Using kICS to Reveal Changed Membrane Diffusion of AQP-9 Treated with Drugs.

The formation of nanodomains in the plasma membrane are thought to be part of membrane proteins regulation and signaling. Plasma membrane proteins are often investigated by analyzing the lateral mobility. k-space ICS (kICS) is a powerful image correlation spectroscopy (ICS) technique and a valuable supplement to fluorescence correlation spectroscopy (FCS). Here, we study the diffusion of aquaporin-9 (AQP9) in the plasma membrane, and the effect of different membrane and cytoskeleton affecting drugs, and therefore nanodomain perturbing, using kICS. We measured the diffusion coefficient of AQP9 after addition of these drugs using live cell Total Internal Reflection Fluorescence imaging on HEK-293 cells. The actin polymerization inhibitors Cytochalasin D and Latrunculin A do not affect the diffusion coefficient of AQP9. Methyl-ß-Cyclodextrin decreases GFP-AQP9 diffusion coefficient in the plasma membrane. Human epidermal growth factor led to an increase in the diffusion coefficient of AQP9. These findings led to the conclusion that kICS can be used to measure diffusion AQP9, and suggests that the AQP9 is not part of nanodomains.

Neurotrophic Factors Protect the Intestinal Barrier from Rotavirus Insult in Mice.

Increased intestinal permeability has been proposed as a mechanism of rotavirus-induced diarrhea. Studies with humans and mice have, however, shown that rotavirus leaves intestinal permeability unaffected or even reduced during diarrhea, in contrast to most bacterial infections. Gastrointestinal permeability is regulated by the vagus nerve and the enteric nervous system, which is composed of neurons and enteric glial cells (EGCs). We investigated whether the vagus nerve, serotonin (5-HT), EGCs, and neurotropic factors contribute to maintaining gut barrier homeostasis during rotavirus infection. Using subdiaphragmatic vagotomized and 5-HT3 receptor knockout mice, we found that the unaffected epithelial barrier during rotavirus infection is independent of the vagus nerve but dependent on 5-HT signaling through enteric intrinsic 5-HT3 receptors. Immunofluorescence analysis showed that rotavirus-infected enterocytes were in close contact with EGCs and enteric neurons and that the glial cell-derived neurotrophic factor (GDNF) was strongly upregulated in enterocytes of infected mice. Moreover, rotavirus and 5-HT activated EGCs (P < 0.001). Using Ussing chambers, we found that GDNF and S-nitrosoglutathione (GSNO) led to denser epithelial barriers in small intestinal resections from noninfected mice (P < 0.01) and humans (P < 0.001) and that permeability was unaffected in rotavirus-infected mice. GSNO made the epithelial barrier denser in Caco-2 cells by increasing the expression of the tight junction protein zona occludens 1 (P < 0.001), resulting in reduced passage of fluorescein isothiocyanate dextran (P < 0.05) in rotavirus-infected monolayers. This is the first report to show that neurotropic factors contribute to maintaining the gut epithelial barrier during viral insult.IMPORTANCE Human and mouse studies have shown that rotavirus infection is associated with low inflammation and unaffected intestinal barrier at the time of diarrhea, properties different from most bacterial and inflammatory diseases of the gut. We showed by in vitro, ex vivo, and in vivo experiments that neurotrophic factors and 5-HT have barrier protective properties during rotavirus insult. These observations advance our understanding of how the gut barrier is protected against rotavirus and suggest that rotavirus affects the gut barrier differently from bacteria. This is the first report to show that neurotrophic factors contribute to maintain the gut epithelial barrier during viral insult.

Retention of EsxA in the Capsule-Like Layer of Mycobacterium tuberculosis Is Associated with Cytotoxicity and Is Counteracted by Lung Surfactant.

Mycobacterium tuberculosis, the pathogen that causes tuberculosis, primarily infects macrophages but withstands the host cell's bactericidal effects. EsxA, also called virulence factor 6-kDa early secretory antigenic target (ESAT-6), is involved in phagosomal rupture and cell death. We provide confocal and electron microscopy data showing that M. tuberculosis bacteria grown without detergent retain EsxA on their surface. Lung surfactant has detergent-like properties and effectively strips off this surface-associated EsxA, which advocates a novel mechanism of lung surfactant-mediated defense against pathogens. Upon challenge of human macrophages with these M. tuberculosis bacilli, the amount of surface-associated EsxA rapidly declines in a phagocytosis-independent manner. Furthermore, M. tuberculosis bacteria cultivated under exclusion of detergent exert potent cytotoxic activity associated with bacterial growth. Together, this study suggests that the surface retention of EsxA contributes to the cytotoxicity of M. tuberculosis and highlights how cultivation conditions affect the experimental outcome.

