Regulation of Focal Adhesion Kinase Activation,Breast Cancer Cell Motility,and Amoeboid Invasion by the RhoA Guanine Nucleotide Exchange Factor Net1

Net1 is a RhoA guanine nucleotide exchange factor (GEF) that is overexpressed in a subset of human cancers and contributes to cancer cell motility and invasion in vitro. However, the molecular mechanism accounting for its role in cell motility and invasion has not been described. In the present work, we show that expression of both Net1 isoforms in breast cancer cells is required for efficient cell motility. Although loss of Net1 isoform expression only partially blocks RhoA activation, it inhibits lysophosphatidic acid (LPA)-stimulated migration as efficiently as knockdown of RhoA itself. However, we demonstrate that the Net1A isoform predominantly controls myosin light-chain phosphorylation and is required for trailing edge retraction during migration. Net1A interacts with focal adhesion kinase (FAK), localizes to focal adhesions, and is necessary for FAK activation and focal adhesion maturation during cell spreading. Net1A expression is also required for efficient invasion through a Matrigel matrix. Analysis of invading cells demonstrates that Net1A is required for amoeboid invasion, and loss of Net1A expression causes cells to shift to a mesenchymal phenotype characterized by high β₁-integrin activity and membrane type 1 matrix metalloproteinase (MT1-MMP) expression. These results demonstrate a previously unrecognized role for the Net1A isoform in controlling FAK activation during planar cell movement and amoeboid motility during extracellular matrix (ECM) invasion.     

Characterization of the interaction between Robo1 and heparin and other glycosaminoglycans

Roundabout 1 (Robo1) is the cognate receptor for secreted axon guidance molecule, Slits, which function to direct cellular migration during neuronal development and angiogenesis. The Slit2–Robo1 signaling is modulated by heparan sulfate, a sulfated linear polysaccharide that is abundantly expressed on the cell surface and in the extracellular matrix. Biochemical studies have further shown that heparan sulfate binds to both Slit2 and Robo1 facilitating the ligand–receptor interaction. The structural requirements for heparan sulfate interaction with Robo1 remain unknown. In this report, surface plasmon resonance (SPR) spectroscopy was used to examine the interaction between Robo1 and heparin and other GAGs and determined that heparin binds to Robo1 with an affinity of ∼650 nM. SPR solution competition studies with chemically modified heparins further determined that although all sulfo groups on heparin are important for the Robo1–heparin interaction, the N-sulfo and 6-O-sulfo groups are essential for the Robo1–heparin binding. Examination of differently sized heparin oligosaccharides and different GAGs also demonstrated that Robo1 prefers to bind full-length heparin chains and that GAGs with higher sulfation levels show increased Robo1 binding affinities.     

Cardiovascular disease after Escherichia coli O157:H7 gastroenteritis

Background:

Escherichia coli O157:H7 is one cause of acute bacterial gastroenteritis, which can be devastating in outbreak situations. We studied the risk of cardiovascular disease following such an outbreak in Walkerton, Ontario, in May 2000.

Methods:

In this community-based cohort study, we linked data from the Walkerton Health Study (2002–2008) to Ontario’s large healthcare databases. We included 4 groups of adults: 3 groups of Walkerton participants (153 with severe gastroenteritis, 414 with mild gastroenteritis, 331 with no gastroenteritis) and a group of 11 263 residents from the surrounding communities that were unaffected by the outbreak. The primary outcome was a composite of death or first major cardiovascular event (admission to hospital for acute myocardial infarction, stroke or congestive heart failure, or evidence of associated procedures). The secondary outcome was first major cardiovascular event censored for death. Adults were followed for an average of 7.4 years.

Results:

During the study period, 1174 adults (9.7%) died or experienced a major cardiovascular event. Compared with residents of the surrounding communities, the risk of death or cardiovascular event was not elevated among Walkerton participants with severe or mild gastroenteritis (hazard ratio [HR] for severe gastroenteritis 0.74, 95% confidence interval [CI] 0.38–1.43, mild gastroenteritis HR 0.64, 95% CI 0.42–0.98). Compared with Walkerton participants who had no gastroenteritis, risk of death or cardiovascular event was not elevated among participants with severe or mild gastroenteritis.

