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激光原子力顯微鏡觀察肌動(dòng)蛋白體外自組織纖維結(jié)構(gòu)多態(tài)性的初步研究

激光生物學(xué)報(bào) ?第13 卷第4 期 2004 年8 月?

張軍1 ,2 ,王遠(yuǎn)亮1 ,CHEN Xin-yong3 ,何創(chuàng)龍1 ,程超1

(1. 重慶大學(xué)教育部生物力學(xué)及組織工程重點(diǎn)實(shí)驗(yàn)室,中國(guó)重慶400044 ;2. 重慶醫(yī)科大學(xué)生物學(xué)教研室,中國(guó)重慶 400016 ;3. Laboratory of Biophysics and Surface Analysis , University of Nottingham , Nottingham NG7 2RD , United Kingdom)

摘要

利用原子力顯微鏡(atomic force microscope , AFM) 技術(shù),研究了肌動(dòng)蛋白體外通過(guò)自組織過(guò)程形成的纖維結(jié)構(gòu)及其多態(tài)性。肌動(dòng)蛋白在體外通過(guò)自組織過(guò)程能夠聚合形成離散的樹(shù)狀分支的纖維叢和具有不同直徑的長(zhǎng)纖維等高級(jí)纖維結(jié)構(gòu),表現(xiàn)出明顯的結(jié)構(gòu)多態(tài)性;與微絲工具藥物鬼筆環(huán)肽干預(yù)下自裝配形成的主要由單根微絲和微絲束等纖維成份構(gòu)成的連續(xù)網(wǎng)絡(luò)結(jié)構(gòu)明顯不同。

關(guān)鍵詞

肌動(dòng)蛋白；自組織；結(jié)構(gòu)多態(tài)性；原子力顯微鏡

中圖分類(lèi)號(hào): Q245 ;Q-334

文獻(xiàn)標(biāo)識(shí)碼: A

文章編號(hào): 1007-7146 (2004) 04-0253-05

Preliminary Observation on the Structural Polymorphism of Actin Filaments in Self-organizing in vitro using Laser Atomic Force Microscope

ZHANG Jun1 , 2 , WANG Yuan-liang1 , Chen Xin-yong3 , HE Chuang-long1 , CHENG Chao1

(1. Key Lab of Biomechanics & Tissue Engineering , Ministry of Education , Chongqing University , Chongqing 400044 , China ;

2. Biology Department , Chongqing University of Medicine Sciences , Chongqing 400016 , China : 3. Laboratory of Biophysics and Surface Analysis , University of Nottingham , Nottingham NG7 2RD , United Kingdom)

Abstract

The laser atomic force microscope , a novel tool for surface structure exploration , is employed to investigate the struc-tural polymorphism of actin filaments by self-organization in vitro. It is demonstrated that the actin can be polymerized into tree-like branch structure and long filaments with different diameters in F-buffer without the addition of F-actin stabilizing reagent , such as phalloidin etc. , which is capable of inhibiting the F-actin from depolymerization. These polymerized filaments clearly exhibit the structural polymorphism and show obvious difference from those in the cross-linked network mainly composed of single F-actin and F-actin bundle in the presence of phalloidin under same experimental conditions in previous experiments.

