Development of an in situ assay for simultaneous detection of the genomic and replicative form of PCV2 using padlock probes and rolling circle amplification

<p>Abstract</p> <p>Background</p> <p>In this study we utilized padlock probes and rolling circle amplification as a mean to detect and study the replication of porcine circovirus type 2 (PCV2) in cultured cells and in infected tissue. Porcine circovirus type 2 is a single-stranded circular DNA virus associated with several severe diseases, porcine circovirus diseases (PCVD) in pigs, such as postweaning multisystemic wasting syndrome. The exact reason and mechanisms behind the trigger of PCV2 replication that is associated with these diseases is not well-known. The virus replicates with rolling circle replication and thus also exists as a double-stranded replicative form.</p> <p>Results</p> <p>By applying padlock probes and rolling circle amplification we could not only visualise the viral genome but also discriminate between the genomic and the replicative strand in situ. The genomic strand existed in higher numbers than the replicative strand. The virus accumulated in certain nuclei but also spread into the cytoplasm of cells in the surrounding tissue. In cultured cells the average number of signals increased with time after infection.</p> <p>Conclusions</p> <p>We have developed a method for detection of both strands of PCV2 in situ that can be useful for studies of replication and in situ detection of PCV2 as well as of DNA viruses in general.</p

H9N2 virus-derived M1 protein promotes H5N6 virus release in mammalian cells: Mechanism of avian influenza virus inter-species infection in humans

H5N6 highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4 not only exhibits unprecedented intercontinental spread in poultry, but can also cause serious infection in humans, posing a public health threat. Phylogenetic analyses show that 40% (8/20) of H5N6 viruses that infected humans carried H9N2 virus-derived internal genes. However, the precise contribution of H9N2 virus-derived internal genes to H5N6 virus infection in humans is unclear. Here, we report on the functional contribution of the H9N2 virus-derived matrix protein 1 (M1) to enhanced H5N6 virus replication capacity in mammalian cells. Unlike H5N1 virus-derived M1 protein, H9N2 virus-derived M1 protein showed high binding affinity for H5N6 hemagglutinin (HA) protein and increased viral progeny particle release in different mammalian cell lines. Human host factor, G protein subunit beta 1 (GNB1), exhibited strong binding to H9N2 virus-derived M1 protein to facilitate M1 transport to budding sites at the cell membrane. GNB1 knockdown inhibited the interaction between H9N2 virus-derived M1 and HA protein, and reduced influenza virus-like particles (VLPs) release. Our findings indicate that H9N2 virus-derived M1 protein promotes avian H5N6 influenza virus release from mammalian, in particular human cells, which could be a major viral factor for H5N6 virus cross-species infection

Development of enabling technologies for single cell analysis with mass spectrometry

Mass spectrometry (MS) is an effective methodology for untargeted, label-free, highly multiplexed analyses of trace compounds based on their mass-to-charge ratios. For biological applications, these properties have generated interest in determining biomarkers of diseased states, detecting drug compounds and metabolites, and observing previously unknown chemical messengers. Recent developments in instrumentation have provided exquisite sensitivity with robust performance. A growing field of single cell chemical analysis has arisen around these figures of merit. While early reports utilized manual isolation and extraction, recent developments in high-throughput sampling have enabled the examination of large populations of cells. One such method includes the analysis of dispersed single cells on a flat surface. When cells are randomly seeded onto the surface, their locations have to be determined by optical imaging to direct acquisition of isolated cells efficiently. A variety of microprobe ionization sources are suitable for such analyses, though smaller probe footprints can utilize more densely seeded samples. This dissertation describes two technologies for performing single cell analysis with mass spectrometry. The first, synchronized desorption electrospray ionization (DESI), facilitates ambient ionization MS with high mass resolution, low duty cycle mass analyzers. The initial report utilized synchronized DESI for mass spectrometry imaging, but interrupting the desorption plume would be useful for profiling several locations on a surface in an arbitrary order for single cell analysis. The second methodology utilizes microscopy images to guide MS profiling. Specifically, image analysis software, called microMS, was developed to perform cell finding and correlate optical coordinates with the physical coordinates in a mass spectrometer. Since most of the functionality of microMS is decoupled from the mass spectrometer, the workflow can be easily extended to a variety of instruments. Using matrix-assisted laser desorption/ionization (MALDI) time of flight (TOF)-MS, rodent pancreatic islet cells were investigated and heterogeneous peptide processing was detected at the single cell level. With secondary ion mass spectrometry, disparate tissue from the mammalian nervous system was differentiated and further stratified into separate populations. A unique feature of such analyses is that only a fraction of the sample is consumed and the location of a cell is constant once the sample is dried. This property greatly simplifies sequential, follow-up analysis. As an example, MALDI-TOF-MS was utilized to rapidly screen a population of islet cells to select alpha and beta cell types. The locations of those cells were then targeted for liquid microjunction extraction in order to examine their metabolite profiles with capillary electrophoresis-MS. Finally, while microscopy-guided MS profiling is accurate enough to target single cells, the methodology is flexible enough to analyze much larger samples, including tissue sections or bacterial colonies. As an application, natural product mutant libraries were screened directly from E. coli colonies using microMS. The suite of technologies and protocols described increases the applicability of many mass spectrometers to characterize a range of cells, colonies and similar objects for their chemical composition

Supramolecular clustering of the cardiac sodium channel Nav1.5 in HEK293F cells, with and without the auxiliary β3-subunit.

