1、主講人：高志芹,第九章 細(xì)胞骨架 chapter 9 cytoskeleton,2,Introduction of cytoskeleton細(xì)胞骨架概述,細(xì)胞骨架的主要特征： A system of filaments found in the cytoplasm of cells. The cytoskeleton is unique to eukaryotic cells (真核細(xì)胞). It is a dynamic(動(dòng)態(tài)的) three-dimensional structure.,推薦細(xì)胞骨架學(xué)習(xí)網(wǎng)址：,3,4,Introduction of cytoskeleton細(xì)胞骨架概述,I

2、t acts as both muscle(肌肉) and skeleton(骨骼). It maintains cell shape, protects the cell. 維持細(xì)胞形態(tài)，承受外力,保護(hù)細(xì)胞。 enables cellular motion, and plays important roles in intracellular transport (the movement of vesicles and organelles, for example) 細(xì)胞運(yùn)動(dòng)、胞內(nèi)物質(zhì)運(yùn)輸 cellular division.細(xì)胞分裂,細(xì)胞骨架的主要功能,5,1.結(jié)構(gòu)與支持；2.胞內(nèi)運(yùn)輸

3、；3.收縮和運(yùn)動(dòng)；4.空間組織,6,Introduction of cytoskeleton細(xì)胞骨架概述,The cytoskeleton is made up of three kinds of protein filaments: Microfilaments(MF, 微絲) intermediate filaments (IF, 中間纖維) microtubules (MT, 微管).,細(xì)胞骨架的類型：,7,MF IF MT,Microtubule, MT: is a polymer of globular tubulin subunits, which are arranged in

4、a cylindrical tube measuring 25 nm in diameter.,Microfilametnt,MF: Is a long, thin fiber approximately 7nm in diameter.,Intermediate filament, IF: is 10 nm in diameter. It is a rope-like assembly of fibrous polypeptides.,Three types of protein fibers in cells,8,what is the cytoskeleton? 細(xì)胞骨架的概念,The

5、cytoskeleton is unique to eukaryotic cells. It is a dynamic three-dimensional structure that fills the cytoplasm. This structure acts as both muscle and skeleton, for movement and stability. 真核細(xì)胞的細(xì)胞質(zhì)中的動(dòng)態(tài)、三維蛋白纖維網(wǎng)絡(luò)結(jié)構(gòu)，就像肌肉和骨骼，與細(xì)胞運(yùn)動(dòng)和穩(wěn)定性有關(guān)。,9,微絲的結(jié)構(gòu)。 微絲的組裝；微絲組裝的特征；影響微絲組裝的因素。 微絲在細(xì)胞中形成的暫時(shí)性結(jié)構(gòu)和永久性結(jié)構(gòu)。 Molecula

6、r motor的定義。 MAP肌球蛋白的結(jié)構(gòu)。 肌纖維的結(jié)構(gòu)組成和肌肉收縮的滑動(dòng)模型。,第一節(jié) 微絲與細(xì)胞運(yùn)動(dòng) 主要內(nèi)容,10,Unit 1 Microfilament and cell motility第一節(jié) 微絲與細(xì)胞運(yùn)動(dòng),Definition(定義和概述) 微絲的組成及其組裝 結(jié)構(gòu)與組成成分 微絲的組裝與動(dòng)力學(xué)特性 影響微絲組裝的特異性藥物 微絲網(wǎng)絡(luò)動(dòng)態(tài)結(jié)構(gòu)的調(diào)節(jié)與細(xì)胞運(yùn)動(dòng) 非肌肉細(xì)胞內(nèi)的微絲結(jié)合蛋白 細(xì)胞皮層 應(yīng)力纖維,細(xì)胞偽足的形成與遷移運(yùn)動(dòng) 微絨毛 胞質(zhì)分裂環(huán) 肌球蛋白：依賴于微絲的分子馬達(dá) 型肌球蛋白 非傳統(tǒng)類型的肌球蛋白 肌細(xì)胞的收縮運(yùn)動(dòng) 肌纖維的結(jié)構(gòu) 肌肉收縮的滑動(dòng)模型,11

7、,Microfilaments (or actin filaments) are the thinnest filaments of the cytoskeleton found in the cytoplasm of all eukaryotic cells. MFs are made of actin and involved in cell motility, twisted chain 7 nm in diameter. 微絲(Microfilament, MF), 又稱肌動(dòng)蛋白纖維(Actin filament)，是指真核細(xì)胞中由肌動(dòng)蛋白(actin)組成，直徑為7nm的骨架纖維，呈

