RT Journal Article
SR Electronic
T1 IntAct: a non-disruptive internal tagging strategy to study actin isoform organization and function
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.10.25.465733
DO 10.1101/2021.10.25.465733
A1 M.C. van Zwam
A1 A. Dhar
A1 W. Bosman
A1 W. van Straaten
A1 S. Weijers
A1 E. Seta
A1 B. Joosten
A1 S. Palani
A1 K. van den Dries
YR 2023
UL http://biorxiv.org/content/early/2023/05/26/2021.10.25.465733.abstract
AB 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. Furthermore, IntAct β-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 γ-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.Competing Interest StatementThe authors have declared no competing interest.