Extracellular Protein Interactions Research Articles

Allantodapsone is a Pan-Inhibitor of Staphylococcus aureus Adhesion to Fibrinogen, Loricrin, and Cytokeratin 10.

ABSTRACTStaphylococcus aureus infections have become a major challenge in health care due to increasing antibiotic resistance. We aimed to design small molecule inhibitors of S. aureus surface proteins to be developed as colonization inhibitors. We identified allantodapsone in an initial screen searching for inhibitors of clumping factors A and B (ClfA and ClfB). We used microbial adhesion assays to investigate the effect of allantodapsone on extracellular matrix protein interactions. Allantodapsone inhibited S. aureus Newman adhesion to fibrinogen with an IC50 of 21.3 μM (95% CI 4.5-102 μM), minimum adhesion inhibitory concentration (MAIC) of 100 μM (40.2 μg/mL). Additionally, allantodapsone inhibited adhesion of Lactococcus lactis strains exogenously expressing the clumping factors to fibrinogen (L. lactis ClfA, IC50 of 3.8 μM [95% CI 1.0–14.3 μM], MAIC 10 μM, 4.0 μg/mL; and L. lactis ClfB, IC50 of 11.0 μM [95% CI 0.9–13.6 μM], MAIC 33 μM, 13.3 μg/mL), indicating specific inhibition. Furthermore, the dapsone and alloxan fragments of allantodapsone did not have any inhibitory effect. Adhesion of S. aureus Newman to L2v loricrin is dependent on the expression of ClfB. Allantodapsone caused a dose dependent inhibition of S. aureus adhesion to the L2v loricrin fragment, with full inhibition at 40 μM (OD600 0.11 ± 0.01). Furthermore, recombinant ClfB protein binding to L2v loricrin was inhibited by allantodapsone (P < 0.0001). Allantodapsone also demonstrated dose dependent inhibition of S. aureus Newman adhesion to cytokeratin 10 (CK10). Allantodapsone is the first small molecule inhibitor of the S. aureus clumping factors with potential for development as a colonization inhibitor.IMPORTANCE S. aureus colonization of the nares and the skin provide a reservoir of bacteria that can be transferred to wounds that can ultimately result in systemic infections. Antibiotic resistance can make these infections difficult to treat with significant associated morbidity and mortality. We have identified and characterized a first-in-class small molecule inhibitor of the S. aureus clumping factors A and B, which has the potential to be developed further as a colonization inhibitor.

EGFR-Dependent Extracellular Matrix Protein Interactions Might Light a Candle in Cell Behavior of Non-Small Cell Lung Cancer.

Lung cancer remains the leading cause of cancer-related death and is associated with a poor prognosis. Lung cancer is divided into 2 main types: the major in incidence is non-small cell lung cancer (NSCLC) and the minor is small cell lung cancer (SCLC). Although NSCLC progression depends on driver mutations, it is also affected by the extracellular matrix (ECM) interactions that activate their corresponding signaling molecules in concert with integrins and matrix metalloproteinases (MMPs). These signaling molecules include cytoplasmic kinases, small GTPases, adapter proteins, and receptor tyrosine kinases (RTKs), particularly the epidermal growth factor receptor (EGFR). In NSCLC, the interplay between ECM and EGFR regulates ECM stiffness, angiogenesis, survival, adhesion, migration, and metastasis. Furthermore, some tumor-promoting ECM components (e.g., glycoproteins and proteoglycans) enhance activation of EGFR and loss of PTEN. On the other hand, other tumor-suppressing glycoproteins and -proteoglycans can inhibit EGFR activation, suppressing cell invasion and migration. Therefore, deciphering the molecular mechanisms underlying EGFR and ECM interactions might provide a better understanding of disease pathobiology and aid in developing therapeutic strategies. This review critically discusses the crosstalk between EGFR and ECM affecting cell behavior of NSCLC, as well as the involvement of ECM components in developing resistance to EGFR inhibition.

Molecular Insight into the Binding Property and Mechanism of Sulfamethoxazole to Extracellular Proteins of Anammox Sludge.

