Lipid Domain Structure of the Plasma Membrane Revealed by Patching of Membrane Components

Lateral assemblies of glycolipids and cholesterol, “rafts,” have been implicated to play a role in cellular processes like membrane sorting, signal transduction, and cell adhesion. We studied the structure of raft domains in the plasma membrane of non-polarized cells. Overexpressed plasma membrane markers were evenly distributed in the plasma membrane. We compared the patching behavior of pairs of raft markers (defined by insolubility in Triton X-100) with pairs of raft/non-raft markers. For this purpose we cross-linked glycosyl-phosphatidylinositol (GPI)-anchored proteins placental alkaline phosphatase (PLAP), Thy-1, influenza virus hemagglutinin (HA), and the raft lipid ganglioside GM1 using antibodies and/or cholera toxin. The patches of these raft markers overlapped extensively in BHK cells as well as in Jurkat T–lymphoma cells. Importantly, patches of GPI-anchored PLAP accumulated src-like protein tyrosine kinase fyn, which is thought to be anchored in the cytoplasmic leaflet of raft domains. In contrast patched raft components and patches of transferrin receptor as a non-raft marker were sharply separated. Taken together, our data strongly suggest that coalescence of cross-linked raft elements is mediated by their common lipid environments, whereas separation of raft and non-raft patches is caused by the immiscibility of different lipid phases. This view is supported by the finding that cholesterol depletion abrogated segregation. Our results are consistent with the view that raft domains in the plasma membrane of non-polarized cells are normally small and highly dispersed but that raft size can be modulated by oligomerization of raft components.     

Identification and Characterization of Rvs162/Rvs167-3, a Novel N-BAR Heterodimer in the Human Fungal Pathogen Candida albicans

Membrane reshaping resides at the core of many important cellular processes, and among its mediators are the BAR (Bin, Amphiphysin, Rvs) domain-containing proteins. We have explored the diversity and function of the Rvs BAR proteins in Candida albicans and identified a novel family member, Rvs167-3 (orf19.1861). We show that Rvs167-3 specifically interacts with Rvs162 to form a stable BAR heterodimer able to bind liposomes in vitro. A second, distinct heterodimer is formed by the canonical BAR proteins Rvs161 and Rvs167. Purified Rvs161/Rvs167 complex also binds liposomes, indicating that C. albicans expresses two functional BAR heterodimers. We used live-cell imaging to localize green fluorescent protein (GFP)-tagged Rvs167-3 and Rvs167 and show that both proteins concentrate in small cortical spots. However, while Rvs167 strictly colocalizes with the endocytic marker protein Abp1, we do not observe any colocalization of Rvs167-3 with sites of endocytosis marked by Abp1. Furthermore, the rvs167-3Δ/Δ mutant is not defective in endocytosis and strains lacking Rvs167-3 or its partner Rvs162 do not display increased sensitivity to high salt concentrations or decreased cell wall integrity, phenotypes which have been observed for rvs167Δ/Δ and rvs161Δ/Δ strains and which are linked to endocytosis defects. Taken together, our results indicate different roles for the two BAR heterodimers in C. albicans: the canonical Rvs161/Rvs167 heterodimer functions in endocytosis, whereas the novel Rvs162/Rvs167-3 heterodimer seems not to be involved in this process. Nevertheless, despite their different roles, our phenotypic analysis revealed a genetic interaction between the two BAR heterodimers, suggesting that they may have related but distinct membrane-associated functions.     

