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.

Correct communication between immune cells and peripheral neurons is crucial for the protection of our bodies. Its breakdown is observed in many common, often painful conditions, including arthritis, neuropathies, and inflammatory bowel or bladder disease. Here, we have characterised the immune response in a mouse model of neuropathic pain using flow cytometry and cell-type-specific RNA sequencing (RNA-seq). We found few striking sex differences, but a very persistent inflammatory response, with increased numbers of monocytes and macrophages up to 3 1/2 months after the initial injury. This raises the question of whether the commonly used categorisation of pain into “inflammatory” and “neuropathic” is one that is mechanistically appropriate. Finally, we collated our data with other published RNA-seq data sets on neurons, macrophages, and Schwann cells in naive and nerve injury states. The result is a practical web-based tool for the transcriptional data mining of peripheral neuroimmune interactions. http://rna-seq-browser.herokuapp.com/

Correct communication between immune cells and peripheral neurons is crucial for the protection of our bodies. Its breakdown is observed in many common, often painful conditions, including arthritis, neuropathies and inflammatory bowel or bladder disease. Here, we have characterised the immune response in a mouse model of neuropathic pain using flow cytometry and cell-type specific RNA sequencing (RNA-seq). We found few striking sex differences, but a very persistent inflammatory response, with increased numbers of monocytes and macrophages up to 3½ months after the initial injury. This raises the question of whether the commonly used categorisation of pain into "inflammatory" and "neuropathic" is one that is mechanistically appropriate. Finally, we collated our data with other published RNA-seq datasets on neurons, satellite glial cells, macrophages and Schwann cells in naïve and nerve injury states. The result is a practical web-based tool for the transcriptional data-mining of peripheral neuroimmune interactions. Figure 8: At one week post PSNL, MHCII+/Ly6C-myeloid cells from sciatic nerve upregulate functions relating to interactions with other immune cells, in favour of more generic pro-inflammatory and homeostatic activities.A) STRING network analysis reveals that 77 of 186 genes upregulated in ipsilateral MHCII+ macrophages at adj. p < 0.05 are likely to be functionally connected with overrepresented processes including antigen presentation, regulation of lymphocytes and myeloid cell activation. B) Conversely, 41 of 93 significantly downregulated genes formed a network that includes transcripts relating to pro-inflammatory function (TNF, complements), regulation of angiogenesis and canonical macrophage markers, like CD163 typically found in resident macrophages. See Suppl. Table 5 for differential expression tables.

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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