Antibiotics are widely found in nitrogen-containing wastewater, which may affect the operation stability of anaerobic ammonium oxidation (anammox)-based biological treatment systems. Extracellular polymeric substances (EPSs) of anammox sludge play a pivotal role in combining with antibiotics; however, the exact role and how the structure of the leading component of EPSs (i.e., extracellular proteins) changes under antibiotic stress remain to be elucidated. Here, the interaction between sulfamethoxazole and the extracellular proteins of anammox sludge was investigated via multiple spectra and molecular simulation. Results showed that sulfamethoxazole statically quenched the fluorescent components of EPSs, and the quenching constant of the aromatic proteins was the largest, with a value of 1.73 × 104 M-1. The overall binding was an enthalpy-driven process, with ΔH = -75.15 kJ mol-1, ΔS = -0.175 kJ mol-1 K-1, and ΔG = -21.10 kJ mol-1 at 35 °C. The O-P-O and C═O groups responded first under the disturbance of sulfamethoxazole. Excessive sulfamethoxazole (20 mg L-1) would decrease the ratio of α-helix/(β-sheet + random coil) of extracellular proteins, resulting in a loose structure. Molecular docking and dynamic simulation revealed that extracellular proteins would provide abundant sites to bind with sulfamethoxazole, through hydrogen bond and Pi-Akyl hydrophobic interaction forces. Once sulfamethoxazole penetrates into the cell surface and combines with the transmembrane ammonium transport domain, it may inhibit the NH4+ transport. Our findings enhance the understanding on the interaction of extracellular proteins and sulfamethoxazole, which may be valuable for deciphering the response property of anammox sludge under the antibiotic stress.

Abnormal expression of FAK and paxillin correlates with oral cancer invasion and metastasis.

Globally, the tenth most common cancer is the oral squamous cell carcinoma (OSCC) and the treatment strategy for improving of OSCC patients survival rate still remains a challenging one. Aberrant regulation of cell to extracellular matrix protein interactions leads to progression of human cancers. The focal adhesion kinase (FAK) and its downstream target paxillin have been implicated in cancer growth, migration, invasion and metastasis of different cancers. However, the clinical significance of FAK and paxillin in OSCC is not well characterized so far. In the present work, we showed that relative mRNA and protein expressions of FAK and paxillin are significantly higher in side population (SP) cells of OSCC cell line SCC-55. Concomitantly, the matrix metalloproteinase-11 (MMP-11) level is also significantly elevated in SP cells. The enhanced expression of paxillin is strongly correlated with increased chemoresistance, proliferation rate, migration and invasion potential of SP cells. In addition, inhibition of paxillin expression by RNAi makes SP cells more sensitive to chemotherapy drugs. Therefore, our results suggest that paxillin over expression might play a significant role in cancer progression, invasion and chemoresistance of OSCC.

Control of oviductal fluid flow by the G-protein coupled receptor Adgrd1 is essential for murine embryo transit

Dysfunction of embryo transport causes ectopic pregnancy which affects approximately 2% of conceptions in the US and Europe, and is the most common cause of pregnancy-related death in the first trimester. Embryo transit involves a valve-like tubal-locking phenomenon that temporarily arrests oocytes at the ampullary-isthmic junction (AIJ) where fertilisation occurs, but the mechanisms involved are unknown. Here we show that female mice lacking the orphan adhesion G-protein coupled receptor Adgrd1 are sterile because they do not relieve the AIJ restraining mechanism, inappropriately retaining embryos within the oviduct. Adgrd1 is expressed on the oviductal epithelium and the post-ovulatory attenuation of tubal fluid flow is dysregulated in Adgrd1-deficient mice. Using a large-scale extracellular protein interaction screen, we identified Plxdc2 as an activating ligand for Adgrd1 displayed on cumulus cells. Our findings demonstrate that regulating oviductal fluid flow by Adgrd1 controls embryo transit and we present a model where embryo arrest at the AIJ is due to the balance of abovarial ciliary action and the force of adovarial tubal fluid flow, and in wild-type oviducts, fluid flow is gradually attenuated through Adgrd1 activation to enable embryo release. Our findings provide important insights into the molecular mechanisms involved in embryo transport in mice.

