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

• Published in: International journal of molecular sciences 2020-04, Vol.21 (8), p.2746
• Main Authors: Malek, Natalia, Mrówczyńska, Ewa, Michrowska, Aleksandra, Mazurkiewicz, Ewa, Pavlyk, Iuliia, Mazur, Antonina Joanna
• Format: Article
• Language: English
• Subjects: Actin; actin isoforms; Actins - analysis; Actins - genetics; Actins - metabolism; beta actin; Cell adhesion & migration; Cell Line, Tumor; Cell Movement - drug effects; Cell Movement - genetics; Cloning; CRISPR; CRISPR-Cas Systems; CRISPR/Cas9(D10A) technique; focal adhesion; Focal Adhesions - drug effects; Focal Adhesions - genetics; Focal Adhesions - metabolism; gamma actin; Gene Editing - methods; Gene Knockout Techniques - methods; Genetic modification; Genome editing; Humans; Isoforms; Lysophospholipids - pharmacology; Melanoma; Melanoma - genetics; Melanoma - metabolism; Motility; Muscles; Mutation; Neoplasm Invasiveness - genetics; Nuclease; Polymerization; Protein Isoforms - metabolism; Proteins; Signal Transduction - drug effects; Signal Transduction - genetics; Stress Fibers - genetics; Stress Fibers - metabolism; Tetradecanoylphorbol Acetate - analogs & derivatives; Tetradecanoylphorbol Acetate - pharmacology
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms21082746

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 (CRISPR clones with inactivated , CR- ) and (CRISPR clones with inactivated , CR- ) 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 or γ actin coded by . 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- 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- and CR- 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.