Phosphorylation of Collapsin Response Mediator Protein 2 on Tyr-479 Regulates CXCL12-induced T Lymphocyte Migration

In the central nervous system, collapsin response mediator protein 2 (CRMP2) is a transducer protein that supports the semaphorin-induced guidance of axons toward their cognate target. However, we previously showed that CRMP2 is also expressed in immune cells and plays a crucial role in T lymphocyte migration. Here we further investigated the molecular mechanisms underlying CRMP2 function in chemokine-directed T-cell motility. Examining Jurkat T-cells treated with the chemokine CXCL12, we found that 1) CXCL12 induces a dynamic re-localization of CRMP2 to uropod, the flexible structure of migrating lymphocyte, and increases its binding to the cytoskeletal protein vimentin; 2) CXCL12 decreases phosphorylation of the glycogen synthase kinase-3β-targeted residues CRMP2-Thr-509/514; and 3) tyrosine Tyr-479 is a new phosphorylation CRMP2 residue and a target for the Src-family kinase Yes. Moreover, phospho-Tyr-479 increased under CXCL12 signaling while phospho-Thr-509/514 decreased. The functional importance of this tyrosine phosphorylation was demonstrated by Y479F mutation that strongly reduced CXCL12-mediated T-cell polarization and motility as tested in a transmigration model and on neural tissue. We propose that differential phosphorylation by glycogen synthase kinase-3β and Yes modulates the contribution of CRMP2 to cytoskeletal reorganization during chemokine-directed T-cell migration. In addition to providing a novel mechanism for T lymphocyte motility, our findings reveal CRMP2 as a transducer of chemokine signaling.T lymphocyte migration is the basis of major immune functions such as responses to infection and inflammation, as well as normal recirculation through the lymphoid organs. Indeed, the role of T-cells depends strongly on their ability to travel between organs via the blood and lymph and to move rapidly within these tissues, by extravasation (1). This latter function is dependent on extracellular signals, among which chemokines play a major role.Chemokines form a superfamily of small proteins that orchestrate lymphocyte polarization and migration (2). These proteins exert their functions by binding specific seven-transmembrane-domain G-protein-coupled receptors on the T-cell surface (3). T-lymphocytes exposed to chemokines, in a soluble or surface-bound gradient, develop a polarized shape, extending at the front, an F-actin-rich lamellipodium, which constitutes the leading edge, and a trailing edge or uropod in which both the microtubule and vimentin networks are retracted during migration. Although F-actin has the well known function of producing the mechanical forces required to generate movement (4), the role of microtubules and vimentin in T-cell migration requires further investigation.Cytoskeletal remodeling is of key importance in migrating cells (5) and is one of the functions carried out by the chemokine stromal cell-derived factor-1α, also named CXCL12. In association with its cognate receptor CXCR4, CXCL12 is a potent chemoattractant for mature T-cells and monocytes (6). Following ligand recognition and binding, CXCR4 signaling starts with the activation of G proteins, followed by various signaling cascade effectors, including MAP² kinases, phosphoinositide 3-kinase, and phospholipase Cγ (7). Although this intracellular signaling cascade has not been completely elucidated, the Src family non-receptor tyrosine kinase Lck and the Syk kinase ZAP-70 have emerged as the main candidates for delivering the input signal following CXCR4 activation (8). Thus, tyrosine kinase activity appears as a central step in CXCR4-dependent chemotaxis.While searching for molecules involved in T-cell motility, we recently identified collapsin response mediator protein 2 (CRMP2) (9), a protein first described in the context of neuronal growth cone advance (10, 11). We demonstrated that CRMP2 regulated both T-cell polarization and spontaneous/chemokine-induced migration of T-lymphocytes. Moreover, CRMP2 was found at the uropod of motile T-cells and has the ability to bind cytoskeletal elements, including vimentin. A correlation between CRMP2 expression levels and cell migratory rates toward a chemokine gradient, including CXCL12, was demonstrated by overexpression and knockdown experiments in T-cells (9). In addition, we recently reported that, in mouse model of neuroinflammation, elevated CRMP2 expression in T lymphocytes correlated with their elevated migratory rates and their ability to target the central nervous system (12). The importance of CXCL12 in the central nervous system and its implication in the pathogenesis of central nervous system disorders, including neuroinflammatory diseases, are well documented (review in Ref. 13). Thus, the aim of the present study was to determine whether and how CRMP2 participates in the transduction pathway induced by CXCL12 on T lymphocytes.     

