Membrane distribution of the glycine receptor α3 studied by optical super-resolution microscopy

In this study, the effect of glycine receptor (GlyR) α3 alternative RNA splicing on the distribution of receptors in the membrane of human embryonic kidney 293 cells is investigated using optical super-resolution microscopy. Direct stochastic optical reconstruction microscopy is used to image both α3K and α3L splice variants individually and together using single- and dual-color imaging. Pair correlation analysis is used to extract quantitative measures from the resulting images. Autocorrelation analysis of the individually expressed variants reveals clustering of both variants, yet with differing properties. The cluster size is increased for α3L compared to α3K (mean radius 92 ± 4 and 56 ± 3 nm, respectively), yet an even bigger difference is found in the cluster density (9,870 ± 1,433 and 1,747 ± 200 μm^?2, respectively). Furthermore, cross-correlation analysis revealed that upon co-expression, clusters colocalize on the same spatial scales as for individually expressed receptors (mean co-cluster radius 94 ± 6 nm). These results demonstrate that RNA splicing determines GlyR α3 membrane distribution, which has consequences for neuronal GlyR physiology and function.     

Can the structure of a riparian forest remnant influence stream water quality? A tropical case study

In rural areas, riparian forest remnants can be very important for the maintenance and improvement of stream water quality. We evaluated if a forest remnant influenced stream water quality, and if these results were influenced by forest structure. We sampled reaches from 140 m upstream of the remnant edge until 600 m within the forest remnant. Electric conductivity (EC) and ammonium concentrations decreased as the stream flowed through the remnant, whereas dissolved oxygen, total phosphorus (P), total dissolved P, organic P, and silicate concentrations increased along the remnant. Variation in forest structure was due to a gradient in forest stratification versus tree height and diameter at breast height, and a gradient in tree density versus basal area. More stratified parts of the forest, with smaller trees, resulted in lower EC values and concentrations of total nitrogen and nitrite, whereas higher density of trees resulted in lower levels of total and dissolved P, creating heterogeneity at very local scales. The overall mean influence of this riparian forest remnant improved stream water quality, suggesting that forest remnants have local effects that can be important when managing stream water quality at larger spatial scales.     

Modulation of apolipoprotein B antigenic determinants in human low density lipoprotein subclasses

To investigate the effect of low density lipoprotein (LDL) heterogeneity on the conformation of LDL apolipoprotein B (apo-B), the immunoreactivities of 6 monoclonal antibodies against LDL apo-B were measured in 3 LDL subfractions isolated by equilibrium density gradient ultracentrifugation. To ensure a broad range of LDL particles, the LDL subfractions were prepared from normal subjects and patients with hyperapobetalipoproteinemia. With 3 of the antibodies, 1D1, 5E11, and 3A10, LDL fractions 1 (the most buoyant), 2 (the intermediate), and 3 (the densest) were equally immunoreactive and competed similarly with reference whole LDL. In contrast, with 3 other antibodies, 2D8, 3F5, and 4G3, fraction 1 was significantly more reactive than fraction 3; that is for each in turn, 290, 250, and 150% more of the densest LDL protein was required to achieve the same displacement as with fraction 1. Further, the immunoreactivities of the 3 LDL fractions with antibodies 2D8, 3F5, and 4G3 were negatively correlated with their LDL cholesterol to LDL protein ratio with r values of 0.727, 0.898, and 0.870, respectively, suggesting that as LDL particle size decreases, the conformation of the LDL apo-B changes progressively. It is of interest that the antigenic determinants recognized by 3F5 and 4G3 are close to the LDL receptor recognition site on LDL apo-B. Therefore, it is possible that the reduced immunoreactivity of these determinants in dense LDL may be the in vitro correlate of the reduced fractional catabolics rate of dense LDL compared to buoyant LDL previously observed in vivo.     

