Knockout of ACTB and ACTG1 with CRISPR/Cas9(D10A) Technique Shows that Non-Muscle β and γ Actin Are Not Equal in Relation to Human Melanoma Cells’ Motility and Focal Adhesion Formation

Abstract

Non-muscle actins have been studied for many decades; however, the reason for the existence of both isoforms is still unclear. Here we show, for the first time, a successful inactivation of the ACTB (CRISPR clones with inactivated ACTB, CR-ACTB) and ACTG1 (CRISPR clones with inactivated ACTG1, CR-ACTG1) genes in human melanoma cells (A375) via the RNA-guided D10A mutated Cas9 nuclease gene editing [CRISPR/Cas9(D10A)] technique. This approach allowed us to evaluate how melanoma cell motility was impacted by the lack of either β actin coded by ACTB or γ actin coded by ACTG1. First, we observed different distributions of β and γ actin in the cells, and the absence of one actin isoform was compensated for via increased expression of the other isoform. Moreover, we noted that γ actin knockout had more severe consequences on cell migration and invasion than β actin knockout. Next, we observed that the formation rate of bundled stress fibers in CR-ACTG1 cells was increased, but lamellipodial activity in these cells was impaired, compared to controls. Finally, we discovered that the formation rate of focal adhesions (FAs) and, subsequently, FA-dependent signaling were altered in both the CR-ACTB and CR-ACTG1 clones; however, a more detrimental effect was observed for γ actin-deficient cells. Our research shows that both non-muscle actins play distinctive roles in melanoma cells’ FA formation and motility.

The origin of the expressed retrotransposed gene ACTBL2 and its influence on human melanoma cells’ motility and focal adhesion formation

“…In one of our previous experiments, we observed that the cells lacking β-or γ-actin had changed the formation of focal adhesions (FAs) 8 . That is why we evaluated the impact of ACTBL2 knockout on FAs formation.…”

“…The functional diversification of non-muscle isoactins and their subcellular localization is still under active research. Recently, we reported that β-and γ-actin are non-redundant regarding their involvement in 2D and 3D motility and focal adhesion formation of human melanoma cells 8 . In one of our earlier studies 9 , we obtained data showing that gelsolin, one of the many actin-binding proteins (ABPs), interacts specifically with β-actin-like protein 2 (actbl2) in human melanoma cells.…”

“…The cells were fixed and stained with antibodies recognizing either α-Parvin or VASP. α-Parvin is a constituent of focal adhesion 53 , and VASP is an actin nucleator also present in FAs 8,49 . α-Parvin in A375 cells is present solely in FAs.…”

Time for rethinking the different β‐actin transgenic mouse models?

“…It is even unclear whether in cells pure filaments of individual forms exist because in vitro it has been shown that both cytoplasmic actins form copolymers (Muller et al, 2013) and the subcellular localization experiments provided evidence for mixed areas in cells where two types of actins are present. A nice example of this is shown in Figure 1 of Malek et al (2020).…”

“…However, mouse embryonic fibroblasts (MEFs) lacking β-actin can migrate if treated with ROCK-inhibitor suggesting that γ-actin (or α-smooth muscle actin) is also capable of driving cell migration (Tondeleir et al, 2012). Similarly, A375 melanoma devoid of β-actin had the same velocity as control cells in 2D migration experiments, whereas these cells lacking γ-actin migrated more slowly (Malek et al, 2020). β-Actin protein has also been located in the nucleus (Falahzadeh, Banaei-Esfahani, & Shahhoseini, 2015), suggesting a role in gene expression, transcription and epigenetic regulation in the nucleus (Hofmann et al, 2004;Olson & Nordheim, 2010;Visa & Percipalle, 2010;Xu, Thuraisingam, Morais, Rola-Pleszczynski, & Radzioch, 2010;Zheng, Han, Bernier, & Wen, 2009).…”