The Suppressor of AAC2 Lethality SAL1 Modulates Sensitivity of Heterologously Expressed Artemia ADP/ATP Carrier to Bongkrekate in Yeast

The ADP/ATP carrier protein (AAC) expressed in Artemia franciscana is refractory to bongkrekate. We generated two strains of Saccharomyces cerevisiae where AAC1 and AAC3 were inactivated and the AAC2 isoform was replaced with Artemia AAC containing a hemagglutinin tag (ArAAC-HA). In one of the strains the suppressor of ΔAAC2 lethality, SAL1, was also inactivated but a plasmid coding for yeast AAC2 was included, because the ArAACΔsal1Δ strain was lethal. In both strains ArAAC-HA was expressed and correctly localized to the mitochondria. Peptide sequencing of ArAAC expressed in Artemia and that expressed in the modified yeasts revealed identical amino acid sequences. The isolated mitochondria from both modified strains developed 85% of the membrane potential attained by mitochondria of control strains, and addition of ADP yielded bongkrekate-sensitive depolarizations implying acquired sensitivity of ArAAC-mediated adenine nucleotide exchange to this poison, independent from SAL1. However, growth of ArAAC-expressing yeasts in glycerol-containing media was arrested by bongkrekate only in the presence of SAL1. We conclude that the mitochondrial environment of yeasts relying on respiratory growth conferred sensitivity of ArAAC to bongkrekate in a SAL1-dependent manner. © 2013 Wysocka-Kapcinska et al

Antibiotic-induced release of small extracellular vesicles (exosomes) with surface-associated DNA

Recently, biological roles of extracellular vesicles (which include among others exosomes, microvesicles and apoptotic bodies) have attracted substantial attention in various fields of biomedicine. Here we investigated the impact of sustained exposure of cells to the fluoroquinolone antibiotic ciprofloxacin on the released extracellular vesicles. Ciprofloxacin is widely used in humans against bacterial infections as well as in cell cultures against Mycoplasma contamination. However, ciprofloxacin is an inducer of oxidative stress and mitochondrial dysfunction of mammalian cells. Unexpectedly, here we found that ciprofloxacin induced the release of both DNA (mitochondrial and chromosomal sequences) and DNA-binding proteins on the exofacial surfaces of small extracellular vesicles referred to in this paper as exosomes. Furthermore, a label-free optical biosensor analysis revealed DNA-dependent binding of exosomes to fibronectin. DNA release on the surface of exosomes was not affected any further by cellular activation or apoptosis induction. Our results reveal for the first time that prolonged low-dose ciprofloxacin exposure leads to the release of DNA associated with the external surface of exosomes

Improved Characterization of EV Preparations Based on Protein to Lipid Ratio and Lipid Properties.

In recent years the study of extracellular vesicles has gathered much scientific and clinical interest. As the field is expanding, it is becoming clear that better methods for characterization and quantification of extracellular vesicles as well as better standards to compare studies are warranted. The goal of the present work was to find improved parameters to characterize extracellular vesicle preparations. Here we introduce a simple 96 well plate-based total lipid assay for determination of lipid content and protein to lipid ratios of extracellular vesicle preparations from various myeloid and lymphoid cell lines as well as blood plasma. These preparations included apoptotic bodies, microvesicles/microparticles, and exosomes isolated by size-based fractionation. We also investigated lipid bilayer order of extracellular vesicle subpopulations using Di-4-ANEPPDHQ lipid probe, and lipid composition using affinity reagents to clustered cholesterol (monoclonal anti-cholesterol antibody) and ganglioside GM1 (cholera toxin subunit B). We have consistently found different protein to lipid ratios characteristic for the investigated extracellular vesicle subpopulations which were substantially altered in the case of vesicular damage or protein contamination. Spectral ratiometric imaging and flow cytometric analysis also revealed marked differences between the various vesicle populations in their lipid order and their clustered membrane cholesterol and GM1 content. Our study introduces for the first time a simple and readily available lipid assay to complement the widely used protein assays in order to better characterize extracellular vesicle preparations. Besides differentiating extracellular vesicle subpopulations, the novel parameters introduced in this work (protein to lipid ratio, lipid bilayer order, and lipid composition), may prove useful for quality control of extracellular vesicle related basic and clinical studies

Changes in the proteome of sea urchin Paracentrotus lividus coelomocytes in response to LPS injection into the body cavity

Background The immune system of echinoderm sea urchins is characterised by a high degree of complexity that is not completely understood. The Mediterranean sea urchin Paracentrotus lividus coelomocytes mediate immune responses through phagocytosis, encapsulation of non-self particles, and production of diffusible factors including antimicrobial molecules. Details of these processes, and molecular pathways driving these mechanisms, are still to be fully elucidated. Principal findings In the present study we treated the sea urchin P. lividus with the bacterial lipopolysaccharide (LPS) and collected coelomocytes at different time-points (1, 3, 6 and 24 hours). We have shown, using label-free quantitative mass spectrometry, how LPS is able to modulate the coelomocyte proteome and to effect cellular pathways, such as endocytosis and phagocytosis, as soon as the immunomodulating agent is injected. The present study has also shown that treatment can modulate various cellular processes such as cytoskeleton reorganisation, and stress and energetic homeostasis. Conclusions Our data demonstrates, through mass spectrometry and the following functional annotation bioinformatics analysis, how the bacterial wall constituent is sufficient to set off an immune response inducing cytoskeleton reorganisation, the appearance of clusters of heat shock proteins (Hsp) and histone proteins and the activation of the endocytic and phagocytic pathways. Data are available via ProteomeXchange with identifier PXD008439

