SPOTS: signaling protein oligomeric transduction structures are early mediators of death receptor-induced apoptosis at the plasma membrane

Fas (CD95, APO-1, TNFRSF6) is a TNF receptor superfamily member that directly triggers apoptosis and contributes to the maintenance of lymphocyte homeostasis and prevention of autoimmunity. Although FADD and caspase-8 have been identified as key intracellular mediators of Fas signaling, it is not clear how recruitment of these proteins to the Fas death domain leads to activation of caspase-8 in the receptor signaling complex. We have used high-resolution confocal microscopy and live cell imaging to study the sequelae of early events in Fas signaling. These studies have revealed a new stage of Fas signaling in which receptor ligation leads to the formation of surface receptor oligomers that we term signaling protein oligomerization transduction structures (SPOTS). Formation of SPOTS depends on the presence of an intact Fas death domain and FADD but is independent of caspase activity. Analysis of cells expressing Fas mutations from patients with the autoimmune lymphoproliferative syndrome (ALPS) reveals that formation of SPOTS can be disrupted by distinct mechanisms in ALPS.     

Genetic mapping at 3-kilobase resolution reveals inositol 1,4,5-triphosphate receptor 3 as a risk factor for type 1 diabetes in Sweden

We mapped the genetic influences for type 1 diabetes (T1D), using 2,360 single-nucleotide polymorphism (SNP) markers in the 4.4-Mb human major histocompatibility complex (MHC) locus and the adjacent 493 kb centromeric to the MHC, initially in a survey of 363 Swedish T1D cases and controls. We confirmed prior studies showing association with T1D in the MHC, most significantly near HLA-DR/DQ. In the region centromeric to the MHC, we identified a peak of association within the inositol 1,4,5-triphosphate receptor 3 gene (ITPR3; formerly IP3R3). The most significant single SNP in this region was at the center of the ITPR3 peak of association (P=1.7 x 10(-4) for the survey study). For validation, we typed an additional 761 Swedish individuals. The P value for association computed from all 1,124 individuals was 1.30 x 10(-6) (recessive odds ratio 2.5; 95% confidence interval [CI] 1.7-3.9). The estimated population-attributable risk of 21.6% (95% CI 10.0%-31.0%) suggests that variation within ITPR3 reflects an important contribution to T1D in Sweden. Two-locus regression analysis supports an influence of ITPR3 variation on T1D that is distinct from that of any MHC class II gene.     

A BAC library for the goldfish Carassius auratus auratus (Cyprinidae, Cypriniformes)

A goldfish (Carassius auratus auratus) bacterial artificial chromosome genomic library (BAC library) was constructed from one aquarium-bred male specimen (tetraploid, 4n=100, genome size=3.52 pg/cell). The library consists of 128,352 positive clones with an average insert size of 150.4 kb, covering the genome 11-fold. All clones were spotted onto nylon filters and thus are available for screening of genomic regions of interest, such as candidate genes, gene families, or large-sized syntenic DNA regions of cyprinid species. Preliminary screens with two genes were conducted with hybridizing probes to the genes RAG1 and lgi1. RAG1 is a single-copy gene in zebrafish and is duplicated in C. a. auratus. We found a very close correlation between the number of positive BAC clones and the expected library coverage. Two copies of lgi1 were found in zebrafish. We have detected four different copies in C. a. auratus, not in the expected abundance, which indicates some variation in the coverage of the BAC library. The preliminary screens indicate that many duplicated genes that resulted from the ancient fish-specific genome duplication persist in the tetraploid goldfish genome. Hence, the BAC library will provide a useful resource for the future work on comparative genomics, polyploidy, diploidization, and evolutionary genomics in fishes.     

