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1.
PLoS One ; 8(6): e69163, 2013.
Article in English | MEDLINE | ID: mdl-23805333

ABSTRACT

Integrin-mediated phagocytosis, an important physiological activity undertaken by professional phagocytes, requires bidirectional signalling to/from αMß2 integrin and involves Rap1 and Rho GTPases. The action of Rap1 and the cytoskeletal protein talin in activating αMß2 integrins, in a RIAM-independent manner, has been previously shown to be critical during phagocytosis in mammalian phagocytes. However, the events downstream of Rap1 are not clearly understood. Our data demonstrate that one potential Rap1 effector, Regulator of G-Protein Signalling-14 (RGS14), is involved in activating αMß2. Exogenous expression of RGS14 in COS-7 cells expressing αMß2 results in increased binding of C3bi-opsonised sheep red blood cells. Consistent with this, knock-down of RGS14 in J774.A1 macrophages results in decreased association with C3bi-opsonised sheep red blood cells. Regulation of αMß2 function occurs through the R333 residue of the RGS14 Ras/Rap binding domain (RBD) and the F754 residue of ß2, residues previously shown to be involved in binding of H-Ras and talin1 head binding prior to αMß2 activation, respectively. Surprisingly, overexpression of talin2 or RAPL had no effect on αMß2 regulation. Our results establish for the first time a role for RGS14 in the mechanism of Rap1/talin1 activation of αMß2 during phagocytosis.


Subject(s)
Macrophage-1 Antigen/metabolism , Phagocytosis/physiology , RGS Proteins/metabolism , Animals , COS Cells , Cell Line , Chlorocebus aethiops , Erythrocytes/cytology , Erythrocytes/metabolism , Macrophages/cytology , Macrophages/metabolism , Microscopy, Fluorescence , Plasmids/genetics , Plasmids/metabolism , Protein Binding , RGS Proteins/antagonists & inhibitors , RGS Proteins/genetics , RNA Interference , RNA, Small Interfering/metabolism , Sheep , Talin/metabolism , rap1 GTP-Binding Proteins/metabolism
2.
Environ Res ; 126: 208-10, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23787133

ABSTRACT

Few studies on natural estrogens have been conducted in agricultural ecosystems. High (up to 58 ng/L) estrone concentrations were measured shortly after applications of manure in a small agricultural watershed. No other estrogens (17ß-estradiol, estrone or estriol) were detected afterward (first three rainfalls after applications). Results suggest that aquatic organisms in agricultural watersheds could be exposed to estrone shortly after manure applications, probably from leaching of land-applied animal wastes, but that this exposure would be short-lived.


Subject(s)
Agriculture , Estrogens/analysis , Manure , Water Pollutants, Chemical/analysis , Rain , Rivers/chemistry
3.
J Environ Sci Health B ; 47(10): 967-74, 2012.
Article in English | MEDLINE | ID: mdl-22938581

ABSTRACT

The objective was to investigate the impact of vegetated filter strips on exported atrazine and deethylatrazine concentrations [dissolved and sorbed to eroded sediments (>1.5 µm)], the deethylatrazine to atrazine ratio in water and sediments, the ratio of sorbed to dissolved herbicides in runoff and subsurface infiltration as well as field equilibrium state under natural climate during two seasons. We hypothesize that sorption equilibrium was not achieved in 2004 because of the short delay (<24 h) between herbicide application and the first rain event. In 2005, observations suggest that possible changing sorption equilibrium conditions were reached (20 days after atrazine application), especially for eroded sediments submitted to changing environmental conditions in subsurface. If confirmed by other experiments, this will raise the question of the representativeness of laboratory-determined soil sorption coefficients to predict the fate of pesticides.


