Dendra2-tagged Lifeact and MAP4 as exchangeable probes for single- molecule fluorescence imaging of cytoskeleton in live cells

摘要

Recently, a new method called image reconstruction by integrating exchangeable single-molecule localization (IRIS) was developed (Kiuchi et al., Nat. Methods 2015, 12, 743-746). IRIS ensures high-density labeling for super-resolution microscopy but can be applied for fixed cells only. Here, we extended the IRIS conception to live cell imaging. We applied Lifeact and MAP4 transiently binding to microfilaments and microtubules, respectively. Green-to-red photoconvertible fluorescent protein Dendra2 was chosen as an efficient tag for single-molecule localization microscopy. Live-cell single-molecule localization imaging with Dendra2-Lifeact and Dendra2-MAP4 was performed. As a result, super-resolved images of actin and tubulin in dynamics in living cells were reconstructed. Importantly. Dendra2-Lifeact provided a higher number of individual localizations and denser labeling of microfilaments in comparison with commonly used Dendra2-actin.