Species and Strain Glycosylation Patterns of PrPSc

Background

A key event in transmissible spongiform encephalopathies (TSEs) is the conversion of the soluble, protease-sensitive glycosylated prion protein (PrP^C) to an abnormally structured, aggregated and partially protease-resistant isoform (PrP^Sc). Both PrP isoforms bear two potential glycosylation sites and thus in a typical western blot with an anti-PrP antibody three distinct bands appear, corresponding to the di-, mono- or unglycosylated forms of the protein. The relative intensity and electrophoretic mobility of the three bands are characteristic of each TSE strain and have been used to discriminate between them.

Methodology/Principal Findings

In the present study we used lectin-based western blotting to evaluate possible variations in composition within sugar chains carried by PrP^Sc purified from subjects affected with different TSEs. Our findings indicate that in addition to the already well-documented differences in electrophoretic mobility and amounts of the glycosylated PrP^Sc forms, TSE strains also vary in the abundance of specific N-linked sugars of the PrP^Sc protein.

Conclusions/Significance

These results imply that PrP glycosylation might fine-tune the conversion of PrP^C to PrP^Sc and could play an accessory role in the appearance of some of the characteristic features of TSE strains. The differences in sugar composition could also be used as an additional tool for discrimination between the various TSEs.     

In-Vitro and In-Vivo Effects of Aldicarb on Survival and Development of Heterodera schachtii

Aqueous solutions of 5-500 μg/ml aldicarb inhibited hatching of Heterodera schachtii. Addition of hatching agents, zinc chloride, or sugarbeet root diffusate, to the aldicarb solutions did not decrease the inhibition of hatching. When cysts were removed from the aldicarb solufions and then treated for 4 wk in sugarbeet root diffusate, larvae hatched and emerged. Treatments of newly hatched larvae of H. schachtii with 5-100 μg/ml aldicarb depressed later development of larvae on sugarbeet (Beta vulgaris). Similar treatments with aldicarb sulfoxide had less effect on larval development, and aldicarb sulfone had no effect. Numbers of treated larvae that survived and developed were inversely proportional to concentration (0.1-5.0 μg/ml) and duration (0-14 days) of aldicarb treatments. Development of H. schachtii on sugarbeet grown in aldicarb-treated soil was inversely proportional to the concentration of aldicarb in the tested range of 0.75 - 3.0 μg aldicarb/g of soil. Transfer of nematode-infected plants to soil with aldicarb retarded nematode development, whereas transfer of plants first grownin treated soil to nematode-infested soil only slightly suppressed nematode development. Development of H. schachtii was inhibited in slices of storage roots of table beet (B. vulgaris), sugarbeet and turnip, (Brassica rapa), that had grown in soil treated with aldicarb.     

Effects of the Ionic Composition and Water Potential of Aqueous Solution on the Activity and Survival of Orrina phyllobia

The activity and survival of Orrina phyllobia fourth-stage juveniles (J4) were examined in aqueous solutions representing 96 combinations of eight predominant soil solution ions at total concentrations of 100, 200, and 1,000 meq/liter. Various water potentials were imposed by the addition of mannitol or polyethylene glycol to ionic solutions. Nematode longevity increased as water potential was decreased. Longevity was approximately doubled at a water potential of -23 × 10⁵ Pa and more than tripled at -60 × 10⁵ Pa. No combination oflons at 200 meq/liter was lethal after a 6-day exposure. Several ion combinations significantly increased longevity at -10 and -23 × 10⁵ Pa. Single cation Na⁺ solutions consistently inhibited activity and more than doubled nematode longevity.     

Differential Virulence of Candida glabrata Glycosylation Mutants

The fungus Candida glabrata is an important and increasingly common pathogen of humans, particularly in immunocompromised hosts. Despite this, little is known about the attributes that allow this organism to cause disease or its interaction with the host immune system. However, in common with other fungi, the cell wall of C. glabrata is the initial point of contact between the host and pathogen, and as such, it is likely to play an important role in mediating interactions and hence virulence. Here, we show both through genetic complementation and polysaccharide structural analyses that C. glabrata ANP1, MNN2, and MNN11 encode functional orthologues of the respective Saccharomyces cerevisiae mannosyltransferases. Furthermore, we show that deletion of the C. glabrata Anp1, Mnn2, and Mnn11 mannosyltransferases directly affects the structure of the fungal N-linked mannan, in line with their predicted functions, and this has implications for cell wall integrity and consequently virulence. C. glabrata anp1 and mnn2 mutants showed increased virulence, compared with wild-type (and mnn11) cells. This is in contrast to Candida albicans where inactivation of genes involved in mannan biosynthesis has usually been linked to an attenuation of virulence. In the long term, a better understanding of the attributes that allow C. glabrata to cause disease will provide insights that can be adopted for the development of novel therapeutic and diagnostic approaches.     

