In JoVE (2)

Other Publications (27)

Articles by Michael Schnoor in JoVE

 JoVE Immunology and Infection

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

1Department of Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies of the National Polytechnic Institute, 2Department of Molecular Biomedicine, Center for Research and Advanced Studies of the National Polytechnic Institute, 3Agency for Knowledge Commercialization, Center for Research and Advanced Studies of the National Polytechnic Institute

JoVE 51668

 JoVE Medicine

Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis

1Department of Molecular Biomedicine, Center for Research and Advanced Studies of the National Polytechnic Institute, 2Department of Physiology, Biophysics and Neurosciences, Center for Research and Advanced Studies of the National Polytechnic Institute, 3Department of Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies of the National Polytechnic Institute

JoVE 55095

Other articles by Michael Schnoor on PubMed

Characterization of the Synthetic Compatible Solute Homoectoine As a Potent PCR Enhancer

Biochemical and Biophysical Research Communications. Sep, 2004  |  Pubmed ID: 15336543

Different substances such as dimethyl sulfoxide, tetramethylene sulfoxide, 2-pyrollidone, and the naturally occurring compatible solute betaine enhance PCR amplification of GC-rich DNA templates with high melting temperatures. In particular, cyclic compatible solutes outperform traditional PCR enhancers. We therefore investigated the effects that cyclic naturally occurring ectoine-type compatible solutes and their synthetic derivatives have on melting temperature of double-stranded DNA (dsDNA) and on PCR amplification of different templates. L-ectoine, betaine, and derivatives of L-ectoine decreased, whereas beta-hydroxyectoine increased, the melting temperature of dsDNA. The ability to decrease the melting temperature was greatest for homoectoine, a new synthetic derivative of l-ectoine. Furthermore, compatible solutes, especially homoectoine, enhanced PCR amplification of GC-rich DNA (72.6% GC content; effective range: 0.1-0.5M).

Spatial Integration of TIP47 and Adipophilin in Macrophage Lipid Bodies

The Journal of Biological Chemistry. Feb, 2005  |  Pubmed ID: 15545278

We studied the distribution of the PAT family proteins TIP47 and adipophilin in lipid bodies of THP-1 cell-derived macrophages using freeze-fracture immunolabeling and other techniques. Lipid bodies in macrophages comprise lipid droplets and extensive, previously scantily characterized sheet-like organelles, which we descriptively call "lipid sails." TIP47 and adipophilin are components of many, but not all, the lipid droplets. Both proteins are not confined to the surface of lipid droplets, as supposed, but are also inside lipid droplet cores. They are not codistributed stoichiometrically in lipid droplets. How TIP47 and adipophilin, which are polar proteins, enter the lipid droplets and are packaged among the hydrophobic neutral lipids of the core is unclear. However, in the lipid layers of the core, these proteins are directed sometimes inward and sometimes outward. Because TIP47 and adipophilin also localize to lipid sails, lipid sails are intimately involved in intracellular lipid metabolism.

Cloning, Cellular Localization, Genomic Organization, and Tissue-specific Expression of the TGFbeta1-inducible SMAP-5 Gene

Gene. May, 2005  |  Pubmed ID: 15922870

SMAP-5 is a member of the five-pass transmembrane protein family localizing in the Golgi apparatus and the endoplasmic reticulum. These proteins have been implicated in intracellular trafficking, in secretion and in vesicular transport. Phylogenetic analyses revealed that SMAP-5 is a member of a small Rab GTPase interacting factor protein family. The human SMAP-5 gene spans about 12.5 kb and comprises 6 exons on chromosomal locus 5q32. The proximal 5'-flanking region of the gene lacks a TATA box and is highly GC rich. Consistent with this, the SMAP-5 gene is expressed in all tissues. The highest level of expression was found in coronary smooth muscle cells, in which expression of the SMAP-5 gene was induced by transforming growth factor beta1, thus indicating that this protein may play an important role in inflammation.

