Superresolution Microscopy Reveals Nanometer-Scale Reorganization of MG53 Associated with Membrane Repair

Abstract

Repair of acute damage to the plasma membrane is an important aspect of normal cellular physiology and disruption of this process can result in pathophysiology in a number of human diseases. MG53, a tripartite motif family protein discovered in our lab, is an essential component of the acute membrane repair machinery. It functions as a sensor of oxidation that occurs at the injured cell membrane sites. Upon membrane damage, intracellular vesicles containing MG53 translocate and accumulate to the injured sites to form membrane repair patches. When cells are at resting states, MG53 distributes both in the cytoplasm and at the cell surface through the phosphatidylserine (PS) binding motif. Through the use of superresolution single-molecule fluorescence microscopy, we showed that MG53 is organized in nanometer-scale clusters of ∼25 nm at the surface of resting cells. At the resealed membrane injury sites, clusters that are as big as 100 nm are observed with higher local protein density, consistent with our previous biochemical studies that MG53 forms oligomers upon oxidation. We also investigate two mutant forms of MG53 - the Cys242 point mutation (C242A) and the leucine zipper motif mutant (LZ12), both of which show defective membrane repair capability due to the disrupted disulfide bond formation and impaired oligomerization respectively. Both C242A and LZ12 mutants show decreased cluster size and local protein density at the surface of resting cells.