复旦大学细胞生物学课件09细胞骨架系统

1、Cytoskeleton SystemA. Conception of Cytoskeleton (Narrow sense) A complex network of interconnected microfilaments, microtubules and intermediate filaments that extends throughout the cytosol.Chapter 9MicrobubulesMicrofilamemtsIntermediate filaments1. IntroductionB. Techniques for studying the cytos

2、keletonFluorescent microscopy and Electron microscopy : Immunofluorescence: fluorescently-labeled antibody Fluorescence: microinject into living cells Video microscopy: in vitro motility assays Electron: Triton X-100, Metal replica Quick freezing-deep etching EMBiochemical analysis (in vitro) Differ

3、ence centrifugation; SDSDrugs and mutations (about functions) C. The self-assembly and dynamic structure of cytoskeletal filamentsEach type of cytoskeletal filament is constructed from smaller protein subunits.The cytoskeleton is a network of three filamentous structures.The cytoskeleton is a dynami

4、c strucrure with many roles. 2. Microfilament, MFA. MFs are made of actin and involved in cell motility.Using ATP, G-actin polymerizes to form MF(F-actin) B. MF assembly and disassemblyCharacteristics:(1) Within a MF, all the actin monomers are oriented in the same direction, so MF has a polarityMyo

5、sin is molecular motor for actins.(2) In vitro, (Polymerization) both ends of the MF grow, but the plus end faster than the minus. Because actin monomers tend to add to a filament s plus end and leave from its minus end(3) Dynamic equilibrium between the G-actin and polymeric forms, which is regulat

6、ed by ATP hydrolysis and G-actin concentration.2.2 Assembly Mechanism of actin polymerization: 3 phases of G-actin polymerization. Critical concentration (Cc). In steady state, G-actin monomers only exchange with subunits at the filament ends but there is no net change in the total mass of filaments

7、. During the elongation state, one end of the filament, the (+) end, elongates five to ten times faster than does the opposite (-) end. This is because Cc value is much lower for G-actin addition at the (+) end than for addition at the (-) end. Figure 6-17 The three phases of G-actin polymerization

8、in vitro.(4) Dynamic equilibrium is required for the cell functions. Some MFs are temporary and others permanent. (5)The nucleation of actin filaments at the PM is frequently regulated by external signals, allowing the cell to change its shape and stiffness rapidly in response to changes in its exte

9、rnal environment. This nucleation is catalyzed by a complex of proteins that includes two actin-related proteins, or ARPs(Arp2 and Arp3). Actin arrays in a cell.C. Specific drugs affect polymer dynamicsCytochalasins: Prevent the addition of new monomers to existing MFs, which eventually depolymerize

10、.Phalloidin:A cyclic peptide from the death cap fungus, blocks the depolymerization of MF Those drugs disrupt the monomer-polymer equilibrium, so are poisonous to cellsD. Actin-binding proteinsThe structures and functions of cytoskeleton are mainly controlled by its binding proteins2.4 microfilament

11、-binding proteins Actin binding proteins control the structure and behavior of actin filament. actin binding proteins e.g. proflin (promote acting assembly), thymosin beta4 (inhibits actin assembly). Some cytosolic proteins control actin polymerization. microfilament-binding proteins 3 different typ

12、es of stalbe actin filament structures： Parallel bundle: MFs isotactic parallel arrange，mainly found in microvillus and filopodium (丝状伪足). Contractile bundle: MFs anti-parallel arrange, mainly found in stress fibers (应力纤维) and contractive ring of mitosis(有丝分裂收缩环 )。 Gel-like network: MFs cross-linked

13、 arrange, most be found in cell cortex (cytosol, 细胞皮层).(2) MF-binding proteins成核蛋白（nucleating proteins）: actin-related proteins, ARPs 单体隔离蛋白（monomer-sequestering protein）: thymosin封端（加帽）蛋白（End-blocking(capping) proteins）:capZ 单体聚合蛋白（monomer-polymerizing proteins）:抑制蛋白（profilin）是一种与ATP-肌动蛋白单体结合的蛋白质 肌