Interpretation:

There was no increase in the risk of cardiovascular disease in the decade following acute infection during a major E. coli O157:H7 outbreak.Escherichia coli O157:H7 is one cause of acute bacterial gastroenteritis, causing 63 000 infections each year and 12 major outbreaks since 2006 in the United States alone.^1,2 This strain was most recently implicated in the outbreak involving beef from XL Foods (September 2012), with 17 confirmed cases across Canada.³ A similar enterohemorrhagic strain E. coli O104:H4 was responsible for an outbreak in Germany in May 2011, causing 3792 cases of gastroenteritis and 43 deaths.^4,5Most patients fully recover from acute gastroenteritis caused by E. coli. However, such an illness may predispose patients to long-term disease. Shiga toxin is produced by E. coli O157:H7; this toxin damages the microvasculature of the kidneys leading to hypertension^6–13 and directly damages the systemic vasculature.^14–16 Infected people may progress from a state of acute inflammation of the vasculature to subclinical chronic inflammation, which could promote atherosclerosis.^17–20In Walkerton, Ontario, in May 2000, heavy rains transported bovine fecal matter into the town’s well, contaminating the inadequately chlorinated municipal water supply with E. coli O157:H7.²¹ Over 2300 people developed acute gastroenteritis, and 7 people died.²² The unique circumstances of this outbreak provided a rare opportunity to study the natural history following exposure to this pathogen in a single cohort.²³ Other outbreaks have been geographically dispersed, making it difficult to track cases.^24,25In Walkerton, affected individuals were followed annually in a clinic to assess their long-term outcomes (Walkerton Health Study, 2002–2008). We previously reported that adults who experienced acute gastroenteritis during the outbreak had a higher than expected incidence of hypertension, chronic kidney disease and self-reported cardiovascular disease in follow-up.²³ However, 46% of participants were lost to follow-up by the end of the study, and there were limitations associated with the assessment of cardiovascular disease by participant recall. Thus, we conducted an expanded and extended follow-up study, linking the Walkerton study data to Ontario’s health care databases. Our objective was to more accurately determine the 10-year risk of major cardiovascular events after exposure to E. coli O157:H7.     

The Glassy Wall Boundary for Simulations of Inhomogeneous Systems

Abstract

For simulations on systems that invlove a physical inhomogeneity at infinite dilution, the usual periodic boundary conditions are inappropriate. In previous studies on such systems, surfaces have been re-introduced in order to contain the particles of the sample. When smooth surfaces are used to contain liquids, undesirable structural artifacts in the liquid are promoted by these surfaces. In this study a rigid but structurally liquid-like containing surface, that we call a glassy wall boundary, is introduced, in an attempt to solve this problem. It serves as a containing surface, but “looks” nearly liquid-like to molecules in the mobile liquid near it. We illustrate the properties of this boundary for a system that consists of an isolated polyion surrounded by SPC water at 300K and ～ 1 gcm^?3. We show that this boundary reduces or eliminates some of the problems caused by a smooth surface.     

Simulations on the Primitive Electrolyte Environment of a High Charge-density Polyelectrolyte. A Sampling Problem and its Solution

Abstract

We show that the classical Metropolis Monte Carlo (MMC) algorithm converges very slowly when applied to the primitive electrolyte environment for a high charge-density polyelectrolyte. This slowness of convergence, which is due to the large density inhomogeneity around the polyelectrolyte, produces noticeable errors in the ion distribution functions for MMC runs of 1.3 × 10⁶ trial steps started from nonequilibrium distributions. We report that an algorithm which we call DSMC (for density-scaled Monte Carlo) overcomes this problem and provides relatively rapid convergence in this application. We suggest that DSMC should be well-suited for other Monte Carlo simulations on physical systems where large density inhomogeneities occur.     

