Cell-derived plasma membrane vesicles as minimal cells for analyzing transmembrane signaling

Cellular signalling is classically investigated by measuring optical or electrical properties of single or populations of living cells. Here we show how cell-derived vesicles can be used for anlaysing transmembrane signalling. The vesicles are derived from live mammalian cells by using either chemicals, or by optical tweezers and they comprise parts of the plasma membrane and cytosol of the mother cell. We measured in vesicles derived from individual cells with single molecule sensitivity the different steps of G protein-coupled receptor mediated signalling like ligand binding to receptors, subsequent G protein activation and finally receptor deactivation by interaction with arrestin. Cell-derived plasma membrane vesicles represent the smallest autonomous containers capable of performing cellular signaling reactions thus functioning like minimal cells. Observing cellular signalling reactions in individual vesicles opens the door for downscaling bioanalysis of cellular functions to the attoliter range, multiplexing single cell analysis and investigating receptor mediated signalling in multiarray format

Protein-Binding Microarray Analysis of Tumor Suppressor AP2α Target Gene Specificity

Cheap and massively parallel methods to assess the DNA-binding specificity of transcription factors are actively sought, given their prominent regulatory role in cellular processes and diseases. Here we evaluated the use of protein-binding microarrays (PBM) to probe the association of the tumor suppressor AP2α with 6000 human genomic DNA regulatory sequences. We show that the PBM provides accurate relative binding affinities when compared to quantitative surface plasmon resonance assays. A PBM-based study of human healthy and breast tumor tissue extracts allowed the identification of previously unknown AP2α target genes and it revealed genes whose direct or indirect interactions with AP2α are affected in the diseased tissues. AP2α binding and regulation was confirmed experimentally in human carcinoma cells for novel target genes involved in tumor progression and resistance to chemotherapeutics, providing a molecular interpretation of AP2α role in cancer chemoresistance. Overall, we conclude that this approach provides quantitative and accurate assays of the specificity and activity of tumor suppressor and oncogenic proteins in clinical samples, interfacing genomic and proteomic assays

Transmembrane Signaling Analysis in Model Membrane Systems

Studying the complex mechanisms of transducing an extracellular signal across a plasma membrane to initiate an intracellular responce is of fundamental importance for a cell’s function. Transmembrane signaling is the key allowing cells to sense and communicate with its environment. Signal transduction is primarily mediated by numerous membrane receptors. In this thesis we investigated the function of G-protein-coupled receptors (GPCR). GPCRs represent the largest and most diverse group of membrane proteins, encoded by more than 800 genes. They allow cells to recognize as diverse extracellular stimuli as photons, organic odorants, nucleotides, nucleosides, peptides, lipids and proteins, conferring to GPCRs a fundamental role in signal transduction. They are broadly distributed across the entire body and thus involved in a wide range of physiological and pathological processes, which makes GPCRs one of the main targets of modern medicines. Activation of GPCRs initiate numerous intracellular signaling pathways, involving a large number of proteins and molecules. They act as a central molecular activators and integrators of a complex network of signaling pathways. While most of the studies have been performed on cells, the complexity of cellular systems has raised the need to develop simpler model systems to assess the role of individual signaling com- ponents. In this thesis we present different model systems to study the functionality of GPCR signal transduction from the ligand binding to the activation of downstream cellular reactions. The first part of the thesis concerns the isolation of single receptors from cultured cells by detergent solubilization followed by purification and the subsequent receptor reconstitution into giant unilamellar vesicles (GUV). Detergent micelle receptors were analyzed by fluorescence correlation spectroscopy (FCS) and surface plasmon resonance (SPR) measurements. We could determine the conditions of functional solubilization of GPCRs by characterizing the amount of obtained receptor-detergent micelles, their homogeneity and the capacity of the receptor to selectively bind ligands. The receptors were then reconstituted into GUVs and ligand binding was monitored by confocal microscopy. In the second part of the thesis, we investigated different aspects properties of native plasma membrane vesicles. These vesicles are derived from live mammalian cells using v cytochalasin B or optical tweezers; they comprise parts of a cell’s plasma membrane and cytosol. After the characterization of their overall composition, we investigated their ability to mediated transmembrane activation of intracellular signaling reactions upon agonist binding to a GPCR. We could successfully monitor the ligand binding to receptor by FCS, the subsequent G-protein activation by intermolecular FRET and the receptor desensitization by monitoring the arrestin recruitment to the plasma membrane. These vesicles thus represent the smallest autonomous container performing cellular signaling reactions thus functioning like a minimized cell. Finally, preliminary experiments demonstrated the potential of native vesicles to serve as novel drug delivery system since they meet most of the requirements for an efficient drug delivery platform

