Effect of microtubule-targeting drugs on cell-cell and cell-matrix junctions in tumor epithelial cells.

In this study, we investigated the effects of microtubule-targeting drugs, which either destabilize (the Vinca alkaloid vincristine) or stabilize (the taxane derivative docetaxel) microtubules, on the cell-cell and cell-matrix adhesive junctions of Caco-2 tumor epithelial cells, using fluorescence imaging and functional assays. We found that, in sub-confluent (but not confluent) cells, vincristine (but not docetaxel) affected cell-cell junction morphology. Furthermore, docetaxel (but not vincristine) attenuated the formation of the peri-junctional actomyosin ring and enhanced the internalization of junctional adhesion molecule-A. However, these effects of vincristine and docetaxel did not translate into appreciable functional changes during the opening and resealing of the cell-cell junctions. We also found that vincristine caused enlargement of focal adhesions (the major cell-matrix junctions) without affecting cell adhesion onto the matrix. Thus, we conclude that the microtubule-targeting drugs interfere to variable degrees with the morphology and/or function of the cell-cell and cell-matrix adhesive junctions. In addition, the results highlight the importance of considering the cellular context and dynamics (e.g. cell confluence and junction opening, respectively), when determining the final effects of microtubule manipulation on cell adhesiveness.

Network analysis of cell adhesion: Adhesomes as context-defined subnetworks.

Complex systems consisting of diverse interlinked elements are often represented as networks and are described according to the principles of network analysis. Among the networks of biological interest, several protein-protein interactomes have been reported in recent years, mostly in conjunction with high-throughput assays and extensive efforts of literature mining. The resulting global networks display well-defined topological properties and provide a comprehensive view of all the biological contexts in which a given interactome is involved. Global networks, however, do not provide enough information about the specific contexts, such as biological processes and subcellular compartments in which the individual interactions occur. Thus, to glean additional insights, it is often advantageous to extract context-defined local subnetworks from the global networks. Our recently published network analysis of the cell-cell adhesome, i.e., the protein-protein interaction subnetwork that underlies both the biological process of cell-cell adhesion and the subcellular compartment of the apical junctions in human epithelial cells, is an example of such context-defined approaches.

S-CMC-Lys protective effects on human respiratory cells during oxidative stress.

The mucoactive drug S-carbocysteine lysine salt monohydrate (S-CMC-Lys) stimulates glutathione (GSH) efflux from respiratory cells. Since GSH is one of the most important redox regulatory mechanisms, the aim of this study was to evaluate the S-CMC-Lys effects on GSH efflux and intracellular concentration during an oxidative stress induced by the hydroxyl radical (xOH). Experiments were performed on cultured human respiratory WI-26VA4 cells by means of patch-clamp experiments in whole-cell configuration and of fluorimetric analyses at confocal microscope. xOH exposure induced an irreversible inhibition of the GSH and chloride currents that was prevented if the cells were incubated with S-CMC-Lys. In this instance, the currents were inhibited by the specific blocker CFTR(inh)-172. CFT1-C2 cells, which lack a functional CFTR channel, were not responsive to S-CMC-Lys, but the stimulatory effect of the drug was restored in LCFSN-infected CFT1 cells, functionally corrected to express CFTR. Fluorimetric measurements performed on the S-CMC-Lys-incubated cells revealed a significant increase of the GSH concentration that was completely hindered after oxidative stress and abolished by CFTR(inh)-172. The cellular content of reactive oxygen species was significantly lower in the S-CMC-Lys-treated cells either before or after xOH exposure. As a conclusion, S-CMC-Lys could exert a protective function during oxidative stress, therefore preventing or reducing the ROS-mediated inflammatory response.

Hypotonicity induces aquaporin-2 internalization and cytosol-to-membrane translocation of ICln in renal cells.

Kidney collecting-duct cells swell in response to changes in medulla osmolality caused by the transition from antidiuresis to diuresis. Regulatory volume decrease (RVD) mechanisms must be activated to face this hypotonic stress. In Aquaporin-2 (AQP2)-expressing renal CD8 cells, hypotonicity decreased cell surface expression of AQP2 and increased the amount of AQP2 localized intracellularly, whereas the total amount of AQP2 phosphorylated at ser-256 decreased. Analysis of cAMP dynamics using fluorescence resonance energy transfer (FRET) showed that hypotonicity causes a reduction of cAMP, consistent with a decrease in phospho-AQP2. Moreover, hypotonicity caused a profound actin reorganization, associated with the loss of stress fibers and formation of F-actin patches (microspikes) at the cell border. Those changes were regulated by the monomeric GTPase Cdc42. Interestingly, expression of the dominant-negative Cdc42 (N17-Cdc42) prevented the hypotonicity-induced microspike formation and the generation of Cl(-) currents. Hypotonicity also caused the relocation from the cytosol to the plasma membrane and increase in interaction with actin of ICln (nucleotide-sensitive chloride current protein), which is essential for the generation of ion currents activated during RVD. Together, the profound actin remodeling, internalization of AQP2 and translocation of ICln to the plasma membrane during hypotonicity may contribute to RVD after cell swelling in renal medulla.