8、雙股螺旋狀 。,Definition of Microfilament微絲的定義,12,Cell motility(細(xì)胞運(yùn)動(dòng)),指細(xì)胞表現(xiàn)出的所有運(yùn)動(dòng)，包括細(xì)胞形態(tài)的改變和細(xì)胞內(nèi)部的運(yùn)動(dòng)。 single-celled organisms move in their environments and/or move things internal in order to seek food, produce energy., 變形蟲(chóng)阿米巴運(yùn)動(dòng),13,Cell motility(細(xì)胞運(yùn)動(dòng)),In multicellular organisms cell motility enables singl

9、e cells and groups of cells to migrate in the embryo to the appropriate locations in the body. when cell motility uncontrolled, leading to cancer and the metastasis of cancerous cells (physical development). Others muscle cell contraction (肌細(xì)胞收縮) nerve axon elongation (神經(jīng)軸突的伸長(zhǎng)) cell-surface protrusi

10、ons (細(xì)胞表面突起) separation of chromosomes, etc.(染色體分離),14,. microfilament constitute and assemble一、微絲的組成及其組裝,Structure and Composition (結(jié)構(gòu)與成分) Assemble and Dynamic instability (組裝與動(dòng)力學(xué)特性) Drugs specific to microfilament assembly (影響微絲組裝的特異性藥物),15,Structure and Composition of MF結(jié)構(gòu)與成分,Composition(成分) MFs

11、are made of actin. （MF由肌動(dòng)蛋白組成） Actin is the most abundant protein in the cell: - 2% of total proteins in a regular cell - 10-20% in muscle cells,actin,-,+,+,-,16,Actin exist in two forms in cell（肌動(dòng)蛋白在細(xì)胞內(nèi)以2種形式存在的存在方式）,Two forms: G-actin: globular monomeric actin(球形肌動(dòng)蛋白單體) 一種以游離態(tài)存在，為球狀肌動(dòng)蛋白(globular ac

12、tin，G-actin)，它有聚合成長(zhǎng)纖維的能力。 F-actin: filamentous polymeric actin（聚合纖維狀肌動(dòng)蛋白）肌動(dòng)蛋白在細(xì)胞中的存在方式： 另一種為結(jié)合狀態(tài)，存在于微絲中，稱纖維狀肌動(dòng)蛋白(filamentous actin，F(xiàn)-actin)。 G-actin與F-actin之間可以相互轉(zhuǎn)換。,17,18,Structure of MF（微絲的結(jié)構(gòu)）,Dual twisted chain 7 nm in diameter 直徑7 nm ，雙股螺旋 Compared to Microtubules, thinner, more flexible, shorte

13、r（比微管細(xì)，可彎曲，較短） Point in same direction（同向排列） Different organisation in different cellular regions 在不同的細(xì)胞聚合成不同的形態(tài),19,Structure of MF（微絲的結(jié)構(gòu)）,20,細(xì)胞內(nèi)微絲的染色方法: 考馬斯亮藍(lán)（Coomassie）染色法,微絲呈現(xiàn)藍(lán)色,21,Assemble and Dynamic instability組裝與動(dòng)力學(xué)特性,Polarity（極性） Polymerization process（聚合過(guò)程） Polymerization model（裝配模型）,22,Pol

14、arity（極性）： Within a MF, all the actin monomers are oriented in the same direction, so MF has a polarity 每個(gè)肌動(dòng)蛋白分子都具有極性，一端為正端(plus end)，另一端為負(fù)端(minus end)。裝配時(shí)呈頭尾相連，故微絲也有極性。,-,23,成核(nucleation)-Arp (actin related protein) 延伸(elongation) 穩(wěn)定狀態(tài)(steady state),Polymerization process（聚合過(guò)程）,Arp參與成核,24,In vitro

15、, both ends of the MF grow, but the plus end faster than the minus. Because actin monomers tend to add to a filaments plus end and leave from its minus end- “Tread-milling”踏車行為 Dynamic equilibrium between the G-actin and polymeric forms, which is regulated by ATP hydrolysis and G-actin concentration

16、.,Polymerization model（裝配模型）,25,影響微絲組裝的因素,促進(jìn)組裝：含ATP、Mg2+、高Na+、K+ 促進(jìn)解聚：含Ca2+、低Na+、K+,26,影響微絲組裝的特異性藥物,Cytochalasins B: Prevent the addition of new monomers to existing MFs, which eventually depolymerize.細(xì)胞松弛素B：可以切斷微絲,并結(jié)合在微絲正極阻抑肌動(dòng)蛋白聚合,因而導(dǎo)致微絲解聚。 Phalloidin:A cyclic peptide from the death cap fungus, blo

