Box
1
At the heart of RME is the receptor, a protein that picks up and transports specific molecules and particles (ligands) across the cell (Fig.1). The surface of each cell is covered with transmembrane receptors. The ‘docking’ of the ligand to the receptor triggers a response in the cell which will be uniquely determined by the nature of the receptor/ligand complex and the type of cell. Following ligand binding, the receptor/ligand complexes enter the cell as coated vesicles (endosomes), which are formed when pits in the membrane pinch off. This internalisation can provide a signal for amplification or attenuation of a specific cellular response. Unfortunately, many harmful toxins and viruses can also be transported by this mechanism. Viruses for example have evolved to exploit RME by adapting their coat proteins so that they will bind strongly to certain receptors and be transported into the cell. The general outcome of this is infection leading to cell death, or multiplication and spreading of the pathogen.
So far most of our knowledge of RME has been achieved using cell-free methods where the cells are broken and constituents isolated prior to observation using e.g. electron microscopy, radiochemical assay, etc. Even though these techniques have provided many valuable insights into the process, they cannot provide accurate kinetic data. The Spectrocopy & Imaging group at the Biomedical College has developed the technique of Time Resolved Microfluorimetry on beamline 13.1 to study RME in real time on whole live cells (Fig.2). This method combines fluorimetry with optical microscopy. Prior to observation, the cell receptors and/or the cargo ligands are labelled with highly fluorescent molecules (fluorophores).
Time-resolved Microfluorimetry has been specifically designed to measure receptor/ligand interactions during signalling and endocytosis Ref: Cell. Mol. Biol. 46, 1103-1112. The instrument is capable of collecting fluorescence lifetime data from a small number of molecules (~103) using very low light levels (<10 mW/cm2) that greatly reduce fluorophore photobleaching and cell photodamage and is available to external users. The microflurimeter employs synchrotron radiation (SR) as a source of pulsed and continuously-tunable UV and visible light and has been specifically designed to measure molecular events in cells using fluorescence resonance energy transfer (FRET) Ref: Rev. Sci. Instrum. 67, 3716-3721, and time-resolved fluorescence anisotropy (TRFA) (see Box 2), Ref: Rev. Sci. Instrum. 69, 540-543. Small but highly fluorescent ‘donor’ and ‘acceptor’ probe molecules (fluorophores) can be covalently attached to a particular protein and this introduced into cells. FRET is the transfer of excited state energy from the donor fluorophore to the acceptor species and reports on distances in the range from about 2 to 10 nm (see Box 1). Changes in FRET between fluorescent-labelled ligands bound to cellular proteins can therefore report on structural changes in these proteins as they act in the cell. TRFA provides a quantitative measurement of the molecular motions of probe-bound proteins in cells with sub-nanosecond resolution, and can report on changes in protein segmental motion and on the constraints on the range of protein rotations imposed by its binding to supramolecular structures. The microfluorimeter can rapidly collect multiple consecutive FRET and TRFA decay profiles from a small number of fluorophore-labelled proteins in a group of about 20 cells.
The microfluorimeter has recently been used to follow the interactions of epidermal growth factor (EGF) with its receptor (EGFR) in human epithelial carcinoma A431 cells. The EGFR is a transmembrane tyrosine kinase that mediates the biological signal of a family of small polypeptide mitogens, which include EGF and transforming growth factor-alpha (TGFa), ultimately leading to cell proliferation. After binding to its receptor, EGF is sequestered by the cells and although the subsequent fate of EGF/receptor complexes is poorly understood, it is clear that interruption to the regulation of this process often results in loss of control leading to tumour formation. The superpositions of donor/acceptor-labelled EGF, TGFa and an antibody fragment (29.1) to the receptor’s ectodomain, as measured by FRET and TRFA, have showed the presence of constitutive oligomeric high-affinity EGFR which undergo a ligand-induced structural change in their ectodomains during signal transduction (Ref: Biophys. J. 82:2415-2427).
This new approach allows the possibility of simultaneously following the fate of receptor/ligand complexes spatially and temporally throughout the cell. It provides the means to monitor specific endocytic events that can be used to pin-point the major contributors in the cells regulatory mechanism. The initial stages of adenovirus (Ad) infection have also been monitored using these methods. Ads enter host cells to deliver their DNA into the cell nucleus and replicate. The first step of entry is the attachment of the virion to a permissive cell via interactions with CAR receptors. The virus is then internalised into a clathrin-coated vesicle following interactions with vitronectin-binding integrins. While considerable progress has been made in identifying cell receptors, the mechanisms for penetration, intracellular targeting, and uncoating remain largely unknown. Understanding these mechanisms is crucial for determining strategies for efficient translocation of DNA, a key requirement in the optimisation of Ad-mediated gene therapy. FRET measurements of the distance between fluorophores bound to the proteins in the viral coat has allowed us to successfully monitor the uncoating of the virus in real-time within the host cells, whilst confocal microscopy allowed the visualisation of the viruses before and after entering their host cells.
Boxes:
Box
1
Figures:
Fig.1.
The pathway of receptor mediated endocytosis.
Fig.2.
The time resolved microfluorimeter installed on beamline 13.1.
VUV-IR
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