Real-time visualization of exo- and endocytosis membrane dynamics with confocal and super-resolution microscopy

Real-time confocal and super-resolution imaging reveals membrane dynamics of exo- and endocytosis, including hemi-fusion, fusion pore opening, expansion, constriction, closure (kiss-and-run), fused-vesicle shrinking (shrink fusion), and flat membrane transition to vesicles via intermediate Λ- and Ω-shape structures. Here, we describe a protocol for imaging these membrane dynamics, including primary culture of bovine adrenal chromaffin cells, fluorescent probe application, patch-clamp to deliver depolarization and evoke exo- and endocytosis, electron microscopy (EM), and real-time confocal and stimulated emission depletion (STED) microscopy. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2016), Shin et al. (2018), and Shin et al. (2021). [Display omitted] •Real-time visualization of membrane dynamics of vesicle fusion and endocytosis•Confocal imaging, STED imaging, and electron microscopy of exo-endocytosis•Fluorescent probes and procedures for labeling plasma membrane and vesicles•Detailed protocol for culturing chromaffin cells from bovine adrenal gland Publisher's note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Real-time confocal and super-resolution imaging reveals membrane dynamics of exo- and endocytosis, including hemi-fusion, fusion pore opening, expansion, constriction, closure (kiss-and-run), fused-vesicle shrinking (shrink fusion), and flat membrane transition to vesicles via intermediate Λ- and Ω-shape structures. Here, we describe a protocol for imaging these membrane dynamics, including primary culture of bovine adrenal chromaffin cells, fluorescent probe application, patch-clamp to deliver depolarization and evoke exo- and endocytosis, electron microscopy (EM), and real-time confocal and stimulated emission depletion (STED) microscopy.