Preliminary Study On The Exocytosis Of Two Different Kinds Of Cell Granules

Normally, vesicles are used to transport cargo molecules in eukaryotic cells. Different types of cargo molecules have different trafficking mechanism. In this paper, we have successfully labeled two different kinds of vesicles with fluorescent protein to study the dynamics, molecular mechanism of their trafficking in PC12 cells.Although vesicles are used in many cells to transport cargo molecules, some cells, such as natural killer (NK) cells, use secretory lysosomes. NK cells are able to kill cancer cell, virus, parasitical bacteria in cells and aging or mutant cells. To clarify the mechanism of the secretion of NK cells is helpful for us to treat some human diseases. We have studied the secretion and regulation mechanism of the secretoty lysosomes in NK cells. Meanwhile, their biogenesis was studied.1. Total Internal Reflection Fluorescence Microscopy (TIRFM) was employed to study the difference in trafficking and secretion between large dense-cored vesicles (LDCVs) and synaptic vesicle-like microvesicles (SLMVs) in PC12 cells. To visualize different vesicles, we specifically labeled LDCVs by fusing neuropeptide Y (NPY) with Discosoma sp. red fluorescent protein variant 2 (DsRed2) and SLMVs by fusing the vesicle acetylcholine transporter (VAChT) with enhanced green fluorescent protein (EGFP). The result showed that there were no significant differences in dynamics for these two kinds of vesicles under the 5mM extracellular calcium conditions, and more SLMVs were located within the subplasmalemmal area than LDCVs.2. Secretion was found in FM1-43 labeled NK cells after 5min of stimulation by 100nM PMA. By optical sectioning technology, the significant increase of the number of secretory lysosomes can be found in acridine orange (AO) labeled NK cells after the activation of porcine endothelial cells or the stimulation of PMA. G?6983, an inhibitor of protein kinase C (PKC), can block this kind of increase, but brefeldin A (BFA) which can break down Golgi can not. The change of the number of secretory lysosomes was further confirmed by the 3-D reconstruction of the NK cells, which showed the distribution of the secretory lysosomes. All of these suggested that secretory lysosomes were not generated from Golgi and they were regulated by PKC signal pathway.