Butyrophilin Controls Milk Fat Globule Secretion

Proceedings of the National Academy of Sciences of the United States of America. Jul, 2006  |  Pubmed ID: 16801554

The molecular mechanism underlying milk fat globule secretion in mammary epithelial cells ostensibly involves the formation of complexes between plasma membrane butyrophilin and cytosolic xanthine oxidoreductase. These complexes bind adipophilin in the phospholipid monolayer of milk secretory granules, the precursors of milk fat globules, enveloping the nascent fat globules in a layer of plasma membrane and pinching them off the cell. However, using freeze-fracture immunocytochemistry, we find these proteins in locations other than those previously inferred. Significantly, butyrophilin in the residual plasma membrane of the fat globule envelope is concentrated in a network of ridges that are tightly apposed to the monolayer derived from the secretory granule, and the ridges coincide with butyrophilin labeling in the globule monolayer. Therefore, we propose that milk fat globule secretion is controlled by interactions between plasma membrane butyrophilin and butyrophilin in the secretory granule phospholipid monolayer rather than binding of butyrophilin-xanthine oxidoreductase complexes to secretory granule adipophilin.

Cell Surface Localization of ABCG1 Does Not Require LXR Activation

Arteriosclerosis, Thrombosis, and Vascular Biology. Nov, 2006  |  Pubmed ID: 17053170

Expression of Perilipin Isoforms in Cell Types Involved in Atherogenesis

Atherosclerosis. Jan, 2007  |  Pubmed ID: 16842797

Cloning, Genomic Organization, and Tissue-specific Expression of the RASL11B Gene

Biochimica Et Biophysica Acta. Jul-Aug, 2007  |  Pubmed ID: 17628721

RASL11B is a member of the small GTPase protein family with a high degree of similarity to RAS proteins. Cloning of RASL11B mRNA and in silico analyses revealed that the human RASL11B gene spans about 4.5 kb and comprises four exons on chromosomal locus 4q12. The proximal 5'-flanking region of the gene lacks a TATA box but is GC-rich and contains a CCAAT box and several Sp1 sites. Consistent with this, the RASL11B gene was found to be expressed in all tissues investigated, with highest levels in placenta and in primary macrophages. The predicted RASL11B protein has no typical prenylation signal, indicating that it is probably not anchored to cellular membranes. RASL11B was induced during maturation of THP-1 monocytic cells into macrophage-like cells and in coronary artery smooth muscle cells after treatment with TGF-beta1. These results indicate that RASL11B may play a role in TGF-beta1-mediated developmental processes and in pathophysiologies such as inflammation, cancer, and arteriosclerosis.

Production of Type VI Collagen by Human Macrophages: a New Dimension in Macrophage Functional Heterogeneity

Journal of Immunology (Baltimore, Md. : 1950). Apr, 2008  |  Pubmed ID: 18390756

Macrophages derived from human blood monocytes perform many tasks related to tissue injury and repair. The main effect of macrophages on the extracellular matrix is considered to be destructive in nature, because macrophages secrete metalloproteinases and ingest foreign material as part of the remodeling process that occurs in wound healing and other pathological conditions. However, macrophages also contribute to the extracellular matrix and hence to tissue stabilization both indirectly, by inducing other cells to proliferate and to release matrix components, and directly, by secreting components of the extracellular matrix such as fibronectin and type VIII collagen, as we have recently shown. We now report that monocytes and macrophages express virtually all known collagen and collagen-related mRNAs. Furthermore, macrophages secrete type VI collagen protein abundantly, depending upon their mode of activation, stage of differentiation, and cell density. The primary function of type VI collagen secreted by macrophages appears to be modulation of cell-cell and cell-matrix interactions. We suggest that the production of type VI collagen is a marker for a nondestructive, matrix-conserving macrophage phenotype that could profoundly influence physiological and pathophysiological conditions in vivo.