Lymphocytes eject interferogenic mitochondrial DNA webs in response to CpG and non-CpG oligodeoxynucleotides of class C.

Circulating mitochondrial DNA (mtDNA) is receiving increasing attention as a danger-associated molecular pattern in conditions such as autoimmunity, cancer, and trauma. We report here that human lymphocytes [B cells, T cells, natural killer (NK) cells], monocytes, and neutrophils derived from healthy blood donors, as well as B cells from chronic lymphocytic leukemia patients, rapidly eject mtDNA as web filament structures upon recognition of CpG and non-CpG oligodeoxynucleotides of class C. The release was quenched by ZnCl2, independent of cell death (apoptosis, necrosis, necroptosis, autophagy), and continued in the presence of TLR9 signaling inhibitors. B-cell mtDNA webs were distinct from neutrophil extracellular traps concerning structure, reactive oxygen species (ROS) dependence, and were devoid of antibacterial proteins. mtDNA webs acted as rapid (within minutes) messengers, priming antiviral type I IFN production. In summary, our findings point at a previously unrecognized role for lymphocytes in antimicrobial defense, utilizing mtDNA webs as signals in synergy with cytokines and natural antibodies, and cast light on the interplay between mitochondria and the immune system.

Interaction of Human Enterochromaffin Cells with Human Enteric Adenovirus 41 Leads to Serotonin Release and Subsequent Activation of Enteric Glia Cells.

Human adenovirus 41 (HAdV-41) causes acute gastroenteritis in young children. The main characteristics of HAdV-41 infection are diarrhea and vomiting. Nevertheless, the precise mechanism of HAdV-41-induced diarrhea is unknown, as a suitable small-animal model has not been described. In this study, we used the human midgut carcinoid cell line GOT1 to investigate the effect of HAdV-41 infection and the individual HAdV-41 capsid proteins on serotonin release by enterochromaffin cells and on enteric glia cell (EGC) activation. We first determined that HAdV-41 could infect the enterochromaffin cells. Immunofluorescence staining revealed that the cells expressed HAdV-41-specific coxsackievirus and adenovirus receptor (CAR); flow cytometry analysis supported these findings. HAdV-41 infection of the enterochromaffin cells induced serotonin secretion dose dependently. In contrast, control infection with HAdV-5 did not induce serotonin secretion in the cells. Confocal microscopy studies of enterochromaffin cells infected with HAdV-41 revealed decreased serotonin immunofluorescence compared to that in uninfected cells. Incubation of the enterochromaffin cells with purified HAdV-41 short fiber knob and hexon proteins increased the serotonin levels in the harvested cell supernatant significantly. HAdV-41 infection could also activate EGCs, as shown in the significantly altered expression of glia fibrillary acidic protein (GFAP) in EGCs incubated with HAdV-41. The EGCs were also activated by serotonin alone, as shown in the significantly increased GFAP staining intensity. Likewise, EGCs were activated by the cell supernatant of HAdV-41-infected enterochromaffin cells.IMPORTANCE The nonenveloped human adenovirus 41 causes diarrhea, vomiting, dehydration, and low-grade fever mainly in children under 2 years of age. Even though acute gastroenteritis is well described, how human adenovirus 41 causes diarrhea is unknown. In our study, we analyzed the effect of human adenovirus 41 infection on human enterochromaffin cells and found it stimulates serotonin secretion in the cells, which is involved in regulation of intestinal secretion and gut motility and can also activate enteric glia cells, which are found in close proximity to enterochromaffin cells in vivo This disruption of gut barrier homeostasis as maintained by these cells following human adenovirus 41 infection might be a mechanism in enteric adenovirus pathogenesis in humans and could indicate a possible serotonin-dependent cross talk between human adenovirus 41, enterochromaffin cells, and enteric glia cells.

Microscopic Analysis of Lysosomal Membrane Permeabilization.

Lysosomes and lysosomal proteases have been found to participate during several forms of cell death pathways including apoptosis. A critical step in the mediation of apoptotic signaling is the release of cathepsins to the cytosol, a process known as lysosomal membrane permeabilization (LMP). In this chapter, we describe immunofluorescence detection of LMP in cell cultures stained for cathepsin B and LAMP-2 using three confocal techniques namely laser scanning, spinning disk, and aperture correlation spinning disk confocal to obtain images. Image analysis is performed using Huygens software for deconvolution. LMP results in a decrease in the fraction of cathepsin B colocalizing with LAMP-2, which is quantified through Manders' colocalization coefficient. Analysis of the images obtained by the three techniques show the same trend but the magnitude of the decrease differs due to the axial resolution. The observations emphasize the use of highest possible resolution when determining colocalization.

