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bioRxiv · 10.1101/2025.05.14.654030

IntAct-U-ExM: Ultrastructure Expansion microscopy of actin networks via an internally-tagged actin

Dhar, A.·Palani Balaji, N. K.·Mullick, S.·Dey, S.·Ghosh, A.·Nair, D. K.·Gadadhar, S.·Palani, S.
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Abstract

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 of diverse actin network types in both yeast and mammalian cells. Using expression of ALFA-tagged IntAct variants in yeast and mammalian cells, we show robust visualization of actin patches, cables, and rings in yeast and diverse actin networks such as actin cortex, stress fibers, filopodia, lamellipodium in mammalian cells at improved resolution. 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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/654030v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@169cd82org.highwire.dtl.DTLVardef@1334af3org.highwire.dtl.DTLVardef@7d9d43org.highwire.dtl.DTLVardef@dfc50b_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Dhar, A., Palani Balaji, N. K., Mullick, S., Dey, S., Ghosh, A., Nair, D. K., Gadadhar, S., Palani, S.. 2025-05-15. IntAct-U-ExM: Ultrastructure Expansion microscopy of actin networks via an internally-tagged actin. https://doi.org/10.1101/2025.05.14.654030

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