Abstract
Different types of pores ubiquitously form in cell membranes, leading to various types of cell death that profoundly influence the fate of inflammation and the disease status. However, these pores have never truly been visualized to date. Atomic force microscopy (AFM), which is emerging as a powerful tool to analyze the mechanical properties of biomolecules and cells, is actually an excellent imaging platform that allows biological samples to be visualized by probing surface roughness at the level of atomic resolution. Here, membrane pore structures were clearly visualized using AFM. This visualization not only describes the aperture and depth of the pore complexes but also highlights differences among the pores formed by perforin and gasdermins in tumor cell membranes and by complement in immune cell membranes. Additionally, this type of visualization also reveals the dynamic process of pore formation, fusion, and repair.
Highlights
Membrane pore formation, an evolutionary pathway that mediates cell death, is of paramount significance in immune surveillance and pathogen clearance, as well as in the pathogenesis of inflammatory diseases[1,2,3,4,5] Several executioner molecules that are capable of drilling holes in the membrane of target cells have already been identified
Visualization of perforin/streptolysin O (SLO)-induced pore formation in the cell membrane Upon recognizing antigens, effector CD8+ cytotoxic T cells (CTLs) interact with target cells such as tumor cells or virus-infected cells and release perforin and granzymes into the intercellular immune synapse, where perforin acts as an executioner to drill a hole in the membrane of target cells[16,17,18]
Fixed OVA-B16 cells treated with perforin showed the formation of pore-like structures of various sizes in the cell membrane (Fig. 1b), allowing us to use fixed cells for the experiments in this study
Summary
Membrane pore formation, an evolutionary pathway that mediates cell death, is of paramount significance in immune surveillance and pathogen clearance, as well as in the pathogenesis of inflammatory diseases[1,2,3,4,5] Several executioner molecules that are capable of drilling holes in the membrane of target cells have already been identified. The concept of cell membrane pore formation is based on results from repeatable biological experiments and is supported by basic imaging of two-dimensional artificial liposomes using cryo-electron or other types of scanning microscopy[10,11,12]. The visualization of true pore formation has never been achieved in cellular membranes, due to the stringent requirement for high resolution (nanometer scale) and a requirement for gentle preparation methods under ambient conditions in which scanning electron microscope is usually not applicable. We report a method to directly visualize membrane pores in tumor and immune cells using AFM
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