14、动蛋白纤维解聚蛋白（actin filament-depolymerizing proteins）: cofilin、ADF以及蚕食蛋白与肌动蛋白纤维的减端结合，大大促进肌动蛋白纤维解聚成单体。 交联蛋白（cross-linking proteins）: ABP280和细丝蛋白,促进形成近于正交相互联系的纤维松散网络 纤维切割蛋白（filament-severing proteins）: gelsolin 膜结合蛋白（membrane-binding proteins）:连接膜与肌动蛋白的蛋白质包括联结蛋白（vinculin），ERM家族的成员包括埃兹蛋白（ezrin）、根蛋白（radixin

15、）和膜突蛋白（moesin） Model of the complementary roles of profilin and thymosin 4 in regulating polymerization of G-actin. Actin filaments are likewise strongly affected by the binding of accessory proteins along their sides.Actin filaments in most cells are stabilized by the binding of tropomyosin, an elo

16、ngated protein. Which can prevent the filament from interacting with other proteins.Another important actin filament binding protein, cofilin, present in all eucaryotic cells, which destabilized actin filaments(also called actin depolymerizing factor). Cofilin binds along the length of the actin fil

17、ament, forcing the filament to twist a little more tightly. In addition, cofilin binding cause a large increase in the rate of actin filament treadmilling.The modular structures of four actin-cross-linking proteinsThe formation of two types of actin filament bundles:Contractile bundle mediated by -a

18、ctinin parallel bundle mediated by fimbrin.Gel-like networkActin filaments are often nucleated at the plasma membrane. The highest density of actin filaments is at the cell periphery forming cell cortex. Filamin cross-links actin filaments into a three-dimensional network with the physical propertie

19、s of a gel.Loss of filamin causes abnormal cell motilityE. Functions of MFs(1) Maintain cell s shape and enforce PM(2) Cell migration (Fibroblast et al)Platelet activation is a controlled sequence of actin filament severing,uncapping, elongation,recapping, and cross-linking.(3) Microvillus: Support

20、the projecting membrane of intestinal epithelial cells(4) Stress fibersComposed of actin filaments and myosin-II Stress FibersFocal contactsFocal contacts MFsResponse to tensionResponse to tension(5) Contractile ring: For cytokinesis(6) Muscle contractionOrganization of skeletal muscle tissueSarcome

21、re皮肤 ?蛋白同伴皮肤 ?蛋白平原皮肤球 ?蛋白Proteins play important roles in muscle contraction Myosin: The actin motor porteinATPaseMyosin II-DimerMainly in muscle cellsThick filamemtsLight-chain phosphorylation and the regulation of the assembly of myosin II into thick filaments Tropomyosin, Tm and Tropnin, TnRopeli

22、ke moleculeRegulate MF to bind to the head of myosinComplex, Ca2+-subunitControl the position of Tm on the surface of MFThick and thin filaments sliding modelExcitation-contraction coupling processAction potentialCa2+ rise in cytosolTnTmSlidingSchematic diagram showing how a Ca2+-release channel in

23、the sarcoplasmic reticulum membrane is thought to be opened by a voltage-sensitive transmembrane protein in the adjacent T-tubule membraneF. Smooth muscle cell(平滑肌细胞 ) contractionSmooth and nonmuscle cell contraction are regulated in a manner distinct from that of skeletal muscle cellsCa2+ riseCa2+

24、-calmodulinBind to MLCKRegulate light chain PhosphorylateMyosin interact with actinContractionSLOW3.Microtubule, MTTubulin heterodimersare the protein building blocks of MTsA. Structures: Arrangement of protofilaments in singlet, double, and triplet MTsSingletDoubleTripletABABCIn cilia and flagellaI

25、n centrioles and basal bodiesB. MTs assemble from microtubule-organizing centers (MTOCs)(1) Interphase: CentrosomeDynamic instability(2) Dividing cell: Mitotic spindleDynamic instability(3) Ciliated cell: Basal bodyStabilityBasal body structureC. Characteristics of MT assemblyDynamic instability due

