IntAct-U-ExM enables super-resolution imaging of isoform-specific actin networks across species

by Anubhav Dhar, Sucheta Dey, Sanjana Mullick, Nishant Kumar Suman, Maxime C. van Zwam, Nishaant Kumar Palani Balaji, Angana Ghosh, Deepak Nair, Koen van den Dries, Sudarshan Gadadhar, Saravanan Palani

Expansion microscopy (ExM) has revolutionized super-resolution imaging in cell biology due to its simple and inexpensive workflow. The use of ExM has revealed several novel insights into the nanoscale architectures of cellular protein complexes, especially the microtubule cytoskeleton in model and non-model systems. Despite tremendous progress in expansion microscopy protocols that preserve cellular ultrastructure (U-ExM), compatible probes for imaging actin isoforms with U-ExM are still lacking and have hindered the study of diverse actin isoforms and networks across model systems. Here, we use IntAct, an internally tagged actin that incorporates into cellular actin networks, to develop and optimize U-ExM for diverse actin structures in yeast, mammalian cells, and primary neurons. Using ALFA-tagged IntAct variants, we achieve robust visualization of actin patches, cables, and rings in yeast, as well as diverse actin architectures including the cortex, stress fibers, filopodia, and lamellipodia in mammalian cells at improved resolution. In primary hippocampal neurons, IntAct efficiently labels actin throughout the soma and neuronal projections, revealing strong enrichment at dendritic spines and synaptic boutons. Notably, we observe a periodic organization of F-actin along axons consistent with the membrane-associated periodic cytoskeleton, thereby resolving the periodic, sub-diffraction actin ring organization. We also detect transient nuclear actin filaments using IntAct-U-ExM underscoring the advantages offered by our approach to image understudied actin structures. Overall, we demonstrate the effectiveness of IntAct-U-ExM for performing super-resolution imaging of various actin structures in an isoform-specific manner and highlight the potential of IntAct to study the nanoscale organization of diverse actin cytoskeletal networks across species.