Protective role of host aquaporin 6 against Hazara virus, a model for Crimean-Congo hemorrhagic fever virus infection.

Crimean-Congo hemorrhagic fever virus (CCHFV) is an arthropod-borne pathogen that causes infectious disease with severe hemorrhagic manifestations in vascular system in humans. The proper function of the cells in the vascular system is critically regulated by aquaporins (AQP), water channels that facilitate fluxes of water and small solutes across membranes. With Hazara virus as a model for CCHFV, we investigated the effects of viruses on AQP6 and the impact of AQP6 on virus infectivity in host cells, using transiently expressed GFP-AQP6 cells, immunofluorescent assay for virus detection, epifluorescent imaging of living cells and confocal microscopy. In GFP-AQP6 expressing cells, Hazara virus reduced both the cellular and perinuclear AQP6 distribution and changed the cell area. Infection of human cell with CCHFV strain IbAR 10200 downregulated AQP6 expression at mRNA level. Interestingly, the overexpression of AQP6 in host cells decreased the infectivity of Hazara virus, speaking for a protective role of AQP6. We suggest the possibility for AQP6 being a novel player in the virus-host interactions, which may lead to less severe outcomes of an infection.

Fluxes of water through aquaporin 9 weaken membrane-cytoskeleton anchorage and promote formation of membrane protrusions.

All modes of cell migration require rapid rearrangements of cell shape, allowing the cell to navigate within narrow spaces in an extracellular matrix. Thus, a highly flexible membrane and a dynamic cytoskeleton are crucial for rapid cell migration. Cytoskeleton dynamics and tension also play instrumental roles in the formation of different specialized cell membrane protrusions, viz. lamellipodia, filopodia, and membrane blebs. The flux of water through membrane-anchored water channels, known as aquaporins (AQPs) has recently been implicated in the regulation of cell motility, and here we provide novel evidence for the role of AQP9 in the development of various forms of membrane protrusion. Using multiple imaging techniques and cellular models we show that: (i) AQP9 induced and accumulated in filopodia, (ii) AQP9-associated filopodial extensions preceded actin polymerization, which was in turn crucial for their stability and dynamics, and (iii) minute, local reductions in osmolarity immediately initiated small dynamic bleb-like protrusions, the size of which correlated with the reduction in osmotic pressure. Based on this, we present a model for AQP9-induced membrane protrusion, where the interplay of water fluxes through AQP9 and actin dynamics regulate the cellular protrusive and motile activity of cells.

Water fluxes through aquaporin-9 prime epithelial cells for rapid wound healing.

Cells move along surfaces both as single cells and multi-cellular units. Recent research points toward pivotal roles for water flux through aquaporins (AQPs) in single cell migration. Their expression is known to facilitate this process by promoting rapid shape changes. However, little is known about the impact on migrating epithelial sheets during wound healing and epithelial renewal. Here, we investigate and compare the effects of AQP9 on single cell and epithelial sheet migration. To achieve this, MDCK-1 cells stably expressing AQP9 were subjected to migration assessment. We found that AQP9 facilitated cell locomotion at both the single and multi-cellular level. Furthermore, we identified major differences in the monolayer integrity and cell size upon expression of AQP9 during epithelial sheet migration, indicating a rapid volume-regulatory mechanism. We suggest a novel mechanism for epithelial wound healing based on AQP-induced swelling and expansion of the monolayer.

Water flux in cell motility: expanding the mechanisms of membrane protrusion.

Transmembrane water fluxes through aquaporins (AQPs) are suggested to play pivotal roles in cell polarization and directional cell motility. Local dilution by water influences the dynamics of the subcortical actin polymerization and directs the formation of nascent membrane protrusions. In this paper, recent evidence is discussed in support of such a central role of AQP in membrane protrusion formation and cell migration as a basis for our understanding of the underlying molecular mechanisms of directional motility. Specifically, AQP9 in a physiological context controls transmembrane water fluxes driving membrane protrusion formation, as an initial cellular response to a chemoattractant or other migratory signals. The importance of AQP-facilitated water fluxes in directional cell motility is underscored by the observation that blocking or modifying specific sites in AQP9 also interferes with the molecular machinery that govern actin-mediated cellular shape changes.

Filopodia are induced by aquaporin-9 expression.

Understanding filopodial formation in motile cells is a pertinent task in cell biology. In the present study we show that expression of the human water channel aquaporin-9 (AQP9) in different cell lines induces the formation of numerous filopodial extensions. Several lines of evidence support the role of aquaporins functioning both as a water channel and signaling participant. The number of filopodia is decreased by site-directed serine substitutions in putative PKC-binding or -phosphorylation sites at amino acid position 11 and 222 in AQP9. The filopodial phenotype obtained with wild-type AQP9 is associated with elevated levels of active Cdc42, while serine-deleted mutants have reduced levels of GTP-Cdc42. Co-transfection with inhibitory N-WASP CRIB completely abolishes wild-type AQP9-induced filopodia formation. Active PKC(zeta) phosphorylates wild-type AQP9 and myristoylated PKC(zeta) pseudosubstrate inhibits the formation of filopodia in AQP9-expressing cells. Expression of wild-type AQP9, but not mock or serine substituted mutants, increases sensitivity to hypo-osmolaric conditions, yielding a rapid morphological rounding of cells and cell death starting as early as 24 h post-transfection. We propose that increased water influx through AQP9 is critically involved in the formation of membrane protrusions, and that AQP9-induced actin polymerization is augmented by activation of Cdc42 and PKC(zeta).