X-radiation enhances the collagen type I strap formation and migration potentials of colon cancer cells.

Rectal cancer treatment still fails with local and distant relapses of the disease. It is hypothesized that radiotherapy could stimulate cancer cell dissemination and metastasis. In this study, we evaluated the effect of X-radiation on collagen type I strap formation potential, i.e. matrix remodeling associated with mesenchymal cell migration, and behaviors of SW480, SW620, HCT116 p53+/+ and HCT116 p53-/- colon cancer cells. We determined a radiation-induced increase in collagen type I strap formation and migration potentials of SW480 and HCT116 p53+/+. Further studies with HCT116 p53+/+, indicated that after X-radiation strap forming cells have an increased motility. More, we detected a decrease in adhesion potential and mature integrin ß1 expression, but no change in non-muscle myosin II expression for HCT116 p53+/+ after X-radiation. Integrin ß1 neutralization resulted in a decreased cell adhesion and collagen type I strap formation in both sham and X-radiated conditions. Our study indicates collagen type I strap formation as a potential mechanism of colon cancer cells with increased migration potential after X-radiation, and suggests that other molecules than integrin ß1 and non-muscle myosin II are responsible for the radiation-induced collagen type I strap formation potential of colon cancer cells. This work encourages further molecular investigation of radiation-induced migration to improve rectal cancer treatment outcome.

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.

Dose-dependent autophagic effect of titanium dioxide nanoparticles in human HaCaT cells at non-cytotoxic levels.

BACKGROUND: Interactions between nanoparticles and cells are now the focus of a fast-growing area of research. Though many nanoparticles interact with cells without any acute toxic responses, metal oxide nanoparticles including those composed of titanium dioxide (TiO2-NPs) may disrupt the intracellular process of macroautophagy. Autophagy plays a key role in human health and disease, particularly in cancer and neurodegenerative diseases. We herein investigated the in vitro biological effects of TiO2-NPs (18 nm) on autophagy in human keratinocytes (HaCaT) cells at non-cytotoxic levels. RESULTS: TiO2-NPs were characterized by transmission electron microscopy (TEM) and dynamic light scattering techniques. Cellular uptake, as evaluated by TEM and NanoSIMS revealed that NPs internalization led to the formation of autophagosomes. TiO2-NPs treatment did not reduce cell viability of HaCaT cells nor increased oxidative stress. Cellular autophagy was additionally evaluated by confocal microscopy using eGFP-LC3 keratinocytes, western blotting of autophagy marker LC3I/II, immunodetection of p62 and NBR1 proteins, and gene expression of LC3II, p62, NBR1, beclin1 and ATG5 by RT-qPCR. We also confirmed the formation and accumulation of autophagosomes in NPs treated cells with LC3-II upregulation. Based on the lack of degradation of p62 and NBR1 proteins, autophagosomes accumulation at a high dose (25.0 µg/ml) is due to blockage while a low dose (0.16 µg/ml) promoted autophagy. Cellular viability was not affected in either case. CONCLUSIONS: The uptake of TiO2-NPs led to a dose-dependent increase in autophagic effect under non-cytotoxic conditions. Our results suggest dose-dependent autophagic effect over time as a cellular response to TiO2-NPs. Most importantly, these findings suggest that simple toxicity data are not enough to understand the full impact of TiO2-NPs and their effects on cellular pathways or function.

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.

Aquaporin 9 phosphorylation mediates membrane localization and neutrophil polarization.

Neutrophils are of prime importance in the host innate defense against invading microorganisms by using two primary mechanisms-locomotion toward and phagocytosis of the prey. Recent research points to pivotal roles for water channels known as AQPs in cell motility. Here, we focused on the role of AQP9 in chemoattractant-induced polarization and migration of primary mouse neutrophils and neutrophil-like HL60 cells. We found that AQP9 is phosphorylated downstream of fMLFR or PMA stimulation in primary human neutrophils. The dynamics of AQP9 were assessed using GFP-tagged AQP9 constructs and other fluorescent markers through various live-cell imaging techniques. Expression of WT or the phosphomimic S11D AQP9 changed cell volume regulation as a response to hyperosmotic changes and enhanced neutrophil polarization and chemotaxis. WT AQP9 and S11D AQP9 displayed a very dynamic distribution at the cell membrane, whereas the phosphorylation-deficient S11A AQP9 failed to localize to the plasma membrane. Furthermore, we found that Rac1 regulated the translocation of AQP9 to the plasma membrane. Our results show that AQP9 plays an active role in neutrophil volume regulation and migration. The display of AQP9 at the plasma membrane depends on AQP9 phosphorylation, which appeared to be regulated through a Rac1-dependent pathway.