17、cks the depolymerization of MF鬼筆環(huán)肽(philloidin)：與微絲側(cè)面結(jié)合,防止MF解聚。,Those drugs disrupt the monomer-polymer equilibrium, so are poisonous to cells 影響微絲裝配動(dòng)態(tài)性的藥物對(duì)細(xì)胞都有毒害，說(shuō)明微絲與單體的動(dòng)態(tài)平衡的重要性。,27,二、微絲網(wǎng)絡(luò)動(dòng)態(tài)結(jié)構(gòu)的調(diào)節(jié)與細(xì)胞運(yùn)動(dòng),非肌肉細(xì)胞內(nèi)的微絲結(jié)合蛋白 細(xì)胞皮層 應(yīng)力纖維 細(xì)胞偽足的形成與遷移運(yùn)動(dòng) 微絨毛 胞質(zhì)分裂環(huán),（一）非肌肉細(xì)胞內(nèi)的微絲結(jié)合蛋白,細(xì)胞內(nèi)的微絲（MF in cell） ： 穩(wěn)定（ 永久性perman

18、ent ）的結(jié)構(gòu)：微絨毛 動(dòng)態(tài)（暫時(shí)性 temporary ）結(jié)構(gòu)：胞質(zhì)分裂環(huán)、細(xì)胞偽足等 Dynamic equilibrium is required for the cell functions. Some MFs are temporary and others permanent.,微絲結(jié)合蛋白(MF associate protein, MAP),有100多種，它們可從不同水平調(diào)節(jié)微絲的組裝或影響微絲結(jié)構(gòu)的穩(wěn)定等。,P269 Fig9-5,微絲結(jié)合蛋白(MF associate protein, MAP)及其功能：,Other proteins promote branching

19、or linking of actin filaments into networks. 促進(jìn)微絲分支或者交聯(lián)成網(wǎng)絡(luò)。,There are a lot of proteins that regulate the actin assembly, filament length and stability in vivo. 一些MAP在體內(nèi)調(diào)節(jié)微絲的長(zhǎng)度和穩(wěn)定性。,MAP的功能：,31,Monomer-sequestering proteins（單體隔離蛋白） : bind with actin monomers and prevent them from polymerizing Banding

20、 proteins（成束蛋白）: generate parallel bundles found in microvilli Capping(end-blocking) proteins（封端蛋白）: stabilize F-Actin. Filament growth is prevented by capping proteins that bind to F-actin filament ends Cross-linking proteins（交聯(lián)蛋白）: cross-link actin filaments into a three-dimensional cytoplasmic ge

21、l. Severing proteins（剪切蛋白）: induce depolymerization,See p269, Fig9-5,32,（二）細(xì)胞皮層(cell cortex) The highest density of actin filaments is at the cell periphery forming cell cortex.細(xì)胞膜下，由高密度的肌動(dòng)蛋白絲和微絲結(jié)合蛋白組成的網(wǎng)狀結(jié)構(gòu)。 Maintain cells shape and enforce PM (維持細(xì)胞的形態(tài)結(jié)構(gòu)，支撐細(xì)胞膜) 參與細(xì)胞運(yùn)動(dòng)(Cell migration), 如胞質(zhì)環(huán)流(cyclosis

22、)、阿米巴運(yùn)動(dòng)(amoiboidmotion）、變皺運(yùn)動(dòng)(ruffled membrane locomotion)以及細(xì)胞吞噬活動(dòng)(phagocytosis)等都與微絲有關(guān)。,培養(yǎng)的上皮細(xì)胞（微絲紅色、微管綠色）,33,（三）Stress fibers ： 是真核細(xì)胞中廣泛存在的微絲束結(jié)構(gòu)。 Composed of actin filaments and myosin-II. 電鏡觀察表明，應(yīng)力纖維由大量微絲構(gòu)成凝膠狀網(wǎng)絡(luò)。,應(yīng)力纖維可提供用以對(duì)抗細(xì)胞表面張力或細(xì)胞與基質(zhì)間的張力，以維持細(xì)胞的扁平鋪展和特異的形狀，并賦予細(xì)胞韌性及強(qiáng)度。,（四）細(xì)胞偽足的形成與遷移運(yùn)動(dòng),細(xì)胞運(yùn)動(dòng)是一個(gè)周期性的過(guò)

