Recruitment of EB1, a master regulator of microtubule dynamics, to the surface of the Theileria annulata schizont

The apicomplexan parasite Theileria annulata transforms infected host cells, inducing uncontrolled proliferation and clonal expansion of the parasitized cell population. Shortly after sporozoite entry into the target cell, the surrounding host cell membrane is dissolved and an array of host cell microtubules (MTs) surrounds the parasite, which develops into the transforming schizont. The latter does not egress to invade and transform other cells. Instead, it remains tethered to host cell MTs and, during mitosis and cytokinesis, engages the cell's astral and central spindle MTs to secure its distribution between the two daughter cells. The molecular mechanism by which the schizont recruits and stabilizes host cell MTs is not known. MT minus ends are mostly anchored in the MT organizing center, while the plus ends explore the cellular space, switching constantly between phases of growth and shrinkage (called dynamic instability). Assuming the plus ends of growing MTs provide the first point of contact with the parasite, we focused on the complex protein machinery associated with these structures. We now report how the schizont recruits end-binding protein 1 (EB1), a central component of the MT plus end protein interaction network and key regulator of host cell MT dynamics. Using a range of in vitro experiments, we demonstrate that T. annulata p104, a polymorphic antigen expressed on the schizont surface, functions as a genuine EB1-binding protein and can recruit EB1 in the absence of any other parasite proteins. Binding strictly depends on a consensus SxIP motif located in a highly disordered C-terminal region of p104. We further show that parasite interaction with host cell EB1 is cell cycle regulated. This is the first description of a pathogen-encoded protein to interact with EB1 via a bona-fide SxIP motif. Our findings provide important new insight into the mode of interaction between Theileria and the host cell cytoskeleton.

Slit2 prevents neutrophil recruitment and renal ischemia-reperfusion injury.

Neutrophils recruited to the postischemic kidney contribute to the pathogenesis of ischemia-reperfusion injury (IRI), which is the most common cause of renal failure among hospitalized patients. The Slit family of secreted proteins inhibits chemotaxis of leukocytes by preventing activation of Rho-family GTPases, suggesting that members of this family might modulate the recruitment of neutrophils and the resulting IRI. Here, in static and microfluidic shear assays, Slit2 inhibited multiple steps required for the infiltration of neutrophils into tissue. Specifically, Slit2 blocked the capture and firm adhesion of human neutrophils to inflamed vascular endothelial barriers as well as their subsequent transmigration. To examine whether these observations were relevant to renal IRI, we administered Slit2 to mice before bilateral clamping of the renal pedicles. Assessed at 18 hours after reperfusion, Slit2 significantly inhibited renal tubular necrosis, neutrophil and macrophage infiltration, and rise in plasma creatinine. In vitro, Slit2 did not impair the protective functions of neutrophils, including phagocytosis and superoxide production, and did not inhibit neutrophils from killing the extracellular pathogen Staphylococcus aureus. In vivo, administration of Slit2 did not attenuate neutrophil recruitment or bacterial clearance in mice with ascending Escherichia coli urinary tract infections and did not increase the bacterial load in the livers of mice infected with the intracellular pathogen Listeria monocytogenes. Collectively, these results suggest that Slit2 may hold promise as a strategy to combat renal IRI without compromising the protective innate immune response.

Lissencephaly-1 promotes the recruitment of dynein and dynactin to transported mRNAs

Microtubule-based transport mediates the sorting and dispersal of many cellular components and pathogens. However, the mechanisms by which motor complexes are recruited to and regulated on different cargos remain poorly understood. Here we describe a large-scale biochemical screen for novel factors associated with RNA localization signals mediating minus end-directed mRNA transport during Drosophila development. We identified the protein Lissencephaly-1 (Lis1) and found that minus-end travel distances of localizing transcripts are dramatically reduced in lis1 mutant embryos. Surprisingly, given its well-documented role in regulating dynein mechanochemistry, we uncovered an important requirement for Lis1 in promoting the recruitment of dynein and its accessory complex dynactin to RNA localization complexes. Furthermore, we provide evidence that Lis1 levels regulate the overall association of dynein with dynactin. Our data therefore reveal a critical role for Lis1 within the mRNA localization machinery and suggest a model in which Lis1 facilitates motor complex association with cargos by promoting the interaction of dynein with dynactin.