Disassembly of Endothelial and Epithelial Junctions During Leukocyte Transmigration

Frontiers in Bioscience : a Journal and Virtual Library. 2008  |  Pubmed ID: 18508684

Leukocyte migration occurs as a response to inflammatory signals and is an efficient host defense mechanism against invading pathogens. This innate defense response includes transendothelial and transepithelial migration of leukocytes to facilitate clearance of inflammatory stimuli. The endothelium lines the vascular system and forms the first barrier for leukocytes as they migrate out of the bloodstream. The epithelium largely separates organs from the external environment and forms a second barrier for leukocytes. These cellular barriers are comprised of complex intercellular junctions of different molecular composition. However, for barrier function to be maintained, these specialized intercellular junctions must not be destroyed during transmigration. Innate immune cells including monocytes, neutrophils and dendritic cells are all capable of a highly regulated transmigration response in order to accomplish their different functions. These cells exploit many common adhesive and signaling cascades to traverse cellular junctions. However, there are unique features of each type of leukocyte and barrier that determine specificity of the response. This review will focus on highlighting the mechanisms that leukocytes exploit to open these junctions.

Evidence for Cross-reactivity of JAM-C Antibodies: Implications for Cellular Localization Studies

Biology of the Cell / Under the Auspices of the European Cell Biology Organization. Aug, 2009  |  Pubmed ID: 19143587

JAM-C (junctional adhesion molecule C) has been implicated in the regulation of leukocyte migration, cell polarity, spermatogenesis, angiogenesis and nerve conduction. JAM-C has been also reported to concentrate at TJs (tight junctions) and desmosomes, although detailed localization studies remain incomplete.

Efficient Non-viral Transfection of THP-1 Cells

Journal of Immunological Methods. May, 2009  |  Pubmed ID: 19345690

Macrophages are an important part of the cellular immune system and play a key role during immune responses. Thus, macrophages are interesting targets in basic and clinical research. Primary monocytes or monocyte-derived macrophages do not proliferate on a suitable scale so that their use for functional studies in vitro is limited. Immortal proliferating cell lines, such as the human THP-1 monocytic leukemia cell line, are therefore often used instead of primary cells. Transfection is a useful tool to study the function of gene products, but transfection of THP-1 monocytes and pre-differentiated THP-1 macrophages with subsequent differentiation into mature THP-1 macrophages using phorbol esters is usually accompanied by a progressive loss of cell viability. In this study, we describe a simple and rapid approach for efficient transfection of THP-1 monocytes and pre-differentiated THP-1 macrophages using a modified Nucleofection-based approach. The protocol maintains cell viability and functionality, thus allowing efficient transfection of THP-1 cells combined with subsequent differentiation of transfected THP-1 cells into mature macrophages.

Commensal Escherichia Coli Reduces Epithelial Apoptosis Through IFN-alphaA-mediated Induction of Guanylate Binding Protein-1 in Human and Murine Models of Developing Intestine

Journal of Immunology (Baltimore, Md. : 1950). Jun, 2010  |  Pubmed ID: 20483731

Appropriate microbial colonization protects the developing intestine by promoting epithelial barrier function and fostering mucosal tolerance to luminal bacteria. Commensal flora mediate their protective effects through TLR9-dependent activation of cytokines, such as type I IFNs (alpha, beta) and IL-10. Although IFN-beta promotes apoptosis, IFN-alpha activates specific antiapoptotic target genes whose actions preserve epithelial barrier integrity. We have recently identified guanylate binding protein-1 (GBP-1) as an antiapoptotic protein, regulated by both type I and type II IFNs, that promotes intestinal epithelial barrier integrity in mature intestine. However, the mechanisms by which commensal bacteria regulate epithelial apoptosis during colonization of immature intestine and the contributions of GBP-1 are unknown. The healthy newborn intestine is initially colonized with bacterial species present in the maternal gastrointestinal tract, including nonpathogenic Escherichia coli. Therefore, we examined the influence of commensal E. coli on cytokine expression and candidate mediators of apoptosis in preweaned mice. Specifically, enteral exposure of 2 wk-old mice to commensal E. coli for 24 h selectively increased both IFN-alphaA and GBP-1 mRNA expression and prevented staurosporine-induced epithelial apoptosis. Exogenous IFN-alphaA treatment also induced GBP-1 expression and protected against staurosporine-induced apoptosis in a GBP-1 dependent manner, both in vitro and ex vivo. These findings identify a role for IFN-alphaA-mediated GBP-1 expression in the prevention of intestinal epithelial apoptosis by commensal bacteria. Thus IFN-alphaA mediates the beneficial effects of commensal bacteria and may be a promising therapeutic target to promote barrier integrity and prevent the inappropriate inflammatory responses seen in developing intestine as in necrotizing enterocolitis.