Discrimination of intact mycobacteria at the strain level: a combined MALDI-TOF MS and biostatistical analysis

New methodologies for surveillance and identification of Mycobacterium tuberculosis are required to stem the spread of disease worldwide. In addition, the ability to discriminate mycobacteria at the strain level may be important to contact or source case investigations. To this end, we are developing MALDI-TOF MS methods for the identification of M. tuberculosis in culture. In this report, we describe the application of MALDI-TOF MS, as well as statistical analysis including linear discriminant and random forest analysis, to 16 medically relevant strains from four species of mycobacteria, M. tuberculosis, M. avium, M. intracellulare, and M. kansasii. Although species discrimination can be accomplished on the basis of unique m/z values observed in the MS fingerprint spectrum, discrimination at the strain level is predicted on the relative abundance of shared m/z values among strains within a species. For the 16 mycobacterial strains investigated in the present study, it is possible to unambiguously identify strains within a species on the basis of MALDI-TOF MS data. The error rate for classification of individual strains using linear discriminant analysis was 0.053 using 37 m/z variables, whereas the error rate for classification of individual strains using random forest analysis was 0.023 using only 18 m/z variables. In addition, using random forest analysis of MALDI-TOF MS data, it was possible to correctly classify bacterial strains as either M. tuberculosis or non-tuberculous with 100% accuracy.     

TRAF6 regulates cell fate decisions by inducing caspase 8-dependent apoptosis and the activation of NF-kappaB

Tumor necrosis factor receptor-associated factor 6 (TRAF6) functions as an adaptor, positively regulating the NF-kappaB pathway. Here we report a new function of human TRAF6, the direct stimulation of apoptosis. The mechanism of apoptosis induction results from the capacity of human TRAF6 to interact and activate caspase 8. Both the C-terminal TRAF domain of human TRAF6, which directly interacts with the death effector domain of pro-caspase 8, and the N-terminal RING domain, which is required for activation of caspase 8, are necessary for the induction of apoptosis. The role of endogenous TRAF6 in regulating apoptosis was confirmed by extinguishing TRAF6 expression with specific small-hairpin RNA that resulted in diminished spontaneous apoptosis and resistance to induced apoptosis. In contrast to the human molecule, murine TRAF6 displayed less ability to induce apoptosis and a greater capacity to stimulate NF-kappaB activity. Human and murine TRAF6 are similar except in the region between zinc finger 5 and the TRAF domains. Reciprocal transfer of this connecting region completely exchanged the ability of human and murine TRAF6 to induce apoptosis and activate NF-kappaB. Unique regions of TRAF6 therefore play an important role in determining cell fate.     

Differential autonomic nervous system response in obese and anorexic chickens (Gallus gallus)

Effect of reserpine on body weight (BW), feed intake (FI), brain and plasma catecholamine and indoleamine concentrations in high- (HWS) and low- (LWS) weight selected lines of chickens was investigated. Chicks from each line were assigned to three treatment groups and injected intraperitoneally with 0, 1.25, or 2.50 mg/kg of reserpine at hatch, and again at 5 weeks-of-age. Chick BW and FI were determined weekly. At 7 weeks-of-age, 12 males and females from each group were sacrificed for neurotransmitter analysis. In the HWS line there was a dose-dependent decrease in BW through 7 weeks-of-age, whereas in the LWS line BW decreased only through the first 2 weeks-of-age. In the LWS line, norepinephrine (NE), epinephrine, and 3,4-dihydroxyphenylacetate concentrations decreased in the brain in a linear and quadratic manner in response to reserpine, but not in the HWS line. Both lines showed linear decreases in dopamine levels in response to reserpine; however, serotonin was not affected by reserpine. Chickens in the HWS line had greater plasma NE, and lower 5-hydroxyindoleacetic acid than those in the LWS line. In conclusion, it appears that chickens from the HWS line were more sensitive to the BW reducing effects of reserpine than those from the LWS line, with the latter appearing to have greater sympathetic nervous system activity.     

