1、第九章 細(xì)胞骨架(Cytoskeleton),細(xì)胞骨架是指存在于真核細(xì)胞中的 蛋白纖維網(wǎng)架體系。,原核細(xì)胞？,細(xì)胞骨架的狹義和廣義的概念：,狹義的細(xì)胞骨架： 僅指細(xì)胞質(zhì)骨架，在細(xì)胞質(zhì)基質(zhì)中包括微絲、微管、中間纖維。,廣義的細(xì)胞骨架： 包括細(xì)胞核骨架、細(xì)胞質(zhì)骨架、細(xì)胞膜骨架和細(xì)胞外基質(zhì)。 在細(xì)胞核中存在核骨架 核纖層體系，核骨架、核纖層與中間纖維在結(jié)構(gòu)上相互連接，是貫穿于細(xì)胞核和細(xì)胞質(zhì)的網(wǎng)架體系。,第九章 細(xì)胞骨架(Cytoskeleton), 細(xì)胞質(zhì)骨架 細(xì)胞核骨架 膜骨架,作業(yè),第一節(jié) 細(xì)胞質(zhì)骨架, 微絲 (microfilament, MF) 微 管 (microtubules) 中間纖維

2、 (intermediate filament, IF) 細(xì)胞骨架結(jié)構(gòu)與功能總結(jié),一、微絲 (microfilament, MF),又稱肌動蛋白纖維 (actin filament), 是指真核細(xì)胞中由肌動蛋白 (actin) 組成、直徑為7nm的骨架纖維。, 成分 裝配 微絲特異性藥物 微絲結(jié)合蛋白 微絲功能,成分,肌動蛋白(actin)是微絲的結(jié)構(gòu)成分，外觀呈啞鈴狀，這種actin又叫G-actin, 將G-actin形成的微絲又稱為F-actin。,裝配,MF是由G-actin單體形成的多聚體，肌動蛋白單體具有極性，裝配時呈頭尾相接，故微絲具有極性，既正極與負(fù)極之別。 體外實(shí)驗(yàn)表明，MF

3、正極與負(fù)極都能生長，生長快的一端為正極，慢的一端為負(fù)極；去裝配時，負(fù)極比正極快。由于G-actin在正極端裝配，負(fù)極去裝配，從而表現(xiàn)為踏車行為。 體內(nèi)裝配時，MF呈現(xiàn)出動態(tài)不穩(wěn)定性，主要取決于F-actin結(jié)合的ATP水解速度與游離的G-actin單體濃度之間的關(guān)系。 MF動態(tài)變化與細(xì)胞生理功能變化相適應(yīng)。在體內(nèi)，有些微絲是永久性的結(jié)構(gòu)，有些微絲是暫時性的結(jié)構(gòu)。,Actin assembly in Vitro Schematic diagram of the preferential addition of actin subunits at the plus end of a microfi

4、lament and their preferential loss from the minus end in an in vitro assay. As a result, subunits treadmill through the filament in vitro.,微絲特異性藥物,細(xì)胞松弛素 (cytochalasins) 可以切斷微絲，并結(jié)合在微絲正極阻抑肌動蛋白聚合，因而導(dǎo)致微絲解聚。 鬼筆環(huán)肽 (philloidin) 與微絲側(cè)面結(jié)合，防止MF解聚。 影響微絲裝配動態(tài)性的藥物對細(xì)胞都有毒害，說明微絲功能的發(fā)揮依賴于微絲與肌動蛋白單體庫間的動態(tài)平衡。這種動態(tài)平衡受actin單體

5、濃度和微絲結(jié)合蛋白的影響。,微絲結(jié)合蛋白,整個骨架系統(tǒng)結(jié)構(gòu)和功能在很大程度上受到不同的細(xì)胞骨架結(jié)合蛋白的調(diào)節(jié)。,actin單體結(jié)合蛋白 這些小分子蛋白與actin單體結(jié)合，阻止其添加到微絲末端，當(dāng)細(xì)胞需要單體時才釋放，主要用于actin裝配的調(diào)節(jié)，如proflin等。 微絲結(jié)合蛋白 微絲結(jié)合蛋白將微絲組織成以下三種主要形式：,微絲結(jié)合蛋白, Contractile bundle：MF反向平行排列，主要發(fā)現(xiàn)于應(yīng)力纖維和有絲分裂收縮環(huán)。 Gel-like network：細(xì)胞皮層 (cell cortex)中微絲排列形式，MF相互交錯排列。, Parallel bundle：MF同向平行排列，主要

