Model membrane platforms to study protein-membrane interactions

Abstract Constituting functional interactions between proteins and lipid membranes is one of the essential features of cellular membranes. The major challenge of quantitatively studying these interactions in living cells is the multitude of involved components that are difficult, if not impossible, to simultaneously control. Therefore, there is great need for simplified but still sufficiently detailed model systems to investigate the key constituents of biological processes. To specifically focus on interactions between membrane proteins and lipids, several membrane models have been introduced which recapitulate to varying degrees the complexity and physicochemical nature of biological membranes. Here, we summarize the presently most widely used minimal model membrane systems, namely Supported Lipid Bilayers (SLBs), Giant Unilamellar Vesicles (GUVs) and Giant Plasma Membrane Vesicles (GPMVs) and their applications for protein-membrane interactions.     

Phosphorus pools and other soil properties in the rhizosphere of wheat and legumes growing in three soils in monoculture or as a mixture of wheat and legume

Background and aims

Phosphorus and nitrogen availability and forms are affected by soil properties as well as by plant species and further modulated by soil microbes. Additionally, close contact of the roots of two plant species may affect concentrations and forms of N and P. The aim of this study was to assess properties related to N and P cycling in the rhizosphere of wheat and legumes grown in monoculture or in wheat/legume mixtures in three soils differing in pH.

Methods

Faba bean, white lupin and wheat were grown in three soils differing in pH (4.8, 7.5 and 8.8) in monoculture or in mixed culture of wheat and legumes. Rhizosphere soil was collected at flowering and analyzed for P pools by sequential fractionation, available N as well as community structure of bacteria, fungi, ammonia oxidizers, N₂-fixers and P mobilizers by polymerase chain reaction (PCR)—denaturing gradient gel electrophoresis (DGGE).

Results

Soil type was the major factor determining plant growth, rhizosphere nutrient dynamics and microbial community structure. Among the crop species, only faba bean had a significant effect on nitrification potential activity (PNA) in all three soils with lower activity compared to the unplanted soil. Soil type and plant spieces affected the community composition of ammonia-oxidizing archaea (AOB), ammonia-oxidizing archaea (AOA), N₂-fixers (nifH), P mobilizers (ALP gene) and fungi, but not that of bacteria. Among the microbial groups, the AOA and nifH community composition were most strongly affected by crop species, cropping system and soil type, suggesting that these groups are quite sensitive to environmental conditions. All plants depleted some labile as well as non-labile P pools whereas the less labile organic P pools (NaOH extractable P pools, acid extractable P pools) accumulated in the rhizosphere of legumes. The pattern of depletion and accumulation of some P pools differed between monoculture and mixed culture as well as among soils.

Conclusions

Plant growth and rhizosphere properties were mainly affected by soil type, but also by crop species whereas cropping system had the least effect. Wheat and the legumes depleted less labile inorganic P pools in some soils whereas less labile organic P pools (NaOH extractable P, acid extractable P) accumulated in the rhizosphere of legumes.     

Potential soil P mobilisation capacity–method development and comparison of rhizosphere soil from different crops

Background

Phosphorus (P) deficiency is wide-spread in agricultural soils. In light of increasing P fertilizer costs, it is of interest to assess the capacity of soil microbes to mobilise native soil P and added P. There is currently no method to assess P mobilisation in situ.

Methods

The soil P mobilisation potential was assessed by incubating low P soil for up to 30?days with poorly available P sources; C and N were added to increase microbial activity and ensure that only P was limiting microbial growth.

Results

The increase in microbial P from day 0 to day 15 showed that microbes were able to mobilise P from FePO₄ and phytate. The P mobilisation potential (sum of microbial and resin P) of the rhizosphere soil decreased in the following order: faba bean > chickpea and white lupin > wheat. After 10?days, up to 80% of the mobilised P was microbial P, whereas after 30?days, almost all P mobilised was resin P.

Conclusions

The method developed in this study is useful assessing not only potential of a soil to mobilise P but also, by using different poorly available P sources, the mechanisms of P mobilisation.     

Intraradical dynamics of two coexisting isolates of the arbuscular mycorrhizal fungus Glomus intraradices sensu lato as estimated by real-time PCR of mitochondrial DNA

Real-time PCR in nuclear ribosomal DNA (nrDNA) is becoming a well-established tool for the quantification of arbuscular mycorrhizal (AM) fungi, but this genomic region does not allow the specific amplification of closely related genotypes. The large subunit of mitochondrial DNA (mtDNA) has a higher-resolution power, but mtDNA-based quantification has not been previously explored in AM fungi. We applied real-time PCR assays targeting the large subunit of mtDNA to monitor the DNA dynamics of two isolates of Glomus intraradices sensu lato coexisting in the roots of medic (Medicago sativa). The mtDNA-based quantification was compared to quantification in nrDNA. The ratio of copy numbers determined by the nrDNA- and mtDNA-based assays consistently differed between the two isolates. Within an isolate, copy numbers of the nuclear and the mitochondrial genes were closely correlated. The two quantification approaches revealed similar trends in the dynamics of both isolates, depending on whether they were inoculated alone or together. After 12 weeks of cultivation, competition between the two isolates was observed as a decrease in the mtDNA copy numbers of one of them. The coexistence of two closely related isolates, which cannot be discriminated by nrDNA-based assays, was thus identified as a factor influencing the dynamics of AM fungal DNA in roots. Taken together, the results of this study show that real-time PCR assays targeted to the large subunit of mtDNA may become useful tools for the study of coexisting AM fungi.     