Subject(s)
Environmental Restoration and Remediation/methods , Herbicides/chemistry , Soil Pollutants/chemistry , Vegetables/chemistry , Adsorption , Environmental Restoration and Remediation/instrumentation , Kinetics
4.
J Environ Sci Health B ; 47(6): 495-504, 2012.
Article in English | MEDLINE | ID: mdl-22494372

ABSTRACT

Livestock manure applied to agricultural land is one of the ways natural steroid estrogens enter soils. To examine the impact of long-term solid beef cattle (Bos Taurus) manure on soil properties and 17ß-estradiol sorption and mineralization, this study utilized a soil that had received beef cattle manure over 35 years. The 17ß-estradiol was strongly sorbed and sorption significantly increased (P < 0.05) with increasing soil organic carbon content (SOC) and with an increasing annual rate of beef cattle manure. The 17ß-estradiol mineralization half-life was significantly negatively correlated, and the total amount of 17ß-estradiol mineralized at 90 days (MAX) was significantly positively correlated with 17ß-estradiol sorption. The long-term rate of manure application had no significant effect on MAX, but the addition of fresh beef cattle manure in the laboratory resulted in significantly (P < 0.05) smaller MAX values. None of the treatments showed MAX values exceeding one-third of the 17ß-estradiol applied.


Subject(s)
Estradiol/chemistry , Manure/analysis , Soil Pollutants/chemistry , Soil/chemistry , Adsorption , Agriculture , Aluminum Silicates/chemistry , Animals , Cattle , Clay
5.
J Cell Sci ; 125(Pt 12): 2825-30, 2012 Jun 15.
Article in English | MEDLINE | ID: mdl-22454526

ABSTRACT

The adaptor protein Nck has been shown to link receptor ligation to actin-based signalling in a diverse range of cellular events, such as changes in cell morphology and motility. It has also been implicated in phagocytosis. However, its molecular role in controlling actin remodelling associated with phagocytic uptake remains to be clarified. Here, we show that Nck, which is recruited to phagocytic cups, is required for Fcγ receptor (FcγR)- but not complement receptor 3 (CR3)-induced phagocytosis. Nck recruitment in response to FcγR ligation is mediated by the phosphorylation of tyrosine 282 and 298 in the ITAM motif in the cytoplasmic tail of the receptor. In the absence of FcγR phosphorylation, there is also no recruitment of N-WASP or Cdc42 to phagocytic cups. Nck promotes FcγR-mediated phagocytosis by recruiting N-WASP to phagocytic cups. Efficient phagocytosis, however, only occurs, if the CRIB domain of N-WASP can also interact with Cdc42. Our observations demonstrate that Nck and Cdc42 collaborate to stimulate N-WASP-dependent FcγR-mediated phagocytosis.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Oncogene Proteins/metabolism , Phagocytosis , Receptors, IgG/metabolism , Wiskott-Aldrich Syndrome Protein, Neuronal/metabolism , Wiskott-Aldrich Syndrome/metabolism , cdc42 GTP-Binding Protein/metabolism , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Motifs , Animals , Humans , Macrophages/metabolism , Mice , Mice, Knockout , Oncogene Proteins/genetics , Protein Binding , Receptors, IgG/chemistry , Receptors, IgG/genetics , Signal Transduction , Wiskott-Aldrich Syndrome/genetics , Wiskott-Aldrich Syndrome/physiopathology , Wiskott-Aldrich Syndrome Protein, Neuronal/genetics , cdc42 GTP-Binding Protein/genetics
6.
J Cell Sci ; 124(Pt 17): 2897-902, 2011 Sep 01.
Article in English | MEDLINE | ID: mdl-21878497