Running the Gauntlet: Inhibitory Effects of Algal Turfs on the Processes of Coral Recruitment

Mortality of corals is increasing due to bleaching, disease and algal overgrowth. In the Caribbean, low rates of coral recruitment contribute to the slow or undetectable rates of recovery in reef ecosystems. Although algae have long been suspected to interfere with coral recruitment, the mechanisms of that interaction remain unclear. We experimentally tested the effects of turf algal abundance on 3 sequential factors important to recruitment of corals: the biophysical delivery of planktonic coral larvae, their propensity to settle, and the availability of microhabitats where they survive. We deployed coral settlement plates inside and outside damselfish Stegastes spp. gardens and cages. Damselfish aggression reduced herbivory from fishes, and cages became fouled with turf algae, both locally increasing algal biomass surrounding the plates. This reduced flushing rates in nursery microhabitats on the plate underside, limiting larvae available for settlement. Coral spat settled preferentially on an early successional crustose coralline alga Titanoderma prototypum but also on or near other coralline algae, biofilms, and calcareous polychaete worm tubes. Post-settlement survival was highest in the fully grazed, lowest algal biomass treatment, and after 27 mo 'spat' densities were 73 % higher in this treatment. The 'gauntlet' refers to the sequence of ecological processes through which corals must survive to recruit. The highest proportion of coral spat successfully running the gauntlet did so under conditions of low algal biomass resulting from increased herbivory. If coral recruitment is heavily controlled at very local scales by this gauntlet, then coral reef managers could improve a reef's recruitment potential by managing for reduced algal biomass.

Secondary succession and dipterocarp recruitment in Bornean rain forest after logging

To understand succession in dipterocarp rain forest after logging, the structure, species composition and dynamics of primary (PF) and secondary (SF) forest at Danum were compared. In 10 replicate 0.16-ha plots per forest type trees >= 10 cm gbh (3.2 cm dbh) were measured in 1995 and 2001. The SF had been logged in 1988, which allowed successional change to be recorded at 8 and 13 years. In 2001, saplings (1.0-3.1 cm dbh) were measured in nested quadrats. The forest types were similar in mean radiation at 2 m height, and in density, basal area and species number of all trees. Among small (10 <= 31.4) and large ( >= 31.4 cm gbh) trees, in both 1995 and 2001, there were 10- and 3-fold more dipterocarps in SF than PF respectively; and averaging over the two dates, there were correspondingly ca. 10- and 18-fold more pioneers. Mortality was ca. 60% higher in SF than PF, largely due to a seven-fold difference for pioneers: for dipterocarps there was little difference. Recruitment was similar in PF and SE Stem growth rates were 37% higher in SF than PF for all trees, although dipterocarps showed the opposite trend. Among saplings, dipterocarps dominated SF with a 10-fold higher density than in PF. For dipterocarps, the light (LH) and medium-heavy (MHH) canopy hardwoods, and the shade-tolerant, smaller-stature other (OTH) species (e.g. Hopea and Vatica) were in the ratios ca. 40:15:45 in SF and 85: < 1:15 in PF. LHs had higher mortality than OTHs in SE In PF ca. 80% of the saplings were LH: in SF ca. 70% were OTH. The predominance of OTHs in SF is explained by the logging of primary rain forest which was in a likely late stage of recovery from natural disturbance, plus the continuing shaded conditions in the understorey promoted by dense pioneer vegetation. At 13 years after logging succession appeared to be inhibited: LHs were being suppressed but MHHs and OTHs persisted. Succession in lowland dipterocarp, rain forests may therefore depend on the successional state of the primary forest when it is logged. A review of logged versus unlogged studies in Borneo highlights the need for more detailed ecological comparisons.

Does low phosphorus supply limit seedling establishment and tree growth in groves of ectomycorrhizal trees in a central African rainforest?

In a forest grove at Korup dominated by the ectomycorrhizal species Microberlinia bisulcata, an experiment tested whether phosphorus (P) was a limiting nutrient. P-fertilization of seven subplots 1995-97 was compared with seven controls. It led to large increases in soil P concentrations. Trees were measured in 1995 and 2000. M. bisulcata and four other species were transplanted into the treatments, and a wild cohort of M. bisulcata seedlings was followed in both. Leaf litter fall from trees and seedlings were analysed for nutrients. Growth of trees was not affected by added P. Transplanted seedlings survived better in the controls than added-P subplots: they did not grow better with added-P.M. bisulcata wildlings survived slightly better in the added-P subplots in yr 1 but not later. Litter fall and transplanted survivors had much higher concentrations of P (not N) in the added-P than control subplots. Under current conditions, it appears that P does not limit growth of trees or hinder seedling establishment, especially of M. bisculcata, in these low-P grove soils.

