37Mechanoelectrical transduction is a cellular signalling pathway where physical stimuli are 38 converted into electro-chemical signals by mechanically activated ion channels. We describe 39 here the presence of mechanically activated currents in melanoma cells that are dependent on 40 TMEM87a, which we have renamed Elkin1. Heterologous expression of this protein in 41 PIEZO1-deficient cells, that exhibit no baseline mechanosensitivity, is sufficient to 42 reconstitute mechanically activated currents. Melanoma cells lacking functional Elkin1 43 exhibit defective mechanoelectrical transduction, decreased motility and increased 44 dissociation from organotypic spheroids. By analysing cell adhesion properties, we 45 demonstrate that Elkin1 deletion is associated with increased cell-substrate adhesion and 46 decreased homotypic cell-cell adhesion strength. We therefore conclude that Elkin1 supports 47 a PIEZO1-independent mechanoelectrical transduction pathway and modulates cellular 48 adhesions and regulates melanoma cell migration and cell-cell interactions. 49 50 130 occurred within the stimulus range, allowing us to use a Boltzmann sigmoidal fit to determine 131 the MA current sensitivity. Half-maximal activation of MA currents was seen with 132 approximately 18 nm of substrate deflection (Effective deflection ED50; standard error = 133 20.5 nm). These data indicate a correlation between migratory properties and the MA current 134 sensitivity to deflections applied at cell-substrate contact points. The robust MA current 135 activation observed in cells cultured on LM511 also provided an excellent system to 136 investigate the molecules required for this mechanoelectrical transduction. 137 5 138 157 1999), indicating that neither likely mediates the deflection-evoked currents in WM266-4 158 cells (Figure 1-figure supplement 2). We then examined the proteomics data for proteins of 159 unknown function with 4 or more predicted transmembrane (TM) domains. We prioritised 160 the investigation of Elkin1 due to its expression in melanoma cells but not healthy 161 melanocytes, its expression in additional mechanosensitive cells (Alveolar Type II cells) and 162 its upregulation in additional human cancers (Human Protein Atlas (Uhlén et al., 2005) 163 available from www.proteinatlas.org). We generated miRNA constructs targeting Elkin1 and 164 found that knockdown of Elkin1 transcript resulted in a dramatic reduction in MA currents to 165 deflections up to 1000 nm (Figure 2A,B). These data suggested that Elkin1 contributes to 166 MA currents in melanoma cells. 167 168 Three human isoforms (representing splice variants) of Elkin1 have been identified: isoforms 169 1 and 3 (555 and 494 aa respectively), contain 6 predicted TM domains (Figure 2C). Isoform 170 2 (181 aa) does not contain any predicted TM domains and was not examined in this study.171 6We cloned hsElkin1-iso1 and hsElkin1-iso3 from WM266-4 cDNA and generated C-terminal 172 GFP fusion constructs. We confirmed the plasma membrane localisation of these two 17...
Ion channels activated by mechanical inputs are important force sensing molecules in a wide array of mammalian cells and tissues. The transient receptor potential channel, TRPV4, is a polymodal, nonselective cation channel that can be activated by mechanical inputs but only if stimuli are applied directly at the interface between cells and their substrate, making this molecule a context-dependent force sensor. However, it remains unclear how TRPV4 is activated by mechanical inputs at the cell-substrate interface, which cell intrinsic and cell extrinsic parameters might modulate the mechanical activation of the channel and how mechanical activation differs from TRPV4 gating in response to other stimuli. Here we investigated the impact of substrate mechanics and cytoskeletal components on mechanically evoked TRPV4 currents and addressed how point mutations associated with TRPV4 phosphorylation and arthropathy influence mechanical activation of the channel. Our findings reveal distinct regulatory modulation of TRPV4 from the mechanically activated ion channel PIEZO1, suggesting the mechanosensitivity of these two channels is tuned in response to different parameters. Moreover, our data demonstrate that the effect of point mutations in TRPV4 on channel activation are profoundly dependent on the gating stimulus.
Metastasis is the primary cause of cancer related mortality and morbidity. The circulating tumor cells (CTCs) are shed by epithelial-origin tumors, intravasate assisted by tumor-associated macrophages (TAMs) and travel with blood. A few CTCs extravasate and seed metastasis. However, to reach their destination CTCs have to adapt to hostile for them circulation environment. Previously, we found that the nanomechanical properties of CTCs isolated from prostate cancer patient blood point at CTCs endurance and invasiveness associated with EMT (epithelial-mesenchymal transition). in turn these properties correlated with the patients' sensitivity to androgen deprivation therapy. Here we present evidence that CTCs uniquely co-isolate with macrophage-like cells similar to tumor-associated macrophages (TAMs). We determined with atomic force microscopy (AFM) that pronounced presence of these immune cells was associated with high softness and high adhesion of CTCs. Such survival-promoting phenotypes were related to mechanical fitness and invasiveness in CTCs and could be especially strong in patients starting hormonal therapy. Next, we aimed to recapitulate the tumor cells -macrophages interactions in cell culture using prostate cancer cell lines. Nanomechanical phenotyping and single-cell proteomic analysis showed that the presence of TAM like cells promoted hybrid EMT that increased the mechanical fitness of CTCs. We postulate that to acquire hybrid EMT, certain CTCs are coached by TAMs during their co-migration through the bloodstream. The resulting well-mechanically fit CTCs survive the critical early stage of circulation thanks to softness bestowing resistance to shear stress, and adhesiveness enabling protective cell clustering.
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