Hypertonicity-induced cation channels in HepG2 cells: architecture and role in proliferation vs. apoptosis.

KEY POINTS: Na+ conducting hypertonicity-induced cation channels (HICCs) are key players in the volume restoration of osmotically shrunken cells and, under isotonic conditions, considered as mediators of proliferation - thereby opposing apoptosis. In an siRNA screen of ion channels and transporters in HepG2 cells, with the regulatory volume increase (RVI) as read-out, δENaC, TRPM2 and TRPM5 were identified as HICCs. Subsequently, all permutations of these channels were tested in RVI and patch-clamp recordings and, at first sight, HICCs were found to operate in an independent mode. However, there was synergy in the siRNA perturbations of HICC currents. Accordingly, proximity ligation assays showed that δENaC was located in proximity to TRPM2 and TRPM5 suggesting a physical interaction. Furthermore, δENaC, TRPM2 and TRPM5 were identified as mediators of HepG2 proliferation - their silencing enhanced apoptosis. Our study defines the architecture of HICCs in human hepatocytes as well as their molecular functions. ABSTRACT: Hypertonicity-induced cation channels (HICCs) are a substantial element in the regulatory volume increase (RVI) of osmotically shrunken cells. Under isotonic conditions, they are key effectors in the volume gain preceding proliferation; HICC repression, in turn, significantly increases apoptosis rates. Despite these fundamental roles of HICCs in cell physiology, very little is known concerning the actual molecular architecture of these channels. Here, an siRNA screening of putative ion channels and transporters was performed, in HepG2 cells, with the velocity of RVI as the read-out; in this first run, δENaC, TRPM2 and TRPM5 could be identified as HICCs. In the second run, all permutations of these channels were tested in RVI and patch-clamp recordings, with special emphasis on the non-additivity and additivity of siRNAs - which would indicate molecular interactions or independent ways of channel functioning. At first sight, the HICCs in HepG2 cells appeared to operate rather independently. However, a proximity ligation assay revealed that δENaC was located in proximity to both TRPM2 and TRPM5. Furthermore, a clear synergy of HICC current knock-downs (KDs) was observed. δENaC, TRPM2 and TRPM5 were defined as mediators of HepG2 cell proliferation and their silencing increased the rates of apoptosis. This study provides a molecular characterization of the HICCs in human hepatocytes and of their role in RVI, cell proliferation and apoptosis.

Multiplexed imaging of intracellular protein networks.

Cellular functions emerge from the collective action of a large number of different proteins. Understanding how these protein networks operate requires monitoring their components in intact cells. Due to intercellular and intracellular molecular variability, it is important to monitor simultaneously multiple components at high spatiotemporal resolution. However, inherent trade-offs narrow the boundaries of achievable multiplexed imaging. Pushing these boundaries is essential for a better understanding of cellular processes. Here the motivations, challenges and approaches for multiplexed imaging of intracellular protein networks are discussed. © 2016 International Society for Advancement of Cytometry.

Diverse patterns of molecular changes in the mechano-responsiveness of focal adhesions.

Focal adhesions anchor contractile actin fibers with the extracellular matrix, sense the generated tension and respond to it by changing their morphology and composition. Here we ask how this mechanosensing is enabled at the protein-network level, given the modular assembly and multitasking of focal adhesions. To address this, we applied a sensitive 4-color live cell imaging approach, enabling monitoring patterns of molecular changes in single focal adhesions. Co-imaging zyxin, FAK, vinculin and paxillin revealed heterogeneities in their responses to Rho-associated kinase (ROCK)-mediated perturbations of actomyosin contractility. These responses were rather weakly correlated between the proteins, reflecting diverse compositional changes in different focal adhesions. This diversity is partially attributable to the location of focal adhesions, their area, molecular content and previous contractility perturbations, suggesting that integration of multiple local cues shapes differentially focal adhesion mechano-responsiveness. Importantly, the compositional changes upon ROCK perturbations exhibited distinct paths in different focal adhesions. Moreover, the protein exhibiting the strongest response to ROCK perturbations varied among different focal adhesions. The diversity in response patterns is plausibly enabled by the modular mode of focal adhesions assembly and can provide them the needed flexibility to perform multiple tasks by combining optimally a common set of multifunctional components.