Seedling resistance, tolerance and escape from herbivores: insights from co-dominant canopy tree species in a resource-poor African rain forest

* Although plants can reduce the impacts of herbivory in multiple ways, these defensive traits are often studied in isolation and an understanding of the resulting strategies is incomplete. * In the study reported here, empirical evidence was simultaneously evaluated for the three main sets of traits available to plants: (i) resistance through constitutive leaf traits, (ii) tolerance to defoliation and (iii) escape in space, for three caesalpiniaceous tree species Microberlinia bisulcata, Tetraberlinia bifoliolata and T. korupensis, which co-dominate groves within the lowland primary rain forest of Korup National Park (Cameroon). * Mesh cages were placed around individual wild seedlings to exclude insect herbivores at 41 paired canopy gap and understorey locations. After following seedling growth and survival for c. 2 years, caged and control treatments were removed, leaves harvested to determine nutrient and phenolic concentrations, leaf mass per area estimated, and seedling performance in gaps followed for a further c. 2 years to quantify tolerance to the leaf harvesting. * The more nutrient-rich leaves of the weakly shade-tolerant M. bisulcata were damaged much more in gaps than the two strongly shade-tolerant Tetraberlinia species, which had higher leaf mass per area and concentrations of total phenols. Conversely, the faster-growing M. bisulcata was better able to tolerate defoliation in terms of height growth (reflushing capacity), but not at maintaining overall leaf numbers, than the other two species. * Across gaps, insect-mediated Janzen–Connell effects were most pronounced for M. bisulcata, less so for T. korupensis, and not detectable for T. bifoliolata. The three species differed distinctly in their secondary metabolic profiles. * Taken together, the results suggested a conceptual framework linking the three sets of traits, one in which the three co-dominant species adopt different strategies towards herbivore pressure depending on their different responses to light availability. This study is one of the first in a natural forest ecosystem to examine resistance to, tolerance of, and escape from herbivory among a group of co-occurring tropical tree species.

Influence of inspiration to expiration ratio on cyclic recruitment and derecruitment of atelectasis in a saline lavage model of acute respiratory distress syndrome*

OBJECTIVE Cyclic recruitment and derecruitment of atelectasis can occur during mechanical ventilation, especially in injured lungs. Experimentally, cyclic recruitment and derecruitment can be quantified by respiration-dependent changes in PaO2 (ΔPaO2), reflecting the varying intrapulmonary shunt fraction within the respiratory cycle. This study investigated the effect of inspiration to expiration ratio upon ΔPaO2 and Horowitz index. DESIGN Prospective randomized study. SETTING Laboratory investigation. SUBJECTS Piglets, average weight 30 ± 2 kg. INTERVENTIONS At respiratory rate 6 breaths/min, end-inspiratory pressure (Pendinsp) 40 cm H2O, positive end-expiratory pressure 5 cm H2O, and FIO2 1.0, measurements were performed at randomly set inspiration to expiration ratios during baseline healthy and mild surfactant depletion injury. Lung damage was titrated by repetitive surfactant washout to induce maximal cyclic recruitment and derecruitment as measured by multifrequency phase fluorimetry. Regional ventilation distribution was evaluated by electrical impedance tomography. Step changes in airway pressure from 5 to 40 cm H2O and vice versa were performed after lavage to calculate PO2-based recruitment and derecruitment time constants (TAU). MEASUREMENTS AND MAIN RESULTS In baseline healthy, cyclic recruitment and derecruitment could not be provoked, whereas in model acute respiratory distress syndrome, the highest ΔPaO2 were routinely detected at an inspiration to expiration ratio of 1:4 (range, 52-277 torr [6.9-36.9 kPa]). Shorter expiration time reduced cyclic recruitment and derecruitment significantly (158 ± 85 torr [21.1 ± 11.3 kPa] [inspiration to expiration ratio, 1:4]; 25 ± 12 torr [3.3 ± 1.6 kPa] [inspiration to expiration ratio, 4:1]; p < 0.0001), whereas the PaO2/FIO2 ratio increased (267 ± 50 [inspiration to expiration ratio, 1:4]; 424 ± 53 [inspiration to expiration ratio, 4:1]; p < 0.0001). Correspondingly, regional ventilation redistributed toward dependent lung regions (p < 0.0001). Recruitment was much faster (TAU: fast 1.6 s [78%]; slow 9.2 s) than derecruitment (TAU: fast 3.1 s [87%]; slow 17.7 s) (p = 0.0078). CONCLUSIONS Inverse ratio ventilation minimizes cyclic recruitment and derecruitment of atelectasis in an experimental model of surfactant-depleted pigs. Time constants for recruitment and derecruitment, and regional ventilation distribution, reflect these findings and highlight the time dependency of cyclic recruitment and derecruitment.