Cortactin Deficiency is Associated with Reduced Neutrophil Recruitment but Increased Vascular Permeability in Vivo

The Journal of Experimental Medicine. Aug, 2011  |  Pubmed ID: 21788407

Neutrophil extravasation and the regulation of vascular permeability require dynamic actin rearrangements in the endothelium. In this study, we analyzed in vivo whether these processes require the function of the actin nucleation-promoting factor cortactin. Basal vascular permeability for high molecular weight substances was enhanced in cortactin-deficient mice. Despite this leakiness, neutrophil extravasation in the tumor necrosis factor-stimulated cremaster was inhibited by the loss of cortactin. The permeability defect was caused by reduced levels of activated Rap1 (Ras-related protein 1) in endothelial cells and could be rescued by activating Rap1 via the guanosine triphosphatase (GTPase) exchange factor EPAC (exchange protein directly activated by cAMP). The defect in neutrophil extravasation was caused by enhanced rolling velocity and reduced adhesion in postcapillary venules. Impaired rolling interactions were linked to contributions of β(2)-integrin ligands, and firm adhesion was compromised by reduced ICAM-1 (intercellular adhesion molecule 1) clustering around neutrophils. A signaling process known to be critical for the formation of ICAM-1-enriched contact areas and for transendothelial migration, the ICAM-1-mediated activation of the GTPase RhoG was blocked in cortactin-deficient endothelial cells. Our results represent the first physiological evidence that cortactin is crucial for orchestrating the molecular events leading to proper endothelial barrier function and leukocyte recruitment in vivo.

The EhCPADH112 Complex of Entamoeba Histolytica Interacts with Tight Junction Proteins Occludin and Claudin-1 to Produce Epithelial Damage

PloS One. 2013  |  Pubmed ID: 23762290

Entamoeba histolytica, the protozoan responsible for human amoebiasis, causes between 30,000 and 100,000 deaths per year worldwide. Amoebiasis is characterized by intestinal epithelial damage provoking severe diarrhea. However, the molecular mechanisms by which this protozoan causes epithelial damage are poorly understood. Here, we studied the initial molecular interactions between the E. histolytica EhCPADH112 virulence complex and epithelial MDCK and Caco-2 cells. By confocal microscopy, we discovered that after contact with trophozoites or trophozoite extracts (TE), EhCPADH112 and proteins forming this complex (EhCP112 and EhADH112) co-localize with occludin and claudin-1 at tight junctions (TJ). Immunoprecipitation assays revealed interaction between EhCPADH112 and occludin, claudin-1, ZO-1 and ZO-2. Overlay assays confirmed an interaction of EhCP112 and EhADH112 with occludin and claudin-1, whereas only EhADH112 interacted also with ZO-2. We observed degradation of all mentioned TJ proteins after incubation with TE. Importantly, inhibiting proteolytic activity or blocking the complex with a specific antibody not only prevented TJ protein degradation but also epithelial barrier disruption. Furthermore, we discovered that TE treatment induces autophagy and apoptosis in MDCK cells that could contribute to the observed barrier disruption. Our results suggest a model in which epithelial damage caused by E. histolytica is initiated by the interaction of EhCP112 and EhADH112 with TJ proteins followed by their degradation. Disruption of TJs then induces increased paracellular permeability, thus facilitating the entry of more proteases and other parasite molecules leading eventually to tissue destruction.