Rapid and sensitive large-scale screening of low affinity extracellular receptor protein interactions by using reaction induced inhibition of Gaussia luciferase

Extracellular protein interactions mediated by cell surface receptors are essential for intercellular communication in multicellular organisms. Assays to detect extracellular interactions must account for their often weak binding affinities and also the biochemical challenges in solubilising membrane-embedded receptors in an active form. Methods based on detecting direct binding of soluble recombinant receptor ectodomains have been successful, but genome-scale screening is limited by the usual requirement of producing sufficient amounts of each protein in two different forms, usually a “bait” and “prey”. Here, we show that oligomeric receptor ectodomains coupled to concatenated units of the light-generating Gaussia luciferase enzyme robustly detected low affinity interactions and reduced the amount of protein required by several orders of magnitude compared to other reporter enzymes. Importantly, we discovered that this flash-type luciferase exhibited a reaction-induced inhibition that permitted the use of a single protein preparation as both bait and prey thereby halving the number of expression plasmids and recombinant proteins required for screening. This approach was tested against a benchmarked set of quantified extracellular interactions and shown to detect extremely weak interactions (KDs ≥ μM). This method will facilitate large-scale receptor interaction screening and contribute to the goal of mapping networks of cellular communication.

Hemostasis in the Lab, Approaching to the Correct Diagnosis in the Coagulopathies

Coagulation is the result of a coordinated interaction of blood proteins, circulating cells, endothelium cells and extracellular matrix proteins in the vessel wall. This process works in conjunction with other mechanisms that have to keep the blood in an effective flow, without hemorrity when damage occurs or without a thrombus when clotting begins. Most of the time, there is not so much information that a clotting test gives us, however, in the face of a real clinical scenario that concurrent with a personal haemorrhage, this is essential to take action. In addition, there is a thrombophilic scenario that has to be investigated. In all of them, it is important to note that doctors have to take into account that the pre-analytical phase plays a fundamental role, so it is very important to look around any situation that may impact the patient's results. There is a wide range of laboratory tests to evaluate different mechanisms of hemostasis, according to the history and characteristic of the clinical patient. With this in mind, there is a right time to ask for the right test, to get a better result for diagnosis and treatment. Analytical quality is the set of actions that are carried out in the laboratory with the aim of obtaining an accurate result, reflecting the real situation of the patient. Grossly, platelets, clotting factors and the fibrinolytic system could be studied, not all patients need all tests, they could be solved with a simple test, but may require a deep focus; that is why the patient with a history of mucocutaneous bleeding has to be studied for thrombocytopaties or VWD; a patient with muscle or joint bleeding has to be a factor deficiency test. We propose an initial approach to detecting the pathological situations essential to make decisions. Each person has different symptoms and different approaches, which is why identification and the right tests give us the greatest opportunity to get the right diagnosis.

Interaction of nano-quantum dots (CdSe@ZnS) and extracellular proteins in activated sludge revealed by bio-nano science

This work describes a novel approach for the purification of extracellular proteins (EP) from activated sludge, and provides an insight into the interaction between NPs and actual EP in the active sludge (AS) system.

High-Content Imaging for Large-Scale Detection of Low-Affinity Extracellular Protein Interactions

Extracellular protein interactions coordinate cellular responses with their local environment and have important roles in pathogen invasion and disease. Due to technical challenges associated with studying binding events at the cell surface, the systematic and reliable identification of novel ligand–receptor pairs remains difficult. Here, we describe the development of a cell-based assay using large-scale transient transfections and high-content imaging (HCI) to detect extracellular binding events. We optimized the parameters for efficient transfection of human cells with cDNA plasmids encoding full-length cell surface receptors in 384-well plates. Using a range of well-characterized structurally diverse low-affinity cell surface interactions, we show that transfected cells probed with highly avid ligands can be used to successfully identify ligand–receptor pairs using an HCI platform and automated image analysis software. To establish the high-throughput potential of this approach, we also screened a pool of ligands against a collection of 2455 cell surface expression clones and found that known ligand–receptor interactions could be robustly and consistently detected across the library using this technology.