Nck recruitment to Eph receptor, EphB1/ELK, couples ligand activation to c-Jun kinase.

Eph family receptor tyrosine kinases signal axonal guidance, neuronal bundling, and angiogenesis; yet the signaling systems that couple these receptors to targeting and cell-cell assembly responses are incompletely defined. Functional links to regulators of cytoskeletal structure are anticipated based on receptor mediated cell-cell aggregation and migratory responses. We used two-hybrid interaction cloning to identify EphB1-interactive proteins. Six independent cDNAs encoding the SH2 domain of the adapter protein, Nck, were recovered in a screen of a murine embryonic library. We mapped the EphB1 subdomain that binds Nck and its Drosophila homologue, DOCK, to the juxtamembrane region. Within this subdomain, Tyr594 was required for Nck binding. In P19 embryonal carcinoma cells, activation of EphB1 (ELK) by its ligand, ephrin-B1/Fc, recruited Nck to native receptor complexes and activated c-Jun kinase (JNK/SAPK). Transient overexpression of mutant EphB1 receptors (Y594F) blocked Nck recruitment to EphB1, attenuated downstream JNK activation, and blocked cell attachment responses. These findings identify Nck as an important intermediary linking EphB1 signaling to JNK.

Hazard index maps for woody material recruitment and transport in alpine catchments

A robust and reliable risk assessment procedure for hydrologic hazards deserves particular attention to the role of transported woody material during flash floods or debris flows. At present, woody material transport phenomena are not systematically considered within the procedures for the elaboration of hazard maps. The consequence is a risk of losing prediction accuracy and of underestimating hazard impacts. Transported woody material frequently interferes with the sediment regulation capacity of open check dams and moreover, when obstruction phenomena at critical crosssections of the stream occur, inundations can be triggered. The paper presents a procedure for the determination of the relative propensity of mountain streams to the entrainment and delivery of recruited woody material on the basis of empirical indicators. The procedure provided the basis for the elaboration of a hazard index map for all torrent catchments of the Autonomous Province of Bolzano/Bozen. The plausibility of the results has been thoroughly checked by a backward oriented analysis on natural hazard events, documented since 1998 at the Department of Hydraulic Engineering of the aforementioned Alpine Province. The procedure provides hints for the consideration of the effects, induced by woody material transport, during the elaboration of hazard zone maps.

Differential recruitment of brain networks during visuospatial and color processing: Evidence from ERP microstates

Recent functional magnetic resonance imaging (fMRI) studies consistently revealed contributions of fronto-parietal and related networks to the execution of a visuospatial judgment task, the so-called "Clock Task". However, due to the low temporal resolution of fMRI, the exact cortical dynamics and timing of processing during task performance could not be resolved until now. In order to clarify the detailed cortical activity and temporal dynamics, 14 healthy subjects performed an established version of the "Clock Task", which comprises a visuospatial task (angle discrimination) and a control task (color discrimination) with the same stimulus material, in an electroencephalography (EEG) experiment. Based on the time-resolved analysis of network activations (microstate analysis), differences in timing between the angle compared to the color discrimination task were found after sensory processing in a time window starting around 200ms. Significant differences between the two tasks were observed in an analysis window from 192ms to 776ms. We divided this window in two parts: an early phase - from 192ms to ∼440ms, and a late phase - from ∼440ms to 776ms. For both tasks, the order of network activations and the types of networks were the same, but, in each phase, activations for the two conditions were dominated by differing network states with divergent temporal dynamics. Our results provide an important basis for the assessment of deviations in processing dynamics during visuospatial tasks in clinical populations.