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.

Thymosin β4 (Tβ4), a multifunctional 44-amino acid polypeptide and a member of actin-binding proteins (ABPs), plays an important role in developmental processes and wound healing. In recent years an increasing number of data has been published suggesting Tβ4's involvement in tumorigenesis. However, Tβ4's role in melanoma tumor development still remains to be elucidated. In our study we demonstrate that Tβ4 is crucial for melanoma adhesion and invasion. For the purpose of our research we tested melanoma cell lines differing in invasive potential. Moreover, we applied shRNAs to silence TMSB4X (gene encoding Tβ4) expression in a cell line with high TMSB4X expression. We found out that Tβ4 is not only a component of focal adhesions (FAs) and interacts with several FAs components but also regulates FAs formation. We demonstrate that Tβ4 level has an impact on FAs' number and morphology. Moreover, manipulation with TMSB4X expression resulted in changes in cells' motility on non-coated and Matrigel TM (resembling basement membrane composition)-coated surfaces and drastically decreased invasion abilities of the cells. Additionally, a correlation between Tβ4 expression level and exhibition of mesenchymal-like [epithelialmesenchymal transition (EMT)] features was discovered. Cells with lowered TMSB4X expression were less EMT-progressed than control cells. Summarizing, obtained results show that Tβ4 by regulating melanoma cells' adhesion has an impact on motility features and EMT. Our study not only contributes to a better understanding of the processes underlying melanoma cells' capacity to create metastases but also highlights Tβ4 as a potential target for melanoma management therapy.

We have recently found that β-actin-like protein 2 (actbl2) forms complexes with gelsolin in human melanoma cells and can polymerize. Phylogenetic and bioinformatic analyses showed that actbl2 has a common origin with two non-muscle actins, which share a separate history from the muscle actins. The actin groups’ divergence started at the beginning of vertebrate evolution, and actbl2 actins are characterized by the largest number of non-conserved amino acid substitutions of all actins. We also discovered that ACTBL2 is expressed at a very low level in several melanoma cell lines, but a small subset of cells exhibited a high ACTBL2 expression. We found that clones with knocked-out ACTBL2 (CR-ACTBL2) or overexpressing actbl2 (OE-ACTBL2) differ from control cells in the invasion, focal adhesion formation, and actin polymerization ratio, as well as in the formation of lamellipodia and stress fibers. Thus, we postulate that actbl2 is the seventh actin isoform and is essential for cell motility.

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Thymosin β4 (Tβ4) is a small, 44-amino acid polypeptide. It has been implicated in multiple processes, including cell movement, angiogenesis, and stemness. Previously, we reported that melanoma cell lines differ in Tβ4 levels. Studies on stable clones with silenced TMSB4X expression showed that Tβ4 impacted adhesion and epithelial-mesenchymal transition progression. Here, we show that the cells with silenced TMSB4X expression exhibited altered actin cytoskeleton’s organization and subcellular relocalization of two intermediate filament proteins: Nestin and Vimentin. The rearrangement of the cell cytoskeleton resulted in changes in the cells’ topology, height, and stiffness defined by Young’s modulus. Simultaneously, only for some A375 clones with a lowered Tβ4 level, we observed a decreased ability to initiate colony formation in soft agar, tumor formation in vivo, and alterations in Nanog’s expression level transcription factor regulating stemness. Thus, we show for the first time that in A375 cells, biomechanical properties are not directly coupled to stemness features, and this cell line is phenotypically heterogeneous.