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

Abstract

Actin plays a central role in many biological processes such as cell division, motility and contractility. In birds and mammals, actin has six, highly conserved isoforms, four of which are primarily present in muscles and two that are ubiquitously expressed across tissues. While each isoform has nonredundant biological functions, we currently lack the tools to investigate the molecular basis for isoform-specificity due to their high similarity and the limited possibilities to manipulate actin. To solve this technical challenge, we developed IntAct, an internally tagged actin system to study actin isoform organization in fixed and living cells. We performed a microscopy-based screen for 11 internal actin positions and identified one residue pair that allows for non-disruptive epitope tag integration. Using knockin cell lines with tags into the ubiquitously expressed β-actin, we demonstrate that IntAct actins are properly expressed and that their filament incorporation is indistinguishable from wildtype. We further show that IntAct actins can be visualized in living cells by exploiting the nanobody-targeted ALFA tag and that they keep their ability to interact with the actin-binding proteins profilin and cofilin. Lastly, we also introduced the tag in the ubiquitously expressed γ-actin and demonstrate that the differential localization observed for actin isoforms remains unaltered for IntAct actins. Together, our data demonstrate that IntAct is a promising tool to study actin isoform localization, dynamics and molecular interactions to finally enable the molecular characterization of actin isoforms in biological processes.