细胞生物学：chap7细胞骨架

1、 Cytoskeleton and Cell MotilityWhat is the cytoskeleton? Cytoskeleton: a network of protein fibers in the cytoplasm of eukaryotic cells that gives shape to a cell, holds and moves organelles, and is typically involved in cell movement. Its made up of three primary protein filaments: Microtubules Mic

2、rofilaments Intermediate filamentsCytoskeleton Functionstructural support maintains shape of cell provides anchorage for organellesmotility cell locomotion cilia, flagella, etc.regulation organizes structures & activities of cell5Dispersivity(弥散性弥散性), integrity(整体性整体性), variability(变动性变动性). Dyna

3、mic structures with filaments able to grow and shrink rapidly.The cytoskeleton is rich content in eukaryotic cells.The cytoskeleton is highly conserved in evolution. All are protein polymers.6characteristics of cytoskeleton Evolutionary perspective Proteins that make up the fibers are very similar i

4、n all living things from bacteria to humans tubulin (all cells) actin (eukaryote cells) Means that they are both ancient and essential for life. broad topic of cytoskeletonNucleoskeletonnuclear laminaextracellular matrix Form the network structure throughout the integration of the nucleus, cytoplasm

5、 and extracellular.Section 1 Microtubules, MTfound in 1963found in 1963 nSlautterbackSlautterback ( (animal) animal) nLedbetter and PorterLedbetter and Porter ( (plant)plant)They function both to determine cell shape and in a variety of cell movements, including some forms of cell locomotion, the in

6、tracellular transport of organelles, and the separation of chromosomes during mitosis. Electronmicrograph (Left) of the cytoplasm of a fibroblast, displaying microtubules (MT). Part 1 The Structure of MicrotubulesMicrotubules are dynamic structures that undergo continual assembly and disassembly wit

7、hin the cell. 1. The Structure of MicrotubulesnMicrotubules are rigid hollow rods Protofilaments Microtubules are polymers of - and -tubulin dimers. The tubulin dimers polymerize end to end in longitudinal rows，termed protofilaments. The protofilaments then bundle into hollow cylindrical filaments，w

8、hich are composed of 13 protofilaments. The Structure of Microtubules Composition of ProtofilamentsMicrotubules 80 95% of total protein- - tubulin - -tubulin -tubulinheterodimer GTP GTPtubulin heterodimer nAn , -tubulin heterodimer is the basic structural unit of microtubules.The The & tubulins

9、are spherical & tubulins are spherical acidic protein, each about acidic protein, each about 55 kDa55 kDa. . They are homologous but not identical. They are homologous but not identical. w a-Tubulin has a bound GTP, that does a-Tubulin has a bound GTP, that does not hydrolyze. not hydrolyze. w -

10、Tubulin may have bound GTP or -Tubulin may have bound GTP or GDP. GDP. Under certain conditions, -tubulin Under certain conditions, -tubulin can hydrolyze its bound GTP to GDP can hydrolyze its bound GTP to GDP plus Pplus Pi i, release P, release Pi i, and exchange the , and exchange the GDP for GTP

11、. GDP for GTP. Composition + end tubulin - end tubulin Assembly process a fast-growing plus end a slow-growing minus end The polarity of MTThe Structure of MicrotubulesSinglet 13 protofilaments Most popular in cytoplasm Unstable2. Arrangement of protofilaments Doublet One complete 13-protofilament m

12、icrotubule (tubule A ) and one additional tubules (tubule B) consisting of 10 protofilaments Cilia and flagella The luminal surface of the oviductTriplet One complete 13-protofilament microtubule (the A tubule) and two additional tubules (B and C) consisting of 10 protofilaments centrioles and basal

13、 bodies stable Part 2 Microtubule Associated proteins (MAPs)StructureStructure of MAPsBasic Microtubule binding domain: Can stabilize or destabilize microtubules.Acidic projection domain: Cross link microtubules with plasma membrane or other cytoskeletal filaments.20 Type of MAPsType of MAPsn Type I