Key words

actin ; self-organization ; structural polymorphism; atomic force microscope

? ? ? ?肌動(dòng)蛋白(Actin) 是真核細(xì)胞內(nèi)最保守、含量最豐富的蛋白質(zhì)之一,作為細(xì)胞骨架和結(jié)構(gòu)的主要成分,具有極其重要的細(xì)胞生理功能[1 ,2 ] 。肌動(dòng)蛋白一般以球形肌動(dòng)蛋白(G-actin) 和纖維性肌動(dòng)蛋白( F-actin , 即微絲) 兩種形式存在。G-actin 的一級(jí)序列通常由375 個(gè)氨基酸殘基組成,分子量約為43KD ,溶液中,在Mg2 + , K+ , Na + 及ATP 誘導(dǎo)下能自聚合形成高分子量、右手雙螺旋結(jié)構(gòu)的F-actin[3 ,4 ] 。在體內(nèi),肌動(dòng)蛋白不僅作為必需成分參與肌球蛋白介導(dǎo)的肌肉收縮[5 ,6 ] ,并可以通過(guò)受調(diào)控的聚合動(dòng)力學(xué)過(guò)程[7 ]或凝膠-溶液轉(zhuǎn)化[8 ] 單獨(dú)介導(dǎo)細(xì)胞運(yùn)動(dòng);而且越來(lái)越多的研究表明,肌動(dòng)蛋白參與了遠(yuǎn)比以前人們認(rèn)為的更加廣泛和復(fù)雜的生命活動(dòng),如細(xì)胞粘附、信號(hào)轉(zhuǎn)導(dǎo)、離子通道調(diào)控等等[9～14 ] 。由于肌動(dòng)蛋白在真核細(xì)胞中擔(dān)負(fù)了多種重要的生理功能,是細(xì)胞內(nèi)最重要的蛋白質(zhì)之一,因而自從被發(fā)現(xiàn)以來(lái),其結(jié)構(gòu)和功能研究成為細(xì)胞生物學(xué)和結(jié)構(gòu)生物學(xué)的重要內(nèi)容。不僅多種晶體形式的G-actin 精細(xì)原子結(jié)構(gòu)已獲得闡明[15～17 ] ,而且高分辨率的F-actin 原子結(jié)構(gòu)模型也通過(guò)纖維X-射線衍射[18 ,19 ] 和電子顯微鏡成像而得到建立[20～22 ] ;雖然人們?cè)诩?dòng)蛋白高分辨率的結(jié)構(gòu)解析研究中獲得了巨大成功,但是對(duì)肌動(dòng)蛋白體外大尺度結(jié)構(gòu)體系卻關(guān)注較少。而肌動(dòng)蛋白在體內(nèi)行使正常生理功能時(shí),是依賴(lài)于大尺度的超分子結(jié)構(gòu)體系,并非以單個(gè)G-actin 或單根F-actin 來(lái)發(fā)揮其細(xì)胞功能。

? ? ? ?為了探索肌動(dòng)蛋白體外大尺度自聚合結(jié)構(gòu)體系,我們利用原子力顯微鏡———一種已廣泛應(yīng)用于肌動(dòng)蛋白結(jié)構(gòu)、功能及動(dòng)力學(xué)研究的新型表面結(jié)構(gòu)分析儀器[23 ] ,對(duì)肌動(dòng)蛋白體外自組織纖維結(jié)構(gòu)進(jìn)行了研究,發(fā)現(xiàn)肌動(dòng)蛋白在沒(méi)有F-actin 穩(wěn)定劑(如鬼筆環(huán)肽) 存在的情況下,能夠通過(guò)自組織過(guò)程形成離散的、樹(shù)狀分支的纖維叢和具有不同直徑的長(zhǎng)纖維結(jié)構(gòu);與Shao Z , 等人實(shí)驗(yàn)中,肌動(dòng)蛋白在鬼筆環(huán)肽介入下形成由單根微絲和微絲束構(gòu)成的大范圍連續(xù)網(wǎng)絡(luò)結(jié)構(gòu)明顯不同[24 ] 。這暗示,作為微絲工具藥物而廣泛應(yīng)用的鬼筆環(huán)肽可能對(duì)肌動(dòng)蛋白大尺度結(jié)構(gòu)形態(tài)有較大的影響。

1 材料和方法

1.1 材料

? ? ? ?G-actin 提取自牛骨骼肌,純化按照Spudich 和Watt 等人的方法[25 ] 。分離的肌動(dòng)蛋白達(dá)電泳單點(diǎn)純,儲(chǔ)存于4 ℃的G-緩沖液中備用;Na2ATP、DTT 購(gòu)自Sigma ,Tris-base 、Tris-HCl 購(gòu)自Promega , 其余試劑均為國(guó)產(chǎn)分析純;CSPM-2000we 型原子力顯微鏡為中國(guó)科學(xué)院化學(xué)研究所本原納米儀器有限公司生產(chǎn),使用NANOPROBETM NP 型Si3N4 探針。

1.2 方法

1.2.1 溶液配制　G-緩沖液( 2mmolPL Tris-Cl ,pH7. 5 , 0. 2mmolPL CaCl2 , 0. 5mmolPL DTT , 0. 2mmolPL ATP) 和F-緩沖液(5mmolPL Tris-Cl , pH7. 5 , 2mmolPLMgCl2 , 100mmolPL KCl , 1mmolPL DTT , 1mmolPLATP) 均按常規(guī)方法制備,所有使用的溶劑和新鮮配制的溶液均通過(guò)<0. 22μm 的濾膜過(guò)濾去除顆粒。

1.2.2 待測(cè)樣品制備　用F-緩沖液將1ml 純化G-actin 精確稀釋至100ml ,使溶液中G-actin的最終濃度為5μgPml ,然后將樣品置于37 ℃恒溫箱中孵育30min 使其聚合。聚合完成后,在新鮮剝離的云母表面滴加5μl 待測(cè)溶液,使其自然脫水制成待測(cè)樣品。