A draft of the human septin interactome

Background

Septins belong to the GTPase superclass of proteins and have been functionally implicated in cytokinesis and the maintenance of cellular morphology. They are found in all eukaryotes, except in plants. In mammals, 14 septins have been described that can be divided into four groups. It has been shown that mammalian septins can engage in homo- and heterooligomeric assemblies, in the form of filaments, which have as a basic unit a hetero-trimeric core. In addition, it has been speculated that the septin filaments may serve as scaffolds for the recruitment of additional proteins.

Methodology/Principal Findings

Here, we performed yeast two-hybrid screens with human septins 1–10, which include representatives of all four septin groups. Among the interactors detected, we found predominantly other septins, confirming the tendency of septins to engage in the formation of homo- and heteropolymeric filaments.

Conclusions/Significance

If we take as reference the reported arrangement of the septins 2, 6 and 7 within the heterofilament, (7-6-2-2-6-7), we note that the majority of the observed interactions respect the “group rule”, i.e. members of the same group (e.g. 6, 8, 10 and 11) can replace each other in the specific position along the heterofilament. Septins of the SEPT6 group preferentially interacted with septins of the SEPT2 group (p<0.001), SEPT3 group (p<0.001) and SEPT7 group (p<0.001). SEPT2 type septins preferentially interacted with septins of the SEPT6 group (p<0.001) aside from being the only septin group which interacted with members of its own group. Finally, septins of the SEPT3 group interacted preferentially with septins of the SEPT7 group (p<0.001). Furthermore, we found non-septin interactors which can be functionally attributed to a variety of different cellular activities, including: ubiquitin/sumoylation cycles, microtubular transport and motor activities, cell division and the cell cycle, cell motility, protein phosphorylation/signaling, endocytosis, and apoptosis.     

Variation in eggshell traits between geographically distant populations of pied flycatchers Ficedula hypoleuca

The expression and impact of maternal effects may vary greatly between populations and environments. However, little is known about large‐scale geographical patterns of variation in maternal deposition to eggs. In birds, as in other oviparous animals, the outermost maternal component of an egg is the shell, which protects the embryo, provides essential mineral resources and allows its interaction with the environment in the form of gas exchange. In this study, we explored variation of eggshell traits (mass, thickness, pore density and pigmentation) across 15 pied flycatcher populations at a large geographic scale. We found significant between‐population variation in all eggshell traits, except in pore density, suggesting spatial variation in their adaptive benefits or in the females’ physiological limitations during egg laying. Between‐ population variation in shell structure was not due to geographic location (latitude and longitude) or habitat type. However, eggshells were thicker in populations that experienced higher ambient temperature during egg laying. This could be a result of maternal resource allocation to the shell being constrained under low temperatures or of an adaptation to reduce egg water loss under high temperatures. We also found that eggshell colour intensity was positively associated with biliverdin pigment concentration, shell thickness and pore density. To conclude, our findings reveal large‐ scale between‐population variation of eggshell traits, although we found little environmental dependency in their expression. Our findings call for further studies that explore other environmental factors (e.g. calcium availability and pollution levels) and social factors like sexual selection intensity that may account for differences in shell structure between populations.     

Direct-Imaging-Based Quantification of Bacillus cereus ATCC 14579 Population Heterogeneity at a Low Incubation Temperature