Live Cell Analysis and Mathematical Modeling Identify Determinants of Attenuation of Dengue Virus 2’-O-Methylation Mutant

Dengue virus (DENV) is the most common mosquito-transmitted virus infecting ~390 million people worldwide. In spite of this high medical relevance, neither a vaccine nor antiviral therapy is currently available. DENV elicits a strong interferon (IFN) response in infected cells, but at the same time actively counteracts IFN production and signaling. Although the kinetics of activation of this innate antiviral defense and the timing of viral counteraction critically determine the magnitude of infection and thus disease, quantitative and kinetic analyses are lacking and it remains poorly understood how DENV spreads in IFN-competent cell systems. To dissect the dynamics of replication versus antiviral defense at the single cell level, we generated a fully viable reporter DENV and host cells with authentic reporters for IFN-stimulated antiviral genes. We find that IFN controls DENV infection in a kinetically determined manner that at the single cell level is highly heterogeneous and stochastic. Even at high-dose, IFN does not fully protect all cells in the culture and, therefore, viral spread occurs even in the face of antiviral protection of naïve cells by IFN. By contrast, a vaccine candidate DENV mutant, which lacks 2’-O-methylation of viral RNA is profoundly attenuated in IFN-competent cells. Through mathematical modeling of time-resolved data and validation experiments we show that the primary determinant for attenuation is the accelerated kinetics of IFN production. This rapid induction triggered by mutant DENV precedes establishment of IFN-resistance in infected cells, thus causing a massive reduction of virus production rate. In contrast, accelerated protection of naïve cells by paracrine IFN action has negligible impact. In conclusion, these results show that attenuation of the 2’-O-methylation DENV mutant is primarily determined by kinetics of autocrine IFN action on infected cells.     

Reliability of Intra-Retinal Layer Thickness Estimates

Purpose

Measurement of intra-retinal layer thickness using optical coherence tomography (OCT) has become increasingly prominent in multiple sclerosis (MS) research. Nevertheless, the approaches used for determining the mean layer thicknesses vary greatly. Insufficient data exist on the reliability of different thickness estimates, which is crucial for their application in clinical studies. This study addresses this lack by evaluating the repeatability of different thickness estimates.

Methods

Studies that used intra-retinal layer segmentation of macular OCT scans in patients with MS were retrieved from PubMed. To investigate the repeatability of previously applied layer estimation approaches, we generated datasets of repeating measurements of 15 healthy subjects and 13 multiple sclerosis patients using two OCT devices (Cirrus HD-OCT and Spectralis SD-OCT). We calculated each thickness estimate in each repeated session and analyzed repeatability using intra-class correlation coefficients and coefficients of repeatability.

Results

We identified 27 articles, eleven of them used the Spectralis SD-OCT, nine Cirrus HD-OCT, two studies used both devices and two studies applied RTVue-100. Topcon OCT-1000, Stratus OCT and a research device were used in one study each. In the studies that used the Spectralis, ten different thickness estimates were identified, while thickness estimates of the Cirrus OCT were based on two different scan settings. In the simulation dataset, thickness estimates averaging larger areas showed an excellent repeatability for all retinal layers except the outer plexiform layer (OPL).

Conclusions

Given the good reliability, the thickness estimate of the 6mm-diameter area around the fovea should be favored when OCT is used in clinical research. Assessment of the OPL was weak in general and needs further investigation before OPL thickness can be used as a reliable parameter.     

Determination of the Diversity of Rhodopirellula Isolates from European Seas by Multilocus Sequence Analysis

