Regulation of the Actin Cytoskeleton by an Interaction of IQGAP Related Protein GAPA with Filamin and Cortexillin I

Filamin and Cortexillin are F-actin crosslinking proteins in Dictyostelium discoideum allowing actin filaments to form three-dimensional networks. GAPA, an IQGAP related protein, is required for cytokinesis and localizes to the cleavage furrow during cytokinesis. Here we describe a novel interaction with Filamin which is required for cytokinesis and regulation of the F-actin content. The interaction occurs through the actin binding domain of Filamin and the GRD domain of GAPA. A similar interaction takes place with Cortexillin I. We further report that Filamin associates with Rac1a implying that filamin might act as a scaffold for small GTPases. Filamin and activated Rac associate with GAPA to regulate actin remodelling. Overexpression of filamin and GAPA in the various strains suggests that GAPA regulates the actin cytoskeleton through interaction with Filamin and that it controls cytokinesis through association with Filamin and Cortexillin

The N-Terminal DH-PH Domain of Trio Induces Cell Spreading and Migration by Regulating Lamellipodia Dynamics in a Rac1-Dependent Fashion

The guanine-nucleotide exchange factor Trio encodes two DH-PH domains that catalyze nucleotide exchange on Rac1, RhoG and RhoA. The N-terminal DH-PH domain is known to activate Rac1 and RhoG, whereas the C-terminal DH-PH domain can activate RhoA. The current study shows that the N-terminal DH-PH domain, upon expression in HeLa cells, activates Rac1 and RhoG independently from each other. In addition, we show that the flanking SH3 domain binds to the proline-rich region of the C-terminus of Rac1, but not of RhoG. However, this SH3 domain is not required for Rac1 or RhoG GDP-GTP exchange. Rescue experiments in Trio-shRNA-expressing cells showed that the N-terminal DH-PH domain of Trio, but not the C-terminal DH-PH domain, restored fibronectin-mediated cell spreading and migration defects that are observed in Trio-silenced cells. Kymograph analysis revealed that the N-terminal DH-PH domain, independent of its SH3 domain, controls the dynamics of lamellipodia. Using siRNA against Rac1 or RhoG, we found that Trio-D1-induced lamellipodia formation required Rac1 but not RhoG expression. Together, we conclude that the GEF Trio is responsible for lamellipodia formation through its N-terminal DH-PH domain in a Rac1-dependent manner during fibronectin-mediated spreading and migration

Long Astral Microtubules and RACK-1 Stabilize Polarity Domains during Maintenance Phase in Caenorhabditis elegans Embryos

Cell polarity is a very well conserved process important for cell differentiation, cell migration, and embryonic development. After the establishment of distinct cortical domains, polarity cues have to be stabilized and maintained within a fluid and dynamic membrane to achieve proper cell asymmetry. Microtubules have long been thought to deliver the signals required to polarize a cell. While previous studies suggest that microtubules play a key role in the establishment of polarity, the requirement of microtubules during maintenance phase remains unclear. In this study, we show that depletion of Caenorhabditis elegans RACK-1, which leads to short astral microtubules during prometaphase, specifically affects maintenance of cortical PAR domains and Dynamin localization. We then investigated the consequence of knocking down other factors that also abolish astral microtubule elongation during polarity maintenance phase. We found a correlation between short astral microtubules and the instability of PAR-6 and PAR-2 domains during maintenance phase. Our data support a necessary role for astral microtubules in the maintenance phase of cell polarity

Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET

Direct visualization and light control of several cellular processes is a challenge, owing to the spectral overlap of available genetically encoded probes. Here we report the most red-shifted monomeric near-infrared (NIR) fluorescent protein, miRFP720, and the fully NIR Forster resonance energy transfer (FRET) pair miRFP670-miRFP720, which together enabled design of biosensors compatible with CFP-YFP imaging and blue-green optogenetic tools. We developed a NIR biosensor for Rac1 GTPase and demonstrated its use in multiplexed imaging and light control of Rho GTPase signaling pathways. Specifically, we combined the Rac1 biosensor with CFP-YFP FRET biosensors for RhoA and for Rac1-GDI binding, and concurrently used the LOV-TRAP tool for upstream Rac1 activation. We directly observed and quantified antagonism between RhoA and Rac1 dependent on the RhoA-downstream effector ROCK; showed that Rac1 activity and GDI binding closely depend on the spatiotemporal coordination between these two molecules; and simultaneously observed Rac1 activity during optogenetic manipulation of Rac1.Peer reviewe

