Miniaturized single-cell analyses for biomedical applications

Numerous diseases affecting living beings have as a cause a modification of gene expression resulting in anomalous expression of proteins in up- or down-regulated fashion, with altered functions or expression of proteins that are not present in the cell in a normal situation. Among the well-known examples is cancer, where a series of DNA damages turn cells out of control of the rest of the body, from which they do not receive or follow control signals. To have a better understanding of the mechanism of these diseases, in particular the genetic diversity existing in a single affected tissue, it is necessary to be able to perform single-cell analyses that unveil the subpopulations of diseased cells leading to adequate medical treatment. In the course of this thesis, we report on the development of single-cell analysis methods which are of interest for medical applications. The first part focuses on the investigation of the viscoelastic properties of cell membranes by observing the back-relaxation of plasma membrane nanotubes which have been pulled out of a cell by an optical tweezer. Applied to the investigation of the viscoelastic properties of individual tumor cells taken from patients, we could show that this method can distinguish the state of progress of skin melanoma. In the second part, we used beads comprising a chemically modified surface to capture specifically one or several proteins inside single-cells. After extraction out of the cell, the affinity bead is transferred in a microfluidic stream of a fluorescently labeled antibody to detect and quantify the protein(s) of interest. The extraction and detection procedures occur inside a microfluidic platform to allow future automatization of the process. The last chapter focuses on the use of cell-derived extracellular vesicles (EVs) as diagnostic and therapeutic agents. With this goal in mind, we explore the potential of EVs as carriers to transfer genetic material into cells. To demonstrate the feasibility of this approach, we encapsulate EVs inside a giant unilamellar vesicle and release their cargo in a time- and space-controlled manner. This method could have therapeutic applications using a patient's self-EVs for gene therapy.

Conformational dynamics of cationic cys-loop receptors

Davor Kosanic

Understanding the functional dynamics of ligand-gated ion-channels (LGIC's) on the molecular level is the aim of this thesis. Members of the protein family of LGIC's are involved in the fast synaptic signal transmission and thereby play a central role in inter-cellular communication in higher multi-cellular organisms. Here, two prototypical members of ligand-gated cys-loop receptors were studied: the homopentameric type 3 serotonin receptor (5HT3AR) and the muscle-type nicotinic acetylcholine receptor (nAchR). The single-channel kinetics of the 5HT3AR was investigated using single-channel patch-clamp and single-molecule fluorescence techniques. Our principal findings are that the 5HT3AR channel shows up to five, agonist activated, nearly equidistant conductance levels, by binding of up to five agonist molecules. Detailed analysis of data obtained using serotonin the native agonist and quipazine-TMR, a fluorescent partial agonist at different ionic conditions has been performed. An allosteric calcium modulation was observed with serotonin but not with the partial agonist indicating receptor activation with altered conformational pathways. In conclusion, a functional model is proposed, describing a two-way conformational activation. Binding of each agonist opens a "conductance filter" within the addressed subunit. Additionally, a global gate, which can be modulated by the presence of calcium ions, determines the channel kinetics. In order to identify at least one of the hypothesized conformational pathways of the 5HT3A receptor activation, a mutation of aspartate to cysteine at the position 298 was introduced into the receptor variant devoid of cysteine residues. Labelling the mutated residue with a fluorescent dye resulted in a fluorescence quenching upon receptor activation. This effect was interpreted as a functional conformational rearrangement. Although no specific time-scale of such a (local or global) change in conformation could be determined on the molecular level, the application of the described approach as a possible test for functionality upon receptor solubilisation is discussed. The simultaneous observation of the discrete ligand binding and the subsequent single-channel gating was discussed for years in the LGIC field as the method of choice for direct and unbiased examination of the channel activation process. Here, current recordings of single-channel gating events were combined with optical single-molecule detection of fluorescent agonist binding. The time delay Δt between ligand binding and gating events of a gain-of-function variant of the foetal and the wild-type adult nAchR were measured on the single molecule level on living cells. For times above approximately 1 ms, the distribution of Δt for both receptor types obeys a power-law with an exponent close to 3/2, corresponding to a normal conformational diffusion. Increasing the time resolution, a monoexponential function describes the obtained data best for times below 1 ms, suggesting that an activation barrier in the free energy landscape of the receptor activation can be overcome within one step. Finally, a novel method for the fast and simple induction of synapse formation was developed. Optical tweezers were used for pulling membrane-nanotubes out of the plasma membrane of a living cell. Connecting these nanotubes to the cell membrane of up to 100 µm distant cells, axonal or dendritic protrusions can be mimicked. Functional electrical synapses, based on gap junctions, were artificially created. The results obtained in this work contribute to the functional characterisation of LGIC's in living cells, yielding models that might be extrapolated as concepts of conformational dynamics of proteins in general.

EPFL2009

HIV-1 Nef protein binds to the cellular protein PACS-1 to downregulate class I major histocompatibility complexes

Didier Trono

Major-histocompatibility-complex (MHC) proteins are used to display, on the surface of a cell, peptides derived from foreign material - such as a virus - that is infecting that cell. Cytotoxic T lymphocytes then recognize and kill the infected cell. The HIV-1 Nef protein downregulates the cell-surface expression of class I MHC proteins, and probably thereby promotes immune evasion by HIV-1. In the presence of Nef, class I MHC molecules are relocalized from the cell surface to the trans-Golgi network (TGN) through as-yet-unknown mechanisms. Here we show that Nef-induced downregulation of MHC-I expression and MHC-I targeting to the TGN require the binding of Nef to PACS-1, a molecule that controls the TGN localization of the cellular protein furin. This interaction is dependent on Nef's cluster of acidic amino acids. A chimaeric integral membrane protein containing Nef as its cytoplasmic domain localizes to the TGN after internalization, in an acidic-cluster- and PACS-1-dependent manner. These results support a model in which Nef relocalizes MHC-I by acting as a connector between MHC-I's cytoplasmic tail and the PACS-1-dependent protein-sorting pathway.

2000

Incorporation of Rhodopsin in Laterally Structured Supported Membranes: Observation of Transducin Activation with Spatially and Time-Resolved Surface Plasmon Resonance

Klaus Peter Hofmann, Horst Vogel

Rhodopsin-transducin coupling was used as an assay to investigate a laterally patterned membrane reconstituted with a receptor and its G protein. It served as a model system to show the feasibility to immobilize G protein-coupled receptors on solid supports and investigate receptor activation and interaction with G proteins by one-dimensional imaging surface plasmon resonance. Supported membranes were formed by the self-assembly of lipids and rhodopsin from detergent soln. onto functionalized gold surfaces. They formed micrometer-sized alternating regions of pure fluid phospholipid bilayers sepd. by bilayers composed of an outer phospholipid leaflet on a gold-attached inner thiolipid. Rhodopsin was found to incorporate preferentially into the phospholipid bilayer regions, whereas transducin was uniformly distributed over the entire outer surface of the supported patterned membrane. The influence of rhodopsin on the dark binding of transducin to lipid membranes was described quant. and compared with previously published data. Coupling reactions with transducin resembled closely the native system, indicating that the native functionality of rhodopsin was preserved in the supported membranes. The spatially varying properties of the membranes resulted in a pattern of rhodopsin activity on the surface. This combination of techniques is very promising for the investigation of the lateral diffusion of transducin, can be extended to include signaling proteins downstream of the G protein, and may be applied to functional screening of other G protein-coupled receptors. In the future, it may also serve as a basis for constructing biosensors based on receptor proteins. [on SciFinder (R)]

1998

Conformational dynamics of cationic cys-loop receptors

Davor Kosanic

EPFL2009