Use of fourier transform infrared spectroscopy analysis of extracellular vesicles isolated from body fluids for diagnosing, prognosing and monitoring pathophysiological states and method therfor

The method analyzes biological fluids collected from patients, and uses attenuated total reflecting (ATR) sensors and spectral analysis by Fourier transform infrared spectroscopy (FTIR) to form a spectral fingerprint of extracellular vesicles (EVs) isolated from said biological fluids

Recombinant expression and functional characterization of the mouse olfactory receptor mOR256-17 in mammalian cells

Olfactory receptors (ORs) constitute the largest family of sensory membrane proteins in mammals. They play a key role within the olfactory system in recognizing and discriminating a nearly unlimited number of structurally diverse odorous molecules. The molecular basis of OR-mediated signal detection and transduction is poorly understood. This is due to difficulties in functional expression of ORs in high yields, preventing structural and biophysical studies at the level of the receptor protein. Here we report on recombinant expression of mouse receptor mOR256-17 yielding 10(6) ORs per cell in transiently transfected mammalian cells. For quantification and optimization of OR expression, we employed different fluorescent probes. Green fluorescent protein fused to the C-terminus of mOR256-17 allowed quantification of total cellular OR biosynthesis, and post-translational fluorescence labeling of a 12-amino acid polypeptide sequence at the N-terminus permitted the selective visualization and quantification of ORs at the plasma membrane using cell flow cytometry. Our dual-color labeling approach is generally applicable to quantification of membrane proteins for mammalian cell-based expression. By screening a large odorant compound library, we discovered a selective spectrum of potent mOR256-17-specific agonists essential for probing the receptor function for future scaled-up productions

Molecular and Dimensional Profiling of Highly Purified Extracellular Vesicles by Fluorescence Fluctuation Spectroscopy

Cells secrete extracellular vesicles (EVs) into their microenvironment that act as mediators of intercellulard communication under physiological conditions and in this context also actively participate in spreading various diseases. Large efforts are currently made to produce reliable EV samples and to develop, improve, and standardize techniques allowing their biophysical characterization. Here, we used ultrafiltration and size-exclusion chromatography for the isolation and a model-free fluorescence fluctuation analysis for the investigation of the physical and biological properties of EVs secreted by mammalian cells. Our purification strategy produced enriched samples of morphologically intact EVs free of extravesicular proteins and allowed labeling of marker molecules on the vesicle surface for single-vesicle analysis with single-molecule sensitivity. This novel approach provides information on the distribution profile of both EV size and relative expression level of a specific exosomal marker, deciphering the overall heterogeneity of EV preparations

Deamidation and Transamidation of Substance P by Tissue Transglutaminase Revealed by Electron-Capture Dissociation Fourier Transform Mass Spectrometry (Chem. Eur. J. 2/2011)

