In our research, we used the gold standard F-actin probe, phalloidin, to perform points accumulation for imaging in nanoscale topography (PAINT) to achieve superresolution imaging of the F-actin nanoarchitecture within the delicate membrane protrusions of dendritic cells. The cover image of the September 17 issue of Biophysical Journal showcases a phalloidin-PAINT superresolution image of the F-actin cytoskeleton of an immature mouse DC2.4 dendritic cell.
The phalloidin-PAINT image of DC2.4 cells shows actin-rich structures, including podosomes and membrane protrusions. F-actin polarization is particularly evident at the leading edge of the cell, where it forms F-actin–rich podosomes. In addition, phalloidin-PAINT reveals the membrane protrusions around the cell body, suggesting the enrichment of F-actin in the membrane protrusions at the cell periphery. The local actin densities in the rest of the cell body appear significantly lower.
The superresolution image featured here was obtained by using Alexa Fluor 647–conjugated phalloidin (phalloidin-AF647). Chemically fixed DC2.4 cells were imaged in a buffer containing phalloidin-AF647 and the chaotropic salt potassium thiocyanate, which facilitated the dissociation of phalloidin from F-actin. The final superresolution image was reconstructed from a 30,000-frame single-molecule localization sequence captured by using total internal reflection fluorescence microscopy.
Membrane protrusions are delicate and prone to damage during solution exchange steps in standard superresolution experiments that involve phalloidin staining. Phalloidin-PAINT eliminates the need for additional labeling and washing steps, thereby better preserving the delicate membrane structures. In addition, phalloidin-PAINT presents a quantitative single-molecule localization microscopy technique for researchers to perform nanoscale characterizations of F-actin in both the cell body and the mechanically delicate F-actin–associated processes found in other cells, including neurons, cancer cells, and immune cells. More details on our work can be found at https://hu.lab.uic.edu/.
— Hirushi Gunasekara, Thilini Perera, Chih-Jia Chao, Joshua Bruno, Badeia Saed, Jesse Anderson, Zongmin Zhao, and Ying S. Hu