14、: Type I: Heat Shock Proteins, MW 27KD, in the neuron axon(轴突)n Type II Type II 22 B：Ganglion cells from the rat retina in cell culture. Labeled with antibody to microtubule-associated protein (MAP-2).Part 3 Microtubule assemblyMicrotubules assemble by polymerization of subunitsAddition and loss onl

15、y at the endsAssembly of GTP bound tubulin is favored over GDP boundAssembly and disassembly depend on critical concentration of subunitsCharacterics: Dynamic instabilityThe process of microtubule assembly：Free Free - - tubulintubulin dimersdimers associate longitudinallyassociate longitudinally（8nm

16、8nm）Formation of Formation of protofilamentprotofilament Spiral Spiral lamellalamella ( (Lateral association of Lateral association of protofilamentsprotofilaments) )Widened to 13 Widened to 13 protofilamentprotofilament Sheets wrap Sheets wrap around to form around to form a hollow tubea hollow tub

17、emicrotubulemicrotubule Elongation by addition of subunitsElongation by addition of subunitsThe polarity of MT ： 24The steps of microtubule assembly Nucleation phase (lag phase) Short oligomers form the core of MT. (The importance of MTOC) Polymerization phase (elongation phase) Speed of tubulin pol

18、ymerization is greater than the depolymerization,then microtubules extend. Steady state phase (equilibrium phase) Assembly and assembly speed is equal.The phases of microtubule assembly 细胞生物学27Nucleation1.MTOC the initial point of MT assembleuMicrotubule organising centre (MTOC) The MTOC is the site

19、 of microtubule nucleation and can be visualized in cells by immunohistochemical detection of -tubulin. Minus end is adjacent to MTOC, plus is a distal end. 微管的聚合从特异性的核心形成位点开始，这些核心形成位点微管的聚合从特异性的核心形成位点开始，这些核心形成位点主要是中心体和纤毛的基体，称为微管组织中心主要是中心体和纤毛的基体，称为微管组织中心功能：帮助大多数细胞微管装配过程中成核功能：帮助大多数细胞微管装配过程中成核.u Basal

20、bodies associated with cilia and certain intercellular junctions u Centrosome associated with spindle formation. A pair of centrioles at the right angle pericentriolar material.29Centrosome and Basal bodies MTOC Nucleation Microtubule nucleation requires the action of a third type of tubulin, -tubul

21、in. The -tubulin combines with several other associated proteins to form a circular structure known as the -tubulin ring complex (-TuRC). This complex acts as a scaffold for / tubulin dimers to begin polymerization. -TuRC : circle structure of 1013 moleculars -TuRC3132 中心体：中心体：中心粒中心粒+ +中心中心粒旁物质粒旁物质T

22、he Orientation of Microtubules in a Cell 2.Microtubule assembly in vitro (Self-Assembly) Tubulin: critical concentration1mg/ml GTP pH ：6.9 Temprature：370C Mg2+ tub in polymerCc Characteristics of MT assembly动态不稳定性动态不稳定性指微管随反应体系中游离指微管随反应体系中游离二聚体的二聚体的浓度变化而发生的生长状态和缩短状态的转变。浓度变化而发生的生长状态和缩短状态的转变。Dynamic i

23、nstability refers to the fact that growing and shrinking microtubules can coexist in the same region of a cell, and that a given microtubule can switch back and forth between growing and shortening phases. GTP cap：Catastrophe:accidental loss of GTP capRescue:regain of GTP cap36dynamic instability(动态

24、不稳定性动态不稳定性)微管动态不稳定性微管动态不稳定性 细胞生物学37Nucleotide hydrolysis and GTP cap The GTP is hydrolysed to GDP soon after assembly. Rate of subunit addition faster than hydrolysis. “GTP CAP”39treadmilling(踏车运动踏车运动)又称轮回现象又称轮回现象, ,是微管组装后处于动态平衡的一种现象。是微管组装后处于动态平衡的一种现象。403.Microtubule assembly in vivo (1) In vivo cel

