The Study On Dynamic Tracking Of Vesicles Trafficking And The Relation Between Vesicles And Lipid Rafts

The vesicles transport is a complicated system in cell in which materials are transfered any abnormity of the system would results in corresponding deseases. Vesicles function as transport carries and can be regulated. Lipid-Raft is a microdomain containing special lipids and proteins between the two layers of cell membranes and plays an important role in vesicles transport. In this thesis, single melecular technique and total internal reflex fluorecence microscope imaging technique were used to investigate the location and mobility characteristics of Glucose Transporter 4 in the real-time dynamic and space location relation between the large dense core vesicles (LDCVs) and two kinds of lipid raft.The study will be very interesting in illustrations of the mechenisms of vesicles transport as well as of diabetes and helpneses in developing the drugs targeting at GLUT4 and benifical in further sighting into the functions of lipid-raft. It is an importance of homeostasis body's glucose. It is known that insulin is important hormone to regulate balance of blood glucose. Insulin helps to transport glucose cross plasma membrane into cells and decrease glucose level in blood. In fact, the effect of insulin is dependent on the effect of GLUT4 in some degree. Therefore, the GLUT4 becomes a key factor of regulation in balance of blood glucose. Evidences show the type2 diabetes is related with the abnormity of transportation of GLUT4. The transport of GLUT4 is regulated by many factors, which include its sequestration mechanism in cells, dynamic sorting, assemble secretory vesicles and structure of membrane lipid. GLUT4 transportation in cells has two recycling pathways. The cycle 1 occurs between the cell surface and endosomes and cycle 2 between the trans-Golgi network (TGN) and endosomes. It seems likely that various recycling loops must be coordinated in regulating the trafficking of intracellular GLUT4. Although different GLUT4 trafficking pathways have been proposed, it is still unclear whether GLUT4 is classified into distinct patterns according to their mobility. It would be clearly helpful to investigate the mobility of a single GLUT4 in living cells. Here we have labeled the GLUT4 by constructing a fusion protein linking EGFP to the C-terminus of mouse GLUT4. The GLUT4-EGFP fusion protein was expressed in 3T3-L1 cells by transient transfection. By using total internal reflection fluorescence microscopy (TIRFM), the corresponding fluorescence images of GLUT4-EGFP fusion protein were recorded in charge coupled device (CCD) at high time resolution. In order to obtain the sub-pixel displacement of GLUT4, we employed a Gaussian-based single particle tracking (SPT) method to resolve the kinetics of vesicle motion. We found that GLUT4 moves in a constrained fashion as if it is tethered by some intracellular structure. The distribution of the three-dimensional diffusion coefficients (D(3)) was a smooth continuum. Our results demonstrate that even the existence of different recycling loops for GLUT4, they still are organized in a continuous range of mobility rather than into separated classes in intact 3T3-L1, but they are organized in two kinds of mobility pools after stimulation by insulin. In addition, we have studied the relationships between the vesicles transport and lipid rafts. Lipid rafts are specific membrane microdomains that are rich in cholesterol, glycosphingolipids and glycosylphosphatidylinositol linked molecules. They play role in the system of protein trafficking. Here we have labeled the Caveolin and flotillin, which are two kinds of marker proteins of rafts by constructing a fusion protein that links EGFP, and labeled the NPY and SNAP25 by constructing a fusion protein that links DsRed. The fusion proteins were expressed in PC12 cells by transient transfection. By using total internal reflection fluorescence microscopy (TIRFM), the corresponding fluorescence images were recorded with charge coupled device (CCD) at high time resolution. We found that Caveolin is not in colocation with NPY during free-stimulation and stimulation with high concentration K+, and neither colocation with SNAP25. Flotillin does not distinctly coexisted with NPY. The results show that rafts may not take part in the transport procedure of LDCVs with NPY. The results are not consistent with that from biochemical approaches. The difference may be interpreted by two ways: on the one hand,it is possibility that rafts take part in small vesicles transport but no do in LDCVs, because the two kinds of vesicles have actually different mechanism of transport; on the other hand different results attribute to different experimental ways. It is very necessary that we must develop new methods to study rafts.