scholarly journals 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

2020 ◽  
Vol 21 (8) ◽  
pp. 2746 ◽  
Author(s):  
Natalia Malek ◽  
Ewa Mrówczyńska ◽  
Aleksandra Michrowska ◽  
Ewa Mazurkiewicz ◽  
Iuliia Pavlyk ◽  
...  

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.

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Natalia Malek ◽  
Aleksandra Michrowska ◽  
Ewa Mazurkiewicz ◽  
Ewa Mrówczyńska ◽  
Paweł Mackiewicz ◽  
...  

AbstractWe 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.


2008 ◽  
Vol 6 (11) ◽  
pp. 1766-1774 ◽  
Author(s):  
Ralf Buettner ◽  
Tania Mesa ◽  
Adina Vultur ◽  
Frank Lee ◽  
Richard Jove

Cancers ◽  
2019 ◽  
Vol 12 (1) ◽  
pp. 33 ◽  
Author(s):  
Hui Liu ◽  
Ann L. B. Seynhaeve ◽  
Rutger W. W. Brouwer ◽  
Wilfred F. J. van IJcken ◽  
Liu Yang ◽  
...  

Melanoma is one of the most aggressive cancers, and patients with distant metastases have dire outcomes. We observed previously that melanoma progression is driven by a high migratory potential of melanoma cells, which survive and proliferate under harsh environmental conditions. In this study, we report that CREPT (cell-cycle related and expression-elevated protein in tumor), an oncoprotein highly expressed in other cancers, is overexpressed in melanoma cells but not melanocytes. Overexpression of CREPT stimulates cell proliferation, migration, and invasion in several melanoma cell lines. Further, we show that CREPT enhances melanoma progression through upregulating and activating Ras homolog family member A (RhoA)-induced actin organization and focal adhesion assembly. Our study reveals a novel role of CREPT in promoting melanoma progression. Targeting CREPT may be a promising strategy for melanoma treatment.


2020 ◽  
Author(s):  
WenHao Zhang ◽  
LiPing Zhou ◽  
Guo Liu

AbstractIn human melanoma cells, resistance to conventional chemotherapy and radiotherapy and rapid metastasis give melanoma a remarkable feature of the most aggressive and lethal. The low response rate of melanoma to existing treatment modalities is a substantial threat to patients and researchers. It is crucial to identify new therapeutic agents for the fatal malignancy melanoma. Vitexin is a flavonoid compound in many traditional Chinese medicines that exhibits antioxidant, anti-inflammatory and anti-tumour activities in many cancer cells. In our study, we elucidated the inhibitory effects of vitexin on invasion and metastasis in human melanoma A375 and C8161 cells in vitro. After vitexin treatment for 24 h or 48 h, the invasive ability and migration of melanoma cells were decreased in a dose- and time-dependent manners. In western blot analysis, we verified that vitexin inhibited the expression levels of MMP-2, MMP-9, vimentin, Slug and Twist which are known as the regulators of protein degradation and promote various cell behaviours such as migration and invasion. To further investigate the target signal that may be influenced by vitexin, immunofluorescence assay was performed to observe STAT3 localization and western blot results showed that vitexin decreased the expression of the phosphorylation of kinases that inducing STAT3 activation. Accordingly, we provide inspiring insight into the basic inhibition mechanism of vitexin, which will soon be an issue due to its scientific potential for further development as a novel anti-tumour agent for the clinical therapy of human melanoma.


IUBMB Life ◽  
2020 ◽  
Vol 72 (5) ◽  
pp. 942-956
Author(s):  
Xiu Shan ◽  
Weijie Dong ◽  
Li Zhang ◽  
Xin Cai ◽  
Yi Zhao ◽  
...  

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