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

Subject Area Biophysics
Cell Biology
Term from 2014 to 2023
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 260752559
 
Final Report Year 2024

Final Report Abstract

Cell membranes, especially the sarcolemma of muscle cells, continually suffer small lesions even during normal exercise. Cells have developed repair mechanisms to avoid cell death upon injury, and several human muscle diseases have been traced back to a malfunctioning membrane repair system. Key aspects of these processes are still poorly understood. We have studied membrane repair with a combination of molecular and cellular studies tightly linked to the development and adaptation of advanced imaging methods (confocal microscopy, single molecule localization microscopy (SMLM), single particle tracking (SPT) and correlative light and electron microscopy (CLEM). We have taken advantage of the transparent zebrafish embryo permitting direct observation of membrane repair of myofibers injured by laser irradiation in the intact animal in real time. These studies were extended to mammalian cells for comparison. As an emergency measure, membrane lesions are provisionally sealed by a micron-sized amorphous repair patch containing proteins including Dysferlin (Dysf) and various Annexins. Subsequently, the bilayer membrane is reconstituted. We found that macrophages remove the repair patch, triggered by the presentation of phosphatidylserine (PS) as an eat-me signal on the cell surface. PS enrichment in the repair patch is mediated by the membrane protein Dysf, which both appear to come from the adjacent undamaged sarcolemma. We identified and investigated, by mutational analysis, a polybasic sequence of five amino acids in Dysf that is responsible for its recruitment and repair function. These results offer a mechanistic explanation for a known Dysf gene mutation causing severe muscle disease in humans. A similar motif in another membrane repair protein, MG53, was also analyzed. Where do materials for the provisional plug and membrane restoration come from? Annexins and other cytosolic proteins simply precipitate at the damage site in response to Ca 2+ influx. Membrane proteins and lipids, however, appear to derive from the adjacent membrane, as vesicles were largely absent in EM and optical images of the wound. We investigated caveolae, nanoscale membrane invaginations that are abundant in muscle (and other) cells, for their role in cell membrane restoration. They cause extensive folding of the membrane and can flatten to free extra membrane area under stress. Using SMLM and SPT to quantify the dynamics of caveolar proteins Caveolin3 and Cavin1, we found that caveolae mobilize and disassemble near a lesion. In a knockout zebrafish mutant lacking caveolae, long-term survival (14 h) of the cells was drastically compromised for injured cells of mutant embryos, which strongly supports a crucial involvement of caveolae in membrane repair. We have significantly advanced our understanding of cell membrane repair, which is a prerequisite for technological exploitation. This knowledge may also be beneficial for therapeutic approaches to muscular dystrophies.

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