Synthetic glycopolymers and natural fucoidans cause human platelet aggregation via PEAR1 and GPIbα

AbstractFucoidans are sulfated fucose-based polysaccharides that activate platelets and have pro- and anticoagulant effects; thus, they may have therapeutic value. In the present study, we show that 2 synthetic sulfated α-l-fucoside-pendant glycopolymers (with average monomeric units of 13 and 329) and natural fucoidans activate human platelets through a Src- and phosphatidylinositol 3-kinase (PI3K)–dependent and Syk-independent signaling cascade downstream of the platelet endothelial aggregation receptor 1 (PEAR1). Synthetic glycopolymers and natural fucoidan stimulate marked phosphorylation of PEAR1 and Akt, but not Syk. Platelet aggregation and Akt phosphorylation induced by natural fucoidan and synthetic glycopolymers are blocked by a monoclonal antibody to PEAR1. Direct binding of sulfated glycopolymers to epidermal like growth factor (EGF)–like repeat 13 of PEAR1 was shown by avidity-based extracellular protein interaction screen technology. In contrast, synthetic glycopolymers and natural fucoidans activate mouse platelets through a Src- and Syk-dependent pathway regulated by C-type lectin-like receptor 2 (CLEC-2) with only a minor role for PEAR1. Mouse platelets lacking the extracellular domain of GPIbα and human platelets treated with GPIbα-blocking antibodies display a reduced aggregation response to synthetic glycopolymers. We found that synthetic sulfated glycopolymers bind directly to GPIbα, substantiating that GPIbα facilitates the interaction of synthetic glycopolymers with CLEC-2 or PEAR1. Our results establish PEAR1 as the major signaling receptor for natural fucose-based polysaccharides and synthetic glycopolymers in human, but not in mouse, platelets. Sulfated α-l-fucoside-pendant glycopolymers are unique tools for further investigation of the physiological role of PEAR1 in platelets and beyond.

Alpha-v–containing integrins are host receptors for the Plasmodium falciparum sporozoite surface protein, TRAP

Malaria-causing Plasmodium sporozoites are deposited in the dermis by the bite of an infected mosquito and move by gliding motility to the liver where they invade and develop within host hepatocytes. Although extracellular interactions between Plasmodium sporozoite ligands and host receptors provide important guidance cues for productive infection and are good vaccine targets, these interactions remain largely uncharacterized. Thrombospondin-related anonymous protein (TRAP) is a parasite cell surface ligand that is essential for both gliding motility and invasion because it couples the extracellular binding of host receptors to the parasite cytoplasmic actinomyosin motor; however, the molecular nature of the host TRAP receptors is poorly defined. Here, we use a systematic extracellular protein interaction screening approach to identify the integrin αvβ3 as a directly interacting host receptor for Plasmodium falciparum TRAP. Biochemical characterization of the interaction suggests a two-site binding model, requiring contributions from both the von Willebrand factor A domain and the RGD motif of TRAP for integrin binding. We show that TRAP binding to cells is promoted in the presence of integrin-activating proadhesive Mn2+ ions, and that cells genetically targeted so that they lack cell surface expression of the integrin αv-subunit are no longer able to bind TRAP. P. falciparum sporozoites moved with greater speed in the dermis of Itgb3-deficient mice, suggesting that the interaction has a role in sporozoite migration. The identification of the integrin αvβ3 as the host receptor for TRAP provides an important demonstration of a sporozoite surface ligand that directly interacts with host receptors.