Human Mena Associates with Rac1 Small GTPase in Glioblastoma Cell Lines

Mammarian enabled (Mena), a member of the Enabled (Ena)/Vasodilator-stimulated phosphoprotein (VASP) family of proteins, has been implicated in cell motility through regulation of the actin cytoskeleton assembly, including lamellipodial protrusion. Rac1, a member of the Rho family GTPases, also plays a pivotal role in the formation of lamellipodia. Here we report that human Mena (hMena) colocalizes with Rac1 in lamellipodia, and using an unmixing assisted acceptor depletion fluorescence resonance energy transfer (u-adFRET) analysis that hMena associates with Rac1 in vivo in the glioblastoma cell line U251MG. Depletion of hMena by siRNA causes cells to be highly spread with the formation of lamellipodia. This cellular phenotype is canceled by introduction of a dominant negative form of Rac1. A Rac activity assay and FRET analysis showed that hMena knock-down cells increased the activation of Rac1 at the lamellipodia. These results suggest that hMena possesses properties which help to regulate the formation of lamellipodia through the modulation of the activity of Rac1

A 300-year record of sedimentation in a small tilled catena in Hungary based on δ13C, δ15N, and C/N distribution

Purpose Soil erosion is one of the most serious hazards that endanger sustainable food production. Moreover, it has marked effects on soil organic carbon (SOC) with direct links to global warming. At the same time, soil organic matter (SOM) changes in composition and space could influence these processes. The aim of this study was to predict soil erosion and sedimentation volume and dynamics on a typical hilly cropland area of Hungary due to forest clearance in the early eighteenth century. Materials and methods Horizontal soil samples were taken along two parallel intensively cultivated complex convex-concave slopes from the eroded upper parts at mid-slope positions and from sedimentation in toe-slopes. Samples were measured for SOC, total nitrogen (TN) content, and SOMcompounds (δ13C, δ15N, and photometric indexes). They were compared to the horizons of an in situ non-eroded profile under continuous forest. On the depositional profile cores, soil depth prior to sedimentation was calculated by the determination of sediment thickness. Results and discussion Peaks of SOC in the sedimentation profiles indicated thicker initial profiles, while peaks in C/N ratio and δ13C distribution showed the original surface to be ~ 20 cm lower. Peaks of SOC were presumed to be the results of deposition of SOC-enriched soil from the upper slope transported by selective erosion of finer particles (silts and clays). Therefore, changes in δ13C values due to tillage and delivery would fingerprint the original surface much better under the sedimentation scenario than SOC content. Distribution of δ13C also suggests that the main sedimentation phase occurred immediately after forest clearance and before the start of intense cultivation with maize. Conclusions This highlights the role of relief in sheet erosion intensity compared to intensive cultivation. Patterns of δ13C indicate the original soil surface, even in profiles deposited as sediment centuries ago. The δ13C and C/N decrease in buried in situ profiles had the same tendency as recent forest soil, indicating constant SOM quality distribution after burial. Accordingly, microbiological activity, root uptake, and metabolism have not been effective enough to modify initial soil properties

Androgen-Induced Cell Migration: Role of Androgen Receptor/Filamin A Association

Background: Androgen receptor (AR) controls male morphogenesis, gametogenesis and prostate growth as well as development of prostate cancer. These findings support a role for AR in cell migration and invasiveness. However, the molecular mechanism involved in AR-mediated cell migration still remains elusive. Methodology/Principal Findings: Mouse embryo NIH3T3 fibroblasts and highly metastatic human fibrosarcoma HT1080 cells harbor low levels of transcriptionally incompetent AR. We now report that, through extra nuclear action, AR triggers migration of both cell types upon stimulation with physiological concentrations of the androgen R1881. We analyzed the initial events leading to androgen-induced cell migration and observed that challenging NIH3T3 cells with 10 nM R1881 rapidly induces interaction of AR with filamin A (FlnA) at cytoskeleton. AR/FlnA complex recruits integrin beta 1, thus activating its dependent cascade. Silencing of AR, FlnA and integrin beta 1 shows that this ternary complex controls focal adhesion kinase (FAK), paxillin and Rac, thereby driving cell migration. FAK-null fibroblasts migrate poorly and Rac inhibition by EHT impairs motility of androgen-treated NIH3T3 cells. Interestingly, FAK and Rac activation by androgens are independent of each other. Findings in human fibrosarcoma HT1080 cells strengthen the role of Rac in androgen signaling. The Rac inhibitor significantly impairs androgen-induced migration in these cells. A mutant AR, deleted of the sequence interacting with FlnA, fails to mediate FAK activation and paxillin tyrosine phosphorylation in androgen-stimulated cells, further reinforcing the role of AR/FlnA interaction in androgen-mediated motility. Conclusions/Significance: The present report, for the first time, indicates that the extra nuclear AR/FlnA/integrin beta 1 complex is the key by which androgen activates signaling leading to cell migration. Assembly of this ternary complex may control organ development and prostate cancer metastasis