ABSTRACT

Phagocytosis is a highly ordered process orchestrated by signalling through Rho GTPases to locally organise the actin cytoskeleton and drive particle uptake. Specific Rho family members that regulate phagocytosis are not known, as the majority of studies have relied on the use of dominant-negative mutants and/or toxins, which can inactivate multiple Rho GTPases. To identify the relevant GTPases for phagocytosis through the Fcγ receptor (FcγR) and complement receptor 3 (CR3), we depleted 20 Rho proteins individually in an RNA interference (RNAi) screen. We find that distinct GTPase subsets are required for actin polymerisation and uptake by macrophages: FcγR-dependent engulfment requires Cdc42 and Rac2 (but not Rac1), whereas CR3 requires RhoA. Surprisingly, RhoG is required for particle uptake through both FcγR and CR3. RhoG has been previously linked to Rac and Cdc42 signalling in different model systems, but not to RhoA. Interestingly, we find that RhoG is also recruited and activated at phagocytic cups downstream of FcγR and CR3, irrespective of their distinct actin structures and mechanisms of internalisation. Thus, the functional links between RhoG and RhoA downstream of CR3-dependent phagocytosis are new and unexpected. Our data suggest a broad role for RhoG in consolidating signals from multiple receptors during phagocytosis.


Subject(s)
Complement C3b/immunology , Macrophages/immunology , Phagocytosis/physiology , Receptors, IgG/immunology , rhoA GTP-Binding Protein/immunology , Animals , COS Cells , Cell Line , Chlorocebus aethiops , Humans , Macrophages/enzymology , Mice , Phagocytosis/genetics , Phagocytosis/immunology , RNA Interference , Sheep , Signal Transduction
7.
Biochem J ; 437(3): 461-7, 2011 Aug 01.
Article in English | MEDLINE | ID: mdl-21605078

ABSTRACT

During αMß2-mediated phagocytosis, the small GTPase Rap1 activates the ß2 integrin by binding to a region between residues 732 and 761. Using COS-7 cells transfected with αMß2, we show that αMß2 activation by the phorbol ester PMA involves Ser(756) of ß2. This residue is critical for the local positioning of talin and biochemically interacts with Rap1. Using the CaM (calmodulin) antagonist W7, we found Rap1 recruitment and the inside-out activation of αMß2 to be affected. We also report a role for CaMKII (calcium/CaM-dependent kinase II) in the activation of Rap1 during integrin activation. These results demonstrate a distinct physiological role for Ser(756) of ß2 integrin, in conjunction with the actions of talin and Rap1, during αMß2 activation in macrophages.


Subject(s)
CD18 Antigens/chemistry , CD18 Antigens/metabolism , Macrophage-1 Antigen/metabolism , Serine/chemistry , rap1 GTP-Binding Proteins/metabolism , Animals , Antibodies , COS Cells , Chlorocebus aethiops , Erythrocytes , Gene Expression Regulation , Humans , Macrophages/drug effects , Macrophages/metabolism , Mutation , Sheep , Talin , Tetradecanoylphorbol Acetate/pharmacology
8.
J Cell Biochem ; 111(4): 999-1009, 2010 Nov 01.
Article in English | MEDLINE | ID: mdl-20665668

ABSTRACT

The small GTPase Rap1 and the cytoskeletal protein talin regulate binding of C3bi-opsonised red blood cells (RBC) to integrin α(M)ß(2) in phagocytic cells, although the mechanism has not been investigated. Using COS-7 cells transfected with α(M)ß(2), we show that Rap1 acts on the ß(2) and not the α(M) chain, and that residues 732-761 of the ß(2) subunit are essential for Rap1-induced RBC binding. Activation of α(M)ß(2) by Rap1 was dependent on W747 and F754 in the ß(2) tails, which are required for talin head binding, suggesting a link between Rap1 and talin in this process. Using talin1 knock-out cells or siRNA-mediated talin1 knockdown in the THP-1 monocytic cell line, we show that Rap1 acts upstream of talin but surprisingly, RIAM knockdown had little effect on integrin-mediated RBC binding or cell spreading. Interestingly, Rap1 and talin influence each other's localisation at phagocytic cups, and co-immunoprecipitation experiments suggest that they interact together. These results show that Rap1-mediated activation of α(M)ß(2) in macrophages shares both common and distinct features from Rap1 activation of α(IIb)ß(3) expressed in CHO cells.


