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

Abstract

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 {beta}-actin that permits non-disruptive tag integration. Next, we used knock-in cell lines to demonstrate that the expression and filament incorporation of IntAct {beta}-actin is indistinguishable from wildtype. Furthermore, IntAct {beta}-actin remains associated with actin-binding proteins profilin, cofilin and formin family members DIAPH1 and FMNL2 and can be targeted in living cells. To demonstrate the usability of IntAct for actin isoform investigations, we also generated IntAct {gamma}-actin cells and show that actin isoform specific distribution remains unaltered in human cells. Moreover, introduction of tagged actin variants in yeast demonstrated an expected variant-dependent incorporation into patches and filaments. Together, our data indicate that IntAct is a versatile tool to study actin isoform localization, dynamics and molecular interactions.