6、發(fā)現(xiàn)于微絨毛與絲狀偽足。,微絲功能, 維持細(xì)胞形態(tài)，賦予質(zhì)膜機(jī)械強(qiáng)度； 細(xì)胞運(yùn)動； 微絨毛 (microvillus)； 應(yīng)力纖維 (stress fiber)； 參與胞質(zhì)分裂； 肌肉收縮 (muscle contraction)。,微絲遍及胞質(zhì)各處，集中分布于質(zhì)膜下，和其結(jié)合蛋白形成網(wǎng)絡(luò)結(jié)構(gòu)，維持細(xì)胞形狀和賦予質(zhì)膜機(jī)械強(qiáng)度，如哺乳動物紅細(xì)胞膜骨架的作用。,細(xì)胞運(yùn)動,成纖維細(xì)胞爬行與微絲裝配和解聚相關(guān)。,Actin filaments in migrating animal cells (A) Schematic drawing of a fibroblast showing flattene

7、d lamellipodia and fine filopodia projecting from its surface, especially in the regions of the leading edge. (B) Details of the arrangement of actin filaments in three regions of the fibroblast are shown, with arrowheads pointing toward the plus end of the filaments. (C) Scanning electron micrograp

8、h showing lamellipodia and filopodia at the leading edge of a human fibroblast migrating in culture.,(A),(B),(C),growing filopodium,Actin filament,是腸上皮細(xì)胞的指狀突起，用以增加腸上皮細(xì)胞表面積，以利于營養(yǎng)的快速吸收。,微絨毛 (microvillus),Membrane- Microfilament linkage,Core actin filaments (crosslinked by fimbrin and villin),Actin fil

9、aments (rootlets),Spectrin connecting fibers,Keratin intermediate filaments,Micrograph of intestinal cell showing microvilli At the core of each 2 m long microvillus, a bundle of actin filaments, cross-linked by fimbrin and villin, stabilizes the fingerlike structure. The plasma membrane surrounding

10、 a microvillus is attached to the sides of the bundle by evenly spaced membrane-microfilament linkages consisting of myosin I. Each bundle continues into the cell as a 0.5 m long rootlet. The rootlets are cross-braced by connecting fibers composed of an intestinal isoform of spectrin, and the bases

11、of rootlets form attachment sites for keratin filaments. These numerous connections anchor the rootlets in a meshwork of filaments and thereby support the upright orientation of the microvilli.,應(yīng)力纖維 (stress fiber),成分： 肌動蛋白、肌球蛋白、原肌球蛋白和-輔肌動蛋白。,功能： 介導(dǎo)細(xì)胞間或細(xì)胞與基質(zhì)表面的粘著。（細(xì)胞貼壁與粘著斑的形成相關(guān)，在形成粘合斑的質(zhì)膜下，微絲緊密平行排列成束，

12、形成應(yīng)力纖維，具收縮功能。）,廣泛存在于真核細(xì)胞中。,收縮環(huán)由大量反向平行排列的微絲組成，其收縮機(jī)制是肌動蛋白和肌球蛋白相對滑動。,肌肉收縮(muscle contraction),肌肉可看作一種特別富含細(xì)胞骨架的效力非常高的能量轉(zhuǎn)換器，它直接將化學(xué)能轉(zhuǎn)變?yōu)闄C(jī)械能。, 肌肉的細(xì)微結(jié)構(gòu)（以骨骼肌為例） 肌小節(jié)的組成 肌肉收縮系統(tǒng)中的有關(guān)蛋白 肌肉收縮的滑動模型 由神經(jīng)沖動誘發(fā)的肌肉收縮基本過程,Levels of Organization of Skeletal Muscle Tissue (a) Muscle tissue is attached by means of tendons to

13、the specific bones it must move. (b) The tissue consists of bundles of muscle fibers, (c) each of which is a long, thin multinucleate cell. (d) Within each cell are many myofibrils. (e) Each myofibril consists of bundles of filaments aligned laterally, giving skeletal muscle its straited appearance.