Subject(s)
Macrophage-1 Antigen/metabolism , Talin/metabolism , rap1 GTP-Binding Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Binding Sites , Cell Line , Complement C3b/metabolism , Enzyme Activation , Erythrocytes/metabolism , Humans , Immunoprecipitation , Macrophage-1 Antigen/chemistry , Membrane Proteins/metabolism , Mice , Mutant Proteins/metabolism , Opsonin Proteins/metabolism , Protein Binding , Protein Subunits/chemistry , Protein Subunits/metabolism , Sheep , Signal Transduction , Transfection
9.
J Environ Sci Health B ; 45(2): 145-51, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20390944

ABSTRACT

Estrogens are a family of feminizing hormones that are excreted by vertebrates. It has been documented that their presence in surface waters, even in the ng/L range, can have detrimental impacts on fish reproduction. Two competitive enzyme-linked immunosorbent assays using rabbit polyclonal antibodies were developed: one for 17beta-estradiol and a second one for 17beta-estradiol (E2)+estrone (E1)+estriol (E3). Two different conjugates were synthesized using the Mixed-anhydride (for the 17beta-estradiol ELISA) and the Mannich (for the E1 + E2 + E3 ELISA) reactions. The 17beta-estradiol ELISA was highly specific with an IC(50) of 243 ng/mL for 17beta-estradiol. The E1 + E2 + E3 ELISA exhibited cross-reactivity with estrone (85%) and estriol (62%) with an IC(50) of 18 ng/mL for 17beta-estradiol. Cross-reactivity was tested against 13 chemically related compounds and both immunoassays showed significant cross-reactivity with two estradiol conjugates: beta estradiol-17-valerate and beta estradiol-3-benzoate (from 57 to 84 %) for which, to our knowledge, there are currently no commercially available ELISA. Characteristics (sensitivity, inter and intra assay variation, and cross-reactivity) of the E1 + E2 + E3 ELISA were further compared to those from a commercial Estriol ELISA. The commercial ELISA was more specific, sensitive and its inter-assay variation was less (9.5% compared to 10% for the E1 + E2 + E3 ELISA) but the E1 + E2 + E3 ELISA had less intra-assay variation (4% compared to 5% for the commercial ELISA). Finally, a solid-phase extraction method compatible with the E1 + E2 + E3 immunoassay demonstrated that this combined approach of extraction and immunoassay had good potential for determining estrogen concentrations in environmental samples such as surface water in urban and agricultural ecosystems.


Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Estradiol , Estriol , Estrone , Animals , Antibodies , Cross Reactions , Estradiol/analysis , Estradiol/immunology , Estriol/analysis , Estriol/immunology , Estrone/analysis , Estrone/immunology , Rabbits , Sensitivity and Specificity , Solid Phase Extraction
10.
Cell Res ; 20(6): 647-64, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20368731

ABSTRACT

Salmonella can invade non-phagocytic cells through its type III secretion system (T3SS-1), which induces a Trigger entry process. This study showed that Salmonella enterica, subspecies enterica serovar Enteritidis can also invade cells via the Rck outer membrane protein. Rck was necessary and sufficient to enable non-invasive E. coli and Rck-coated beads to adhere to and invade different cells. Internalization analysis of latex beads coated with different Rck peptides showed that the peptide containing amino acids 140-150 promoted adhesion, whereas amino acids between 150 and 159 modulated invasion. Expression of dominant-negative derivatives and use of specific inhibitors demonstrated the crucial role of small GTPases Rac1 and Cdc42 in activating the Arp2/3 complex to trigger formation of actin-rich accumulation, leading to Rck-dependent internalization. Finally, scanning and transmission electron microscopy with Rck-coated beads and E. coli expressing Rck revealed microvillus-like extensions that formed a Zipper-like structure, engulfing the adherent beads and bacteria. Overall, our results provide new insights into the Salmonella T3SS-independent invasion mechanisms and strongly suggest that Rck induces a Zipper-like entry mechanism. Consequently, Salmonella seems to be the first bacterium found to be able to induce both Zipper and Trigger mechanisms to invade host cells.


