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

Malek, Natalia; Mrówczyńska, Ewa; Michrowska, Aleksandra; Mazurkiewicz, Ewa; Pavlyk, Iuliia; Mazur, Antonina Joanna

doi:10.3390/ijms21082746

Open AccessArticle

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

by

Natalia Malek

^†,

Ewa Mrówczyńska

^†

,

Aleksandra Michrowska

,

Ewa Mazurkiewicz

,

Iuliia Pavlyk

and

Antonina Joanna Mazur

^*

Department of Cell Pathology, Faculty of Biotechnology, University of Wroclaw, 50-383 Wroclaw, Poland

^*

Author to whom correspondence should be addressed.

^†

These authors contributed equally to this work.

Int. J. Mol. Sci. 2020, 21(8), 2746; https://doi.org/10.3390/ijms21082746

Submission received: 14 February 2020 / Revised: 10 April 2020 / Accepted: 10 April 2020 / Published: 15 April 2020

(This article belongs to the Special Issue Molecular Biology of Melanoma)

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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.

Keywords: beta actin; gamma actin; actin isoforms; melanoma; CRISPR/Cas9(D10A) technique; focal adhesion; adhesion; migration; LPA; PMA

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MDPI and ACS Style

Malek, N.; Mrówczyńska, E.; Michrowska, A.; Mazurkiewicz, E.; Pavlyk, I.; Mazur, A.J. 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. Int. J. Mol. Sci. 2020, 21, 2746. https://doi.org/10.3390/ijms21082746

AMA Style

Malek N, Mrówczyńska E, Michrowska A, Mazurkiewicz E, Pavlyk I, Mazur AJ. 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. International Journal of Molecular Sciences. 2020; 21(8):2746. https://doi.org/10.3390/ijms21082746

Chicago/Turabian Style

Malek, Natalia, Ewa Mrówczyńska, Aleksandra Michrowska, Ewa Mazurkiewicz, Iuliia Pavlyk, and Antonina Joanna Mazur. 2020. "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" International Journal of Molecular Sciences 21, no. 8: 2746. https://doi.org/10.3390/ijms21082746

APA Style

Malek, N., Mrówczyńska, E., Michrowska, A., Mazurkiewicz, E., Pavlyk, I., & Mazur, A. J. (2020). 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. International Journal of Molecular Sciences, 21(8), 2746. https://doi.org/10.3390/ijms21082746

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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

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