Voltage-gated sodium channels comprise an ion-selective α-subunit and one or more associated β-subunits. The β3-subunit (encoded by the SCN3B gene) is an important physiological regulator of the heart-specific sodium channel, Nav1.5. We have previously shown that when expressed alone in HEK293F cells, the full-length β3-subunit forms trimers in the plasma membrane. We extend this result with biochemical assays and use the proximity ligation assay (PLA) to identify oligomeric β3-subunits, not just at the plasma membrane, but throughout the secretory pathway. We then investigate the corresponding clustering properties of the α-subunit and the effects upon these of the β3-subunits. The oligomeric status of the Nav1.5 α-subunit in vivo, with or without the β3-subunit, has not been previously investigated. Using super-resolution fluorescence imaging, we show that under conditions typically used in electrophysiological studies, the Nav1.5 α-subunit assembles on the plasma membrane of HEK293F cells into spatially localized clusters rather than individual and randomly dispersed molecules. Quantitative analysis indicates that the β3-subunit is not required for this clustering but β3 does significantly change the distribution of cluster sizes and nearest-neighbor distances between Nav1.5 α-subunits. However, when assayed by PLA, the β3-subunit increases the number of PLA-positive signals generated by anti-(Nav1.5 α-subunit) antibodies, mainly at the plasma membrane. Since PLA can be sensitive to the orientation of proteins within a cluster, we suggest that the β3-subunit introduces a significant change in the relative alignment of individual Nav1.5 α-subunits, but the clustering itself depends on other factors. We also show that these structural and higher-order changes induced by the β3-subunit do not alter the degree of electrophysiological gating cooperativity between Nav1.5 α-subunits. Our data provide new insights into the role of the β3-subunit and the supramolecular organization of sodium channels, in an important model cell system that is widely used to study Nav channel behavior.We would like to thank the Gurdon Institute Imaging Facility for use of their microscope and general assistance. This work was supported by a British Heart Foundation grant (PG/14/79/31102) to APJ and CLHH, The Wellcome Trust, award number: 105727/Z/14/Z to CLHH and a Medical Research Council grant (MR/K015591/1) to CLF, RAL, and STFC

New insights into the DT40 B cell receptor cluster using a proteomic proximity labeling assay.

In the vertebrate immune system, each B-lymphocyte expresses a surface IgM-class B cell receptor (BCR). When cross-linked by antigen or anti-IgM antibody, the BCR accumulates with other proteins into distinct surface clusters that activate cell signaling, division, or apoptosis. However, the molecular composition of these clusters is not well defined. Here we describe a quantitative assay we call selective proteomic proximity labeling using tyramide (SPPLAT). It allows proteins in the immediate vicinity of a target to be selectively biotinylated, and hence isolated for mass spectrometry analysis. Using the chicken B cell line DT40 as a model, we use SPPLAT to provide the first proteomic analysis of any BCR cluster using proximity labeling. We detect known components of the BCR cluster, including integrins, together with proteins not previously thought to be BCR-associated. In particular, we identify the chicken B-lymphocyte allotypic marker chB6. We show that chB6 moves to within about 30-40 nm of the BCR following BCR cross-linking, and we show that cross-linking chB6 activates cell binding to integrin substrates laminin and gelatin. Our work provides new insights into the nature and composition of the BCR cluster, and confirms SPPLAT as a useful research tool in molecular and cellular proteomics.JSR supported by Grants BB/J021091 and H024085/1 from the Biotechnology and Biological Sciences Research Council (UK). SWH & RWF supported by Grant G0500707 from the Medical Research Council (UK) and Grant 094470/Z/10/Z from the Wellcome Trust. BS & PEF supported by the DePaul University Research Council. This work was supported in part by grants from the Chinese Ministry of Science and Technology 973 Program (2012CB911000 and 2013CB910700) and the National Natural Science Foundation of China (31110103914, 31070656, 31000342, and 31270794).This is the final version of the article. It was first available from ASBMB via http://dx.doi.org/10.1074/jbc.M113.52957

Andy's Algorithms: new automated digital image analysis pipelines for FIJI.