In the biogeography of microorganisms, the habitat size of an attached-living bacterium has never been investigated. We approached this theme with a multilocus sequence analysis (MLSA) study of new strains of Rhodopirellula sp., an attached-living planctomycete. The development of an MLSA for Rhodopirellula baltica enabled the characterization of the genetic diversity at the species level, beyond the resolution of the 16S rRNA gene. The alleles of the nine housekeeping genes acsA, guaA, trpE, purH, glpF, fumC, icd, glyA, and mdh indicated the presence of 13 genetically defined operational taxonomic units (OTUs) in our culture collection. The MLSA-based OTUs coincided with the taxonomic units defined by DNA-DNA hybridization experiments. BOX-PCR supported the MLSA-based differentiation of two closely related OTUs. This study established a taxon-area relationship of cultivable Rhodopirellula species. In European seas, three closely related species covered the Baltic Sea and the eastern North Sea, the North Atlantic region, and the southern North Sea to the Mediterranean. The last had regional genotypes, as revealed by BOX-PCR. This suggests a limited habitat size of attached-living Rhodopirellula species.The biogeography of microorganisms describes the habitat size of the species and the distribution of microorganisms on Earth. The experimental approaches depend on the focus of the studies. Habitats are often analyzed by environmental microbiologists with genetic-fingerprinting techniques, with up to 200 bands or fragments representing the whole community. Although the taxonomic resolution of these operational taxonomic units (OTUs) is limited, the studies revealed a community biogeography (22). Medical microbiologists analyze the alleles of housekeeping genes of microorganisms to gain insight into the epidemiology of pathogens, the population biogeography (2). This strain-specific, fine-scale taxonomic resolution within a species is well suited to observance of recent dispersal events. At the species level, multilocus sequence typing (MLST) and analysis (MLSA), which were developed for intraspecies and intragenus specific studies, respectively, consist of the sequences of several (at least seven) housekeeping gene fragments concatenated to an approximately 5-kilobase alignment (17). Recent MLSA studies revealed its applicability to marine isolates and the analysis of biogeographic patterns: Alteromonas macleodii isolates could be grouped in an epipelagic and an abyssal clade (6), and strains of Pseudomonas aeruginosa were genetically well separated into groups of coastal and oceanic origin (8). However, for Salinibacter ruber strains, biogeographical distinctness was not resolved in an MLSA study but showed allopatry in a metabolic analysis (31). Several studies used MLSA together with DNA-DNA hybridization (DDH) for the delineation of new species, e.g., for Vibrio and Ensifer spp. (20, 36).In the biogeography of microorganisms, the experimental proof of a local genetic evolution was first revealed at sample sites that were physically separated by over 18,000 km (39). Large populations and the small size of microbes have been considered as facilitators for dispersal over long distances, eventually establishing cosmopolitan microbial populations. On the other hand, the smallest spatial scale of a microbial species in an open system has not been investigated. Attached-living bacteria disperse only during a distinct, short time span in their lives. This limitation of the dispersal time stimulated this study of the biogeography of Rhodopirellula baltica in European seas.R. baltica is a planctomycete with typical morphological features. The peptidoglycanless bacteria have an intracellular compartmentation: the riboplasm with the nucleoid is separated by a membrane from the surrounding paryphoplasm. Cells attach with a holdfast substance to surfaces or, in culture, to themselves, forming typical rosettes. Proliferation occurs by budding, and offspring cells live free in the water column: they are motile with a flagellum until they settle on the sediment (4).Seventy recently isolated strains affiliated according to the 16S rRNA gene analysis with R. baltica SH1^T as the closest validly described species (40). The 16S rRNA gene sequences do not offer sufficient information at the species level. A dissimilarity of the 16S rRNA genes of more than 3%, recently reduced to 1.3% (34, 35), indicates that the strains under consideration belong to two species. These thresholds yielded in our strain collection, according to an ARB-based calculation, five or eight operational taxonomic units besides the species R. baltica (40). For strains with highly identical sequences, whole-genome DDH experiments have to be performed to identify the affiliation to established species. Recently, multilocus sequence analyses have emerged as a possible alternative method. Our strain collection comprised many strains with a 16S rRNA gene sequence very closely related to that of R. baltica SH1^T. To gain insight into the genetic identity of the isolates on the species level and the habitat sizes of the species, we developed a multilocus sequence analysis and applied it to the strain collection. The MLSA results were calibrated with a DDH study. The closely related strains were additionally characterized by BOX-PCR, a fingerprinting method (15). Transmission electron microscopy (EM) was performed on some isolates to support the identification as Planctomycetes and to visualize morphological differences between strains.     