23、程，其中包括運(yùn)動(dòng)方向前端細(xì)胞部分的“突出”和后端細(xì)胞部分的“收縮”。 肌動(dòng)蛋白纖維的多聚化驅(qū)動(dòng)細(xì)胞向前“突出”，而這些纖維的空間組織情況則決定“突出”結(jié)構(gòu)的性狀。 尖刺狀的絲狀偽足（filopodia）中包含長(zhǎng)束狀的肌動(dòng)蛋白纖維， 片狀的板狀偽足(lamellipodia)中包含分支狀的肌動(dòng)蛋白纖維。,（四）細(xì)胞偽足的形成與遷移運(yùn)動(dòng),細(xì)胞變形運(yùn)動(dòng)可分4步： 微絲纖維生長(zhǎng)，使細(xì)胞表面突出，形成片足； 在片足與基質(zhì)接觸的位置形成粘著斑； 在myosin的作用下微絲纖維滑動(dòng)，使細(xì)胞主體前移； 解除細(xì)胞后方的粘著點(diǎn),細(xì)胞變形運(yùn)動(dòng),（四）細(xì)胞偽足的形成與遷移運(yùn)動(dòng),a human fibroblast m

24、igrating in culture.,38,（五）Microvillus: Support the projecting membrane of intestinal epithelial cells. 同向平行排列的微絲,39,(六) Contractile ring For cytokinesis（參與細(xì)胞分裂） 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(dòng)蛋白和肌球蛋白相對(duì)滑動(dòng)。,Actin arrays in a cell.,41,三、肌球蛋白：依賴于微絲的分子馬達(dá),Molecular motors are biological molecular machines th

25、at are the essential agents of movement in living organisms. 分子馬達(dá)：一類由生物分子組成，具有馬達(dá)（發(fā)動(dòng)機(jī)）功能的分子機(jī)器 三類家族成員： Kinesin Dynein myosin,42,肌球蛋白(myosin),傳統(tǒng)類型的肌球蛋白(conventional myosin)： 型肌球蛋白 非傳統(tǒng)類型的肌球蛋白(unconventional myosin)：others,43,44,Myosin 的結(jié)構(gòu),2條重鏈4條輕鏈 頭部有actin 和 ATP結(jié)合位點(diǎn),ATP-binding sits,45,Myosin 參與肌細(xì)胞的收縮運(yùn)動(dòng)

26、 骨骼肌細(xì)胞的收縮單位是肌原纖維。,四、肌細(xì)胞的收縮運(yùn)動(dòng),肌(原)纖維(myofibril)的結(jié)構(gòu)： 肌原纖維由粗肌絲(thick myofilament)和細(xì)肌絲(thin myofilament)組成。 粗肌絲 的成分是肌球蛋白(myosin)， 細(xì)肌絲的主要成分是 肌動(dòng)蛋白(G-actin)、 原肌球蛋白(tropomyosin, Tm)、 肌鈣蛋白(troponin, Tn),46,神經(jīng)肌肉接頭處的閘門通道,1,2,3,4,5,47,sliding filament model,Release釋放,Attachment接合,Cocked直立,force generation力產(chǎn)生,re

27、-attachment再接合,48,接合(attachment):肌球蛋白頭部與肌動(dòng)蛋白絲緊密結(jié)合并且ATP很快與肌球蛋白結(jié)合。 釋放(release):ATP結(jié)合于肌球蛋白頭部，從而誘導(dǎo)肌動(dòng)蛋白結(jié)合位點(diǎn)上的肌球蛋白發(fā)生構(gòu)象變化，使肌球蛋白頭部對(duì)細(xì)肌絲的親和力下降。 直立（cocked）:肌球蛋白頭部的ATP結(jié)合位點(diǎn)關(guān)閉，啟動(dòng)明顯的構(gòu)象變化，ATP位移并水解，水解后的ADP和Pi仍結(jié)合在肌球蛋白上。 力產(chǎn)生（force generation）:肌球蛋白頭部微弱結(jié)合到細(xì)肌絲的一個(gè)新的結(jié)合位點(diǎn)上，相繼釋放無(wú)機(jī)Pi、 ADP ，肌球蛋白構(gòu)象變化，又恢復(fù)原來(lái)的構(gòu)象，結(jié)果肌球蛋白與肌動(dòng)蛋白緊密結(jié)合，并產(chǎn)

