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

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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.