Fast Single-Particle Tracking of Membrane Proteins Combined with Super-Resolution Imaging of Actin Nanodomains

Membrane protein dynamics regulates cell functions by initiating downstream signaling cascades. The cell membrane is compartmentalized into nanodomains by actin structures, restricting lateral protein diffusion. Single-particle tracking offers high spatiotemporal resolution for studying protein dynamics in living cells. However, directly observing actin filaments that form barriers of nanodomains for fast protein diffusion is challenging due to their size being below the diffraction limit. Single-molecule localization microscopy resolves these structures but requires imaging in fixed cells. We integrated fast single-particle tracking with single-molecule localization microscopy to generate a dataset of membrane protein dynamics and actin filaments within the same cells at the nanoscales. Optimizing the fixation protocol enabled transition from live-cell tracking to fixed-cell super-resolution imaging. Data for the high-affinity IgE receptor, a transmembrane protein, and the GPI-anchored protein, an outer leaflet protein, was collected at 490 Hz. After fixation, actin filaments were imaged using dSTORM. The treatment of actin structures with phalloidin and PMA generated a dataset of distinct actin architectures for studying their potential influence on membrane protein dynamics.