1.2.3 原子力顯微鏡成像　樣品制備完成后不經(jīng)過(guò)任何物理和化學(xué)處理,立即在室溫下(25℃) 用原子力顯微鏡的接觸模式成像。原子力顯微鏡采集的圖像保存為BMP 格式,儲(chǔ)存在電腦中做進(jìn)一步的分析。上述所有操作均在超凈工作條件下進(jìn)行,以避免污染。

2 結(jié)果

2.1 樹(shù)狀分支結(jié)構(gòu)的纖維叢

? ? ? ?肌動(dòng)蛋白在體外通過(guò)自組織能夠聚合形成離散的、具有典型分形結(jié)構(gòu)特征的樹(shù)狀分支纖維叢結(jié)構(gòu),但不能形成大區(qū)域的連續(xù)網(wǎng)絡(luò)結(jié)構(gòu)(Fig. 1) ;與微絲穩(wěn)定劑鬼筆環(huán)肽存在時(shí),形成由單根微絲和微絲束組成的、大范圍的連續(xù)交聯(lián)網(wǎng)絡(luò)結(jié)構(gòu)明顯不同[24 ] 。

? ? ? ?實(shí)驗(yàn)中,肌動(dòng)蛋白通過(guò)自組織形成的樹(shù)狀分支纖維叢由多級(jí)纖維分支形成,次級(jí)纖維成份間的相互作用和微絲動(dòng)力學(xué)過(guò)程的動(dòng)態(tài)平衡可能是樹(shù)狀分支結(jié)構(gòu)產(chǎn)生的必要條件,但詳細(xì)的聚合動(dòng)力學(xué)機(jī)制還有待進(jìn)一步的研究。

2.2 不同直徑的纖維結(jié)構(gòu)

? ? ? ?肌動(dòng)蛋白可通過(guò)自組織過(guò)程聚合形成不同直徑(7nm～35nm) 的長(zhǎng)纖維,實(shí)驗(yàn)中觀察到的最細(xì)纖維的結(jié)構(gòu)參數(shù)與單根無(wú)鬼筆環(huán)肽結(jié)合時(shí)的F-actin[19 ]一致(Fig. 2 a) ;此外,還有大量具有較大直徑和分支結(jié)構(gòu)的纖維出現(xiàn),表明有不同于單根微絲和微絲束的高級(jí)纖維結(jié)構(gòu)產(chǎn)生(Fig. 2 b～d) 。但高級(jí)纖維結(jié)構(gòu)的構(gòu)成模式還有待深入研究,初步判斷觀察到的高級(jí)結(jié)構(gòu)可能是由若干單根微絲經(jīng)過(guò)(多級(jí)) 螺旋或其它構(gòu)成方式形成的有序結(jié)構(gòu)。

肌動(dòng)蛋白AFM圖像

Fig. 1 　Tree-like branch structure with fractal features.

The structure seems to be composed of multiple order helix filaments with branches , which actually indicate the possible mechanism for formation of high order actin filaments by self-organization.
肌動(dòng)蛋白AFM圖像

Fig.2 Filaments with different diameters.

(a) The slimmest single filament is most similar to F-actin in size with ～7 nm width at half height and ～35 nmpitch ; (b) the filaments have much wider diameter than the slimmest F-actin ; (c) The branch filament could be con-stituted of two enlaced slimmer filaments , forming the branch in the joining point of another filament ; (d) Two fila-ments are aligned side by side , and thought that there might exist certain interaction between them , which could be theearly step for further assembly of giant filaments. The unit of scale ruler is nm.

2.3 自組織纖維結(jié)構(gòu)的3D-重建

? ? ? ?利用計(jì)算機(jī)3D成像技術(shù)對(duì)自組織纖維結(jié)構(gòu)進(jìn)行了3D 構(gòu)建以獲取有用的表面結(jié)構(gòu)細(xì)節(jié),結(jié)果表明不同類(lèi)型的纖維結(jié)構(gòu)具有類(lèi)似的表面構(gòu)象和拓?fù)湫再|(zhì),提示各種類(lèi)型的纖維結(jié)構(gòu)可能來(lái)源于相同的聚合動(dòng)力學(xué)過(guò)程。

2.3.1 樹(shù)狀分支結(jié)構(gòu)的3D-重建　對(duì)樹(shù)狀分支結(jié)構(gòu)的纖維叢局部區(qū)域進(jìn)行了3D-重建,發(fā)現(xiàn)其表面存在明顯的螺紋結(jié)構(gòu)(Fig. 3) ,表明粗纖維可能由更細(xì)的構(gòu)成成份纏繞螺旋而成;但分支節(jié)點(diǎn)的結(jié)構(gòu)還未能清晰地解析,其構(gòu)成方式需進(jìn)一步研究。

