Aleksandra Makowiecka

Skin melanocytes reside on the basement membrane (BM), which is mainly composed of laminin, collagen type IV, and proteoglycans. For melanoma cells, in order to invade into the skin, melanocytes must cross the BM. It has been reported that changes in the composition of the BM accompany melanocytes tumorigenesis. Previously, we reported high gelsolin (GSN)—an actin-binding protein—levels in melanoma cell lines and GSN’s importance for migration of A375 cells. Here we investigate whether melanoma cells migrate differently depending on the type of fibrous extracellular matrix protein. We obtained A375 melanoma cells deprived of GSN synthesis and tested their migratory properties on laminin, collagens type I and IV, fibronectin, and Matrigel, which resembles the skin’s BM. We applied confocal and structured illuminated microscopy (SIM), gelatin degradation, and diverse motility assays to assess GSN’s influence on parameters associated with cells’ ability to protrude. We show that GSN is important for melanoma cell migration, predominantly on laminin, which is one of the main components of the skin’s BM.

Thymosin β4 (Tβ4) is a small, 44-amino acid polypeptide. It has been implicated in multiple processes, including cell movement, angiogenesis, and stemness. Previously, we reported that melanoma cell lines differ in Tβ4 levels. Studies on stable clones with silenced TMSB4X expression showed that Tβ4 impacted adhesion and epithelial-mesenchymal transition progression. Here, we show that the cells with silenced TMSB4X expression exhibited altered actin cytoskeleton’s organization and subcellular relocalization of two intermediate filament proteins: Nestin and Vimentin. The rearrangement of the cell cytoskeleton resulted in changes in the cells’ topology, height, and stiffness defined by Young’s modulus. Simultaneously, only for some A375 clones with a lowered Tβ4 level, we observed a decreased ability to initiate colony formation in soft agar, tumor formation in vivo, and alterations in Nanog’s expression level transcription factor regulating stemness. Thus, we show for the first time that in A375 cells, biomechanical properties are not directly coupled to stemness features, and this cell line is phenotypically heterogeneous.

The understanding of melanoma malignancy mechanisms is essential for patient survival, because melanoma is responsible for ca. 75% of deaths related to skin cancers. Enhanced formation of invadopodia and extracellular matrix (ECM) degradation are two important drivers of cell invasion, and actin dynamics facilitate protrusive activity by providing a driving force to push through the ECM. We focused on the influence of epidermal growth factor (EGF), hepatocyte growth factor (HGF) and transforming growth factor β (TGFβ) on melanoma cell invasiveness, since they are observed in the melanoma microenvironment. All three factors stimulated invasion of A375 and WM1341D cells derived from primary tumor sites. In contrast, only EGF and HGF stimulated invasion of WM9 and Hs294T cells isolated from lymph node metastases. Enhanced formation of invadopodia and ECM degradation underlie the increased amount of invasive cells after stimulation with the tested agents. Generally, a rise in invasive potential was accompanied by a decrease in actin polymerization state (F:G ratio). The F:G ratio remained unchanged or was even increased in metastatic cell lines treated with TGFβ. Our findings indicate that the effects of stimulation with EGF, HGF and TGFβ on melanoma cell invasiveness could depend on melanoma cell progression stage.

Actin is a highly conserved protein that is expressed in all eukaryotic cells and has essential functions in the cytoplasm and the nucleus. Nuclear actin is involved in transcription by all three RNA polymerases, chromatin remodelling, RNA processing, intranuclear transport, nuclear export and in maintenance of the nuclear architecture. The nuclear actin level and polymerization state are important factors regulating nuclear processes such as transcription. Our study shows that, in contrast to the cytoplasm, the majority of endogenous nuclear actin is unpolymerized in human melanoma A375 cells. Most mammalian cells express the two non-muscle β- and γ-actin isoforms that differ in only four amino acids. Despite their sequence similarity, studies analysing the cytoplasmic functions of these isoforms demonstrated that β- and γ-actins show differences in localization and function. However, little is known about the involvement of the individual actin isoforms in nuclear processes. Here, we used the human melanoma A375 cell line to analyse actin isoforms in regard to their nuclear localization. We show that both β- and γ-non-muscle actin isoforms are present in nuclei of these cells. Immunolocalization studies demonstrate that both isoforms co-localize with RNA polymerase II and hnRNP U. However, we observe differences in the ratio of cytoplasmic to nuclear actin distribution between the isoforms. We show that β-actin has a significantly higher nucleus-to-cytoplasm ratio than γ-actin.

Gelsolin is one of the most intensively studied actin-binding proteins. However, in the literature comprehensive studies of GSN expression during development have not been performed yet in all model organisms. In zebrafish, gelsolin is a dorsalizing factor that modulates bone morphogenetic proteins signaling pathways, whereas knockout of the gelsolin coding gene, GSN is not lethal in murine model. To study the role of gelsolin in development of higher vertebrates, it is crucial to estimate GSN expression pattern during development. Here, we examined GSN expression in the developing chicken embryo. We applied numerous methods to track GSN expression in developing embryos at mRNA and protein level. We noted a characteristic GSN expression pattern. Although GSN transcripts were present in several cell types starting from early developmental stages, a relatively high GSN expression was observed in eye, brain vesicles, midbrain, neural tube, heart tube, and splanchnic mesoderm. In older embryos, we observed a high GSN expression in the cranial ganglia and dorsal root ganglia. A detailed analysis of 10-day-old chicken embryos revealed high amounts of gelsolin especially within the head region: in the olfactory and optic systems, meninges, nerves, muscles, presumptive pituitary gland, and pericytes, but not oligodendrocytes in the brain. Obtained results suggest that GSN is expressed at high levels in some tissues of ectodermal origin including all neural crest derivatives. Additionally, we describe that silencing of GSN expression in brain vesicles leads to altered morphology of the mesencephalon. This implies gelsolin is crucial for chicken brain development.