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.
Highlights
The actin cytoskeleton is essential for the proper functioning of many cellular processes, including maintenance of the cell shape, chemotaxis, cell movement, adhesion, transport of cellular organelles, mitosis, replication, transcription, and even DNA repair [1,2]
Prior to subjecting the cells to the CRISPR/Cas9(D10A) procedure, we examined the expression level of both non-muscle actins in melanoma cells
Upon normalization of the results to A375 cells there were statistically significant differences in ACTB and ACTG1 expression for other melanoma cells lines when compared to A375 cells, the differences were not so high varying form ca. 0.5- to 2-fold
Summary
The actin cytoskeleton is essential for the proper functioning of many cellular processes, including maintenance of the cell shape, chemotaxis, cell movement, adhesion, transport of cellular organelles, mitosis, replication, transcription, and even DNA repair [1,2]. In higher vertebrates, including mammals, there are four muscle isoforms and two non-muscle isoforms of actin [7,8,9,10], and actins have been intensively studied for several decades, there are still controversies about the functional diversification of non-muscle isoactins (β and γ actin), which differ by only four amino acids within their N-termini [7,8] Their different properties are explained, on the one hand, by protein features that manifest through dissimilar polymerization dynamics [11], divergent interactions with ABPs [7,12], and discrepancies in posttranslational modifications [13]. By inactivation the genes encoding the β (ACTB) and γ (ACTG1) actins with the help of the RNA-guided D10A mutated Cas nuclease gene editing, CRISPR/Cas9(D10A), system in A375 cells, we report, for the first time, that the lack of γ actin had the most severe consequences on A375 cells and these non-muscle actins are unequal in their features, their influence on the formation of focal adhesions (FAs) and protrusive structures and, the motility of melanoma cells
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