Mass spectrometry of extracellular vesicles

The review briefly summaries main features of extracellular vesicles, a joint terminology for exosomes, microvesicles, and apoptotic vesicles. These vesicles are in the center of interest in biology and medical sciences, and form a very active field of research. Mass spectrometry (MS), with its specificity and sensitivity, has the potential to identify and characterize molecular composition of these vesicles; but as yet there are only a limited, but fast-growing, number of publications that use MS workflows in this field. MS is the major tool to assess protein composition of extracellular vesicles: qualitative and quantitative proteomics approaches are both reviewed. Beside proteins, lipid and metabolite composition of vesicles might also be best assessed by MS techniques; however there are few applications as yet in this respect. The role of alternative analytical approaches, like gel-based proteomics and antibody-based immunoassays, are also mentioned. The objective of the review is to give an overview of this fast-growing field to help orient MS-based research on extracellular vesicles

Plant roots release small extracellular vesicles with antifungal activity

Extracellular Vesicles (EVs) play pivotal roles in cell-to-cell and inter-kingdom communication. Despite their relevant biological implications, the existence and role of plant EVs released into the environment has been unexplored. Herein, we purified round-shaped small vesicles (EVs) by differential ultracentrifugation of a sampling solution containing root exudates of hydroponically grown tomato plants. Biophysical analyses, by means of dynamic light scattering, microfluidic resistive pulse sensing and scanning electron microscopy, showed that the size of root-released EVs range in the nanometric scale (50–100 nm). Shot-gun proteomics of tomato EVs identified 179 unique proteins, several of which are known to be involved in plant-microbe interactions. In addition, the application of root-released EVs induced a significant inhibition of spore germination and of germination tube development of the plant pathogens Fusarium oxysporum, Botrytis cinerea and Alternaria alternata. Interestingly, these EVs contain several proteins involved in plant defense, suggesting that they could be new components of the plant innate immune system

The ArAac protein expressed in yeasts is sensitive to BKA.

<p>In the table on top the yeast strains and their expression of adenine nucleotide carriers and <i>SAL1</i> is indicated; ‘no’ signifies absence of the gene indicated in the left-most column; ‘<b>Yes</b>’ signifies the presence of the gene indicated in the left-most column. In the panels, reconstructed time courses of rhodamine 123 fluorescence (expressed as % of m) as functions of time are shown. In the top three panels mitochondria from strains RKY67-1C (black and grey traces) and MWY79/15 (green and magenta traces) express <i>SAL1</i>. In the bottom three panels, mitochondria from strains MWY84/4 (black traces) and MWY83/5 (green traces) do not express <i>SAL1</i>. The pH of the media is indicated on the top of each panel. EtOH signifies ethanol.</p

Strains used in this work.

<p>Strains used in this work.</p

Viability of modified and control yeast strains in response to BKA (1 M for the glucose-containing plates, and 20 nM for the glycerol-containing plates).

<p>Yeast strains were grown on media indicated in the panels in the absence or presence of BKA at 28°C. Each lane contains spots with initially 10⁶, 10⁵, 10⁴, 10³ and 10² cells (top to bottom). The genotypes of the strains are given in the table shown on the top of the figure: ‘no’ signifies absence of the gene indicated in the left-most column; ‘Yes’ signifies the presence of the gene indicated in the left-most column. 1 (MWY80) - <i>aac1Δ aac2Δ aac3Δ</i>, 2 (MWY85/9) - <i>sal1Δ</i>, 3 (MR6) – <i>WT</i>, 4 (RKY67-1C), 5 (RKY67-1D) – two independent clones <i>aac1Δ aac3Δ AAC2</i>, 6 (MWY79/15), 7 (MWY79/17) - two independent clones <i>aac1Δ aac3Δ ArAAC</i>, 8 (MWY84/4) - <i>aac1Δ aac3Δ AAC2 sal1Δ</i>, 9 (MWY83/5), 10 (MWY83/1) - two independent clones <i>aac1Δ aac3Δ ArAAC sal1Δ [AAC2].</i> Plates were scanned after 4 (glucose) or 6 (glycerol) days of incubation.</p

Primary sequence coverage of <i>ArAAC</i> expressed in yeast and <i>ArAAC</i> expressed in <i>Artemia franciscana</i> as determined from all mass spectrometry experiments described in ‘Materials and Methods’.

<p>Sequence coverage was based on searching peptide tandem spectra against the <i>Artemia franciscana</i> protein from NCBI, as described in the text. A: Primary sequence coverage of <i>Artemia franciscana</i> adenine nucleotide translocator protein (gi|308390607) expressed in <i>Artemia franciscana</i>. 70% Sequence coverage was obtained by identifying 210/301 residues (green) in the protein. B: Primary sequence coverage of <i>Artemia franciscana</i> adenine nucleotide translocator protein (gi|308390607) expressed in <i>Saccharomyces cerevisiae</i> (strain MWY79/15). 62% Sequence coverage was obtained by identifying 188/301 residues (green).</p