Caspases at the crossroads of immune-cell life and death

Caspases are responsible for crucial aspects of inflammation and immune-cell death that are disrupted in a number of genetic autoimmune and autoinflammatory diseases. The caspase family of proteases can be divided into pro-apoptotic and pro-inflammatory members based on their substrate specificity and participation in separate signalling cascades. However, as discussed here, evidence has emerged over the past few years that a number of the caspases thought to be involved solely in apoptosis also contribute to specific aspects of immune-cell development, activation and differentiation, and can even protect cells from some forms of cell death.     

PfAlbas constitute a new eukaryotic DNA/RNA-binding protein family in malaria parasites

In Plasmodium falciparum, perinuclear subtelomeric chromatin conveys monoallelic expression of virulence genes. However, proteins that directly bind to chromosome ends are poorly described. Here we identify a novel DNA/RNA-binding protein family that bears homology to the archaeal protein Alba (Acetylation lowers binding affinity). We isolated three of the four PfAlba paralogs as part of a molecular complex that is associated with the P. falciparum-specific TARE6 (Telomere-Associated Repetitive Elements 6) subtelomeric region and showed in electromobility shift assays (EMSAs) that the PfAlbas bind to TARE6 repeats. In early blood stages, the PfAlba proteins were enriched at the nuclear periphery and partially co-localized with PfSir2, a TARE6-associated histone deacetylase linked to the process of antigenic variation. The nuclear location changed at the onset of parasite proliferation (trophozoite-schizont), where the PfAlba proteins were also detectable in the cytoplasm in a punctate pattern. Using single-stranded RNA (ssRNA) probes in EMSAs, we found that PfAlbas bind to ssRNA, albeit with different binding preferences. We demonstrate for the first time in eukaryotes that Alba-like proteins bind to both DNA and RNA and that their intracellular location is developmentally regulated. Discovery of the PfAlbas may provide a link between the previously described subtelomeric non-coding RNA and the regulation of antigenic variation.     

Abscisic acid and CO2 signalling via calcium sensitivity priming in guard cells, new CDPK mutant phenotypes and a method for improved resolution of stomatal stimulus-response analyses

Background

Stomatal guard cells are the regulators of gas exchange between plants and the atmosphere. Ca²⁺-dependent and Ca²⁺-independent mechanisms function in these responses. Key stomatal regulation mechanisms, including plasma membrane and vacuolar ion channels have been identified and are regulated by the free cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt).

Scope

Here we show that CO₂-induced stomatal closing is strongly impaired under conditions that prevent intracellular Ca²⁺ elevations. Moreover, Ca²⁺ oscillation-induced stomatal closing is partially impaired in knock-out mutations in several guard cell-expressed Ca²⁺-dependent protein kinases (CDPKs) here, including the cpk4cpk11 double and cpk10 mutants; however, abscisic acid-regulated stomatal movements remain relatively intact in the cpk4cpk11 and cpk10 mutants. We further discuss diverse studies of Ca²⁺ signalling in guard cells, discuss apparent peculiarities, and pose novel open questions. The recently proposed Ca²⁺ sensitivity priming model could account for many of the findings in the field. Recent research shows that the stomatal closing stimuli abscisic acid and CO₂ enhance the sensitivity of stomatal closing mechanisms to intracellular Ca²⁺, which has been termed ‘calcium sensitivity priming’. The genome of the reference plant Arabidopsis thaliana encodes for over 250 Ca²⁺-sensing proteins, giving rise to the question, how can specificity in Ca²⁺ responses be achieved? Calcium sensitivity priming could provide a key mechanism contributing to specificity in eukaryotic Ca²⁺ signal transduction, a topic of central interest in cell signalling research. In this article we further propose an individual stomatal tracking method for improved analyses of stimulus-regulated stomatal movements in Arabidopsis guard cells that reduces noise and increases fidelity in stimulus-regulated stomatal aperture responses ( Box 1). This method is recommended for stomatal response research, in parallel to previously adopted blind analyses, due to the relatively small and diverse sizes of stomatal apertures in the reference plant Arabidopsis thaliana.

