Super-Resolution Imaging of Plasma Membrane Lesions Inflicted by 405-nm Laser Light

Abstract

Vertebrate cells routinely experience small plasma membrane lesions induced, for example, by mechanical stress. It is well known that especially muscle cells possess efficient membrane self-repair mechanisms, and their failure leads to death of the myofiber, depletion of muscle stem cells and myopathy. Membrane repair involves a well-coordinated interplay among many different proteins, including dysferlin, calpains, annexins and MG53. To study these processes, the cell membrane is damaged in a controlled fashion, by applying mechanical force (with a needle or beads), by electroporation or by using focused laser illumination. Laser damage is arguably the most convenient strategy, especially in combination with fluorescence microscopy imaging of the ensuing events. Here we have used a 405-nm laser to locally damage cell membranes of thin adherent cells (HeLa cells) as well as thick tissues (zebrafish). Damage formation was observed as a function of time by using super-resolution localization microscopy on samples expressing fusion proteins with photoactivatable mEosFP markers. We have also measured changes in calcium level and reactive oxygen species by using Fluo-4 and CellROX Deep Red fluorescent indicators.