25、l,microtubule assembly polymerization (1) In vivo cell,microtubule assembly polymerization rate is 5 to 10 times higher than in vitro.rate is 5 to 10 times higher than in vitro.(2) Microtubules(2) Microtubules extend or shorten the changes extend or shorten the changes tend to occur on the positive

26、side.tend to occur on the positive side.(3) Dynamic instability is in high degree of order.(3) Dynamic instability is in high degree of order. TuRC组织微管形成的能力，受组织微管形成的能力，受CDK激酶的调节激酶的调节4142Direct observation of the dynamic instability of microtubules in a living cell4.Other factors effecting MT assembl

27、yv Drugs affect the assembly of MTs(1) Colchicine and VinblastineBinding 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.Part 4 Functions of MicrotubulesMaint

28、ain cell shapeThe growthing microtubules of the nerve cellsForming centrioles, cilia and flagella(1) centrioles The centriole organise the centrosome matrix centrosome: centriole + surrounding materialcentriole + surrounding material Ensures duplication during each cell cycle. Structure: Centrioles

29、are made of microtubules:9 sets of triplets(1 complete and 2 incomplete) CentrioleCentriole is the is the importanteimportante component of component of MTOC,andMTOC,and it i it involved in cell division.nvolved in cell division. CentrioleCentriole contain contain ATPATP enzyme.enzyme.48(2) Cilia an

30、d Flagella Swimming “organelles” Same structure. Cilia: More per cell and shorter Flagella: Few per cell and longer (Sperm)nstructure axoneme top: 9 doublet are in incompletion (轴丝轴丝) mobile jib: 9 doublet +2 central MT basal body ：9 + 0 triplet50Basal body of Cilia /Flagella Basal body at the base

31、of cilia and flagella. 9+0, triplet microtubule, Similar in structure to centrioles. main proteins of axoneme in cilia and flagella doublet MT doublet MT dynein arm: dynein arm: two arms, submicrotubule two arms, submicrotubule ABAB nexinnexin( (微管连接蛋白微管连接蛋白) )：在相邻微管间起连接作用：在相邻微管间起连接作用 radial spokera

32、dial spoke( (放射辐条放射辐条): ): submicrotubule A central MTsubmicrotubule A central MT central sheathcentral sheath( (中央中央鞘鞘）52Intracellular vesicles travel along microtubules by using motor protein. Such as transport along axon3. Intracellular transport4Transport along axon Vesicles transported along MT

33、 from cell body to dendrite. Different proteins are transported at different rates along axons. transport of pigment granules55Chameleon Fish（原产地为亚洲南部的印度）（原产地为亚洲南部的印度）的色素细胞中色素的色素细胞中色素分子的分散与聚集分子的分散与聚集 kinesin dynein myosinThree types of motor proteinsMicrotubule motor proteinsMicrofilament motor prot

34、eins Motor Proteins use microtubulesstructure： tetramer protein, including each two heavy chain and light chain. structural domain: head(2),petiole(1),tail(2)The globular heads of kinesin molecule bind a microtubule and act as ATP-hydrolyzing，force-generating “engines”. Conserved sequenceA tail (-he

35、lical) which can dimerise: Bind to cargo.58（1）kinesin（驱动蛋白）59Human Kinesin Proteins45 human kinesin genes.At least 12 kinesins are involved in mitosis and cytokinesis. Spindle Formation Eg5, Kif2A and KifC1 Chromosome Congression and Alignment KifC1, MCAK, CENP-E, Kif14, Kif18, and Kid Anaphase Spin

36、dle Dynamics Kif4A and Kif4B Cytokinesis Kif4A, Kif4B, MKLP1 and MKLP26061 functional characteristics of kinesin directivity ：Kinesin is a plus end-directed microtubular motor. () ( )蛋白的运动是一步一步地进行的蛋白的运动是一步一步地进行的 Each step is approximately 8 nm in length, which is also the spacing between tubulin dim

