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

由于剪切力，细胞膜，特别是肌肉细胞的肌膜会受到小的损伤。这些膜裂口被修复贴片迅速封闭，以防止细胞死亡。虽然近年来我们已经了解了很多关于膜修复的知识，但对膜修复机制的许多关键方面仍然知之甚少。以前的研究几乎都集中在组织培养细胞或大卵子和合体胚胎的膜修复上，为了研究自然组织背景下的肌膜修复，我们利用透明的斑马鱼胚胎来实时观察完整动物损伤的肌纤维修复。为了将这些过程可视化到单分子水平，我们实施了允许我们使用超分辨率成像的技术。此外，我们开发了一种新的设备，允许将荧光显微镜和修复补片的电子断层扫描相结合的相关工作流程。我们先前已经证明，去铁蛋白(Dysf)介导磷脂酰丝氨酸(PS)在修复补丁中的浓缩。PS与巨噬细胞接触，巨噬细胞在修复过程中移除补丁。我们的初步数据表明，Dysf和PS是从邻近未受损的肌膜招募到病变部位的。鉴于观察到Dysf可以与空泡蛋白相互作用，我们假设小窝是Dysf和PS被招募到修复补丁的储存库。该提案的总体目标是了解导致修复补丁形成的分子机制。具体地说，关键问题是：1.PS和Dysf是否从邻近未受损伤的质膜中的储存库移动到修复补丁？它们是作为新生成的小泡平行于质膜移动，还是在肌膜平面上移动？2.小窝中的Dysf和PS是否会在损伤后释放这些分子？3.斑马鱼中的Dysf是否像哺乳动物一样在损伤之前或对损伤做出反应？这是膜贮藏器中Dysf的调节或正确的空间存储的一部分吗？我们将通过分子和细胞研究的跨学科组合来解决这些问题，分子和细胞研究与开发和适应物理方法密切相关，以实时以最高分辨率在细胞和完整的动物中成像和量化潜在的过程。这些结果将促进我们对基本的膜修复过程的理解。生物膜具有极强的蛋白质功能化，具有很高的生物技术潜力。了解如何维持生物膜的微妙结构是技术开发的先决条件。此外，这项工作将阐明与某些肌营养不良症有关的一些蛋白质的功能，从而可能间接有助于治疗。

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.