An ADP-ribosyltransferase 3 (ART3) variant is associated with reduced sperm counts in Czech males: case/control association study replicating results from the Japanese population

OBJECTIVES: In about 50% of male infertility the underlying pathogenesis remains unknown. A recent Japanese study provided evidence that the rs6836703: G>A single-nucleotide polymorphism (SNP) from the ADP-ribosyltransferase 3 (ART3) gene is significantly associated with non-obstructive azoospermia. However, the functional significance of this association is unknown and replication studies in unrelated populations are thus necessary. Design: In this study, 257 fertile Czech controls of proven paternity and 98 sub-/infertile patients selected according to stringent exclusion / inclusion criteria were genotyped by High Resolution Melting (HRM) of small amplicons. Setting: This study was performed at University Hospital Motol - Laboratory of reproductive genetics using routinely analyzed cases. Results: Significant differences in allele distribution between fertile and sub-/infertile men were found (OR=1.78, 95% CI: 1.17-2.70; p=0.007). Following sub-stratification of cases according to their sperm counts we found that observed differences in allele distributions were increased in oligozoospermic men with sperm counts of <15 million sperm/mL (OR=1.98, 95% CI: 1.28-3.07; p=0.002). This difference was also reflected in genotype distributions between fertile and sub-/infertile men (p=0.008), and fertile versus oligozoospermic men (p=0.004). Conclusions: Our study serves as a first replication of the original Japanese report and opens new avenues of research. Compared to the Japanese patient cohort, we provided evidence that the analyzed ART3 variant is associated with quantitative impairment of spermatogenesis.     

Duetting Behaviour in the Leafhopper Aphrodes makarovi (Hemiptera: Cicadellidae)

Mate recognition and location in Cicadellidae is mediated exclusively via substrate-borne vibrational signals. In the present study we investigated vibrational signals and mate searching behaviour of the leafhopper Aphrodes makarovi. We studied mating behaviour and exchange of vibrational signals between live insects and in playback experiments. Males emitted long and complex calling signals composed of several sections. Female reply was long and always overlapped the end of the male call. The exchange of male and female vibrational signals was a complex and dynamic interaction during which both partners modified their signals according to partner’s reply. The duration of female reply was influenced by the duration of the male call to which she was responding, while the duration of male call was influenced by the duration of the previous female reply. Such relationship suggests the role of sexual selection in the evolution of male vibrational signals.     

Transportomics: screening for substrates of ABC transporters in body fluids using vesicular transport assays

The ATP-binding cassette (ABC) genes encode the largest family of transmembrane proteins. ABC transporters translocate a wide variety of substrates across membranes, but their physiological function is often incompletely understood. We describe a new method to study the substrate spectrum of ABC transporters: We incubate extracts of mouse urine with membrane vesicles prepared from Spodoptera frugiperda Sf9 insect cells overproducing an ABC transporter and determine the compounds transported into the vesicles by LC/MS-based metabolomics. We illustrate the power of this simple "transportomics" approach using ABCC2, a protein present at sites of uptake and elimination. We identified many new substrates of ABCC2 in urine. These included glucuronides of plant-derived xenobiotics, a class of compounds to which humans are exposed on a daily basis. Moreover, we show that the excretion of these compounds in vivo depends on ABCC2: compared to wild-type mice, the urinary excretion of several glucuronides was increased up to 20-fold in Abcc2(-/-) mice. Transportomics has broad applicability, as it is not restricted to urine and can be applied to other ATP-dependent transport proteins as well.     

The BRCA1 alternative splicing variant Δ14-15 with an in-frame deletion of part of the regulatory serine-containing domain (SCD) impairs the DNA repair capacity in MCF-7 cells

The BRCA1 gene codes for a protein involved in the DNA double strand break (DDSB) repair. Alongside the dominant full-length splicing form of BRCA1, numerous endogenously expressed alternative splicing variants of unknown significance have been described in various tissues. Some of them retain the original BRCA1 reading frame but lack several critical BRCA1 structural domains, suggesting an altered function of the resulting protein in the BRCA1-regulated processes. To characterize the effect of the BRCA1Δ14-15 splicing variant (with an in-frame deletion affecting the regulatory serine-containing domain) on the DDSB repair, we constructed the MCF-7 clones stably expressing the analyzed variant with/without a shRNA-mediated downregulation of the endogenous full-length wild-type BRCA1 expression. Our results show that the expression of the BRCA1Δ14-15 variant delays the γ-radiation-induced DDSB repair, alters the kinetics of irradiation-induced foci formation/decomposition and reduces the non-homologous end-joining capacity in MCF-7 cells. Therefore, the BRCA1Δ14-15 is not able to functionally replace the full-length wt BRCA1 in the DDSB repair. Our findings indicate that the endogenously expressed BRCA1 alternative splicing variants may negatively influence genome stability and support the growing evidence of the pathological potential of the sequence variants generated by an altered or misregulated alternative splicing in the process of mammary malignant transformation.