The lactose operon from Lactobacillus casei is involved in the transport and metabolism of the human milk oligosaccharide core-2 N-acetyllactosamine

The lactose operon (lacTEGF) from Lactobacillus casei strain BL23 has been previously studied. The lacT gene codes for a transcriptional antiterminator, lacE and lacF for the lactose-specific phosphoenolpyruvate: phosphotransferase system (PTSLac) EIICB and EIIA domains, respectively, and lacG for the phospho-β-galactosidase. In this work, we have shown that L. casei is able to metabolize N-acetyllactosamine (LacNAc), a disaccharide present at human milk and intestinal mucosa. The mutant strains BL153 (lacE) and BL155 (lacF) were defective in LacNAc utilization, indicating that the EIICB and EIIA of the PTSLac are involved in the uptake of LacNAc in addition to lactose. Inactivation of lacG abolishes the growth of L. casei in both disaccharides and analysis of LacG activity showed a high selectivity toward phosphorylated compounds, suggesting that LacG is necessary for the hydrolysis of the intracellular phosphorylated lactose and LacNAc. L. casei (lacAB) strain deficient in galactose-6P isomerase showed a growth rate in lactose (0.0293 ± 0.0014 h-1) and in LacNAc (0.0307 ± 0.0009 h-1) significantly lower than the wild-type (0.1010 ± 0.0006 h-1 and 0.0522 ± 0.0005 h-1, respectively), indicating that their galactose moiety is catabolized through the tagatose-6P pathway. Transcriptional analysis showed induction levels of the lac genes ranged from 130 to 320-fold in LacNAc and from 100 to 200-fold in lactose, compared to cells growing in glucose

Childhood lead exposure in France: benefit estimation and partial cost-benefit analysis of lead hazard control

<p>Abstract</p> <p>Background</p> <p>Lead exposure remains a public health concern due to its serious adverse effects, such as cognitive and behavioral impairment: children younger than six years of age being the most vulnerable population. In Europe, the lead-related economic impacts have not been examined in detail. We estimate the annual costs in France due to childhood exposure and, through a cost benefit analysis (CBA), aim to assess the expected social and economic benefits of exposure abatement.</p> <p>Methods</p> <p>Monetary benefits were assessed in terms of avoided national costs. We used results from a 2008 survey on blood-lead (B-Pb) concentrations in French children aged one to six years old. Given the absence of a threshold concentration being established, we performed a sensitivity analysis assuming different hypothetical threshold values for toxicity above 15 μg/L, 24 μg/L and 100 μg/L. Adverse health outcomes of lead exposure were translated into social burden and economic costs based on literature data from literature. Direct health benefits, social benefits and intangible avoided costs were included. Costs of pollutant exposure control were partially estimated in regard to homes lead-based paint decontamination, investments aiming at reducing industrial lead emissions and removal of all lead drinking water pipes.</p> <p>Results</p> <p>The following overall annual benefits for the three hypothetical thresholds values in 2008 are: €22.72 billion, €10.72 billion and €0.44 billion, respectively. Costs from abatement ranged from €0.9 billion to 2.95 billion/year. Finally, from a partial CBA of lead control in soils and dust the estimates of total net benefits were € 3.78 billion, € 1.88 billion and €0.25 billion respectively for the three hypothesized B-Pb effect values.</p> <p>Conclusions</p> <p>Prevention of childhood lead exposure has a high social benefit, due to reduction of B-Pb concentrations to levels below 15 μg/L or 24 μg/L, respectively. Reducing only exposures above 100 μg/L B-Pb has little economic impact due to the small number of children who now exhibit such high exposure levels. Prudent public policies would help avoiding future medical interventions, limit the need for special education and increase future productivity, and hence lifetime income for children exposed to lead.</p