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...

Mutations in succinate dehydrogenase (SDH) lead to the development of tumors in a restricted subset of cell types, including chromaffin cells and paraganglia.The molecular basis for this specificity is currently unknown. We show that loss of SDH activity in a chromaffin cell model does not perturb complex I function, retaining the ability to oxidize NADH within the electron transport chain. This activity supports continued oxidation of substrates within the tricarboxylic acid (TCA) cycle. However, due to the block in the TCA cycle at SDH, the high glutamine oxidation activity is only maintained through an efflux of succinate. We also show that although the mitochondria of SDH-deficient cells are less active per se, their higher mass per cell results in an overall respiratory rate that is comparable with wild-type cells. Finally, we observed that when their mitochondria are uncoupled, SDH-deficient cells are unable to preserve their viability, suggesting that the mitochondrial metabolic network is unable to compensate when exposed to additional stress. We therefore show that in contrast to models of SDH deficiency based on epithelial cells, a chromaffin cell model retains aspects of metabolic "health," which could form the basis of cell specificity of this rare tumor type. | KĽUČKOVÁ et al | MATERIALS AND METHODS | Cell culture and chemicalsPreviously characterized immortalized mouse chromaffin cell lines deficient in Sdhb (Sdhb −/− CL6 and CL8) as well K E Y W O R D S electron transport chain, metabolism, mitochondria, pheochromocytoma, succinate dehydrogenase

It has become increasingly evident that T cell functions are subject to translational control in addition to transcriptional regulation. Here, by using live imaging of CD8 + T cells isolated from the Lifeact-EGFP mouse, we show that T cells exhibit a gain in fluorescence intensity following engagement of cognate tumour target cells. The GFP signal increase is governed by Erk1/2-dependent distal T cell receptor (TCR) signalling and its magnitude correlates with IFN-γ and TNF-α production, which are hallmarks of T cell activation. Enhanced fluorescence was due to increased translation of Lifeact-EGFP protein, without an associated increase in its mRNA. Activationinduced gains in fluorescence were also observed in naïve and CD4 + T cells from the Lifeact-EGFP reporter, and were readily detected by both flow cytometry and live cell microscopy. This unique, translationally controlled reporter of effector T cell activation simultaneously enables tracking of cell morphology, F-actin dynamics and activation state in individual migrating T cells. It is a valuable addition to the limited number of reporters of T cell dynamics and activation, and opens the door to studies of translational activity and heterogeneities in functional T cell responses in situ.