胞的肌膜，由于剪切应力而遭受小的损伤。这些膜撕裂处被修复补丁迅速密封，以防止细胞死亡。尽管近年来我们已经了解了很多关于膜修复的知识，但其机制的许多关键方面仍然知之甚少。以往的研究几乎都集中在组织培养细胞或大卵和合胞胚胎的膜修复上。为了研究自然组织环境下的肌上皮修复，我们利用透明的斑马鱼胚胎，实时观察完整动物损伤肌纤维的修复情况。为了将这些过程可视化到单分子水平，我们采用了超分辨率成像技术。此外，我们开发了一种新的设备，允许将荧光显微镜与修复补丁的电子断层扫描相结合的相关工作流程。我们之前已经表明，Dysferlin （Dysf）介导磷脂酰丝氨酸（PS）在修复补丁中的富集。PS与巨噬细胞结合，巨噬细胞在修复过程中移除贴片。我们的初步数据表明，Dysf和PS是从相邻未受损的肌膜募集到病变部位的。鉴于观察到Dysf可以与小泡蛋白相互作用，我们假设小泡是将Dysf和PS招募到修复补丁的储存库。该建议的总体目标是了解导致修复斑块形成的分子机制。具体来说，关键问题是：1。PS和Dysf是否从相邻未受损的质膜中的储层移动到修复补丁？它们是作为新生成的囊泡平行于质膜运动还是在肌膜平面上运动？2. 小泡是Dysf和PS的储存库，在损伤后释放这些分子吗？3. 与哺乳动物一样，斑马鱼的Dysf是在受伤前处理还是在受伤后处理？这是调控的一部分，还是Dysf在膜储层中正确的空间存储？我们将通过分子和细胞研究的跨学科结合来解决这些问题，这些研究与物理方法的发展和适应密切相关，以便在细胞和完整动物中以最高分辨率实时成像和量化潜在过程。这些结果将促进我们对基本膜修复过程的理解。生物膜具有特殊的蛋白质功能化功能，具有很高的生物技术潜力。了解生物膜的微妙结构是如何维持的是技术开发的先决条件。此外，这项工作将阐明与某些肌肉萎缩症有关的许多蛋白质的功能，从而可能间接有助于治疗。