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Knockout of ACTB and ACTG1 with CRISPR/Cas9(D10A) Technique Shows that Non-Muscle ? and ? Actin Are Not Equal in Relation to Human Melanoma Cells' Motility and Focal Adhesion Formation.

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 ? actin coded by ACTB or ? actin coded by ACTG1. First, we observed different distributions of ? and ? 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 ? actin knockout had more severe consequences on cell migration and invasion than ? 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 ? actin-deficient cells. Our research shows that both non-muscle actins play distinctive roles in melanoma cells' FA formation and motility.

SUBMITTER: Malek N

PROVIDER: S-EPMC7216121 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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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.

Malek Natalia N Mrówczyńska Ewa E Michrowska Aleksandra A Mazurkiewicz Ewa E Pavlyk Iuliia I Mazur Antonina Joanna AJ

International journal of molecular sciences 20200415 8

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 t ...[more]

PMID: 32326615

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Project description:BACKGROUND:Dystonia-deafness syndrome is a well-known clinical entity, with sensorineural deafness typically manifesting earlier than dystonia. ACTB p.Arg183Trp heterozygosity has been reported in six patients to cause combined infant-onset deafness and dystonia manifesting in adolescence or young adulthood. Three of these have received beneficial pallidal stimulation. Brain imaging to assess striatal function has not been reported previously, however. Nor has a comprehensive hypothesis been presented for how the pleiotropic manifestations of this specific beta-actin gene mutation originate developmentally. CASE PRESENTATION:A 19-year-old girl with congenital mild dysmorphic facial features, cochlear implants for infant-onset deafness, and mild cognitive and emotional disability, presented with an adolescent-onset, severe generalized dystonia. Brain MRI and multiple single gene sequencing were inconclusive. Due to life-threatening dystonia, we implanted a neurostimulation device, targeting the postero-ventral internal pallidum bilaterally. The Burke-Fahn-Marsden Dystonia Rating Scale motor/disability scores improved from 87/25 to 21/13 at 2.5 months postoperatively, 26/14 at 3 years, and 30/14 at 4 years. Subsequent whole exome sequencing identified heterozygosity for the ACTB p.Arg183Trp variant. Brain imaging included 123I-ioflupane single photon emission computed tomography (Dopamine Transporter-SPECT), SPECT with 123I-epidepride (binds to dopamine type 2-receptors) and 18 Fluoro-Deoxy-Glucose (FDG)-PET. Both Epidepride-SPECT and FDG-PET showed reduced tracer uptake in the striatum bilaterally, particularly in the putamen. DaT-SPECT was slightly abnormal. CONCLUSIONS:In this patient with dystonia-deafness syndrome caused by ACTB p.Arg183Trp heterozygosity, unprecedented brain imaging findings strongly indicate striatal neuronal/dopaminergic dysfunction as the underlying cause of the dystonia. Pallidal stimulation provided a substantial improvement of the severe generalized dystonia, which is largely sustained at 4-year follow-up, and we advise this treatment to be considered in such patients. We hypothesize that the pleiotropic manifestations of the dystonia-deafness syndrome caused by this mutation derive from diverse developmental functions of beta-actin in neural crest migration and proliferation (facial dysmorphogenesis), hair cell stereocilia function (infant-onset deafness), and altered synaptic activity patterns associated with pubertal changes in striatal function (adolescent-onset dystonia). The temporal differences in developmental onset are likely due to varying degrees of susceptibility and of compensatory upregulation of other actin variants in the affected structures.

Dataset Information

Knockout of ACTB and ACTG1 with CRISPR/Cas9(D10A) Technique Shows that Non-Muscle ? and ? Actin Are Not Equal in Relation to Human Melanoma Cells' Motility and Focal Adhesion Formation.

Publications

Knockout of <i>ACTB</i> and <i>ACTG1</i> 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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