Bacillus cereus ATCC 14579 was cultured in microcolonies on Anopore strips near its minimum growth temperature to directly image and quantify its population heterogeneity at an abusive refrigeration temperature. Eleven percent of the microcolonies failed to grow during low-temperature incubation, and this cold-induced population heterogeneity could be partly attributed to the loss of membrane integrity of individual cells.Bacillus cereus is a food poisoning- and food spoilage-causing organism that can be found in a large variety of foods (4, 23). There are two illnesses associated with B. cereus, namely, emetic and diarrheal intoxication (17, 24). Most of the strains related to cases or outbreaks of B. cereus food-borne poisoning were shown to be unable to grow at 7°C (1, 12). The average temperatures of domestic refrigerators have been investigated in various surveys around the world and often ranged from 5°C to 7°C, but extreme values exceeded 10°C to 12°C (5, 16). Inadequate chilling was indeed reported in various incidents of B. cereus food-borne illness (7, 8, 18, 19), pointing to the importance of appropriate refrigeration of foods contaminated with B. cereus to control its growth and toxin production in foods (9).Several studies have demonstrated that microorganisms can show diversity in their population stress response, even in an apparently homogeneous stress environment (6, 11, 21, 22). However, only very limited data describing the heterogeneity in growth performance of individual cells from food-borne pathogens cultured at low temperatures are available (10). Because inadequate chilling of food is one of the factors that contribute to the number of incidents of B. cereus food-borne illness, there is a need for better understanding of its growth performance at lowered incubation temperatures. In this study, we used the direct-imaging-based Anopore technology (6, 13-15) to quantitatively describe the population heterogeneity of B. cereus ATCC 14579 cells at 12°C. The minimum temperature for the growth of B. cereus ATCC 14579 in brain heart infusion (BHI) broth is 7.5°C (personal communication from F. Carlin), but various food-borne-associated B. cereus isolates were shown to be unable to grow at 10°C (1). Therefore, in this study, a culturing temperature of 12°C was chosen, to mimic temperature abuse of refrigerated foods. In addition, the membrane integrity of individual cells was assessed using both membrane permeant and impermeant nucleic acid dyes in order to get more insight into cellular characteristics that may contribute to heterogeneity in growth response.     

Peroxidase Profiling Reveals Genetic Linkage between Peroxidase Gene Clusters and Basal Host and Non-Host Resistance to Rusts and Mildew in Barley

Background

Higher plants possess a large multigene family encoding secreted class III peroxidase (Prx) proteins. Peroxidases appear to be associated with plant disease resistance based on observations of induction during disease challenge and the presence or absence of isozymes in resistant vs susceptible varieties. Despite these associations, there is no evidence that allelic variation of peroxidases directly determines levels of disease resistance.

Methodology/Principal Findings

The current study introduces a new strategy called Prx-Profiling. We showed that with this strategy a large number of peroxidase genes can be mapped on the barley genome. In order to obtain an estimate of the total number of Prx clusters we followed a re-sampling procedure, which indicated that the barley genome contains about 40 peroxidase gene clusters. We examined the association between the Prxs mapped and the QTLs for resistance of barley to homologous and heterologous rusts, and to the barley powdery mildew fungus. We report that 61% of the QTLs for partial resistance to P. hordei, 61% of the QTLs for resistance to B. graminis and 47% of the QTLs for non-host resistance to other Puccinia species co-localize with Prx based markers.

Conclusions/Significance

We conclude that Prx-Profiling was effective in finding the genetic location of Prx genes on the barley genome. The finding that QTLs for basal resistance to rusts and powdery mildew fungi tend to co-locate with Prx clusters provides a base for exploring the functional role of Prx-related genes in determining natural differences in levels of basal resistance.     

Reelin and stk25 have opposing roles in neuronal polarization and dendritic Golgi deployment

The Reelin ligand regulates a Dab1-dependent signaling pathway required for brain lamination and normal dendritogenesis, but the specific mechanisms underlying these actions remain unclear. We find that Stk25, a modifier of Reelin-Dab1 signaling, regulates Golgi morphology and neuronal polarization as part of an LKB1-Stk25-Golgi matrix protein 130 (GM130) signaling pathway. Overexpression of Stk25 induces Golgi condensation and multiple axons, both of which are rescued by Reelin treatment. Reelin stimulation of cultured neurons induces the extension of the Golgi into dendrites, which is suppressed by Stk25 overexpression. In vivo, Reelin and Dab1 are required for the normal extension of the Golgi apparatus into the apical dendrites of hippocampal and neocortical pyramidal neurons. This demonstrates that the balance between Reelin-Dab1 signaling and LKB1-Stk25-GM130 regulates Golgi dispersion, axon specification, and dendrite growth and provides insights into the importance of the Golgi apparatus for cell polarization.