14、 (f) The unit of contraction along each myofibril is the sarcomere, in which thick filaments interdigitate with thin filaments. The thick and thin filaments consist primarily of myosin and actin, respectively.,肌肉收縮系統(tǒng)中的有關(guān)蛋白, 肌球蛋白(myosin) 所有actin-dependent motor proteins都屬于該家族，其頭部具ATP酶活力，沿微絲從負(fù)極到正極進(jìn)行運(yùn)動

15、。 Myosin 主要分布于肌細(xì)胞，有兩個球形頭部結(jié)構(gòu)域（具有ATPase活性）和尾部鏈，多個Myosin尾部相互纏繞，形成myosin filament, 即粗肌絲。 原肌球蛋白 (tropomyosin, Tm) 由兩條平行的多肽鏈形成-螺旋構(gòu)型，位于肌動蛋白螺旋溝內(nèi)，結(jié)合于細(xì)絲，調(diào)節(jié)肌動蛋白與肌球蛋白頭部的結(jié)合。 肌鈣蛋白 (Troponin, Tn)為復(fù)合物，包括三個亞基：TnC (Ca2+敏感性蛋白）能特異與Ca2+結(jié)合；TnT（與原肌球蛋白結(jié)合）；TnI（抑制肌球蛋白ATPase活性）。,由神經(jīng)沖動誘發(fā)的肌肉收縮基本過程, 動作電位的產(chǎn)生； Ca2+的釋放； 原肌球蛋白位移； 肌動

16、蛋白絲與肌球蛋白絲的相對滑動； Ca2+的回收。,二、微管 (Microtubules), 微管結(jié)構(gòu)與組成 裝配 微管特異性藥物 微管組織中心(MTOC) 微管結(jié)合蛋白(MAP) 微管功能,微管結(jié)構(gòu)與組成,微管可裝配成單管、二聯(lián)管（纖毛和鞭毛中）、三聯(lián)管（中心粒和基體中）。,(A),(B),(C),(D),(E),裝配, 裝配方式； 所有的微管都有確定的極性； 微管裝配是一個動態(tài)不穩(wěn)定過程。,- 微管蛋白和- 微管蛋白形成二聚體，二聚體先形成環(huán)狀核心(ring)，經(jīng)過側(cè)面增加二聚體而擴(kuò)展為螺旋帶，二聚體平行于長軸重復(fù)排列形成原纖維 (protofilament)。當(dāng)螺旋帶加寬至13根原纖維時，

17、即合攏形成一段微管。, 動力學(xué)不穩(wěn)定性產(chǎn)生的原因： 微管兩端具 GTP 帽（取決于微管蛋白濃度），微管將繼續(xù)組裝，反之，無GDP帽則解聚。, 微管裝配的動力學(xué)不穩(wěn)定性是指微管裝配生長與快速去裝配的一個交替變換的現(xiàn)象；,微管特異性藥物,秋水仙素 (colchicine) 阻斷微管蛋白組裝成微管，可破壞紡錘體結(jié)構(gòu)。 紫杉酚 (taxol) 能促進(jìn)微管的裝配，并使已形成的微管穩(wěn)定。 為行使正常的微管功能，微管動力學(xué)不穩(wěn)定性是其功能正常發(fā)揮的基礎(chǔ)。,微管組織中心 (MTOC), 概念 常見微管組織中心 中心體 (centrosome) 基體 (basal body),微管在生理狀態(tài)或?qū)嶒?yàn)處理解聚后重新

18、裝配的發(fā)生處稱為微管組織中心 (microtubule organizing center, MTOC)。,常見微管組織中心,間期細(xì)胞 MTOC: 中心體（動態(tài)微管） 分裂細(xì)胞 MTOC: 有絲分裂紡錘體極（動態(tài)微管） 鞭毛纖毛細(xì)胞 MTOC: 基體（永久性結(jié)構(gòu)）,中心體 (centrosome),中心體 (centrosome) 結(jié)構(gòu)； 中心體復(fù)制周期； 管蛋白：位于中心體周圍的基質(zhì)中，環(huán)形結(jié)構(gòu)，結(jié)構(gòu)穩(wěn)定，為微管蛋白二聚體提供起始裝配位點(diǎn)，所以又叫成核位點(diǎn)。,(a),(b),(c),Relation of centrosome duplication to the cell cycle Af

