Establishment of cell array system and investigation of the downstream signaling of Syndecan-2 in filopodia and lamellipodia formation

• Main Authors: Ya-Ting Lei, 雷雅婷
• Other Authors: Yi-Ping Hsueh
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/12880463342643036766

碩士 === 國立陽明大學 === 微生物及免疫學研究所 === 93 === Cell microarray assays have been established by reverse transfection, a modified transfection method also known as solid-phase transfection, which is a systematic and high-throughput approach for studying the function of genes on a genome-wide scale. Cell microarray was therefore adapted for different cell lines, such as COS, HEK293T, rat pheochromocytoma PC12, and neuroblsatoma Neuro-2A cells. To verify the system for screening, we examine the morphology of different cells, which were transfected with defined cDNAs. Over-expression of syndecan-2, syndecan-3 or CINAP promoted filopodia formation of PC-12 cells. In contrast, over-expression of CASK or Tbr-1 had no effect on cell morphology of PC-12 cells. The results not only support that the system established is ready for genome-wide screening but also indicate that syndecan-2, syndecan-3 and CINAP play a role in cell morphology. Syndecan-2 is one of the members of syndecan family, a major heparan sulfate proteoglycan on plasma membrane (HSPG). Syndecans mediate cell-cell and cell-matrix adhesion thereby controlling cell movement and morphology. Syndecan-2 induced not only filopodia but also lamellipodia. The effect of syndecan-2 on cell morphology was reproducible in different cell lines. Rac1 dominant negative mutant blocked syndecan-2 induced phenotype, supporting that Rac1 is necessary for syndecan-2 induced phenotype. In addition to Rac1, several evidences support that PKA is the downstream signaling molecule of syndecan-2. First of all, PKA inhibitors – KT5720 and H89 – prevented the formation of filopodia and lamellipodia. Secondary, addition of forskolin in HEK293T cells also induced filopodia formation. Finally, PKA activity was slightly increased in syndecan-2 over-expressed cells. The cytoplasmic domain of syndecan-2 responsible for filopodia and lamellipodia was mapped by the C-terminal deletion mutants of syndecan-2. The C1 and V regions of syndecan-2 were found to be important for filopodia formation, and they are also responsible for the enhancement of PKA activity. Our results support that via the C1 and V region, syndecan-2 activates PKA pathway and induces filopodia and lamellipodia formation.