Patterning and Lifetime of Plasma Membrane-Localized Cellulose Synthase Is Dependent on Actin Organization in Arabidopsis Interphase Cells

The actin and microtubule cytoskeletons regulate cell shape across phyla, from bacteria to metazoans. In organisms with cell walls, the wall acts as a primary constraint of shape, and generation of specific cell shape depends on cytoskeletal organization for wall deposition and/or cell expansion. In higher plants, cortical microtubules help to organize cell wall construction by positioning the delivery of cellulose synthase (CesA) complexes and guiding their trajectories to orient newly synthesized cellulose microfibrils. The actin cytoskeleton is required for normal distribution of CesAs to the plasma membrane, but more specific roles for actin in cell wall assembly and organization remain largely elusive. We show that the actin cytoskeleton functions to regulate the CesA delivery rate to, and lifetime of CesAs at, the plasma membrane, which affects cellulose production. Furthermore, quantitative image analyses revealed that actin organization affects CesA tracking behavior at the plasma membrane and that small CesA compartments were associated with the actin cytoskeleton. By contrast, localized insertion of CesAs adjacent to cortical microtubules was not affected by the actin organization. Hence, both actin and microtubule cytoskeletons play important roles in regulating CesA trafficking, cellulose deposition, and organization of cell wall biogenesis.Plant cells are surrounded by a flexible yet durable extracellular matrix that makes up the cell wall. This structure offers mechanical strength that counters osmotically driven turgor pressure, is an important factor for water movement in plants, acts as a physical barrier against pathogens (Somerville et al., 2004), and is a determining factor for plant cell morphogenesis. Hence, the cell wall plays a central role in plant biology.Two main types of cell walls can typically be distinguished: the primary and the secondary cell wall. The major load-bearing component in both of these cell walls is the β-1,4-linked glucan polymer cellulose (Somerville et al., 2004). Cellulose polymers are synthesized by plasma membrane (PM)-localized cellulose synthase (CesA) complexes (Mueller and Brown, 1980), which contain several CesA subunits with similar amino acid sequences (Mutwil et al., 2008a). The primary wall CesA complexes are believed to be assembled in the Golgi and are subsequently delivered to the PM via vesicular trafficking (Gutierrez et al., 2009), sometimes associated with Golgi pausing (Crowell et al., 2009). Furthermore, the primary wall CesA complexes are preferentially inserted into the PM at sites that coincide with cortical microtubules (MTs), which subsequently guide cellulose microfibril deposition (Gutierrez et al., 2009). Hence, the cortical MT array is a determinant for multiple aspects of primary wall cellulose production.The actin cytoskeleton plays a crucial role in organized deposition of cell wall polymers in many cell types, including cellulose-related polymers and pectins in tip-growing cells, such as pollen tubes and root hairs (Hu et al., 2003; Chen et al., 2007). Thus, actin-depolymerizing drugs and genetic manipulation of ACTIN genes impair directed expansion of tip-growing cells and long-distance transport of Golgi bodies with vesicles to growing regions (Ketelaar et al., 2003; Szymanski, 2005). In diffusely growing cells in roots and hypocotyls, loss of anisotropic growth has also been observed in response to mutations to vegetative ACTIN genes and to actin-depolymerizing and -stabilizing drugs (Baluska et al., 2001; Kandasamy et al., 2009). While actin is clearly important for cell wall assembly, it is less clear what precise roles it plays.One well-known function of actin in higher plants is to support intracellular movement of cytoplasmic organelles via actomyosin-based motility (Geisler et al., 2008; Szymanski, 2009). During primary wall synthesis in interphase cells, treatment with the actin assembly inhibitor latrunculin B (LatB) led to inhibition of Golgi motility and pronounced inhomogenities in CesA density at the PM (Crowell et al., 2009; Gutierrez et al., 2009) that coincided with the density of underlying and immobile Golgi bodies (Gutierrez et al., 2009). These results suggested that Golgi motility is important for CesA distribution (Gutierrez et al., 2009). The actin cytoskeleton also appears to be important for secondary wall cellulose microfibril deposition. For example, longitudinal actin filaments (AFs) define the movement of secondary wall CesA-containing Golgi bodies in developing xylem vessels (Wightman and Turner, 2008). In addition, it has been proposed that the AFs also can regulate the delivery of the secondary wall CesA complex to the PM via pausing of the Golgi (Wightman and Turner, 2008). It is therefore clear that actin organization is important for CesA distribution and for the pattern of cellulose microfibril deposition.Despite the above findings, very few reports have undertaken detailed studies to elucidate the role of the actin cytoskeleton in the distribution and trafficking of specific proteins in plant cells. Here, we have investigated the intracellular trafficking of CesA-containing vesicles and delivery of CesAs to the PM, in the context of the actin cytoskeleton. We quantitatively demonstrate that the organization of the actin cytoskeleton regulates CesA-containing Golgi distribution and the exocytic and endocytic rate of the CesAs. However, actin organization has no effect on the localized insertion of CesAs at sites of MTs at the PM.     