26、 to the structural differences between a growing and a shrinking microtubule end.GTP cap;Catastrophe: accidental loss of GTP cap;Rescue: regain of GTP cap Why the centrosome can act as MTOCStructureNo centrioles in Plant and fungi MT are nucleated by a protein complex containing ?-tubulinThe centros

27、ome is the major MTOC of animal cells Drugs affect the assembly of MTs(1) ColchicineBinding to tubulin dimers, prevent MTs polymerization(2) TaxolBinding to MTs, stabilize MTsThese compounds are called antimitotic drugs, and have application in medical practice as anticancer drugs Microtuble-associa

28、ted proteins (MAPs)MAPs modulate MT structure, assembly, and functionMotor MAPsNonmotor MAPsTau: In axon, cause MTs to form tight bundlesMAP2: In dendrites, cause MTs to form looser bundlesControl organizationThe importance of MAPs for neurite formationLike axonLike dendrite Organization of MT bundl

29、es by MAPs . Spacing of MTs depends on MAPsInsect cell expressing MAP2Insect cell expressing tauFrom J. Chen et al. 1992. Nature 360: 674The effects of proteins that bind to MT ends(A)The transition between Mt growth and Mt shrinking is controlled in cells by special proteins.(B)Capping proteins hel

30、p to localize Mt in budding yeast cell.?蛋白5. Functions of MTs1. Maintain cell shape2. Motor proteins and intracellular transport（1）Motor proteins: 3 superfamily Kinesin dependent-MT (2KHC+2KLC), N-/M-Kinesin向正极运动；C-kinesin向负极运动 头部具MT 和ATP结合位点；迄今已报道600余种 Cytoplasmic Dynein dependent-MT (2/3HC+ more L

31、C) 14um/s ; C端为马达结构域（结合ATP）;N端为易变尾部 Axonemal arm dyneins: 与纤毛鞭毛运动有关 Myosin dependent-MFIntracellular transport of membrane-bounded vesicles, proteins: Directionality(2) Kinesin is a plus-end directed MT motor protein Hand-over-hand model1985年发现的驱动蛋白分子-“常规驱动蛋白”（conventional kinesin），只是相关蛋白超家族中的一个成员，驱

32、动蛋白-相关蛋白（kinesin-related proteins）超家族简称为KRPs或KLPs（kinesin-like proteins）。根据基因组序列分析，估计哺乳动物能产生50种以上不同的KLPs。KLPs的头部都含有相关的氨基酸序列，反映出它们具有共同的进化祖先，并且在沿微管运动方面具有相似的作用。比较而言，KLPs的尾部有较大变异，具有各种不同的序列，反映出不同的驱动蛋白成员运送不同的货物。 Comparison of the mechanochemical cycles of kinesin and myosin II.Motor proteins generate forc

33、e by coupling ATP hydrolysis to conformational changes.?星期三解释的 1 分子 ATP, 先前 ?8 nm, 相当于 ?秒 1 um.(3) Dynein is a minus-end directed motor proteinAxonemal and cytoplasimic dyneinsMW=1500KDa 胞质动力蛋白至少有两种作用：1. 在有丝分裂期间作为纺锤体定位和染色体移动的产力装置 。2. 作为在胞质中定位高尔基复合体和将小泡和细胞器移向减端的微管马达蛋白。Mediate: 动力蛋白激活蛋白复合体 （dynactin c

34、omplex） Intracellular transport in nerve cellsMt organization in fibroblasts and neurons.Movement of pigment granules: color adjustment The placement of organelles3. Movement of mitotic spindle and chromosomes4. Cilia and flagella: Structure and movementSize and length: The same diameter, flagella a

35、re often much longerMovement: Cilia: Beating; Flagella: Bending motionCiliary dyneinStructure:Microtubule sliding causes cilia/flagella to bendDyneinsCrosslinks and spokesIntermediate filaments, IFs IFs are the most abundant and stable components of the cytoskeleton1. IFs assemble from fibrous subunitsAssembly Characteristics of IFsMonomers: Fibrous proteinsAntiparallel tetramer: No polarityAlmost no IF monomers within cell But IFs are still dynamic polymers in the cell IF typing serves as a diag