Mammals have 6 highly conserved actin isoforms with nonredundant biological functions. The molecular basis of isoform specificity, however, remains elusive due to a lack of tools. Here, we describe the development of IntAct, an internal tagging strategy to study actin isoforms in fixed and living cells. We identified a residue pair in beta-actin that permits tag integration and used knock-in cell lines to demonstrate that IntAct beta-actin expression and filament incorporation is indistinguishable from wild type. Furthermore, IntAct beta-actin remains associated with common actin-binding proteins (ABPs) and can be targeted in living cells. We demonstrate the usability of IntAct for actin isoform investigations by showing that actin isoform-specific distribution is maintained in human cells. Lastly, we observed a variant-dependent incorporation of tagged actin variants into yeast actin patches, cables, and cytokinetic rings demonstrating cross species applicability. Together, our data indicate that IntAct is a versatile tool to study actin isoform localization, dynamics, and molecular interactions.

• PMID: 38466773
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Journal Article

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van Zwam MC, Dhar A, Bosman W, van Straaten W, Weijers S, Seta E, Joosten B, van Haren J, Palani S, van den Dries K

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