| Summary: | 碩士 === 國立陽明大學 === 生物化學研究所 === 92 === Rab3A is a small GTP-binding protein and is implicated in regulated exocytosis. It is thought that Rab3A is a negative regulator of exocytosis and exerts its action by interacting with many effectors including rabphilin3A, calmodulin, RIM. However, it is not known where, when, and how Rab3A regulates exocytosis. In my study, the fusion proteins of Rab3A and its mutants with various fluorescent proteins were constructed and over-expressed in PC12 cells. Then, an on-line total internal reflection fluorescence microscopic system was used to examine the subcellular localization and monitor the movement of these fusion proteins during exocytosis.
The fluorescence of EGFP-wild type Rab3A show punctuate patterns as expected because that Rab3A is known to associate with the secretory vesicles. The C-terminal truncated Rab3A and GDP form Rab3A mutant (Rab3A T36N) show diffuse patterns in the cytosol but the distribution of secretory vesicles appears to be normal, as shown by the distribution of the marker of secretory vesicles, neuropeptide Y. Interestingly, although the GTP form Rab3A mutant (Rab3A Q81L) shows punctuate pattern the number of vesicle near cell surface was more than that of the control cells. The results suggest that C-terminal is responsible for the interaction of Rab3A and vesicles and that vesicle interacts with GTP form Rab3A.
Changes in the fluorescence intensity of Rab3A and its mutants in individual PC12 cells upon stimulation were then monitored and analyzed. In general, the fluorescence of Rab3A decreased upon stimulation. The decrease is less in cells overexpressed GTP form, GDP form and C-terminal truncated Rab3A. The fluorescence of some cells show a transient increase before the decrease. This transient increase has never been observed in the GDP from Rab3A-expressed cells and was delayed in the cells that expressed GTP form Rab3A. For the mutant that lacks the interaction with rabphilin3A (Rab3A F59S), the transient increase was also delayed but to a less extent than that expressed the GTP from Rab3A. These results indicated that Rab3A is involved in the movement of vesicles toward cell membrane, and the final steps of exocytosis.
|
|---|
