The use of quantitative imaging to investigate regulators of membrane trafficking in Arabidopsis stomatal closure

Expansion of gene families facilitates robustness and evolvability of biological processes but impedes functional genetic dissection of signalling pathways. To address this, quantitative analysis of single cell responses can help characterize the redundancy within gene families. We developed high‐throughput quantitative imaging of stomatal closure, a response of plant guard cells, and performed a reverse genetic screen in a group of Arabidopsis mutants to five stimuli. Focussing on the intersection between guard cell signalling and the endomembrane system, we identified eight clusters based on the mutant stomatal responses. Mutants generally affected in stomatal closure were mostly in genes encoding SNARE and SCAMP membrane regulators. By contrast, mutants in RAB5 GTPase genes played specific roles in stomatal closure to microbial but not drought stress. Together with timed quantitative imaging of endosomes revealing sequential patterns in FLS2 trafficking, our imaging pipeline can resolve non‐redundant functions of the RAB5 GTPase gene family. Finally, we provide a valuable image‐based tool to dissect guard cell responses and outline a genetic framework of stomatal closure.      

Individual variation in early‐life telomere length and survival in a wild mammal

Individual variation in survival probability due to differential responses to early‐life environmental conditions is important in the evolution of life histories and senescence. A biomarker allowing quantification of such individual variation, and which links early‐life environmental conditions with survival by providing a measure of conditions experienced, is telomere length. Here, we examined telomere dynamics among 24 cohorts of European badgers (Meles meles). We found a complex cross‐sectional relationship between telomere length and age, with no apparent loss over the first 29 months, but with both decreases and increases in telomere length at older ages. Overall, we found low within‐individual consistency in telomere length across individual lifetimes. Importantly, we also observed increases in telomere length within individuals, which could not be explained by measurement error alone. We found no significant sex differences in telomere length, and provide evidence that early‐life telomere length predicts lifespan. However, while early‐life telomere length predicted survival to adulthood (≥1 year old), early‐life telomere length did not predict adult survival probability. Furthermore, adult telomere length did not predict survival to the subsequent year. These results show that the relationship between early‐life telomere length and lifespan was driven by conditions in early‐life, where early‐life telomere length varied strongly among cohorts. Our data provide evidence for associations between early‐life telomere length and individual life history, and highlight the dynamics of telomere length across individual lifetimes due to individuals experiencing different early‐life environments.     

A Ubiquitin-Binding Domain that Binds a Structural Fold Distinct from that of Ubiquitin

1. Download : Download high-res image (246KB)
2. Download : Download full-size image

     

Mucosal lymphatic-derived gammadelta T cells respond early to experimental Salmonella enterocolitis by increasing expression of IL-2R alpha

To better understand the roles of gammadelta T cells in mucosal infection, we utilized Salmonella enterica serovar Typhimurium (Salmonella serovar Typhimurium) infection in cattle as it closely approximates Salmonella serovar Typhimurium-induced enterocolitis in humans. Protein and gene expression in alphabeta and gammadelta T cells derived from lymphatic ducts draining the gut mucosa in Salmonella serovar Typhimurium-infected calves were analyzed. In calves with enterocolitis, general gene expression trends in gammadelta T cells suggested subtle activation and innate response, whereas alphabeta T cells were relatively quiescent following Salmonella serovar Typhimurium infection. An increase in IL-2R alpha expression on gammadelta T cells from infected calves and results from in vitro assays suggested that gammadelta T cells were primed by Salmonella serovar Typhimurium LPS to better respond to IL-2 and IL-15. Together with gene expression trends in vivo, these data support early priming activation of target tissue gammadelta T cells during Salmonella serovar Typhimurium infection.     

Covalent attachment of proteins to solid supports and surfaces via Sortase-mediated ligation

Background

There is growing interest in the attachment of proteins to solid supports for the development of supported catalysts, affinity matrices, and micro devices as well as for the development of planar and bead based protein arrays for multiplexed assays of protein concentration, interactions, and activity. A critical requirement for these applications is the generation of a stable linkage between the solid support and the immobilized, but still functional, protein.

Methodology

Solid supports including crosslinked polymer beads, beaded agarose, and planar glass surfaces, were modified to present an oligoglycine motif to solution. A range of proteins were ligated to the various surfaces using the Sortase A enzyme of S. aureus. Reactions were carried out in aqueous buffer conditions at room temperature for times between one and twelve hours.

Conclusions

The Sortase A transpeptidase of S. aureus provides a general, robust, and gentle approach to the selective covalent immobilization of proteins on three very different solid supports. The proteins remain functional and accessible to solution. Sortase mediated ligation is therefore a straightforward methodology for the preparation of solid supported enzymes and bead based assays, as well as the modification of planar surfaces for microanalytical devices and protein arrays.     

Does a High‐protein Diet Improve Weight Loss in Overweight and Obese Children?

Objective: To evaluate the effect of a high‐protein diet on anthropometry, body composition, subjective appetite, and mood sensations in overweight and obese children attending a residential weight‐loss camp. Research Methods and Procedures: Children (120; BMI, 33.1 ± 5.5 kg/m²; age, 14.2 ± 1.9 years) were randomly assigned to either a standard or high‐protein diet group (15% vs. 22.5% protein, respectively). All children were assessed at baseline and at the end of the camp for anthropometry, body composition, blood pressure, biochemical variables (n = 27), and subjective appetite and mood sensations (n = 50). Results: Attendance at the weight‐loss camp resulted in significant improvements in most measures. Campers lost 5.5 ± 2.9 kg in body weight (p < 0.001) and 3.8 ± 5.4 kg in fat mass (p < 0.001) and reduced their BMI standard deviation score by 0.27 ± 0.1 (p < 0.001) and their waist circumference by 6.6 ± 2.8 cm (p < 0.001). Subjective sensations of hunger increased significantly over the camp duration, but no other changes in appetite or mood were observed. There were no significant differences between the two diets on any physical or subjective measures. Discussion: Weight‐loss camps are effective in assisting children to lose weight and improve on a range of health outcomes, independently of the protein content of the diet. The implications of an increase in hunger associated with weight loss needs to be considered. Further work is warranted to investigate whether higher levels of dietary protein are feasible or effective in longer‐term weight‐loss interventions of this type.     

Synergistic action of the Saccharomyces cerevisiae homologous recombination factors Rad54 and Rad51 in chromatin remodeling

Rad54, a member of the Swi2/Snf2 protein family, works in concert with the RecA-like recombinase Rad51 during the early and late stages of homologous recombination. Rad51 markedly enhances the activities of Rad54, including the induction of topological changes in DNA and the remodeling of chromatin structure. Reciprocally, Rad54 promotes Rad51-mediated DNA strand invasion with either naked or chromatinized DNA. Here, using various Saccharomyces cerevisiae rad51 and rad54 mutant proteins, mechanistic aspects of Rad54/Rad51-mediated chromatin remodeling are defined. Disruption of the Rad51-Rad54 complex leads to a marked attenuation of chromatin remodeling activity. Moreover, we present evidence that assembly of the Rad51 presynaptic filament represents an obligatory step in the enhancement of the chromatin remodeling reaction. Interestingly, we find a specific interaction of the N-terminal tail of histone H3 with Rad54 and show that the H3 tail interaction domain resides within the amino terminus of Rad54. These results suggest that Rad54-mediated chromatin remodeling coincides with DNA homology search by the Rad51 presynaptic filament and that this process is facilitated by an interaction of Rad54 with histone H3.