Survival of Paecilomyces lilacinus in Selected Carriers and Related Effects on Meloidogyne incognita on Tomato

Laboratory and microplot experiments were conducted to determine the influence of carrier and storage of Paecilomyces lilacinus on its survival and related protection of tomato against Meloidogyne incognita. Spores of P. lilacinus were prepared in five formulations: alginate pellets (pellets), diatomaceous earth granules (granules), wheat grain, soil, and soil plus chitin. Fungal viability was high in wheat and granules, intermediate in pellets, and low in soil and chitin-amended soil stored at 25 ± 2 C. In 1985 P. lilacinus in field microplots resulted in about a 25% increase in tomato yield and 25% gall suppression, compared with nematodes alone. Greatest suppression of egg development occurred in plots treated with P. lilacinus in pellets, wheat grain, and granules. In 1986 carryover protection of tomato against M. incognita resulted in about a threefold increase in tomato fruit yield and 25% suppression of gall development, compared with plants treated with nematodes alone. Higher numbers of fungus-infected egg masses occurred in plots treated with pellets (32%) than in those treated with chitin-amended soil (24%), wheat (16%), granules (12%), or soil (7%). Numbers of fungal colony-forming units per gram of soil in plots treated with pellets were 10-fold greater than initial levels estimated at planting time in 1986.     

Influence of Selected Cultural Practices on Winter Survival of Pratylenchus brachyurus and Subsequent Effects on Soybean Yield

Planting date of soybean, Glycine max, influenced winter survival of Pratylenchus brachyurus in microplots at two locations in North Carolina. Delayed planting resulted in a linear decrease (P = 0.05) in the numbers of P. brachyurus at soybean harvest. Effects of planting date on nematode numbers persisted over winter, indicating that survival in the absence of a host is density independent. Compared with winter fallow, winter wheat, Triticum aestivum, reduced winter survival of P. brachyurus. Subsequent soybean yields were suppressed by the overwintering population of this nematode at one location but not at another.     

Differential Pathogenicity of Four Pratylenchus neglectus Populations on Alfalfa

A Pratylenchus neglectus population from lltah (UT3) was more virulent to Lahontan alfalfa than other P. neglectus populations from Utah (UT1, UT2) and Wyoming (WY). All alfalfa plants survived at 24 ± 3 C when inoculated with WY, UT1, or UT2 at initial populations (Pi) of 500, 1,000, and 5,000 nematodes per plant. At Pi 10,000 with WY, UT1, or UT2, plant mortality was 15, 15, and 20%, respectively; at Pi 5,000 and 10,000 with UT3, plant mortality was 10 and 40%. The WY, UT1, and UT2 populations reduced (P ≤ 0.05) root growth at Pi 10,000 only, and UT3 reduced (P ≤ 0.05) root growth at Pi 1,000, 5,000, and 10,000. At Pi 5,000, shoot dry weights were reduced by 10-23% by WY, 14-29% by UT1, 12-25% by UT2, and 20-48% by UT3 at 15-30 C. The UT3 population reduced (P ≤ 0.05) root dry weight at 20-30 C at Pi 1,000 and 5,000. The WY, UT1, and UT-2 populations did not reduce (P ≥ 0.05) root growth at any temperature or Pi. The UT3 nematode reproductive indices were greater than those of the other nematode populations at all Pi and increased with temperature.     

Effect of Temperature on Infection and Survival of Rotylenchulus reniformis

From infestation of lettuce with preinfective females to egg deposition, populations of Rotylenchulus reniformis from Baton Rouge, Louisiana; Lubbock and Weslaco, Texas; and Mayaguez, Puerto Rico, required 41, 13, 7, and 7 days at 15, 20, 25, and 34 C, respectively. No nematode infection occurred at 10 C with any R. reniformis population, and the population from Puerto Rico did not reproduce at 15 C. Nematode survival was not influenced by temperature, since populations from Texas and Louisiana survived for 6 months without a host at - 5 , - 1 , 4, and 25 C. Survival of R. reniformis was substantially influenced by soil moisture. Soil moistures greater than 7% (< 1 bar) aided nematode survival at storage temperature of 25 C, whereas moisture adversely affected nematode survival below freezing. Soil moisture below 4% (> 15 bars) favored nematode survival below freezing but adversely affected nematodes in soils stored at 25 C. Soil moisture effects on nematode survival were less accentuated at 4 and 0 C.     

