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 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 Statement
The authors have declared no competing interest.
Footnotes
Figure 1 revised: We have extended our colocalization analysis demonstrating that the T229/A230 internally tagged actin has the unique ability to be integrated into stress fibers. The closely related A230/A231 and A231/S232 variant are only present in lamellipodia and filopodia but fail to localize to stress fibers. Figure 3 revised: We now show that IntAct actins associate with the formin family members DIAPH1 and FMNL2. Figure 5 revised: We now show that IntAct actins are integrated into actin patches and, to a varying extent depending on the actin variant used, in actin cables of S. Cerevisae. These experiments highlight the possibility of extending IntAct to other species. Supplementary Figures revised: Included overexpression experiments with all mammalian actin isoforms and show their integration into actin-based structures including lamellipodia, filopodia and stress fibers. Included high resolution images of lamellipodia and stress fibers to show that the architecture of these structures is unaffected in the homozygous ALFA-beta-actin IntAct cells. Included high resolution images of lamellipodia to show that in the presence of the ALFA-Nb-GFP fusion construct, the architecture of lamellipodia is unaffected. Included data on the CRISPR/Cas9-mediated knockin of two tags, ALFA tag in beta-actin and the FLAG tag in gamma-actin, to simultaneously tag two isoforms in one cells. Included Supporting material for the incorporation of actins into patches and cables of S. cerevisiae.
