Study of the physiological role of KIF3A and KIF5B–using PC12 cell as a model system

• Main Authors: Ko-Wei Sye, 謝克威
• Other Authors: Chung-Chih Lin
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/mm67q3

碩士 === 國立陽明大學 === 生命科學暨基因體科學研究所 === 97 === Kinesins constitute a superfamily of microtubule-based motor proteins. Some of them regulate polarized transport of organelles and proteins in nerve system to result in differential morphology and functions in subcellular regions of neurons. Because nerve growth factor (NGF) induces na��ve PC12 cells to acquire neuronal-like differential morphology and functions among cell body and neurites. Therefore, KIFs seem to play a role in NGF-mediated differentiation of PC12 cells. This study is to understand whether KIF3 and KIF5, which possibly regulate transport calcium pools and vesicles, involves in the effects of NGF on calcium homeostasis and exocytotic activity of PC12 cells. Overexpression of shKIF5B instead of shKIF3A or DN-KIF3A-EGFP reduced NGF-induced neurite outgrowth. NGF-differentiated PC12 cells overexpressing either EGFP as control, or DN-KIF3A or shKIF5B were stimulated by ATP to evoke [Ca2+]i increase, and changes of [Ca2+]i in cell body and neurites of these cells were visualized by calcium imaging. DN-KIF3A and shKIF3A reduced resting [Ca2+]i in cell bodies and neurites of NGF-differentiated PC12 cells, but difference of resting calcium among cell bodies and neurites is not affected, i.e. higher resting [Ca2+]i in neurite than in cell body. To test whether mitochondria may be re-distributed by DN-KIF3A to affect calcium homeostasis in PC12 cells, subcellular distribution of mitochondria was visualized by DsRed-Mito. There is no significant difference between control and DN-KIF3A-overexpressing cells. Moreover, only TG (thapsigargin, ER calcium pump inhibitor) instead of CCCP (carbonyl cyanide m-chlorophenylhydrazone, inhibitor to deplete mitochondrial calcium pool) reduces differential resting [Ca2+]i among neuritis and cell bodies and the effects of DN-KIF3A on resting [Ca2+]i, and support that KIF3A regulates resting [Ca2+]i is not mediated by mitochondria calcium pools. Such reduction of resting [Ca2+]i by shKIF3A results in decreased LDCVs (Large dense core vesicle) exocytosis. Similar to shKIF3A, shKif5B reduces resting calcium level only in NGF-treated cells instead of na��ve cells, even morphology of NGF-treated and na��ve KIF5B knock down cells are similar. In conclusion, KIF3A and KIF5B play a role in calcium homeostasis only in NGF-treated PC12 cells. But the mechanism in how KIFs regulate calcium homeostasis and exocytosis needs to be further studied.