Cortactin Regulates the Activity of Small GTPases and ICAM-1 Clustering in Endothelium: Implications for the Formation of Docking Structures

Tissue Barriers. Jan, 2013  |  Pubmed ID: 24665381

Cortactin is an actin-binding molecule that regulates various cellular processes requiring actin dynamics. We recently described cortactin-deficient mice and despite its pivotal role for actin remodeling in vitro, these mice are surprisingly healthy. Analyzing cortactin functions in endothelium under inflammatory conditions, we found that cortactin is required for endothelial barrier functions and leukocyte extravasation in vivo. Importantly, these effects were not regulated by defective actin dynamics but instead by a failure to activate the small GTPases Rap1 and RhoG in endothelial cells. Defective RhoG signaling led to reduced ICAM-1 clustering that supported the interaction with leukocytes. These clusters originally seen as rings surrounding adherent leukocytes actually represented in many cases ICAM-1 containing protrusions as they were described before as docking structures. Thus, cortactin is essential for the formation of endothelial docking structures as well as for leukocyte adhesion and extravasation.

Small GTPases of the Ras Superfamily Regulate Intestinal Epithelial Homeostasis and Barrier Function Via Common and Unique Mechanisms

Tissue Barriers. Dec, 2013  |  Pubmed ID: 24868497

The intestinal epithelium forms a stable barrier protecting underlying tissues from pathogens in the gut lumen. This is achieved by specialized integral membrane structures such as tight and adherens junctions that connect neighboring cells and provide stabilizing links to the cytoskeleton. Junctions are constantly remodeled to respond to extracellular stimuli. Assembly and disassembly of junctions is regulated by interplay of actin remodeling, endocytotic recycling of junctional proteins, and various signaling pathways. Accumulating evidence implicate small G proteins of the Ras superfamily as important signaling molecules for the regulation of epithelial junctions. They function as molecular switches circling between an inactive GDP-bound and an active GTP-bound state. Once activated, they bind different effector molecules to control cellular processes required for correct junction assembly, maintenance and remodelling. Here, we review recent advances in understanding how GTPases of the Rho, Ras, Rab and Arf families contribute to intestinal epithelial homeostasis.

The Role of Actin-binding Proteins in the Control of Endothelial Barrier Integrity

Thrombosis and Haemostasis. Jan, 2015  |  Pubmed ID: 25183310

The endothelial barrier of the vasculature is of utmost importance for separating the blood stream from underlying tissues. This barrier is formed by tight and adherens junctions (TJ and AJ) that form intercellular endothelial contacts. TJ and AJ are integral membrane structures that are connected to the actin cytoskeleton via various adaptor molecules. Consequently, the actin cytoskeleton plays a crucial role in regulating the stability of endothelial cell contacts and vascular permeability. While a circumferential cortical actin ring stabilises junctions, the formation of contractile stress fibres, e. g. under inflammatory conditions, can contribute to junction destabilisation. However, the role of actin-binding proteins (ABP) in the control of vascular permeability has long been underestimated. Naturally, ABP regulate permeability via regulation of actin remodelling but some actin-binding molecules can also act independently of actin and control vascular permeability via various signalling mechanisms such as activation of small GTPases. Several studies have recently been published highlighting the importance of actin-binding molecules such as cortactin, ezrin/radixin/moesin, Arp2/3, VASP or WASP for the control of vascular permeability by various mechanisms. These proteins have been described to regulate vascular permeability under various pathophysiological conditions and are thus of clinical relevance as targets for the development of treatment strategies for disorders that are characterised by vascular hyperpermeability such as sepsis. This review highlights recent advances in determining the role of ABP in the control of endothelial cell contacts and vascular permeability.