Single-molecule force spectroscopy of cartilage aggrecan self-adhesion

We investigated self-adhesion between highly negatively charged aggrecan macromolecules extracted from bovine cartilage extracellular matrix by performing atomic force microscopy (AFM) imaging and single-molecule force spectroscopy (SMFS) in saline solutions. By controlling the density of aggrecan molecules on both the gold substrate and the gold-coated tip surface at submonolayer densities, we were able to detect and quantify the Ca²⁺-dependent homodimeric interaction between individual aggrecan molecules at the single-molecule level. We found a typical nonlinear sawtooth profile in the AFM force-versus-distance curves with a molecular persistence length of l_p = 0.31 ± 0.04 nm. This is attributed to the stepwise dissociation of individual glycosaminoglycan (GAG) side chains in aggrecans, which is very similar to the known force fingerprints of other cell adhesion proteoglycan systems. After studying the GAG-GAG dissociation in a dynamic, loading-rate-dependent manner (dynamic SMFS) and analyzing the data according to the stochastic Bell-Evans model for a thermally activated decay of a metastable state under an external force, we estimated for the single glycan interaction a mean lifetime of τ = 7.9 ± 4.9 s and a reaction bond length of x_β = 0.31 ± 0.08 nm. Whereas the x_β-value compares well with values from other cell adhesion carbohydrate recognition motifs in evolutionary distant marine sponge proteoglycans, the rather short GAG interaction lifetime reflects high intermolecular dynamics within aggrecan complexes, which may be relevant for the viscoelastic properties of cartilage tissue.     

Temperature and the formation of radiation-induced chromosome aberrations. II. The temperature dependence of lesion repair and lesion interaction

Dicentric chromosome aberration yields have been measured after single-exposure and split-dose irradiations of human lymphocytes with 150 kV X-rays. Various temperature programmes between 4 and 37 degrees C were applied before, during and after irradiations and in the radiation-free interval. It was found that chromatin lesion repair was completely suppressed at 21 degrees C and below, whereas lesion formation is reduced only below 17 degrees C. The interaction between repairable lesions which leads to exchange-type aberrations is also suppressed by low temperatures. Hypothermic suppression of chromatin lesion repair and interaction is fully reversible at least up to 12 h of maintenance of the 'stored' state of these lesions.     

Identification of the IL-17 receptor related molecule IL-17RC as the receptor for IL-17F

The proinflammatory cytokines IL-17A and IL-17F have a high degree of sequence similarity and share many biological properties. Both have been implicated as factors contributing to the progression of inflammatory and autoimmune diseases. Moreover, reagents that neutralize IL-17A significantly ameliorate disease severity in several mouse models of human disease. IL-17A mediates its effects through interaction with its cognate receptor, the IL-17 receptor (IL-17RA). We report here that the IL-17RA-related molecule, IL-17RC is the receptor for IL-17F. Notably, both IL-17A and IL-17F bind to IL-17RC with high affinity, leading us to suggest that a soluble form of this molecule may serve as an effective therapeutic antagonist of IL-17A and IL-17F. We generated a soluble form of IL-17RC and demonstrate that it effectively blocks binding of both IL-17A and IL-17F, and that it inhibits signaling in response to these cytokines. Collectively, our work indicates that IL-17RC functions as a receptor for both IL-17A and IL-17F and that a soluble version of this protein should be an effective antagonist of IL-17A and IL-17F mediated inflammatory diseases.     

Variation in ovule and seed size and associated size-number trade-offs in angiosperms

Unlike pollen and seed size, the extent and causes of variation in ovule size remain unexplored. Based on 45 angiosperm species, we assessed whether intra- and interspecific variation in ovule size is consistent with cost minimization during ovule production or allows maternal plants to dominate conflict with their seeds concerning resource investment. Despite considerable intraspecific variation in ovule volume (mean CV = 0.356), ovule production by few species was subject to a size-number trade-off. Among the sampled species, ovule volume varied two orders of magnitude, whereas seed volume varied four orders of magnitude. Ovule volume varied positively among species with flower mass and negatively with ovule number. Tenuinucellate ovules were generally larger that crassinucellate ovules, and species with apical placentation (which mostly have uniovulate ovaries) had smaller ovules than those with other placentation types. Seed volume varied positively among species with fruit mass and seed development time, but negatively with seed number. Seeds grew a median 93-fold larger than the ovules from which they originated. Our results provide equivocal evidence that selection minimizes ovule size to allow efficient resource allocation after fertilization, but stronger evidence that ovule size affords maternal plants an advantage in parent-offspring conflict.