Selectin-independent adhesion during ovarian cancer metastasis

PurposeOvarian cancer (OvCa) progression mainly takes place by intraperitoneal spread. Adhesion of tumor cells to the mesothelial cells which form the inner surface of the peritoneum is a crucial step in this process. Cancer cells use in principle different molecules of the leukocyte adhesion cascade to facilitate adhesion. This cascade is initiated by selectin-ligand interactions followed by integrin - extracellular matrix protein interactions. Here we address the question whether all tumor cells predominantly employ selectin-dependent leukocyte-like adhesion cascade (SDAC) or whether they use integrin mediated adhesion for OvCa progression as well. MethodsA comparative transcriptomic analysis of the human OvCa cell lines OVCAR8 and SKOV3 was performed. Intraperitoneal xenograft model of OVCAR8 cells was used to determine whether there is a correlation between SDAC gene expression and the metastatic potential of the control cells and the cells overexpressing c-Fos. Transcriptomic analysis of OVCAR8 and SKOV3 samples was performed using microarrays. ResultsOne-third of the protein-coding genes involved in SDAC exhibited lower expression levels in OVCAR8 than in SKOV3 cells. In contrast to SKOV3 cells, c-Fos overexpression in OVCAR8 cells did not significantly influence the expression of SDAC genes. Intraperitoneal xenograft model of OVCAR8 cells unexpectedly demonstrated that the aggressiveness of OVCAR8 tumors was not depended on the c-Fos expression level and was comparable to that of SKOV3 control tumors. Gene expression analysis of tumors suggests that SKOV3-derived tumor progression was mainly depended on SDAC. Progression of OVCAR8 tumors relied on other cell adhesion molecules that do not interact with selectins. ConclusionsHigh expression of c-Fos in ovarian cancer cells is not always associated with reduced metastatic potential. Low expression level of SDAC genes may not ensure low OvCa metastatic potential hence alternative adhesion mechanisms involving laminin-integrin interactions exist as well.

Role of LTBP4 in alveolarization, angiogenesis, and fibrosis in lungs.

Deficiency of the extracellular matrix protein latent transforming growth factor-β (TGF-β)-binding protein-4 (LTBP4) results in lack of intact elastic fibers, which leads to disturbed pulmonary development and lack of normal alveolarization in humans and mice. Formation of alveoli and alveolar septation in pulmonary development requires the concerted interaction of extracellular matrix proteins, growth factors such as TGF-β, fibroblasts, and myofibroblasts to promote elastogenesis as well as vascular formation in the alveolar septae. To investigate the role of LTBP4 in this context, lungs of LTBP4-deficient (Ltbp4-/-) mice were analyzed in close detail. We elucidate the role of LTBP4 in pulmonary alveolarization and show that three different, interacting mechanisms might contribute to alveolar septation defects in Ltbp4-/- lungs: 1) absence of an intact elastic fiber network, 2) reduced angiogenesis, and 3) upregulation of TGF-β activity resulting in profibrotic processes in the lung.

Technologies for Proteome-Wide Discovery of Extracellular Host-Pathogen Interactions.

Pathogens have evolved unique mechanisms to breach the cell surface barrier and manipulate the host immune response to establish a productive infection. Proteins exposed to the extracellular environment, both cell surface-expressed receptors and secreted proteins, are essential targets for initial invasion and play key roles in pathogen recognition and subsequent immunoregulatory processes. The identification of the host and pathogen extracellular molecules and their interaction networks is fundamental to understanding tissue tropism and pathogenesis and to inform the development of therapeutic strategies. Nevertheless, the characterization of the proteins that function in the host-pathogen interface has been challenging, largely due to the technical challenges associated with detection of extracellular protein interactions. This review discusses available technologies for the high throughput study of extracellular protein interactions between pathogens and their hosts, with a focus on mammalian viruses and bacteria. Emerging work illustrates a rich landscape for extracellular host-pathogen interaction and points towards the evolution of multifunctional pathogen-encoded proteins. Further development and application of technologies for genome-wide identification of extracellular protein interactions will be important in deciphering functional host-pathogen interaction networks, laying the foundation for development of novel therapeutics.

Extracellular CADM1 interactions influence insulin secretion by rat and human islet β-cells and promote clustering of syntaxin-1.