E-cadherin Is Important for the Maintenance of Intestinal Epithelial Homeostasis Under Basal and Inflammatory Conditions

Digestive Diseases and Sciences. Apr, 2015  |  Pubmed ID: 25772779

Endothelial Actin-binding Proteins and Actin Dynamics in Leukocyte Transendothelial Migration

Journal of Immunology (Baltimore, Md. : 1950). Apr, 2015  |  Pubmed ID: 25848070

The endothelium is the first barrier that leukocytes have to overcome during recruitment to sites of inflamed tissues. The leukocyte extravasation cascade is a complex multistep process that requires the activation of various adhesion molecules and signaling pathways, as well as actin remodeling, in both leukocytes and endothelial cells. Endothelial adhesion molecules, such as E-selectin or ICAM-1, are connected to the actin cytoskeleton via actin-binding proteins (ABPs). Although the contribution of receptor-ligand interactions to leukocyte extravasation has been studied extensively, the contribution of endothelial ABPs to the regulation of leukocyte adhesion and transendothelial migration remains poorly understood. This review focuses on recently published evidence that endothelial ABPs, such as cortactin, myosin, or α-actinin, regulate leukocyte extravasation by controlling actin dynamics, biomechanical properties of endothelia, and signaling pathways, such as GTPase activation, during inflammation. Thus, ABPs may serve as targets for novel treatment strategies for disorders characterized by excessive leukocyte recruitment.

Antioxidative Diet Supplementation Reverses High-fat Diet-induced Increases of Cardiovascular Risk Factors in Mice

Oxidative Medicine and Cellular Longevity. 2015  |  Pubmed ID: 25922641

Obesity is a worldwide epidemic that is characterized not only by excessive fat deposition but also by systemic microinflammation, high oxidative stress, and increased cardiovascular risk factors. While diets enriched in natural antioxidants showed beneficial effects on oxidative stress, blood pressure, and serum lipid composition, diet supplementation with synthetic antioxidants showed contradictive results. Thus, we tested in C57Bl/6 mice whether a daily dosage of an antioxidative mixture consisting of vitamin C, vitamin E, L-arginine, eicosapentaenoic acid, and docosahexaenoic acid (corabion) would affect cardiovascular risk factors associated with obesity. Obese mice showed increased serum triglyceride and glucose levels and hypertension after eight weeks of being fed a high-fat diet (HFD). Importantly, corabion ameliorated all of these symptoms significantly. Oxidative stress and early signs of systemic microinflammation already developed after two weeks of high-fat diet and were significantly reduced by daily doses of corabion. Of note, the beneficial effects of corabion could not be observed when applying its single antioxidative components suggesting that a combination of various nutrients is required to counteract HFD-induced cardiovascular risk factors. Thus, daily consumption of corabion may be beneficial for the management of obesity-related cardiovascular complications.

Crossing the Vascular Wall: Common and Unique Mechanisms Exploited by Different Leukocyte Subsets During Extravasation

Mediators of Inflammation. 2015  |  Pubmed ID: 26568666

Leukocyte extravasation is one of the essential and first steps during the initiation of inflammation. Therefore, a better understanding of the key molecules that regulate this process may help to develop novel therapeutics for treatment of inflammation-based diseases such as atherosclerosis or rheumatoid arthritis. The endothelial adhesion molecules ICAM-1 and VCAM-1 are known as the central mediators of leukocyte adhesion to and transmigration across the endothelium. Engagement of these molecules by their leukocyte integrin receptors initiates the activation of several signaling pathways within both leukocytes and endothelium. Several of such events have been described to occur during transendothelial migration of all leukocyte subsets, whereas other mechanisms are known only for a single leukocyte subset. Here, we summarize current knowledge on regulatory mechanisms of leukocyte extravasation from a leukocyte and endothelial point of view, respectively. Specifically, we will focus on highlighting common and unique mechanisms that specific leukocyte subsets exploit to succeed in crossing endothelial monolayers.