28、生機(jī)械力，拉動(dòng)細(xì)肌絲相對(duì)于肌球蛋白的滑動(dòng)。,49,Section 2 MT and its functions,微管的結(jié)構(gòu)組成與極性 （structure, composition and polarity of MT） 微管的組裝和去組裝 (assemble and dis assemble of MT) 微管組織中心(MTOC) 微管的動(dòng)力學(xué)特性 微管結(jié)合蛋白對(duì)微管網(wǎng)絡(luò)結(jié)構(gòu)的調(diào)節(jié) 微管對(duì)細(xì)胞結(jié)構(gòu)的組織作用 細(xì)胞內(nèi)依賴于微管的物質(zhì)運(yùn)輸 鞭毛和纖毛的結(jié)構(gòu)與功能 紡錘體和染色體運(yùn)動(dòng),50,一、微管的結(jié)構(gòu)組成與極性 structure, composition and polarity of MT

29、,structure are straight, hollow cylinders whose wall is made up of a ring of 13 “protofilaments原纖維; have a diameter of about 25 nm; composition are built by the assembly of dimers of alpha tubulin and beta tubulin;,51,The dimeric tubulin subunits,alpha tubulin and beta tubulin,、微管蛋白亞基均為球形酸性蛋白，分子量相同，

30、但氨基酸組成和多肽鏈序列上有差異。 、微管蛋白在細(xì)胞質(zhì)中的二聚體(heterodimer)形式存在,單體則很快被降解。,52,alpha tubulin and beta tubulin,Both - and -tubulin can bind GTP - Tubulin has a bound molecule of GTP, that does not hydrolyze. -Tubulin can hydrolyze its bound GTP GDP + Pi and exchange with GTP,E,N,53,one end of a microtubule is ringed

31、 by -tubulin - this is known as the minus end the opposite end is ringed by -tubulin - this is known as the plus end there is a greater tendency for subunits to add at the plus end,Polarity,54,二、微管的組裝和去組裝 (assemble and dis assemble of MT),（一）微管的體外組裝與踏車行為 （二）作用于微管的特異性藥物,三、微管組織中心 (MTOC) （一）中心體 （二）基體和其

32、它微管組織中心,55,(一) 微管的體外組裝與踏車行為 MT assemble invitro and treadmilling model,56,The microtubules polymerize in vitro,The conditions required for microtubule assemble in vitro are: and -tubulin subunits; Mg2+; GTP and incubated at 37. The hydrolysis of GTP of course is facilitated at a temperature of 37 an

33、d stopped at temperatures of 4.,體外微管組裝的條件,體外微管的組裝,57,Grows more rapidly from the + end,During elongation, bi-directional polymerization of a polar microtubule continues and grows more rapidly from the + end. concentrations of tubulin subunits are high, and polymerization is faster than depolymerized

34、 Tubulin polymerization is inhibited by raised Ca2+, and low temperature.,正端生長(zhǎng)得快,58,The microtubules polymerize in vivo,The arrangement of microtubules in cells is determined by microtubule-organizing centers (MTOCs).,P284,體內(nèi)微管的組裝,59,（二）作用于微管的特異性藥物 Drugs that disrupt microtubules,Some drugs (all of

35、which inhibit mitosis) affect polymerization or depolymerization of tubulin: Colchicine(秋水仙素) binds tubulin () end directed force exerted by dynein (light green) pull asters toward the poles in combination with the (+) end directed motors in the spindle. pull separated chromotids to the poles of the

36、 cell.,92,九、紡錘體和染色體運(yùn)動(dòng) Movement of spindle and chromosome,93,九、紡錘體和染色體運(yùn)動(dòng) Movement of spindle and chromosome,ATP驅(qū)動(dòng)染色體運(yùn)動(dòng)，致MT解聚 MT解聚驅(qū)使染色體運(yùn)動(dòng),94,九、紡錘體和染色體運(yùn)動(dòng) Movement of spindle and chromosome,染色體的運(yùn)動(dòng)依賴紡錘體微管的組裝和去組裝。 動(dòng)粒微管與動(dòng)粒之間的滑動(dòng)主要是依靠結(jié)合在動(dòng)粒部位的驅(qū)動(dòng)蛋白(kinesin)和動(dòng)力蛋白(dynein)沿微管的運(yùn)動(dòng)來(lái)完成 極微管在紡錘體中部交錯(cuò)，在極微管之間分布有雙極馬達(dá)蛋白 雙極驅(qū)

37、動(dòng)蛋白四聚體沿微管向正極運(yùn)動(dòng)時(shí)，紡錘體二極間距離延長(zhǎng)。 反之紡錘體距離縮短。,95,Section 3 Intermediate filaments, IFs,一、中間絲的主要類型和組成成份 IFs are the most abundant and stable components of the cytoskeleton,一、中間絲的主要類型和組成成份,See P301, Tab 9-2: 6類+未分類,96,The domain organization of intermediate filament protein monomers.,一、中間絲的主要類型和組成成份,Most intermediate fil