Contact between β-cells is necessary for their normal function. Identification of the proteins mediating the effects of β-cell-to-β-cell contact is a necessary step toward gaining a full understanding of the determinants of β-cell function and insulin secretion. The secretory machinery of the β-cells is nearly identical to that of central nervous system (CNS) synapses, and we hypothesize that the transcellular protein interactions that drive maturation of the two secretory machineries upon contact of one cell (or neural process) with another are also highly similar. Two such transcellular interactions, important for both synaptic and β-cell function, have been identified: EphA/ephrin-A and neuroligin/neurexin. Here, we tested the role of another synaptic cleft protein, CADM1, in insulinoma cells and in rat and human islet β-cells. We found that CADM1 is a predominant CADM isoform in β-cells. In INS-1 cells and primary β-cells, CADM1 constrains insulin secretion, and its expression decreases after prolonged glucose stimulation. Using a coculture model, we found that CADM1 also influences insulin secretion in a transcellular manner. We asked whether extracellular CADM1 interactions exert their influence via the same mechanisms by which they influence neurotransmitter exocytosis. Our results suggest that, as in the CNS, CADM1 interactions drive exocytic site assembly and promote actin network formation. These results support the broader hypothesis that the effects of cell-cell contact on β-cell maturation and function are mediated by the same extracellular protein interactions that drive the formation of the presynaptic exocytic machinery. These interactions may be therapeutic targets for reversing β-cell dysfunction in diabetes.

P-selectin is a host receptor for Plasmodium MSP7 ligands.

BackgroundPlasmodium parasites typically elicit a non-sterile but protective immune response in human host populations, suggesting that the parasites actively modulate normal immunological mechanisms. P-selectin is a cell surface receptor expressed in mammals, that is a known component of the inflammatory response against pathogens and has been previously identified as a host factor that influences malaria-associated pathology both in human patients and rodent infection models.MethodsTo better understand the molecular mechanisms underlying the involvement of P-selectin in the pathogenesis of malaria, a systematic extracellular protein interaction screen was used to identify Plasmodium falciparum merozoite surface protein 7 (MSP7) as a binding partner of human P-selectin. This interaction, and those occurring between P-selectin and Plasmodium MSP7 homologues, was characterized biochemically.ResultsPlasmodium falciparum MSP7 and P-selectin were shown to bind each other directly via the N-terminus of PfMSP7 and the P-selectin C-type lectin and EGF-like domains. Orthologous proteins in the murine parasite Plasmodium berghei (PbMSRP1 and PbMSRP2) and mouse P-selectin also interacted. Finally, P-selectin, when complexed with MSP7, could no longer bind to its endogenous carbohydrate ligand, Sialyl-LewisX.ConclusionsNovel interactions were identified between Plasmodium MSP7 protein family members and host P-selectin receptors. Since PfMSP7 could prevent interactions between P-selectin and its leukocyte ligands, these results provide a possible mechanism for the known immunomodulatory effects of both MSP7 and P-selectin in malaria infection models.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0750-z) contains supplementary material, which is available to authorized users.

Unusual remodeling of the hyalinization band in vulval lichen sclerosus by type V collagen and ECM 1 protein

OBJECTIVES:The vulva is the primary site affected in lichen sclerosus, a chronic dermatosis in women that is histologically characterized by a zone of collagen remodeling in the superior dermis. The normal physiological properties of the vulva depend on the assembly of collagen types I (COLI), III (COLIII) and V (COLV), which form heterotypic fibers, and extracellular matrix protein interactions. COLV regulates the heterotypic fiber diameter, and the preservation of its properties is important for maintaining normal tissue architecture and function. In the current work, we analyzed the expression of COLV and its relationship with COLI, COLIII, elastic fibers and extracellular matrix protein 1 in vulvar biopsies from patients with lichen sclerosus.METHODS:Skin biopsies from 21 patients with lichen sclerosus, classified according to Hewitt histological criteria, were studied and compared to clinically normal vulvar tissue (N=21). Morphology, immunohistochemistry, immunofluorescence, 3D reconstruction and morphometric analysis of COLI, COLIII, COLV deposition, elastic fibers and extracellular matrix 1 expression in a zone of collagen remodeling in the superior dermis were performed.RESULTS:A significant decrease of elastic fibers and extracellular matrix 1 protein was present in the hyalinization zone of lichen sclerosus compared to healthy controls. The non-homogeneous distribution of collagen fibers visualized under immunofluorescence in the hyalinization zone of lichen sclerosus and control skin was confirmed by histomorphometry. Lichen sclerosus dermis shows a significant increase of COLI, COLIII and COLV expression compared to the healthy controls. Significant inverse associations were found between elastic fibers and COLV and between COLV and extracellular matrix 1 expression. A direct association was found between elastic fiber content and extracellular matrix 1 expression. Tridimensional reconstruction of the heterotypic fibers of the lichen sclerosus zone of collagen remodeling confirmed the presence of densely clustered COLV.CONCLUSIONS:Increased deposition of abnormal COLV and its correlation with extracellular matrix 1 and elastic fibers suggest that COLV may be a trigger in the pathogenesis of lichen sclerosus.

