Preliminary Study On The Role Of Lipid Raft's Peculiar Proteins In Vesicular Transportation

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 investigeted the location and mobility characteristics of caveolin and flotillin 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 and benifical in further sighting into the functions of lipid-raft. In the present study,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, labeled the NPY by constructing a fusion protein that links DsRed, and labeled the SNAP25 by constructing a fusion protein that links TDimer2. 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 and SNAP25. 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. In addition, We used TIRFM and observed the motion of vesicles containing caveolin-1-EGFP were more active after stimulation with 60mM K+ in caveolin-1-EGFP transfected PC12 cells.At the same time,we have studied the dynamics of the vesicles including caveolin.