Subject(s)
Bacterial Outer Membrane Proteins/physiology , Endocytosis/physiology , Salmonella Infections, Animal/metabolism , Salmonella Infections, Animal/microbiology , Salmonella enteritidis/pathogenicity , Amino Acid Sequence , Animals , Bacterial Adhesion/physiology , Bacterial Outer Membrane Proteins/chemistry , Cell Line , Cell Membrane/chemistry , Cell Membrane/metabolism , Cells, Cultured , Mice , Monomeric GTP-Binding Proteins/metabolism , NIH 3T3 Cells , Salmonella Infections, Animal/pathology , Salmonella enteritidis/chemistry , Salmonella enteritidis/physiology
11.
J Environ Qual ; 39(2): 617-29, 2010.
Article in English | MEDLINE | ID: mdl-20176835

ABSTRACT

In many areas of intensive corn production, atrazine and metolachlor are among the most commonly found herbicides in surface and ground water. This 2-yr study compared the impact of grass and grass+tree buffer strips on the exported masses of atrazine, metolachlor, and a degradation product of atrazine, desethylatrazine (DEA). The experimental system consisted of four replicate plots in a three-way completely randomized design (no buffer zone, grass buffer zone, and grass+tree buffer strips). The field plots were 5 m wide and 30 m long and grown in corn. The grass and grass+tree buffer strips were 5 m and had the same grass vegetation except for eight young hybrid poplars. Over the 2-yr study, surface runoff and subsurface infiltration water (under the buffer strip) were collected after the initial three rainfall events after herbicide application. Dissolved atrazine, metolachlor, and DEA were analyzed by gas chromatography/mass spectrometry. The presence of buffer strips decreased the exported masses of atrazine and metolachlor in surface runoff. A three-way ANOVA with treatment (type of buffer strip), water (surface runoff or subsurface infiltration), and time between herbicide application and rainfall event as factors showed a significant reduction (40-60% in 2004 and 75-95% in 2005) in the total (surface runoff+infiltrated water) exported masses of atrazine and metolachlor in the presence of buffer strips. Rainfall events after herbicide application were different between the 2 yr and greatly affected the flow distribution (e.g., subsurface infiltration) and the leached herbicide concentrations. No significant difference in the capacity to reduce herbicide exports was observed between grass and grass+tree buffer strip treatments; the poorly developed young poplar biomass at the time of the study may partly explain this observation.


Subject(s)
Herbicides/analysis , Poaceae , Populus , Water Pollutants, Chemical/analysis , Water Pollution/prevention & control , Acetamides/analysis , Atrazine/analysis , Trees , Water/analysis , Weather
12.
J Biol Chem ; 285(6): 4087-4098, 2010 Feb 05.
Article in English | MEDLINE | ID: mdl-19926792

ABSTRACT

Pathogenic Yersinia species neutralize innate immune mechanisms by injecting type three secretion effectors into immune cells, altering cell signaling. Our study elucidates how one of these effectors, YopO, blocks phagocytosis. We demonstrate using different phagocytic models that YopO specifically blocks Rac-dependent Fcgamma receptor internalization pathway but not complement receptor 3-dependent uptake, which is controlled by Rho activity. We show that YopO prevents Rac activation but does not affect Rac accumulation at the phagocytic cup. In addition, we show that plasma membrane localization and the guanine-nucleotide dissociation inhibitor (GDI)-like domain of YopO cooperate for maximal anti-phagocytosis. Although YopO has the same affinity for Rac1, Rac2, and RhoA in vitro, it selectively interacts with Rac isoforms in cells. This is due to the differential localization of the Rho family G proteins in resting cells; Rac isoforms partially exist as a GDI-free pool at the membrane of resting cells, whereas RhoA is trapped in the cytosol by RhoGDIalpha. We propose that YopO exploits this basic difference in localization and availability to selectively inhibit Rac-dependent phagocytosis.