Tissue transglutaminase (tTGase) catalyzes both deamidation and transamidation of peptides and proteins by using a peptidyl glutamine as primary substrate. A precise consensus sequence for the enzyme is unknown and the ratio between deamidated and transamidated (or crosslinked) reaction products is highly substrate-dependent. Due to its overlapping body distribution with tTGase and ease of manipulation with tandem mass spectrometry, we used the neuropeptide substance P as a model to investigate the associated enzymatic kinetics and reaction products. Online liquid-chromatography Fourier-transform ion-cyclotron-resonance mass spectrometry (FT-ICR MS) combined with electron-capture dissociation (ECD) was employed to study the tTGase-induced modifications of substance P. A particular strength of ECD for peptide-enzyme reaction product monitoring is its ability to distinguish isomeric amino acids, for example, Glu and iso-Glu, by signature product ions. Our studies show that the primary reaction observed is deamidation, with the two consecutive glutamine residues converted sequentially into glutamate: first Gln(5), and subsequently Gln(6). We then applied ECD FT-ICR MS to identify the transamidation site on an enzymatically cross-linked peptide, which turned out to correspond to Gln(5). Three populations of substance-P dimers were detected that differed by the number of deamidated Gin residues. The higher reactivity of Gln(5) over Gln(6) was further confirmed by cross-linking SP with monodansylcadaverine (MDC). Overall, our approach described herein is of a general importance for mapping both enzymatically induced post-translational protein modifications and cross-linking. Finally, in vitro Ca-signaling assays revealed that the main tTGase reaction product, the singly deamidated SP (RPKPEQFFGLM-NH2), has increased agonist potency towards its natural receptor, thus confirming the biologically relevant role of deamidation

Molecular screening of cancer-derived exosomes by surface plasmon resonance spectroscopy

We reporton a generic method to detect and identify the molecular profile of exosomes either derived from cultured cell lines or isolated from biofluids. Exosomes are nanovesicles shed by cells into their microenvironment and carry the molecular identity of their mother cells. These vesicles are actively involved in intercellular communication under physiological conditions and ultimately in the spread of various diseases such as cancer. As they are accessible in most biofluids (e.g., blood, urine, or saliva), these biological entities are promising tools for cancer diagnostics, offering a non-invasive and remote access to the molecular state of the disease. The composition of exosomes derived from cancer cells depends on the sort and state of the tumor, requiring a screening of multiple antigens to fully characterize the disease. Here, we exploited the capacity of surface plasmon resonance biosensing to detect simultaneously multiple exosomal and cancer biomarkers on exosomes derived from breast cancer cells. We developed an immunosensor surface which provides efficient and specific capture of exosomes, together with their identification through their distinct molecular profiles. The successful analysis of blood samples demonstrated the suitability of our bioanalytical procedure for clinical use

Molecular screening of cancer-derived exosomes by surface plasmon resonance spectroscopy

We report on a generic method to detect and iden- tify the molecular profile of exosomes either derived from cultured cell lines or isolated from biofluids. Exosomes are nanovesicles shed by cells into their microenvironment and carry the molecular identity of their mother cells. These vesi- cles are actively involved in intercellular communication un- der physiological conditions and ultimately in the spread of various diseases such as cancer. As they are accessible in most biofluids (e.g., blood, urine, or saliva), these biological entities are promising tools for cancer diagnostics, offering a non- invasive and remote access to the molecular state of the dis- ease. The composition of exosomes derived from cancer cells depends on the sort and state of the tumor, requiring a screen- ing of multiple antigens to fully characterize the disease. Here, we exploited the capacity of surface plasmon resonance bio- sensing to detect simultaneously multiple exosomal and can- cer biomarkers on exosomes derived from breast cancer cells. We developed an immunosensor surface which provides efficient and specific capture of exosomes, together with their identification through their distinct molecular profiles. The successful analysis of blood samples demonstrated the suit- ability of our bioanalytical procedure for clinical us

The Structure of the Mouse Serotonin 5-HT3 Receptor in Lipid Vesicles

The function of membrane proteins is best understood if their structure in the lipid membrane is known. Here, we determined the structure of the mouse serotonin 5-HT3 receptor inserted in lipid bilayers to a resolution of 12 Å without stabilizing antibodies by cryo electron tomography and subtomogram averaging. The reconstruction reveals protein secondary structure elements in the transmembrane region, the extracellular pore, and the transmembrane channel pathway, showing an overall similarity to the available X-ray model of the truncated 5-HT3 receptor determined in the presence of a stabilizing nanobody. Structural analysis of the 5-HT3 receptor embedded in a lipid bilayer allowed the position of the membrane to be determined. Interactions between the densely packed receptors in lipids were visualized, revealing that the interactions were maintained by the short horizontal helices. In combination with methodological improvements, our approach enables the structural analysis of membrane proteins in response to voltage and ligand gating