19、ter the pair of parent centrioles (red) within the centrosome matrix separate slightly, a daughter centriole (blue) buds from each and elongates. By G2 growth of the daughter centrioles is complete, but the two pairs remain within a single centrosomal complex. Early in mitosis, the centrosome splits

20、, and each centriole pair migrates to opposite ends of the cell. That new centrioles arise de novo, as shown here, rather than by fission of a parent centriole and regrowth has been demonstrated in experiments with biotin-labeled tubulin.,(A),(B),基體 (basal body),位于鞭毛和纖毛根部的類似中心體的結(jié)構(gòu)稱為基體 (basal body) 或

21、基粒； 中心粒和基體均具有自我復(fù)制性質(zhì)。,(a),(b),Basal bodies and axonemes (a) Electron micrograph of a longitudinal section through the basal bodies of a number of cilia at the apical surface of the epithelial cells of a rabbit oviduct. (b) schematic diagram of the structural relationship between the microtubules of t

22、he basal body and the axoneme of a cilium or flagellum.,* Yellow: microtubule-binding domain; Pink: projection domain; Green: 18 amino acids repeats.,Experimental demonstration that spacing of microtubules (MTs) depends on microtubule associated proteins (MAPs) Insect cells transfected with DNA expr

23、essing either long armed MAP2 protein or short armed Tau protein grow long axonlike processes. Shown here are electron micrographs of cross sections through the processes induced by expression of MAP2 (left) or Tau (right) in transfected cells. Note that the spacing between microtubules in MAP2-cont

24、aining cells is larger than in Tau-containing cells. Both cell types contain approximately the same number of microtubules, but the effect of MAP2 is to enlarge the caliber of the axonlike process.,微管功能,維持細(xì)胞形態(tài) 細(xì)胞內(nèi)物質(zhì)的運(yùn)輸 細(xì)胞器的定位 鞭毛 (flagella) 運(yùn)動和纖毛 (cilia) 運(yùn)動 紡錘體與染色體運(yùn)動,維持細(xì)胞形態(tài),用秋水仙素處理細(xì)胞破壞微管，導(dǎo)致細(xì)胞變圓，說明微管對

25、維持細(xì)胞的不對稱形狀是重要的。對于細(xì)胞突起部分，如纖毛、鞭毛、軸突的形成和維持，微管亦起關(guān)鍵作用。,細(xì)胞內(nèi)物質(zhì)的運(yùn)輸, Motor proteins 神經(jīng)元軸突運(yùn)輸?shù)念愋?運(yùn)輸模式 色素顆粒的運(yùn)輸,真核細(xì)胞內(nèi)部是高度區(qū)域化的體系，細(xì) 胞中合成的物質(zhì)、一些細(xì)胞器等必須經(jīng)過細(xì) 胞內(nèi)運(yùn)輸過程。這種運(yùn)輸過程與細(xì)胞骨架體 系中的微管及其Motor protein有關(guān)。,Motor proteins,目前已鑒定的Motor proteins多達(dá)數(shù)十種。根據(jù)其結(jié)合的骨架纖維以及運(yùn)動方向和攜帶的轉(zhuǎn)運(yùn)物不同而分為不同類型。 胞質(zhì)中微管motor protein分為兩大類： 驅(qū)動蛋白 (kinesin)： 通常朝

26、微管的正極方向運(yùn)動； 動力蛋白 (cytoplasmic dynein)： 朝微管的負(fù)極運(yùn)動。 Kinesin與Dynein的分子結(jié)構(gòu)； Kinesin與Dynein的運(yùn)輸方式。,Kenesin,(a),Kenesin,(a) Structure of a kinesin molecule, which consists primarily of heavy chains that wrap around each other in the region of the stalk. The force-generating heads bind to the microtubule, and

27、the tail binds to the cargo being transported. Having a molecular mass of approximately 380 kDa, kinesin is considerably smaller than the other motor proteins, myosin (muscle myosin, 520 kDa) and dynein (over 1,000 kDa). (b) schematic diagram of a kenisin molecule moving a vesicle along a microtubul

28、ar track. The two heads of the molecule are thought to move in a “hand-over-hand” manner, which ensures that the motor is never released from its strack.,(b),Cytoplasmic dynein,Cytoplasmic dynein,Cytoplasmic dynein (a) Structure of a cytoplasmic dynein molecule, which consists of two large, force-ge