Analysis of splice variants for the C. elegans orthologue of human neuroligin reveals a developmentally regulated transcript

Neuroligins are synaptic adhesion molecules and important determinants of synaptic function. They are expressed at postsynaptic sites and involved in synaptic organization through key extracellular and intracellular protein interactions. They undergo trans-synaptic interaction with presynaptic neurexins. Distinct neuroligins use differences in their intracellular domains to selectively recruit synaptic scaffolds and this plays an important role in how they encode specialization of synaptic function. Several levels of regulation including gene expression, splicing, protein translation and processing regulate the expression of neuroligin function. We have used in silico and cDNA analyses to investigate the mRNA splicing of the Caenorhabditis elegans orthologue nlg-1. Transcript analysis highlights the potential for gene regulation with respect to both temporal expression and splicing. We found nlg-1 splice variants with all the predicted exons are a minor species relative to major splice variants lacking exons 13 and 14, or 14 alone. These major alternatively spliced variants change the intracellular domain of the gene product NLG-1. Interestingly, exon 14 encodes a cassette with two distinct potential functional domains. One is a polyproline SH3 binding domain and the other has homology to a region encoding the binding site for the scaffolding protein gephyrin in mammalian neuroligins. This suggests differential splicing impacts on NLG-1 competence to recruit intracellular binding partners. This may have developmental relevance as nlg-1 exon 14 containing transcripts are selectively expressed in L2–L3 larvae. These results highlight a developmental regulation of C. elegans nlg-1 that could play a key role in the assembly of synaptic protein complexes during the early stages of nervous system development.

Characterizing the role of integrin alpha8 in mediating CD4+ T lymphocyte migration across the blood–brain barrier in multiple sclerosis

Multiple sclerosis (MS) is characterized by active migration of peripheral blood (PB) leukocytes across the blood–brain barrier (BBB) into the central nervous system (CNS). Integrins, which are heterodimeric alpha/beta transmembrane proteins, facilitate this process by mediating cell–cell or cell–extracellular matrix protein interactions. Specifically, the beta1 integrin family has been associated with migration of PB lymphocytes across the BBB, as supported by the clinical efficacy of Natalizumab, a monoclonal antibody targeting the alpha4 subunit of integrin alpha4beta1.

Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within pancreatic islets and act in a coordinated fashion to maintain glucose homeostasis. It is becoming increasingly clear that interactions between transmembrane proteins on the surfaces of adjacent beta cells are important determinants of beta-cell function. Elucidation of the roles of particular transcellular interactions by knockdown, knockout or overexpression studies in cultured beta cells or in vivo necessitates direct perturbation of mRNA and protein expression, potentially affecting beta-cell health and/or function in ways that could confound analyses of the effects of specific interactions. These approaches also alter levels of the intracellular domains of the targeted proteins and may prevent effects due to interactions between proteins within the same cell membrane to be distinguished from the effects of transcellular interactions. Here a method for determining the effect of specific transcellular interactions on the insulin secreting capacity and responsiveness of beta cells is presented. This method is applicable to beta-cell lines, such as INS-1 cells, and to dissociated primary beta cells. It is based on coculture models developed by neurobiologists, who found that exposure of cultured neurons to specific neuronal proteins expressed on HEK293 (or COS) cell layers identified proteins important for driving synapse formation. Given the parallels between the secretory machinery of neuronal synapses and of beta cells, we reasoned that beta-cell functional maturation might be driven by similar transcellular interactions. We developed a system where beta cells are cultured on a layer of HEK293 cells expressing a protein of interest. In this model, the beta-cell cytoplasm is untouched while extracellular protein-protein interactions are manipulated. Although we focus here primarily on studies of glucose-stimulated insulin secretion, other processes can be analyzed; for example, changes in gene expression as determined by immunoblotting or qPCR.