Quantification of cellular antigens and their interactions via antibody-based detection methods are widely used in scientific research. Accurate high-throughput quantitation of these assays using general image analysis software can be time consuming and challenging, particularly when attempted by users with limited image processing and analysis knowledge. To overcome this, we have designed Andy's Algorithms, a series of automated image analysis pipelines for FIJI, that permits rapid, accurate and reproducible batch-processing of 3,3'-diaminobenzidine (DAB) immunohistochemistry, proximity ligation assays (PLAs) and other common assays. Andy's Algorithms incorporates a step-by-step tutorial and optimization pipeline to make batch image analysis simple for the untrained user and adaptable across laboratories. Andy's algorithms provide a simpler, faster, standardized work flow compared to existing programs, while offering equivalent performance and additional features, in a free to use open-source application of FIJI. Andy's Algorithms are available at GitHub, publicly accessed at https://github.com/andlaw1841/Andy-s-Algorithm

RAMP3 determines rapid recycling of atypical chemokine receptor-3 for guided angiogenesis.

Receptor-activity-modifying proteins (RAMPs) are single transmembrane-spanning proteins which serve as molecular chaperones and allosteric modulators of G-protein-coupled receptors (GPCRs) and their signaling pathways. Although RAMPs have been previously studied in the context of their effects on Family B GPCRs, the coevolution of RAMPs with many GPCR families suggests an expanded repertoire of potential interactions. Using bioluminescence resonance energy transfer-based and cell-surface expression approaches, we comprehensively screen for RAMP interactions within the chemokine receptor family and identify robust interactions between RAMPs and nearly all chemokine receptors. Most notably, we identify robust RAMP interaction with atypical chemokine receptors (ACKRs), which function to establish chemotactic gradients for directed cell migration. Specifically, RAMP3 association with atypical chemokine receptor 3 (ACKR3) diminishes adrenomedullin (AM) ligand availability without changing G-protein coupling. Instead, RAMP3 is required for the rapid recycling of ACKR3 to the plasma membrane through Rab4-positive vesicles following either AM or SDF-1/CXCL12 binding, thereby enabling formation of dynamic spatiotemporal chemotactic gradients. Consequently, genetic deletion of either ACKR3 or RAMP3 in mice abolishes directed cell migration of retinal angiogenesis. Thus, RAMP association with chemokine receptor family members represents a molecular interaction to control receptor signaling and trafficking properties.This work was supported by NIH Grants RO1-DK099156, RO1-HD060860, and RO1-HL129086 (to K.M.C.); American Heart Association Innovator Award 16IRG27260077 (to K.M.C.); NIH Grant F32-HL134279 (to D.I.M.); American Heart Association Grant 15POST25270006 (to R.B.D.); NIH Grant F31-HL143836 (to N.R.N.); Biotechnology and Biological Sciences Research Council (BBSRC) Grant BB/M00015X/2 (to G.L.); and BBSRC Doctoral Training Partnership Grant BB/JO14540/1 (to M.H.)

VE-cadherin facilitates BMP-induced endothelial cell permeability and signaling

Several vascular disorders, such as aberrant angiogenesis, atherosclerosis and pulmonary hypertension, have been linked to dysfunctional BMP signaling. Vascular hyperpermeability via distortion of endothelial cell adherens junctions is a common feature of these diseases, but the role of BMPs in this process has not been investigated. BMP signaling is initiated by binding of ligand to, and activation of, BMP type I (BMPRI) and type II (BMPRII) receptors. Internalization of VE-cadherin as well as c-Src kinase-dependent phosphorylation have been implicated in the loosening of cell-cell contacts, thereby modulating vascular permeability. Here we demonstrate that BMP6 induces hyperpermeabilization of human endothelial cells by inducing internalization and c-Src-dependent phosphorylation of VE-cadherin. Furthermore, we show BMP-dependent physical interaction of VE-cadherin with the BMP receptor ALK2 (BMPRI) and BMPRII, resulting in stabilization of the BMP receptor complex and, thereby, the support of BMP6-Smad signaling. Our results provide first insights into the molecular mechanism of BMP-induced vascular permeability, a hallmark of various vascular diseases, and provide the basis for further investigations of BMPs as regulators of vascular integrity, both under physiological and pathophysiological conditions

New protein-protein interactions of mitochondrial connexin 43 in mouse heart

Connexin 43 (Cx43), the gap junction protein involved in cell-to-cell coupling in the heart, is also present in the subsarcolemmal fraction of cardiomyocyte mitochondria. It has been described to regulate mitochondrial potassium influx and respiration and to be important for ischaemic preconditioning protection, although the molecular effectors involved are not fully characterized. In this study, we looked for potential partners of mitochondrial Cx43 in an attempt to identify new molecular pathways for cardioprotection. Mass spectrometry analysis of native immunoprecipitated mitochondrial extracts showed that Cx43 interacts with several proteins related with mitochondrial function and metabolism. Among them, we selected for further analysis only those present in the subsarcolemmal mitochondrial fraction and known to be related with the respiratory chain. Apoptosis-inducing factor () and the beta-subunit of the electron-transfer protein (), two proteins unrelated to date with Cx43, fulfilled these conditions, and their interaction with Cx43 was proven by direct and reverse co-immunoprecipitation. Furthermore, a previously unknown molecular interaction between and was established, and protein content and sub-cellular localization appeared to be independent from the presence of Cx43. Our results identify new protein-protein interactions between -Cx43, -Cx43 and - as possible players in the regulation of the mitochondrial redox state