Experimental Colitis Is Attenuated by Cardioprotective Diet Supplementation That Reduces Oxidative Stress, Inflammation, and Mucosal Damage

Oxidative Medicine and Cellular Longevity. 2016  |  Pubmed ID: 26881044

Inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease (CD) are multifactorial, relapsing disorders of the gastrointestinal tract. However, the etiology is still poorly understood but involves altered immune responses, epithelial dysfunction, environmental factors, and nutrition. Recently, we have shown that the diet supplement corabion has cardioprotective effects due to reduction of oxidative stress and inflammation. Since oxidative stress and inflammation are also prominent risk factors in IBD, we speculated that corabion also has beneficial effects on experimental colitis. Colitis was induced in male mice by administration of 3.5% (w/v) dextran sulfate sodium (DSS) in drinking water for a period of 3 or 7 days with or without daily gavage feeding of corabion consisting of vitamin C, vitamin E, L-arginine, and eicosapentaenoic and docosahexaenoic acid. We found that corabion administration attenuated DSS-induced colon shortening, tissue damage, and disease activity index during the onset of colitis. Mechanistically, these effects could be explained by reduced neutrophil recruitment, oxidative stress, production of proinflammatory cytokines, and internalization of the junctional proteins ZO-1 and E-cadherin leading to less edema formation. Thus, corabion may be a useful diet supplement for the management of chronic inflammatory intestinal disorders such as IBD.

Recruitment of Immune Cells into Inflamed Tissues: Consequences for Endothelial Barrier Integrity and Tissue Functionality

Mediators of Inflammation. 2016  |  Pubmed ID: 26989330

A New Nucleocytoplasmic RhoGAP Protein Contributes to Control the Pathogenicity of Entamoeba Histolytica by Regulating EhRacC and EhRacD Activity

Cellular Microbiology. Nov, 2016  |  Pubmed ID: 27107405

Small GTPases are signalling molecules that regulate important cellular processes. GTPases are deactivated by GTPase-activating proteins (GAPs). While human GAPs have been intensively studied, no GAP has yet been characterized in Entamoeba histolytica. In this study, we identified and characterized a novel nucleocytoplasmic RhoGAP in E. histolytica termed EhRhoGAPnc. In silico analyses of the domain structure revealed a previously undescribed peptide region within the carboxy-terminal region of EhRhoGAPnc capable of interacting with phosphatidic acid and phosphatidylinositol 3,5-bisphosphate. The full structural GAP domain showed increase GAP activity compared with the minimum region able to display GAP activity, as analysed both by experimental assays and molecular dynamics simulations. Furthermore, we identified amino acid residues that promote interactions between EhRhoGAPnc and its target GTPases EhRacC and EhRacD. Immunofluorescence studies revealed that EhRhoGAPnc colocalized with EhRacC and EhRacD during uroid formation but not during erythrophagocytosis. Interestingly, during erythrophagocytosis of red blood cells, EhRhoGAPnc colocalized with phosphatidic acid and phosphatidylinositol 3,5-bisphosphate. Overexpression of EhRhoGAPnc in E. histolytica led to inhibition of actin adhesion plate formation, migration, adhesion of E. histolytica to MDCK cells and consequently to an impairment of the cytopathic activity.

Loss of Cortactin Causes Endothelial Barrier Dysfunction Via Disturbed Adrenomedullin Secretion and Actomyosin Contractility

Scientific Reports. Jun, 2016  |  Pubmed ID: 27357373

Changes in vascular permeability occur during inflammation and the actin cytoskeleton plays a crucial role in regulating endothelial cell contacts and permeability. We demonstrated recently that the actin-binding protein cortactin regulates vascular permeability via Rap1. However, it is unknown if the actin cytoskeleton contributes to increased vascular permeability without cortactin. As we consistently observed more actin fibres in cortactin-depleted endothelial cells, we hypothesised that cortactin depletion results in increased stress fibre contractility and endothelial barrier destabilisation. Analysing the contractile machinery, we found increased ROCK1 protein levels in cortactin-depleted endothelium. Concomitantly, myosin light chain phosphorylation was increased while cofilin, mDia and ERM were unaffected. Secretion of the barrier-stabilising hormone adrenomedullin, which activates Rap1 and counteracts actomyosin contractility, was reduced in plasma from cortactin-deficient mice and in supernatants of cortactin-depleted endothelium. Importantly, adrenomedullin administration and ROCK1 inhibition reduced actomyosin contractility and rescued the effect on permeability provoked by cortactin deficiency in vitro and in vivo. Our data suggest a new role for cortactin in controlling actomyosin contractility with consequences for endothelial barrier integrity.