Rotavirus stimulates release of serotonin (5-HT) from human enterochromaffin cells and activates brain structures involved in nausea and vomiting.

Rotavirus (RV) is the major cause of severe gastroenteritis in young children. A virus-encoded enterotoxin, NSP4 is proposed to play a major role in causing RV diarrhoea but how RV can induce emesis, a hallmark of the illness, remains unresolved. In this study we have addressed the hypothesis that RV-induced secretion of serotonin (5-hydroxytryptamine, 5-HT) by enterochromaffin (EC) cells plays a key role in the emetic reflex during RV infection resulting in activation of vagal afferent nerves connected to nucleus of the solitary tract (NTS) and area postrema in the brain stem, structures associated with nausea and vomiting. Our experiments revealed that RV can infect and replicate in human EC tumor cells ex vivo and in vitro and are localized to both EC cells and infected enterocytes in the close vicinity of EC cells in the jejunum of infected mice. Purified NSP4, but not purified virus particles, evoked release of 5-HT within 60 minutes and increased the intracellular Ca²âº concentration in a human midgut carcinoid EC cell line (GOT1) and ex vivo in human primary carcinoid EC cells concomitant with the release of 5-HT. Furthermore, NSP4 stimulated a modest production of inositol 1,4,5-triphosphate (IP3), but not of cAMP. RV infection in mice induced Fos expression in the NTS, as seen in animals which vomit after administration of chemotherapeutic drugs. The demonstration that RV can stimulate EC cells leads us to propose that RV disease includes participation of 5-HT, EC cells, the enteric nervous system and activation of vagal afferent nerves to brain structures associated with nausea and vomiting. This hypothesis is supported by treating vomiting in children with acute gastroenteritis with 5-HT3 receptor antagonists.

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).

Hg2+ and small-sized polyethylene glycols have inverse effects on membrane permeability, while both impair neutrophil cell motility.

Toxic effects after exposure to mercury are well documented in human. Little is, however, known about how Hg(2+) affect host defense in general and neutrophil functions in particular. We show here that exposure of human neutrophils to HgCl(2) dose-dependently impairs chemoattractant-stimulated motility. Long-term exposure (5-10 min) to Hg(2+) yields a rapid influx of extracellular Ca(2+) followed by leakage of cytosolic fluorophores, as assessed using fura-2 and ratio imaging microscopy. The inhibition on motility was partly reversible, since pre-treated neutrophils placed in an Hg(2+)-free environment displayed higher migration rates. The Hg(2+)-induced fluxes were prevented by addition of small-sized polyethylene glycols (PEG 200-400), which also dose-dependently inhibited neutrophil transmigration. Localized, minute micropipette additions of Hg(2+) or PEG caused retraction of the leading edge and redirection of cell migration. Since Hg(2+) increases and PEGs decrease membrane permeability in a partially competitive manner, we suggest that the known aquaporin-inhibitor Hg(2+) alters membrane permeability by affecting the bidirectional flux through the leukocyte aquaporin-9 (AQP9) while small-sized PEGs yield decreased membrane permeability by becoming trapped in the promiscuous channel. The local additions of Hg(2+) or PEG probably force other cell regions to take over from those with blocked AQPs. Hence, the cells turn direction of motility away from the micromanipulator needle.

Neutrophil leukocyte motility requires directed water influx.

The ability of neutrophils to sense and move to sites of infection is essential for our defense against pathogens. For motility, lamellipodium extension and stabilization are prerequisites, but how cells form such membrane protrusions is still obscure. Using contrast-enhanced video microscopy and Transwell assays, we show that water-selective aquaporin channels regulate lamellipodium formation and neutrophil motility. Addition of anti-aquaporin-9 antibodies, HgCl(2), or tetraethyl ammonium inhibited the function(s) of the channels and blocked motility-related shape changes. On human neutrophils, aquaporin-9 preferentially localized to the cell edges, where N-formyl peptide receptors also accumulated, as assessed with fluorescence microscopy. To directly visualize water fluxes at cell edges, cells were loaded with high dilution-sensitive, self-quenching concentrations of fluorophore. In these cells, motile regions always displayed increased fluorescence compared with perinuclear regions. Our observations provide the first experimental support for motility models where water fluxes play a pivotal role in cell-volume increases accompanying membrane extensions.