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IntAct: a non-disruptive internal tagging strategy to study actin isoform organization and function

Actin plays a central role in many cell biological processes including division and motility. Mammals have six, highly conserved actin isoforms with nonredundant biological functions, yet the molecular basis of isoform specificity remains elusive due to a lack of tools. Here, we describe the development of IntAct, an internal tagging strategy to study actin isoform function in fixed and living cells. We first identified a residue pair in β-actin that permits non-disruptive tag integration. Next, we used knock-in cell lines to demonstrate that the expression and filament incorporation of IntAct β-actin is indistinguishable from wildtype. Also, IntAct β-actin remains associated with the actin-binding proteins profilin and cofilin and can be targeted in living cells. To demonstrate isoform-specificity, we generated IntAct γ-actin cells and demonstrate that characteristic actin isoform localization remains unaltered. Together, our data demonstrate that IntAct is a versatile tool to study actin isoform localization, dynamics and molecular interactions.