The Pro-inflammatory Cytokines IFNγ/TNFα Increase Chromogranin A-positive Neuroendocrine Cells in the Colonic Epithelium

The Biochemical Journal. Nov, 2016  |  Pubmed ID: 27538402

The gastrointestinal tract is the largest hormone-producing organ in the body due to a specialized cell population called enteroendocrine cells (EECs). The number of EECs increases in the mucosa of inflammatory bowel disease patients; however, the mechanisms responsible for these changes remain unknown. Here, we show that the pro-inflammatory cytokines interferon γ (IFNγ) and tumor necrosis factor α (TNFα) or dextran sulfate sodium (DSS)-induced colitis increase the number of EECs producing chromogranin A (CgA) in the colonic mucosa of C57BL/6J mice. CgA-positive cells were non-proliferating cells enriched with inactive phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and autophagy markers. Moreover, inhibition of Akt and autophagy prevented the increase in CgA-positive cells after IFNγ/TNFα treatment. Similarly, we observed that CgA-positive cells in the colonic mucosa of patients with colitis expressed Akt and autophagy markers. These findings suggest that Akt signaling and autophagy control differentiation of the intestinal EEC lineage during inflammation.

EhNPC1 and EhNPC2 Proteins Participate in Trafficking of Exogenous Cholesterol in Entamoeba Histolytica Trophozoites: Relevance for Phagocytosis

PLoS Pathogens. Dec, 2016  |  Pubmed ID: 28002502

Entamoeba histolytica, the highly phagocytic protozoan causative of human amoebiasis lacks the machinery to synthesize cholesterol. Here, we investigated the presence of NPC1 and NPC2 proteins in this parasite, which are involved in cholesterol trafficking in mammals. Bioinformatics analysis revealed one Ehnpc1 and two Ehnpc2 genes. EhNPC1 appeared as a transmembrane protein and both EhNPC2 as peripheral membrane proteins. Molecular docking predicted that EhNPC1 and EhNPC2 bind cholesterol and interact with each other. Genes and proteins were identified in trophozoites. Serum pulse-chase and confocal microscopy assays unveiled that after trophozoites sensed the cholesterol source, EhNPC1 and EhNPC2 were organized around the plasma membrane in a punctuated pattern. Vesicles emerged and increased in number and size and some appeared full of cholesterol with EhNPC1 or EhNPC2 facing the extracellular space. Both proteins, but mostly EhNPC2, were found out of the cell associated with cholesterol. EhNPC1 and cholesterol formed networks from the plasma membrane to the nucleus. EhNPC2 appeared in erythrocytes that were being ingested by trophozoites, co-localizing with cholesterol of erythrocytes, whereas EhNPC1 surrounded the phagocytic cup. EhNPC1 and EhNPC2 co-localized with EhSERCA in the endoplasmic reticulum and with lysobisphosphatidic acid and EhADH (an Alix protein) in phagolysosomes. Immunoprecipitation assays confirmed the EhNPC1 and EhNPC2 association with cholesterol, EhRab7A and EhADH. Serum starved and blockage of cholesterol trafficking caused a low rate of phagocytosis and incapability of trophozoites to produce damage in the mouse colon. Ehnpc1 and Ehnpc2 knockdown provoked in trophozoites a lower intracellular cholesterol concentration and a diminished rate of phagocytosis; and Ehnpc1 silencing also produced a decrease of trophozoites movement. Trafficking of EhNPC1 and EhNPC2 during cholesterol uptake and phagocytosis as well as their association with molecules involved in endocytosis strongly suggest that these proteins play a key role in cholesterol uptake.

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