29、nerating globular heads (each composed of a dynein heavy chains), a stalk, and a number of smaller subunits at the base of the molecule. These latter subunits are presumed to mediate the binding of the motor protein to the cargo being transported. (b) Schematic diagram of two vesicles moving in oppo

30、site directions along the same microtubule, one powered by kinesin moving toward the plus end of the track; and the other by cytoplasmic dynein moving toward the minus end of the track. In the model shown here, each vesicle contains both types of motor proteins, but the kinesin molecules are inactiv

31、ated in the upper vesicle and the dynein molecules are inactivated in the lower vesicle. Both motor proteins are attached to the vesicle membrane by an intermediary: kinesin is attached by the transmembrane protein kinectin, and dynein by a soluble protein complex called dynactin.,A general model fo

32、r kinesin- and dynein-mediated transport in a typical cell The array of microtubules, with their (+) ends pointing toward the cell periphery, radiates from an MTOC in the Golgi region. Kenesin-dependent anterograde transport conveys mitochondria (carried by KIF1B), lysosomes, and an assortment of me

33、mbrane vesicles to the endoplasmic reticulum (ER) or cell periphery. Cytosolic dynein-dependent retrograde transport conveys elements of the ER, late endosomes, and lysosomes to the cell center.,神經(jīng)元軸突運(yùn)輸?shù)念愋?深綠：微管 淺蘭：內(nèi)質(zhì)網(wǎng) 黃色：高爾基體,上圖：內(nèi)質(zhì)網(wǎng)抗體染色 下圖：微管抗體染色,上圖：高爾基抗體染色 下圖：微管抗體染色,(A),(B),(C),鞭毛 (flagella) 運(yùn)動和纖毛

34、 (cilia) 運(yùn)動, 纖毛和鞭毛的運(yùn)動形式； 纖毛與鞭毛的結(jié)構(gòu)； 纖毛運(yùn)動機(jī)制。,受力運(yùn)動,恢復(fù)運(yùn)動,三、中間纖維 (IF),纖維直徑10nm，介于粗肌絲和細(xì)肌絲之間，故被命名為中間纖維。IF幾乎分布于所有動物細(xì)胞中并形成網(wǎng)絡(luò)結(jié)構(gòu)，特別是在需要承受機(jī)械壓力的細(xì)胞中含量相當(dāng)豐富。如上皮細(xì)胞中，除了胞質(zhì)外，在內(nèi)核膜下的核纖層也屬于IF。,中間纖維的裝配 中間纖維的成分與分布 中間纖維結(jié)合蛋白 (IFAP) 及其判定標(biāo)準(zhǔn) 中間纖維的功能,中間纖維的裝配,中間纖維裝配過程； IF裝配與MF、MT裝配相比，有以下幾個特點(diǎn)： IF裝配的單體是纖維狀蛋白 (MF, MT的單體呈球形）； 反向平行的四聚體

35、導(dǎo)致 IF 不具有極性； IF在體外裝配時不需要核苷酸或結(jié)合蛋白的輔助，在體內(nèi)裝配后，細(xì)胞中幾乎不存在IF單體（但I(xiàn)F的存在形式也可以受到細(xì)胞調(diào)節(jié)，如核纖層的裝配與解聚）。,Experimental demonstration of the dynamic character of intermediate filaments,Experimental demonstration of the dynamic character of intermediate filaments,These photographs show the results of an experiment in wh

36、ich biotin-labeled type I keratin was microinjected into cultured epithelial cells and localized 20 minutes later using immunofluorescence. The photograph in a shows the localization of the injected biotinylated keratin (as evealed by anti-biotin antibodies) that had become incorporated into filamen

37、ts during the 20 minute period following injection. The photograph in b shows the distribution of intermediate filaments in the cell as revealed by anti-keratin antibodies. The dotlike pattern of fluorescence in a indicates that the injected subunits are incorporated into the existing filaments at s