Box 1. Improved resolution of stimulus-induced stomatal movements in guard cells by tracking of individual stomatal apertures

Arabidopsis guard cells have become a prime model system for analysing signal transduction, since early research combining genetic and ion channel analyses in this system (Ichida et al., 1997; Pei et al., 1997, 1998; Roelfsema and Prins, 1997). Arabidopsis stomata are small relative to other stomatal model systems and stomatal apertures of various plant types including Arabidopsis are known to show variability in the size of individual stomatal complexes and also variability in the opening apertures of stomata of similar size in a given leaf (Gorton et al., 1988; Mott and Buckley, 2000; Mott and Peak, 2007). Thus stomatal aperture measurements are expected to show a clear degree of statistical variation. Use of blind experiments, in which the genotype and, when possible, the stimulus being applied to guard cells is unknown to the experimenter (Murata et al., 2001) has been employed by several laboratories, has become a standard in the field and has aided in addressing the above limitations of the range of stomatal aperture sizes found under any given condition.Research in our laboratory has shown that a major additional improvement in experiments can be made, by adding imaging of the same individual stomatal apertures over time (Allen et al., 2001; Mori et al., 2006; Vahisalu et al., 2008; Siegel et al., 2009), while performing blind experiments. In such ‘stomatal tracking’ experiments the lower side of a leaf is attached to a glass coverslip in an extracellular incubation medium (Webb et al., 2001; Young et al., 2006). The mesophyll and upper leaf epidermis are removed surgically for better optical resolution of stomatal apertures in the intact lower leaf epidermis (Young et al., 2006). For stimulus-induced stomatal closing analyses, a field of well-opened stomata is located and images are captured (e.g. using Scion Image software) for later analyses and data storage. The bottom (dry side) of coverslips can be marked with colour marker pens to label grids in the regions where apertures where imaged, for finding these same stomata subsequently if needed. Images of the same stomatal apertures are taken over time and can be stored for later analyses of individual stomatal apertures and for deposition of image files. While this approach has been used as a standard for imposed Ca²⁺ oscillation studies (Allen et al., 2001; Mori et al., 2006; Vahisalu et al., 2008; Fig. 4), we have found that this method also substantially improves stomatal movement response analyses to any given stimulus (Siegel et al., 2009; see Figs 1 and 4 and, Box Fig. 1). For example, while individual stomata are known to have diverse apertures (e.g. Box Fig. 1C), the relative responses of wide open stomata and smaller stomatal apertures to ABA or to CO₂ were comparable (Fig. 1 and Box Fig. 1; Siegel et al., 2009). Note that this method has previously been proposed and used in Vicia faba (Gorton et al., 1988), for which stomata exhibit relatively weak ABA and CO₂ responses, compared with, for example, Arabidopsis. We propose that this simple image-capturing approach, together with blind analyses, be used as a standard for stomatal response research in arabidopsis. Our research experience with this method shows that this approach will aid in greatly improving resolution and robustness and in defining the functions of individual Ca²⁺-independent and Ca²⁺-dependent components and mechanisms in stomatal response analyses. Open in a separate windowBox Fig. 1.ABA-induced stomatal closing of individually tracked stomatal apertures. (A) Average individually tracked stomatal apertures in the presence of 50 µm Ca²⁺ (open triangles) and in the presence of 200 nm free Ca²⁺ (open squares) in the bath solution from three experiments are shown and were normalized to the stomatal apertures at time = 0. (B, C) ABA-induced stomatal closing in the presence of 50 µm Ca²⁺ in five individually tracked stomatal apertures. In (A; open triangles) normalized stomatal apertures of the same stomata depicted in (B) and (C) are shown. Methods used in these experiments tracking individual stomatal apertures are described in Siegel et al. (2009). ABA-induced stomatal closing experiments are reproduced from Siegel et al. (2009) with permission of the publisher.