Nck and Cdc42 co-operate to recruit N-WASP to promote FcγR-mediated phagocytosis.

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.

RhoG is required for both FcγR- and CR3-mediated phagocytosis.

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.

Impact of recent manure applications on natural estrogen concentrations in streams near agricultural fields.

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.

Ser756 of ß2 integrin controls Rap1 activity during inside-out activation of αMß2.

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.

Impact of vegetated filter strips on sorbed herbicide concentrations and sorption equilibrium in agricultural plots.

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.

Solid beef cattle manure application impacts on soil properties and 17ß-estradiol fate in a clay loam soil.

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.

Sequestering of Rac by the Yersinia effector YopO blocks Fcgamma receptor-mediated phagocytosis.

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.

Regulation of Wiskott-Aldrich syndrome protein and related molecules.

Wiskott-Aldrich syndrome protein (WASP), the gene mutated in the haematological disorder Wiskott-Aldrich syndrome, is the founding member of a family of conserved cytoskeletal regulators. WASP family proteins regulate actin dynamics through binding and activation of the Arp2/3 complex, which nucleates new actin filaments. Recently, a huge amount of information on WASPs has become available, both in terms of the function of individual domains, identification of their binding partners, and the unravelling of complex regulatory mechanisms. Together, these new findings place WASP-related molecules at the crossroads between distinct signalling pathways, which they integrate into coherent cytoskeletal responses.

Two distinct cytoplasmic regions of the beta2 integrin chain regulate RhoA function during phagocytosis.

AlphaMbeta2 integrins mediate phagocytosis of opsonized particles in a process controlled by RhoA, Rho kinase, myosin II, Arp2/3, and actin polymerization. AlphaMbeta2, Rho, Arp2/3, and F-actin accumulate underneath bound particles; however, the mechanism regulating Rho function during alphaMbeta2-mediated phagocytosis is poorly understood. We report that the binding of C3bi-opsonized sheep red blood cells (RBCs) to alphaMbeta2 increases Rho-GTP, but not Rac-GTP, levels. Deletion of the cytoplasmic domain of beta2, but not of alphaM, abolished Rho recruitment and activation, as well as phagocytic uptake. Interestingly, a 16-amino acid (aa) region in the membrane-proximal half of the beta2 cytoplasmic domain was necessary for activating Rho. Three COOH-terminal residues (aa 758-760) were essential for beta2-induced accumulation of Rho at complement receptor 3 (CR3) phagosomes. Activation of Rho was necessary, but not sufficient, for its stable recruitment underneath bound particles or for uptake. However, recruitment of active Rho was sufficient for phagocytosis. Our data shed light on the mechanism of outside-in signaling, from ligated integrins to the activation of Rho GTPase signaling.

Rap1 controls activation of the α(M)ß(2) integrin in a talin-dependent manner.

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.

A mechanical bottleneck explains the variation in cup growth during FcgammaR phagocytosis.

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.

Newly formed E-cadherin contacts do not activate Cdc42 or induce filopodia protrusion in human keratinocytes.

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.

An essential role for talin during alpha(M)beta(2)-mediated phagocytosis.

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.

Impact of grass and grass with poplar buffer strips on atrazine and metolachlor losses in surface runoff and subsurface infiltration from agricultural plots.

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.

Development of competitive ELISAs for 17beta-estradiol and 17beta-estradiol +estrone+estriol using rabbit polyclonal antibodies.

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.

Regulation by phosphorylation. Yet another twist in the WASP story.

Cell migration and other complex cellular processes involve a variety of signaling molecules and require the integration of multiple signals into a coherent cytoskeletal response. Two papers in the May issue of Molecular Cell now demonstrate that phosphorylation plays a critical role in WASP function as a regulator of Arp2/3-mediated actin polymerization.

Regulation of WASP/WAVE proteins: making a long story short.

Despite their homology, the regulation of WASP and WAVE, activators of Arp2/3-dependent actin polymerization, has always been thought to be different. Several recent studies have revealed new aspects of their regulation, highlighting its complexity and the crucial role of post-translational modifications. New data also suggest additional functions for WASP family proteins, pushing us to reconsider existing models.

EspJ of enteropathogenic and enterohaemorrhagic Escherichia coli inhibits opsono-phagocytosis.

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.

Subversion of actin dynamics by EPEC and EHEC.

During the course of infection, enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) subvert the host cell signalling machinery and hijack the actin cytoskeleton to tighten their interaction with the gut epithelium, while avoiding phagocytosis by professional phagocytes. Much progress has been made recently in our understanding of how EPEC and EHEC regulate the pathways leading to local activation of two regulators of actin cytoskeleton dynamics, the Wiskott-Aldrich syndrome protein (N-WASP) and the Arp2/3 complex. A recent highlight is the unravelling of functions for effector proteins (particularly Tir, TccP, Map and EspG/EspG2) that are injected into the host cell by a type III secretion system.

Rho-kinase and myosin-II control phagocytic cup formation during CR, but not FcgammaR, phagocytosis.

Phagocytosis through Fcgamma receptor (FcgammaR) or complement receptor 3 (CR) requires Arp2/3 complex-mediated actin polymerization, although each receptor uses a distinct signaling pathway. Rac and Cdc42 are required for actin and Arp2/3 complex recruitment during FcgammaR phagocytosis, while Rho controls actin assembly at CR phagosomes. To better understand the role of Rho in CR phagocytosis, we tested the idea that a known target of Rho, Rho-kinase (ROK), might control phagocytic cup formation and/or engulfment of particles. Inhibitors of ROK (dominant-negative ROK and Y-27632) and of the downstream target of ROK, myosin-II (ML7, BDM, and dominant-negative myosin-II), were used to test this idea. We found that inhibition of the Rho --> ROK --> myosin-II pathway caused a decreased accumulation of Arp2/3 complex and F-actin around bound particles, which led to a reduction in CR-mediated phagocytic engulfment. FcgammaR-mediated phagocytosis, in contrast, was independent of Rho or ROK activity and was only dependent on myosin-II for particle internalization, not for actin cup formation. While myosins have been previously implicated in FcgammaR phagocytosis, to our knowledge, this is the first demonstration of a role for myosin-II in CR phagocytosis.