Investigation On Motion, Congregation And Internalization Of Insulin-Insulin Receptor Complexes In Living Cells At Single Molecule Level

In chapter one, single molecule detection (SMD) techniques are reviewed briefly. These techniques are powerful tools for investigation of dynamics and kinetics of molecules. Single-molecule visualization with a high spatial and temporal resolution provides a direct way to observe biological events inside or/and outside cells at the single-molecule level. Fluorescence microscopy is rapidly expanding single molecule detection techniques into all fields of cell and molecular biology. Total internal reflection fluorescence microscopy (TIRFM), laser scanning confocal fluorescence microscopy (LSCFM), near-field scanning optical microscopy (NSOM), multi-photon excitation microscopy (MPEM), fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM) and fluorescence correlation spectroscopy (FCS) are introduced. A brief summary of their applications is also provided, followed by presentations of the instrumentation of the technique.In chapter two, we developed a simple intracellular fluorescence microscopy for acquisition of high-resolution images of single biomolecules labeled with quantum dots on apical plasma membrane, in cell interior and on basal plasma membrane of living cells. Since a lot of biological process is not restricted at the cell surface, but proceeds in the whole cell from the cell surface to the cell interior, this method has more advantageous than the other single molecule techniques, such as laser scanning confocal fluorescence microscopy and epi-illumination fluorescence microscopy. In the intracellular fluorescence microscopy, the incident angle of a laser beam is adjusted to produce total internal reflection at the apical surface of a single cell. In this case, the whole cell is illuminated by laser, and the fluorescent molecules outside the cell are not excited. Therefore, the background is reduced and the signal-noise-ratio is improved notably. Through visualizing single quantum dots labeled to insulin molecules on the apical plasma membrane, in the cell interior and on the basal plasma membrane of the cell, it is demonstrated that the method has following advantages: 1. The image on the apical plasma membrane of a living cell can be taken, which makes investigations of the biological events on the cellular membrane possible without the interference from the coverslip.2. The resolution of single biomolecules on fluorescence images taken at different positions of the cytoplasm is much better than that taken by using Epi-FM.3. The single molecule images with a high-resolution on the basal plasma membrane can also be obtained.4. Imaging at different positions of a living cell with a high temporal and spatial resolution can be carried out conveniently using the method. It is superior for studying the dynamics and kinetics of biomolecules in cell interior at single molecule level.In chapter three, the intracellular fluorescence microscopy provided in the chapter two are used for studying the early events of insulin signaling in living cells at single molecule level, combining with the other detection methods such as epi-illumination fluorescence microscopy and laser scanning confocal fluorescence microscopy. Insulin binding leads to its receptor (IR) activation and the initiation of signal transduction pathways. The early processes include the moving, congregation and internalization of IR-insulin complexes, which have been studied by morphological methods and biochemical methods. However, there were no reports about real time tracking and number of ligand-receptors in endosomes by now. Here, we observed these early processes using single molecule fluorescence detection. These are:1. The lateral moving and the congregation of IR-insulin complexes on the plasma membrane are traced at single molecule level. Different numbers of IR-insulin in congeries and the different congregation processes can be observed. First, insulin in the solution binds to its receptor (IR) on the cell membrane to produce a complex (IR-insulin). Then, congregation of two IR-insulin complexes happens. After that, a free insulin molecule in the solution binds to an IR and congregates with this dual-IR-insulin congregation. Thus a triple-IR-insulin congregation is formed. The results show the variation and the asynchronism of the congregation processes, which can not be revealed using conventional methods.2. The number of IR-insulin complexes in a congregation is calculated to be 2 to 14. The number of the congregation with more IR-insulin complexes is less than that with fewer IR-insulin complexes.3. It was found that the congregations on the apical surface are more than that on the basal surface. A possible reason is that the coverslip, on which the cell is attached, restricts moving of IR on the plasma membrane.4. It is demonstrated that the phenomena mentioned above are induced by the activation of insulin to IR as comparing with the behavior of IR in the present of IR antibody.In chapter 4, intracellular fluorescence microscopy provided in the chapter two is applied for tracking the internalization and the endosomes of IR-insulin induced by insulin for living HL-7702 cells. For the importance of the insulin-mediated internalization of IR into emdosomes, which plays a crucial role in effecting and regulating signal transduction in addition to modulating the levels of circulating insulin and the cellular concentration of IR in target tissues, many studies about internalization and endosomes transportation have been reported. In these investigations, electron microscopy and chemical and biological methods are widely used. However, using the static imaging methods and assemble average detection with destroying of the cell structure and endosomes transport systems, the individual biological events cannot be revealed. We trace these events in living cells. These are:1. Forming of IR-insulin endosomes is induced by the activation of insulin to IR, not the circle processes of IR based on the images of single IR-insulin complexes or IR-insulin endosomes, which are taken by the intracellular fluorescence microscopy, and comparing with the cell behavior in the present IR antibody.2. After single IR-insulin complexes on the plasma membrane increases, endosomes are formed at the juxtamembranal cytoplasm. Then, some big endosomes appears near the karyon.3. The different internalization rates of IR-insulin complex from the plasma membrane to the cell interior are found.4. The motion of the endosomes is irregular and restricted in a moving region.