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標題: | 第七號生長休止基因在神經發育上的機制探討 Studies on the molecular mechanism of Gas7 in neuronal differentiation |
作者: | Jhong-Jhe You 游忠哲 |
指導教授: | 林淑端(Sue Lin-Chao) |
關鍵字: | Gas7,N-WASP,PCH (Pombe Cdc15 homology),神經發育,CDK5RAP2,中心粒, Gas7,N-WASP,PCH (Pombe Cdc15 homology),neuronal maturation,CDK5RAP2,centrosome, |
出版年 : | 2010 |
學位: | 博士 |
摘要: | 第七號生長休止基因(Gas7)是由Bernner等研究人員在利用低含量血清培養或生長至融合而變成生長休止狀態的纖維母細胞株NIH3T3當中所發現的。該基因目前知道大量表現於小鼠的睪丸及大腦中。在大腦內,該基因特別表現於海馬迴、大腦皮層和小腦的Purkinje神經細胞之中。進一步研究指出對於人工培養完整分化的Purkinje神經細胞以及利用NGF所誘導分化成神經細胞的PC12細胞株都顯示出該基因的必要性。生化分析與細胞生物學的研究則顯示出該基因所表現的蛋白能夠與肌動蛋白直接產生交互作用,並且該蛋白的C端部分能夠促進肌動蛋白的聚合反應。最後,能夠進一步影響細胞內部肌動蛋白骨架的改變及調控。但是對於Gas7基因在神經細胞當中的功能與作用仍不了解。在本研究中則利用生化分析及神經細胞生物學來探討Gas7基因在神經細胞當中的功能。第一部分,研究發現到一個新的Gas7交互作用蛋白,N-WASP。在此部分發現到Gas7蛋白能與N-WASP蛋白藉由WW-Pro的作用方式來直接進行交互作用。並透過這樣的作用方式來進一步影響肌動蛋白骨架的改變。進一步的證據則顯示出Gas7與N-WASP蛋白之間的交互作用對於海馬迴神經細胞的突觸正常生長是相當重要的。第二部分,藉由了解與Gas7交互作用的蛋白來進一步了解Gas7蛋白在神經細胞中可能的角色。該蛋白利用酵母菌雜合系統所篩選出來的蛋白,稱為CDK5RAP2。在本研究中發現到CDK5RAP2蛋白屬於一個中心粒蛋白。並且發現該蛋白在中心粒中是作為一個骨架來提供一個位置使其他不同的蛋白進行交互做用。在細胞中,倘若剔除CDK5RAP2的表現,會導致中心粒無法發展成熟,進一步影響到細胞的複製或分裂。該蛋白也在人工培養的海馬迴神經細胞與大腦皮質神經細胞中有發現。但該蛋白在神經細胞中的功能則有待進一步研究。綜合以上,本研究清楚的進一步證明Gas7如何透過N-WASP蛋白在海馬迴神經細胞中來調控神經發育過程。另外,我們也提出Gas7可能透過CDK5RAP2蛋白來調控神經細胞內的微小管骨架的改變,並進而影響神經的發育或神經細胞移動的可能性。 Gas7 (Growth arrest-specific gene 7) was isolated in growth-arrested NIH3T3 cells upon serum starvation or growth confluence. Gas7 is highly expressed in the mouse testis and brain, mostly in the hippocampus, cortex, and Purkinje neurons of cerebellum. Previous data showed that Gas7 is required for the neuronal differentiation of terminally differentiated Purkinje cells and differentiation of PC12 upon NGF stimulation. Biochemical studies also showed that Gas7 functions with actin to enhance actin polymerization and crosslinking the actin filaments through Gas7 C-termini, resulting in membrane ruffles in the cells. However, the role of Gas7 in the neurons remained unclear. Here, we tried to determine the role of Gas7 in neurons through determination of its interacting proteins. First, N-WASP was discovered as a novel Gas7-interacting protein through its interaction with WISH, another Gas7-interacting protein. We obtained clear evidence that Gas7 functions directly with N-WASP, via WW-Pro interaction, to regulate actin dynamics in a Cdc42-independent manner in the cells. Our data show that this specific interaction is indispensable for regular neurite outgrowth of hippocampal neurons. Second, another Gas7-interacting protein, Gas7CIP1/CDK5RAP2, from yeast two-hybrid system screening was characterized to assess indirectly the role of Gas7 in the cells. The data showed that CDK5RAP2 is centrosome-associated protein and acts as a scaffold to recruit γ-tubulin. CDK5RAP2 contributes to the maturation of centrosome and affects the cell cycle. Furthermore, we found that CDK5RAP2 is distributed in the centrosome of developing hippocampal neurons during early stages of development. The centrosome regulates neuronal differentiation or migration processes through the regulation of microtubule dynamics in neurons. Therefore, our data implicate that CDK5RAP2 may play a role in the regulation of microtubule dynamics to affect the neuronal differentiation or migration, especially nucleokinesis. However, how CDK5RAP2 functions in the neurons still awaits further investigation. Overall, this study clearly showed how Gas7 functions with N-WASP to regulate the neurite outgrowth of hippocampal neurons, thus providing the possibility that Gas7 may function together with CDK5RAP2 to regulate microtubule dynamics in neurons to control neuronal differentiation or migration. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/8677 |
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