Subject(s)
Bacterial Proteins/metabolism , Phagocytosis , Protein Serine-Threonine Kinases/metabolism , Receptors, IgG/metabolism , rac1 GTP-Binding Protein/metabolism , Animals , Bacterial Proteins/genetics , Binding Sites , Blotting, Western , COS Cells , Cell Line , Cell Membrane/metabolism , Chlorocebus aethiops , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Guanine Nucleotide Dissociation Inhibitors/genetics , Guanine Nucleotide Dissociation Inhibitors/metabolism , Humans , Macrophages/cytology , Macrophages/metabolism , Mice , Microscopy, Confocal , Mutation , Protein Binding , Protein Serine-Threonine Kinases/genetics , Receptors, IgG/genetics , Transfection , Yersinia enterocolitica/genetics , Yersinia enterocolitica/metabolism , rac1 GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/metabolism
13.
Mol Syst Biol ; 5: 298, 2009.
Article in English | MEDLINE | ID: mdl-19690567

ABSTRACT

Phagocytosis is the process by which cells internalize particulate material, and is of central importance to immunity, homeostasis and development. Here, we study the internalization of immunoglobulin G-coated particles in cells transfected with Fcgamma receptors (FcgammaRs) through the formation of an enveloping phagocytic cup. Using confocal microscopy, we precisely track the location of fluorescently tagged FcgammaRs during cup growth. Surprisingly, we found that phagocytic cups growing around identical spherical particles showed great variability even within a single cell and exhibited two eventual fates: a cup either stalled before forming a half-cup or it proceeded until the particle was fully enveloped. We explain these observations in terms of a mechanical bottleneck using a simple mathematical model of the overall process of cup growth. The model predicts that reducing F-actin concentration levels, and hence the deforming force, does not necessarily lead to stalled cups, a prediction we verify experimentally. Our analysis gives a coherent explanation for the importance of geometry in phagocytic uptake and provides a unifying framework for integrating the key processes, both biochemical and mechanical, occurring during cup growth.


Subject(s)
Receptors, IgG/chemistry , Actins/chemistry , Animals , COS Cells , Cell Membrane/metabolism , Chlorocebus aethiops , Diffusion , Green Fluorescent Proteins/chemistry , Humans , Microscopy, Confocal/methods , Models, Biological , Models, Theoretical , Phagocytosis , Signal Transduction , Systems Biology/methods
14.
J Cell Sci ; 122(Pt 16): 2996-3004, 2009 Aug 15.
Article in English | MEDLINE | ID: mdl-19638416

ABSTRACT

At the onset of mitosis, most adherent cells undergo cell retraction characterised by the disassembly of focal adhesions and actin stress fibres. Mitosis takes place in rounded cells, and the two daughter cells spread again after cytokinesis. Because of the well-documented ability of the small GTPase Rap1 to stimulate integrin-dependent adhesion and spreading, we assessed its role during mitosis. We show that Rap1 activity is regulated during this process. Changes in Rap1 activity play an essential role in regulating cell retraction and spreading, respectively, before and after mitosis of HeLa cells. Indeed, endogenous Rap1 is inhibited at the onset of mitosis; conversely, constitutive activation of Rap1 inhibits the disassembly of premitotic focal adhesions and of the actin cytoskeleton, leading to delayed mitosis and to cytokinesis defects. Rap1 activity slowly increases after mitosis ends; inhibition of Rap1 activation by the ectopic expression of the dominant-negative Rap1[S17A] mutant prevents the rounded cells from spreading after mitosis. For the first time, we provide evidence for the direct regulation of adhesion processes during mitosis via the activity of the Rap1 GTPase.