38、ites throughout their length, rather than at their ends. (Bar, 10m.),中間纖維的成分與分布,IF成分比MF、MT復(fù)雜，種類多，具有組織特異性； IF在形態(tài)上相似，而化學(xué)組成有明顯的差別； 中間纖維類型與分布； 中間纖維蛋白的表達(dá)具有嚴(yán)格的組織特異性。,Properties and distribution of the major mammalian intermediate filament proteins,1. 在細(xì)胞內(nèi)與中間纖維共分布； 2. 抗高鹽與非離子去垢劑抽提，與中間纖維共同分離； 3. 與中間纖維經(jīng)歷相同的解

39、聚和重裝配周期； 4. 在體外能與中間纖維結(jié)合。,中間纖維結(jié)合蛋白 (IFAP) 及其判定標(biāo)準(zhǔn),中間纖維的功能,增強(qiáng)細(xì)胞抗機(jī)械壓力的能力； 角蛋白纖維參與橋粒的形成和維持； 結(jié)蛋白纖維是肌肉 Z 盤的重要結(jié)構(gòu)組分； 對于維持肌肉細(xì)胞的收縮裝置起重要作用； 神經(jīng)元纖維在神經(jīng)細(xì)胞軸突運(yùn)輸中起作用； 參與傳遞細(xì)胞內(nèi)機(jī)械的或分子的信息； 中間纖維與mRNA的運(yùn)輸有關(guān)。,keratin filament,desmosome connecting two cells,open channel,Key to Cytoskeletal Functions,(a) Epithelial cell,(b) Ner

40、ve cell,(c) Dividing cell,In which: (1) Structure and Support; (2) Intracellular Transport; (3) Contractility and Motility; (4) Spatial Organization.,第二節(jié) 細(xì)胞核骨架, 核基質(zhì) (Nuclear Matrix) 染色體骨架 (chromosome scaffold) 核纖層 (Nuclear Lamina),一、Nuclear Matrix, 形態(tài)結(jié)構(gòu) 成分 核骨架結(jié)合序列 功能,形態(tài)結(jié)構(gòu),核骨架分級抽提法（由Penman實(shí)驗(yàn)室建立）： 先用非

41、離子去垢劑處理細(xì)胞，溶解膜結(jié)構(gòu)系統(tǒng), 胞質(zhì)中可溶性成分隨之流失，存留細(xì)胞骨架體系；再用Tween40和脫氧膽酸鈉處理，胞質(zhì)中的微管、微絲與一些蛋白結(jié)構(gòu)被溶去，胞質(zhì)中只有中間纖維網(wǎng)能完好存留；然后用核酸酶與0.25mol/L硫酸銨處理，染色質(zhì)中DNA、RNA和組蛋白被抽提，最終核內(nèi)呈現(xiàn)一個精細(xì)發(fā)達(dá)的核骨架網(wǎng)絡(luò)，結(jié)合非樹脂包埋-去包埋劑電鏡制樣方法，可清晰地顯示“核骨架-核纖層-中間纖維”結(jié)構(gòu)體系。,Attachment of nuclear matrix fibers to the nuclear lamina,成分,核骨架不象胞質(zhì)骨架那樣由非常專一的蛋白成分組成，核骨架的成分比較復(fù)雜，主要成分

42、是核骨架蛋白及核骨架結(jié)合蛋白，并含有少量RNA。 核骨架蛋白 骨架結(jié)合蛋白 其它,核骨架結(jié)合序列(matrix associated region, MAR),DNA序列中的MAR, 與核骨架蛋白的結(jié)合不為高鹽溶液的抽提所破壞，在基因表達(dá)調(diào)控中有作用； 核骨架結(jié)合序列的基本特征； MAR 的功能； 通過與核骨架蛋白的結(jié)合，將 DNA 放射環(huán)錨定在核骨架上； 作為許多功能性基因調(diào)控蛋白的結(jié)合位點(diǎn)。,核骨架結(jié)合序列的基本特征,富含AT; 富含DNA解旋元件 (DNA unwinding elements); 富含反向重復(fù)序列 (Inverted Repeats); 含有轉(zhuǎn)錄因子結(jié)合位點(diǎn)。,功能,核骨架與DNA復(fù)制； 核骨架與基因表達(dá)； 大量研究工作表明，真核細(xì)胞中RNA的轉(zhuǎn)錄和加工均與核骨架有關(guān)。具有轉(zhuǎn)錄活性的基因是結(jié)合在核骨架上的；RNA聚合酶在核