Palmitate and Lipopolysaccharide Trigger Synergistic Ceramide Production in Primary Macrophages

Macrophages play a key role in host defense and in tissue repair after injury. Emerging evidence suggests that macrophage dysfunction in states of lipid excess can contribute to the development of insulin resistance and may underlie inflammatory complications of diabetes. Ceramides are sphingolipids that modulate a variety of cellular responses including cell death, autophagy, insulin signaling, and inflammation. In this study we investigated the intersection between TLR4-mediated inflammatory signaling and saturated fatty acids with regard to ceramide generation. Primary macrophages treated with lipopolysaccharide (LPS) did not produce C16 ceramide, whereas palmitate exposure led to a modest increase in this sphingolipid. Strikingly, the combination of LPS and palmitate led to a synergistic increase in C16 ceramide. This response occurred via cross-talk at the level of de novo ceramide synthesis in the ER. The synergistic response required TLR4 signaling via MyD88 and TIR-domain-containing adaptor-inducing interferon beta (TRIF), whereas palmitate-induced ceramide production occurred independent of these inflammatory molecules. This ceramide response augmented IL-1β and TNFα release, a process that may contribute to the enhanced inflammatory response in metabolic diseases characterized by dyslipidemia.     

Characterization of the Staphylococcus aureus rRNA Methyltransferase Encoded by orfX,the Gene Containing the Staphylococcal Chromosome Cassette mec (SCCmec) Insertion Site

The gene orfX is conserved among all staphylococci, and its complete sequence is maintained upon insertion of the staphylococcal chromosome cassette mec (SCCmec) genomic island, containing the gene encoding resistance to β-lactam antibiotics (mecA), into its C terminus. The function of OrfX has not been determined. We show that OrfX was constitutively produced during growth, that orfX could be inactivated without altering bacterial growth, and that insertion of SCCmec did not alter gene expression. We solved the crystal structure of OrfX at 1.7 Å and found that it belongs to the S-adenosyl-l-methionine (AdoMet)-dependent α/β-knot superfamily of SPOUT methyltransferases (MTases), with a high structural homology to YbeA, the gene product of the Escherichia coli 70 S ribosomal MTase RlmH. MTase activity was confirmed by demonstrating the OrfX-dependent methylation of the Staphylococcus aureus 70 S ribosome. When OrfX was crystallized in the presence of its AdoMet substrate, we found that each monomer of the homodimeric structure bound AdoMet in its active site. Solution studies using isothermal titration calorimetry confirmed that each monomer bound AdoMet but with different binding affinities (K_d = 52 ± 0.4 and 606 ± 2 μm). In addition, the structure shows that the AdoMet-binding pocket, formed by a deep trefoil knot, contains a bound phosphate molecule, which is the likely nucleotide methylation site. This study represents the first characterization of a staphylococcal ribosomal MTase and provides the first crystal structure of a member of the α/β-knot superfamily of SPOUT MTases in the RlmH or COG1576 family with bound AdoMet.     

CR1-mediated ATP Release by Human Red Blood Cells Promotes CR1 Clustering and Modulates the Immune Transfer Process

Humans and other higher primates are unique among mammals in using complement receptor 1 (CR1, CD35) on red blood cells (RBC) to ligate complement-tagged inflammatory particles (immune complexes, apoptotic/necrotic debris, and microbes) in the circulation for quiet transport to the sinusoids of spleen and liver where resident macrophages remove the particles, but allow the RBC to return unharmed to the circulation. This process is called immune-adherence clearance. In this study we found using luminometric- and fluorescence-based methods that ligation of CR1 on human RBC promotes ATP release. Our data show that CR1-mediated ATP release does not depend on Ca²⁺ or enzymes previously shown to mediate an increase in membrane deformability promoted by CR1 ligation. Furthermore, ATP release following CR1 ligation increases the mobility of the lipid fraction of RBC membranes, which in turn facilitates CR1 clustering, and thereby enhances the binding avidity of complement-opsonized particles to the RBC CR1. Finally, we have found that RBC-derived ATP has a stimulatory effect on phagocytosis of immune-adherent immune complexes.