Search

Tumour cell detachment from the primary tumour is an early and crucial step of the metastatic cascade. At the single cell level, it was already shown that migrating melanoma cells establish both intra‐ and extracellular pH gradients and that the Na+/H+ exchanger NHE1 accumulates at the leading edges to strengthen cell‐matrix interactions. However, less is known about the role of NHE1 in collective cell migration and the specific pH microenvironment at tumour cell–cell contacts. We used MV3 melanoma cells transfected with a NHE1‐expressing vector or a control vector. NHE1 localization at cell–cell contacts was assessed via immunofluorescence imaging. Collective migration was analysed by live‐cell imaging. The NHE1 activity and the perimembranous pH were measured both intra‐ and extracellularly by ratiometric fluorescence microscopy. NHE1 clearly localizes at cell–cell contacts. Its overexpression further increases migratory speed and translocation in multidirectional pathway analyses. NHE1 overexpressing MV3 cells also move further away from their neighbouring cells during wound closure assays. pH measurements revealed that the NHE1 is highly active at cell–cell contacts of melanoma cells. NHE1‐mediated pH dynamics at such contact sites are more prominent in NHE1‐overexpressing melanoma cells. Our findings highlight the contribution of the NHE1 towards modulation and plasticity of melanoma cell–cell contacts. We propose that its localization and functional activity at cell–cell contacts promotes evasion of single melanoma cells from the primary tumour. [ABSTRACT FROM AUTHOR]

Abstract: NTPDase2, a member of the CD39/NTPDase family, is an ecto‐nucleotidase anchored to the plasma membrane by two transmembrane domains, with a catalytic site facing the extracellular space and preferentially hydrolyzing nucleoside triphosphates. While NTPDase2 is expressed in many cell types, its unique functionality, mobility and dynamics at the cell membrane remain unexplored. We therefore constructed a recombinant NTPDase2 linked to the yellow fluorescent protein (EYFP) to investigate its dynamics by confocal microscopy. The present study shows that the expression of EYFP‐NTPDase2 in different cell lines does not affect its proliferation, migration and adhesion to extracellular matrices (ECM). Moreover, in human embryonic kidney cells 293 (HEK293) grown on collagen type I and fibronectin, EYFP‐NTPDase2 fluorescence is greater in free plasma membrane regions than in cell–cell contacts, in comparison with cells grown on other substrates. Differences in the time required for fluorescence recovery after photobleaching (FRAP) in free membrane regions and cell–cell contacts indicate that the mobility of EYFP‐NTPDase2 depends on the matrix to which the cells are attached. © 2018 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

Cell-cell contact-mediated Notch signaling is essential for mesenchymal stem cell (MSC) chondrogenesis during development. However, subsequent deactivation of Notch signaling is also required to allow for stem cell chondrogenic progression. Recent literature has shown that Notch signaling can also influence Wnt/β-catenin signaling, critical for MSC differentiation, through perturbations in cell-cell contacts. Traditionally, abundant cell-cell contacts, consistent with development, are emulated in vitro using pellet cultures for chondrogenesis. However, cells are often encapsulated within biomaterials-based scaffolds, such as hydrogels, to improve therapeutic cell localization in vivo. To explore the role of Notch and Wnt/β-catenin signaling in the context of hydrogel-encapsulated MSC chondrogenesis, we compared signaling and differentiation capacity of MSCs in both hydrogels and traditional pellet cultures. We demonstrate that encapsulation within poly(ethylene glycol) hydrogels reduces cell-cell contacts, and both Notch (7.5-fold) and Wnt/β-catenin (84.7-fold) pathway activation. Finally, we demonstrate that following establishment of cell-cell contacts and transient Notch signaling in pellet cultures, followed by Notch signaling deactivation, resulted in a 1.5-fold increase in MSC chondrogenesis. Taken together, these findings support that cellular condensation, and establishment of initial cell-cell contacts is critical for MSC chondrogenesis, and this process is inhibited by hydrogel encapsulation. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 1291-1302, 2015. [ABSTRACT FROM AUTHOR]

Claudins are the critical transmembrane proteins in tight junctions. Claudin-5, for instance, prevents paracellular permeation of small molecules. However, the molecular interaction mechanism is unknown. Hence, the claudin-claudin interaction and tight junction strand formation were investigated using systematic single mutations. Claudin-5 mutants transfected into tight junction-free cells demonstrated that the extracellular loop 2 is involved in strand formation via trans-interaction, but not via polymerization, along the plasma membrane of one cell. Three phenotypes were obtained: the tight junction type (wild-type-like trans- and cis-interaction; the disjunction type (blocked trans-interaction); the intracellular type (disturbed folding). Combining site-directed mutagenesis, live-cell imaging-, electron microscopy-, and molecular modeling data led to an antiparallel homodimer homology model of the loop. These data for the first time explain how two claudins hold onto each other and constrict the paracellular space. The intermolecular interface includes aromatic (F147, Y148, Y158) and hydrophilic (Q156, E159) residues. The aromatic residues form a strong binding core between two loops from opposing cells. Since nearly all these residues are conserved in most claudins, our findings are of general relevance for all classical claudins. On the basis of the data we have established a novel molecular concept for tight junction formation. [ABSTRACT FROM AUTHOR]