Subject(s)
Cell Movement , Mitosis , rap1 GTP-Binding Proteins/metabolism , Actins/metabolism , Cell Adhesion , Cell Shape , Cytoskeleton/metabolism , Down-Regulation , Enzyme Activation , HeLa Cells , Humans , Mutant Proteins/metabolism , Signal Transduction
15.
Biol Cell ; 102(1): 13-24, 2009 Sep 23.
Article in English | MEDLINE | ID: mdl-19583567

ABSTRACT

BACKGROUND INFORMATION: The appropriate regulation of cell-cell adhesion is an important event in the homoeostasis of different cell types. In epithelial cells, tight adhesion mediated by E-cadherin receptors is essential for the differentiation and functionality of epithelial sheets. Upon assembly of cadherin-mediated cell-cell contacts, it is well established that the small GTPases Rho and Rac are activated and are necessary for junction stability. However, the role of the small GTPase Cdc42 in cadherin adhesion is less clear. Cdc42 can be activated by E-cadherin in a breast tumour cell line, but the requirement for Cdc42 function for new junction assembly or maintenance has been contradictory. Cdc42 participation in cell-cell contacts has been inferred from the presence of filopodia, the typical F-actin structure induced by Cdc42 activation, as cells approach each other to establish cell-cell contacts. Yet, under these conditions, the contribution of migration to filopodia protrusion cannot be excluded and the results are difficult to interpret. RESULTS: In the present study, we set out to address (a) whether Cdc42 is activated by new E-cadherin cell-cell contacts when junction assembly occurs without prior migration and (b) whether Cdc42 function is necessary for cadherin stability. We found that junction formation in confluent keratinocytes or upon E-cadherin clustering decreased Cdc42-GTP levels. In the absence of serum- and migration-induced Cdc42 activation, we demonstrated that cell-cell contacts do not induce filopodia or require Cdc42 function to assemble. CONCLUSION: We conclude that Cdc42 does not participate in the early events that initiate stable cadherin adhesion in keratinocytes. Yet, it is feasible that Cdc42 may be activated at later time points or by other receptors. Cdc42 can then participate in additional functions during polarization, such as Golgi re-positioning or basolateral trafficking.


Subject(s)
Cadherins/metabolism , Intercellular Junctions/metabolism , Keratinocytes/metabolism , Pseudopodia/metabolism , cdc42 GTP-Binding Protein/metabolism , Animals , Cell Adhesion/physiology , Cell Line , Cells, Cultured , Fluorescent Antibody Technique , Humans , Mice , Signal Transduction
16.
J Cell Sci ; 121(11): 1773-83, 2008 Jun 01.
Article in English | MEDLINE | ID: mdl-18492791

ABSTRACT

By linking actin dynamics to extracellular components, integrins are involved in a wide range of cellular processes that are associated with or require cytoskeletal remodelling and cell-shape changes. One such function is integrin-dependent phagocytosis, a process that several integrins are capable of mediating and that allows the binding and clearance of particles. Integrin-dependent phagocytosis is involved in a wide range of physiological processes, from the clearance of microorganisms and apoptotic-cell removal to extracellular-matrix remodelling. Integrin signalling is also exploited by microbial pathogens for entry into host cells. Far from being a particular property of specific integrins and specialised cells, integrin-dependent uptake is emerging as a general, intrinsic ability of most integrins that is associated with their capacity to signal to the actin cytoskeleton. Integrin-mediated phagocytosis can therefore be used as a robust model in which to study integrin regulation and signalling.


Subject(s)
Actins/metabolism , Cytoskeleton/metabolism , Integrins/metabolism , Phagocytosis/physiology , Animals , Bacterial Adhesion/physiology , Cell Adhesion/physiology , Cell Membrane/metabolism , Humans , Protein Binding/physiology , Signal Transduction/physiology
17.
Cell Microbiol ; 10(5): 1104-15, 2008 May.
Article in English | MEDLINE | ID: mdl-18201246