Some Factors Affecting Survival of Desiccation by Infective Juveniles of Orrina phyllobia

The survival of desiccation by J4 Orrina phyllobia was examined at controlled relative humidities. When nematodes were transferred from water to air at 10% relative humidity (rh), 80% died within 30 minutes. When nematodes were transferred from water to air with rh at 70% or greater for ca. 15 minutes prior to being transferred to 10% rh, more than 90% of them survived desiccation. This phenomenon is referred to as preconditioning and occurred at much faster rates (2-30 minutes) than has been observed for other nematode species (24 hours). Differences in preconditioning rates may be due to technique-dependent variations in boundary layer resistance around nematodes during desiccation.     

Differential Effects of Typha Litter and Plants on Invasive Lythrum Salicaria Seedling Survival and Growth

Invasive species are a problem because of their detrimental ecological and economic effects. Increased disturbance caused by human impacts is hypothesized as a primary factor promoting the spread of invaders. Plants and plant litter can have important effects on plant colonization and community composition by affecting seedling survival and growth. I examined the hypothesis that invasion of non-native Lythrum salicaria in Typha-dominated marshes is disturbance-dependent. If so, the removal of Typha plants or litter would increase the survival and growth of L. salicaria seedlings. Additionally, the removal of both plants and litter could result in an additive or synergistic effect on the establishment of L. salicaria. Alternatively, L. salicaria may be a successful invader because it has a high capacity to establish and grow regardless of neighbours. In this case, L. salicaria would be expected to perform well even in plants and litter. Strategies for managing L. salicaria will depend on which factors promote invasion. I measured the differential effects of plants and litter, alone and in combination, on the survival and growth of L. salicaria seedlings transplanted into marshes. The presence of plants and litter did not affect seedling survival in relatively dry wetland sites, indicating that L. salicaria seedlings have the capacity to persist in the presence of neighbouring Typha spp. competitors. However, removal of both plants and litter allowed increased growth of L. salicaria seedlings in drier wetlands. Therefore, growth was facilitated by disturbance that removed all vegetation. Small disturbances (0.6 m²) decreased competitive suppression by native Typha spp. neighbours and resulted in significant increases in growth. Disturbance of wetlands at risk of invasion by L. salicaria should be avoided.     

Glycosylation increases the thermostability of human aquaporin 10 protein

Human aquaporin10 (hAQP10) is a transmembrane facilitator of both water and glycerol transport in the small intestine. This aquaglyceroporin is located in the apical membrane of enterocytes and is believed to contribute to the passage of water and glycerol through these intestinal absorptive cells. Here we overproduced hAQP10 in the yeast Pichia pastoris and observed that the protein is glycosylated at Asn-133 in the extracellular loop C. This finding confirms one of three predicted glycosylation sites for hAQP10, and its glycosylation is unique for the human aquaporins overproduced in this host. Nonglycosylated protein was isolated using both glycan affinity chromatography and through mutating asparagine 133 to a glutamine. All three forms of hAQP10 where found to facilitate the transport of water, glycerol, erythritol, and xylitol, and glycosylation had little effect on functionality. In contrast, glycosylated hAQP10 showed increased thermostability of 3-6 °C compared with the nonglycosylated protein, suggesting a stabilizing effect of the N-linked glycan. Because only one third of hAQP10 was glycosylated yet the thermostability titration was mono-modal, we suggest that the presence of at least one glycosylated protein within each tetramer is sufficient to convey an enhanced structural stability to the remaining hAQP10 protomers of the tetramer.     

Effect of Temperature on Development and Survival of the Mermithid Filipjevimermis leipsandra

The development and survival of egg, juvenile (parasitic), preovipositing, and ovipositing stages of the parasitic mermithid Filipjevimermis leipsandra were determined at eight temperatures. Infection of the host, Diabrotica balteata, peaked at 30 C and fell off sharply at warmer or cooler temperatures. No development, oviposition, or infection occurred at < 20 C. Fecundity was highest at 25 C, and 50% of the eggs were laid in the first 4-7 days of oviposition.