2.3.2 長(zhǎng)纖維結(jié)構(gòu)的3D-重建　從長(zhǎng)纖維結(jié)構(gòu)的3D-重建圖中,可觀察到不同直徑的纖維表面存在與樹(shù)狀分支結(jié)構(gòu)表面類(lèi)似的螺紋結(jié)構(gòu)( Fig. 4) 。雖然長(zhǎng)纖維結(jié)構(gòu)和分支結(jié)構(gòu)具有不同的表觀形態(tài),但是二者在構(gòu)成方式、聚合動(dòng)力學(xué)過(guò)程以及有序結(jié)構(gòu)多態(tài)性形成的分子機(jī)制上可能存在本質(zhì)的聯(lián)系。

肌動(dòng)蛋白自組織纖維結(jié)構(gòu)AFM圖像

Fig.3 3D-reconstruction of branch structure.The spiral flight can be clearly observed on the fila-ments surface , which demonstrates that the giant fila-ments could come from the thinner filament by intertwist-ing with one another in self-organization dynamics. Theunit of scale ruler is nm.

3 討論

? ? ? ?實(shí)驗(yàn)表明,作為微絲工具藥物而廣泛應(yīng)用的鬼筆環(huán)肽可能對(duì)體外肌動(dòng)蛋白大尺度聚合結(jié)構(gòu)有較大的影響。在體外簡(jiǎn)單熱力學(xué)體系中,肌動(dòng)蛋白自組織為單純的、無(wú)干預(yù)的熱力學(xué)過(guò)程,完全受其內(nèi)在的熱力學(xué)特性所驅(qū)動(dòng)。利用肌動(dòng)蛋白聚合動(dòng)力學(xué)的踏車(chē)模型可以較好地對(duì)肌動(dòng)蛋白自組織行為進(jìn)行定性的分析:在F-緩沖液中,肌動(dòng)蛋白的聚合過(guò)程在經(jīng)過(guò)成核期、生長(zhǎng)期之后能夠迅速進(jìn)入動(dòng)態(tài)平衡。此時(shí),結(jié)合態(tài)的纖維型肌動(dòng)蛋白殘基與游離的球型肌動(dòng)蛋白含量保持動(dòng)態(tài)平衡。微絲的總體長(zhǎng)度處于相對(duì)恒定的狀態(tài),受總長(zhǎng)度的限制,單根微絲長(zhǎng)度有限,微絲間的相互作用較難發(fā)生,形成交聯(lián)的幾率很小,因此在自組織過(guò)程中,肌動(dòng)蛋白難以聚合形成連續(xù)的交聯(lián)網(wǎng)絡(luò)纖維結(jié)構(gòu),只能形成離散的纖維結(jié)構(gòu);在微絲穩(wěn)定劑(如鬼筆環(huán)肽) 介入時(shí),肌動(dòng)蛋白聚合在經(jīng)過(guò)成核期進(jìn)入生長(zhǎng)期之后,肌動(dòng)蛋白體外自裝配由于微絲解聚合過(guò)程被抑制而受到干預(yù),正常的聚合P解聚合動(dòng)態(tài)平衡受到破壞,導(dǎo)致自裝配向聚合單方向進(jìn)行,使溶液中的游離肌動(dòng)蛋白和寡聚體濃度很低, 此時(shí)幾乎所有的肌動(dòng)蛋白均聚合為微絲,微絲的總長(zhǎng)度接近最大,單根微絲相對(duì)較長(zhǎng),彼此之間的相互作用較易發(fā)生,因而容易形成大范圍的交聯(lián)網(wǎng)絡(luò)結(jié)構(gòu)。

肌動(dòng)蛋外肌動(dòng)蛋白大尺度聚AFM圖像

Fig.4 3D-reconstruction of two parallel filaments with different diameters. On the surface of the filaments , similar topological and conformational structure traits can be discerned ;suggesting that all the filaments observed in the experi-ment could result from the same thermodynamic process.The unit of scale ruler is nm.

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作者簡(jiǎn)介

張軍：男，1973 年10 月17 日出生,四川南充人,博士研究生。重慶醫(yī)科大學(xué)任教,從事細(xì)胞生物學(xué)、生物物理與生物化學(xué)領(lǐng)域科研工作。已有7 篇學(xué)術(shù)論文被SCI , Medline , CA等國(guó)際檢索系統(tǒng)收錄。

Biography

ZHANG Jun : male , born in the 17th of October 1973. He is a Ph D graduate and also teaches in the Chongqing University of Medical Sciences , majoring in cell biology , biochemistry and biophysics. He has published 7 articles that have been cited by SCI , Medline and CA.