Functional studies of tetraspanin glycoprotein M6Aa and M6Ab in PC-12 cells and zebrafish embryo

• Main Authors: Kai-Yun Huang, 黃凱筠
• Other Authors: Ming-Ting Lee
• Format: Others
• Language: en_US
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/02093741313256912594

博士 === 國立臺灣大學 === 生化科學研究所 === 100 === M6A is a member of the proteolipid protein (PLP/DM20) family and expressed specifically in neurons. M6A was first identified in the adult mouse brain as an antigen reacting with the monoclonal M6 antibody that was shown to cause disruption of normal neurite extension in cultured neurons from mouse cerebellum. There are duplicated forms of zebrafish M6A proteins, M6Aa and M6Ab. In in vivo experiments, we used both PC-12 cells and zebrafish to investigate the role of zebrafish M6Aa and M6Ab in filopodium formation and neurite outgrowth. We provide evidence demonstrating that zebrafish M6Aa and M6Ab were able to promote extensive filopodium formation in NGF-treated PC-12 cells, similar to the function of mammalian M6A. Furthermore, we showed that phosphorylation at serine 263 of zebrafish M6Ab contributed to this induction. Abolishing serine 263 phosphorylation site on M6Ab would greatly affect the extensive filopodium formation in PC-12 cells. On the other hand, only S263D could induce neurite outgrowth, while the wild-type M6Ab also induced neurite outgrowth only in the presence of a constitutively active CaMKII β1 in zebrafish embryos. The expression profile of M6Aa and M6Ab were characterized by whole-mount in situ hybridization and RT-PCR. Expression patterns are similar between M6Aa and M6Ab. RT-PCR results show that M6Aa and M6Ab are both abundant in brain and eye in zebrafish adult tissues. We observed that M6Aa and M6Ab are highly expressed in eye, telencephalon and hiddbrain from 24 to 96 hours post fertilization (hpf), through the whole-mount in situ hybridization. Morpholino-mediated knockdown of either M6Aa or M6Ab expression caused similar defects in brain, anteroposterior (AP) axes and tail development in zebrafish embryo. Knock-down of M6Aa or M6Ab causes decrease in Ca2+ influx and increase in the CaMKII kinase activity which results in increased expression of Ca2+ regulation-related genes. Taken together, our results reveal that the phosphorylation status of zebrafish M6Ab at serine 263 is critical in regulating filopodium formation. Morpholino knockdown of M6Aa or M6Ab triggers apoptosis, decreases Ca2+ influx and increase in CaMKII kinase activity suggesting that M6A protein could act as a Ca2+ regulator and is prerequisite for zebrafish neuronal development.