Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22364
標題: 出生後小鼠大腦組織細胞中與肌動蛋白絲重組有關的結合蛋白表現量的探討。
Expression of actin binding proteins associated with the reorganization of actin filament in postnatal mouse brain.
作者: 陳俊雄
Jin-Hsiung
關鍵字: 微絲細胞骨架
actin polymerization/depolymerization
聚合/裂解
細胞極性
細胞分裂
細胞運動
細胞形狀維持
真核細胞
核酸序列
高度保留
分子區域
氨基酸
真菌
哺乳動物
細胞間連結
卵細胞
减數分裂
專一表現
基因產物
核酸定位
胚體
發育階段
大腦初始構造
背脊腦室區
背根神經結
嗅葉
大腦皮質
海馬迴
小腦
基因表現
免疫抗體
蛋白表現免疫蛋白共同沉澱
cell shape
mobility
muscle contraction
formin
gene mutation
limb deformity
chick embryo
budding yeast
actin capping protein
cell division
nucleotide sequence
highly conseved regions
FH1 and FH2 domain
praline-rich sequence
profilin
actin binding protein
nucleotide-actin exchange rate
coiled-coil domain
gene knock out (KO) mice
meiosis
oocytes
formin homologous proteins (formin-2)
embryonic and adult brain
neuronal cells
mRNA expression
postnatal brains
postnatal brain development
antibody against mouse formin proteins
co-immunoprecipitations
protein-protein interactions
semi-quantitative RT-PCR
immunoblotting
neurogenesis
出版社: 生命科學系所
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摘要: 肌動蛋白分子進行微絲細胞骨架的聚合/裂解過程,驅動細胞極性的調控以及影響細胞分裂、細胞運動、與細胞形狀的維持。真核細胞也因此發展出許多不同的機制來調控這種細胞內微絲分子的整合以及細胞功能的表現。Formin蛋白基因在1990年由Woychik等人所發現,有許多不同的分子異構物;可以調控細胞內肌動蛋白微絲骨架重組的作用。根據核酸序列分析,formin蛋白擁有FH1跟FH2這兩個高度保留的分子區域;FH1分子區域含有許多proline 氨基酸可與另一種調控肌動蛋白微絲骨架聚合的蛋白profilin產生交互作用,而FH2區域則與肌動蛋白直接作用,增加微絲骨架聚合的速度。Formin蛋白分子調控機制的研究大多在真菌等低等的真核生物,譬如:對真菌細胞分裂扮演著相當重要的角色。反之,在高等的真核細胞 (譬如:哺乳動物),則發現formin基因對細胞間的連接以及細胞的運動有貢獻,基因的缺失會造成動物肢體發育的殘缺,以及造成動物卵細胞進行减數分裂時的障礙。Formin-2為一種最近在小鼠以及人神經組織細胞內專一表現的基因產物。利用核酸定位的實驗發現:在胚體神經系統的發育階段有高度基因的表現:胚胎發育的第9.5天開始表現於大腦的初始構造;第10.5天開始有基因表現於脊椎腦室區的內側跟外側,以及表現於背根神經節等區域。另外,出生後的大腦也有文獻報告指出:基因表現在嗅葉、大腦皮質、海馬迴、小腦等神經構造。然而,目前並沒有細胞內蛋白作用的研究報告,也因此並不清楚formin-2在神經系統相關的細胞構造內表現的生物意義。在我們的前實驗發現:formin-2在腦中的基因表現量似乎會在小鼠出生後一個月後有逐漸減少的現象。由於formin-2基因表現量與小鼠大腦成熟的時間點有吻合的現象,因此本研究論文旨在證實:formin-2基因表現量在小鼠大腦成熟後有減少的現象。同時,利用其它調控肌動蛋白微絲骨架的蛋白(e.g profilin and caldesmon) 作為對照。另外,利用市售可以辨識小鼠formin-2蛋白的免疫抗體,可以進一步檢視出生後小鼠大腦成熟前後組織細胞內formin-2蛋白表現的變化。最後,利用免疫蛋白共同沉澱法來進一步探討:在小鼠出生後大腦發育成熟時,formin-2是否在神經組織細胞內與肌動蛋白以及其他相關蛋白產生交互作用。實驗結果證實:小鼠出生後,大約在一個月後,腦部神經組織細胞內只有formin-2這個調控微絲骨架的蛋白基因表現量有明顯降低的情形產生,而其餘的調控蛋白(e.g. caldesmon, profilin, and tropomyosin) 基因表現量並沒有明顯的改變。降低formin-2基因表現量,可能與出生後動物腦部成熟發育時停止神經元細胞增生有關。
It is generally accepted that dynamic reorganization of actin polymerization and depolymerization is the basic mechanism for maintaining cell shape, facilitating mobility and regulating muscle contraction. In 1990, Woychik and his colleague first identified “formin” as a limb formation gene. This gene mutation would cause a limb deformity in chick embryo. In budding yeast formin was reported to act as an actin capping protein to regulate actin polymerization/ depolymerization, and to take part in the control of cell division. Based on the nucleotide sequence, formin has been deduced to contain the two highly conserved regions, FH1 and FH2 domains in the C-terminal end of the molecule. FH1 domain is a proline-rich sequence for the interaction with profilin, another actin binding protein to catalyze the nucleotide-actin exchange rate. FH2 domain is a coiled-coil domain and has the binding site for the actin filament. Studies with formin gene knock out (KO) mice showed that the KO mice displayed the deficient meiosis of oocytes, suggesting the formin may control the actin filament reorganization and cell division. More recently, studies also showed that the formin homologous protein (formin-2) was expressed in embryonic and adult brain. However, physiological functions of formin-2 in the neuronal cells are not currently understood. In our preliminary experiments, we found that formin-2 mRNA expression levels decreased in the postnatal brains of mouse at the ages from 1 month to 1.5 years. It seems that the reduction of formin-2 gene expression in the postnatal brain may be functionally related to the halt of the neuronal cell division at the mature brain (1 to 3 month). This study was aimed to demonstrate if the formin-2 gene expression decreased the postnatal brain development by comparing the expression levels of other actin-binding proteins such as profiln, caldesmon, and tropomyosin. Since antibody against mouse formin protein was just available recently, we were able to confirm further the protein levels of formin in the brain of mouse during the postnatal development. Finally, we carried out the experiments of co-immunoprecipitation to further examine the protein-protein interactions of formin-2 with actin and other associated proteins in the brain. The data obtained with the semi-quantitative RT-PCR showed that the ratio of formin-2 mRNA to beta-actin mRNA in mouse brain tissues decreased after P30 (Postnatal day 30) since maturation of the brain. Using the immunoblotting with antibodies against mouse formin-2, tropomyosin, and caldesmon also indicated a time-decreased protein levels of formin-2, but not of the others (e.g caldesmon, profilin, and tropomyosin) in mouse brain tissues after P30. In addition, the co-immunoprecitation experiments showed that only in the postnatal brain tissues of one-month-old mouse detected the interactions of formin-2 with actin, profilin. All together, results obtained from this study indicated that the postnatal brain development has an effect on the expression of formin-2 in mouse, which may be related to neurogenesis of the brain.
URI: http://hdl.handle.net/11455/22364
其他識別: U0005-1906200611270000
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1906200611270000
Appears in Collections:生命科學系所

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