ABSTRACT

A key strategy in microbial pathogenesis is the subversion of the first line of cellular immune defences presented by professional phagocytes. Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC respectively) remain extracellular while colonizing the gut mucosa by attaching and effacing mechanism. EPEC use the type three secretion system effector protein EspF to prevent their own uptake into macrophages. EPEC can also block in trans the internalization of IgG-opsonized particles. In this study, we show that EspJ is the type three secretion system effector protein responsible for trans-inhibition of macrophage opsono-phagocytosis by both EPEC and EHEC. While EspF plays no role in trans-inhibition of opsono-phagocytosis, espJ mutants of EPEC or EHEC are unable to block uptake of opsonized sheep red blood cells (RBC), a phenotype that is rescued upon complementation with the espJ gene. Importantly, ectopic expression of EspJ(EHEC) in phagocytes is sufficient to inhibit internalization of both IgG- and C3bi-opsonized RBC. These results suggest that EspJ targets a basic mechanism common to these two unrelated phagocytic receptors. Moreover, EspF and EspJ target independent aspects of the phagocytic function of mammalian macrophages in vitro.


Subject(s)
Enterohemorrhagic Escherichia coli/metabolism , Escherichia coli O157/metabolism , Escherichia coli Proteins/metabolism , Macrophages/microbiology , Opsonin Proteins/immunology , Phagocytosis , Animals , COS Cells , Carrier Proteins/metabolism , Chlorocebus aethiops , Intracellular Signaling Peptides and Proteins , Macrophage-1 Antigen/immunology , Transfection
19.
Mol Biol Cell ; 18(3): 976-85, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17202407

ABSTRACT

The cytoskeletal, actin-binding protein talin has been previously implicated in phagocytosis in Dictyostelium discoideum and mammalian phagocytes. However, its mechanism of action during internalization is not understood. Our data confirm that endogenous talin can occasionally be found at phagosomes forming around IgG- and C3bi-opsonized red blood cells in macrophages. Remarkably, talin knockdown specifically abrogates uptake through complement receptor 3 (CR3, CD11b/CD18, alpha(M)beta(2) integrin) and not through the Fc gamma receptor. We show that talin physically interacts with CR3/alpha(M)beta(2) and that this interaction involves the talin head domain and residues W747 and F754 in the beta(2) integrin cytoplasmic domain. The CR3/alpha(M)beta(2)-talin head interaction controls not only talin recruitment to forming phagosomes but also CR3/alpha(M)beta(2) binding activity, both in macrophages and transfected fibroblasts. However, the talin head domain alone cannot support phagocytosis. Our results establish for the first time at least two distinct roles for talin during CR3/alpha(M)beta(2)-mediated phagocytosis, most noticeably activation of the CR3/alpha(M)beta(2) receptor and phagocytic uptake.


Subject(s)
Macrophage-1 Antigen/metabolism , Phagocytosis , Talin/metabolism , Amino Acid Sequence , Animals , COS Cells , Chlorocebus aethiops , Erythrocytes/metabolism , Green Fluorescent Proteins/metabolism , Humans , Immunoglobulin G/immunology , Macrophage-1 Antigen/chemistry , Mice , Molecular Sequence Data , Phenylalanine , Protein Binding , Protein Structure, Tertiary , Protein Transport , Receptors, IgG/immunology , Recombinant Fusion Proteins/metabolism , Sheep , Tryptophan
20.
J Bacteriol ; 188(8): 3110-5, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16585770

ABSTRACT

EspF of enteropathogenic Escherichia coli targets mitochondria and subverts a number of cellular functions. EspF consists of six putative Src homology 3 (SH3) domain binding motifs. In this study we identified sorting nexin 9 (SNX9) as a host cell EspF binding partner protein, which binds EspF via its amino-terminal SH3 region. Coimmunoprecipitation and confocal microscopy showed specific EspF-SNX9 interaction and non-mitochondrial protein colocalization in infected epithelial cells.


Subject(s)
Carrier Proteins/metabolism , Escherichia coli Proteins/metabolism , Amino Acid Motifs , Carrier Proteins/chemistry , Epithelial Cells/chemistry , Epithelial Cells/microbiology , Escherichia coli Proteins/chemistry , HeLa Cells , Humans , Immunoprecipitation , Intracellular Signaling Peptides and Proteins , Microscopy, Confocal , Protein Binding , Sorting Nexins , Vesicular Transport Proteins , src Homology Domains
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