37、ers along a protofilament移动的速度与移动的速度与ATPATP的浓度有关的浓度有关速度高时速度高时, ,可达到每秒可达到每秒900nm900nmEg. Cell body to synapse.types cytoplasmic dyneins axon dyneins structure： Cytoplasmic dynein is a huge protein: molecular mass of approximately 1.5 million daltons Composed of two identical heavy chains and a variet

38、y of intermediate and some light chains. ATP hydrolysis to generate movement. To move along a microtubule toward the polymers minus end. () ( )63（2）dynein（动力蛋白）多亚基蛋白多亚基蛋白动力蛋白动力蛋白驱动蛋白与动力蛋白驱动蛋白与动力蛋白 function take part in cell division. To promote the separation of chromosomes transport vesicle and var

39、ious kinds menbrane-binding organelles. Motor proteins that power the cilia and the flagella of eukaryotic cells.Microtubules mediated material transportationKinesin-dept.transport: conveys Mit(by KIF),Lys,MvER or periphery.Cytosolic dynein-dept transport: conveys elements of ER, late endosome, Lysc

40、ell centernER and golgi use MT as a track for proper distribution.nGolgi fragments while ER collapse to centre when the cells treated with drugs that destroy MT.4. positioning and moving of organellesComparison of kinesins and dyneins Kinesins: tether ER-derived membranes to preformed microtubule tr

41、acks, and walk toward the microtubule plus ends dyneins : positioning the Golgi apparatus and moving Golgi vesicles along microtubule tracks 5. the movement of chromosome and cell division participating in hedgehog, JNK, Wnt ,ERK signal pathway .6. cellar signal transductionSection 2 Microfilament,

42、MF Microfilaments or actin filaments are the thinnest filaments of the cytoskeleton. MF are solid rods made up of globular proteins called Actin. Electronmicrograph (Left) of the cytoplasm of a fibroblast, displaying actin microfilaments (MF) .Part 1 Composition and Structure of MicrofilamentnMicrof

43、ilaments are composed of globular subunits, G-actin. Monomers of the protein actin polymerize to form long, thin fibers, F-actin .nThey are approximately 8 nm in diameter and the thinnest of the cytoskeletal filaments. Structure of actin monomerActin encoded by highly conserved gene family. There ar

44、e three classes of actin: actin : muscle cells, actin： muscle and nonmusclecells cells actin：Structure of actin monomerl 375 amino-acid residue. 43 kDa globular protein with 2 domains(4 subdomains)l Three dimensional structure is a clam.l Each molecule has Mg2+ and ATP or ADP in the ATP fold (a deep

45、 cleft between subdomains 2 and 4)ActinlBecause each actin subunit has polarity and all the subunits of an actin filament are pointed in the same direction, the entire microfilament has polarity. Plus end: grow fast.Minus end: grow slowly The structure of MFlTwisted strings of beads on a double heli

46、x structurelLinear chain of G-actinslDiameter 5-9 nm ATP可与可与actin结合形成结合形成ATP-actin, ATP-actin与与F-actin的亲和力高。的亲和力高。 ATP-actin与与F-actin结合后结合后actin构构象可发生变化而致象可发生变化而致ATP水解成水解成ADP-actin。 ADP-actin与与F-actin的亲和力低的亲和力低 ADP-actin可被置换成可被置换成ATP-actin77ADP/ATP exchangenThe actin monomer can exchange bound ADP f

47、or ATP. Part 2 Microfilament associated proteinsActin-binding proteins:They associate with monomers or filaments influence the organization of actin filaments in cells.There are more 100 kinds of Actin-binding proteins in cells.Microfilament Associated Proteins Monomer sequestering protein Cross-lin

48、king protein End blocking protein Filament-severing protein Actin filament depolymerization protein Membrane-binding protein微丝结合蛋白的功能微丝结合蛋白的功能调节调节G-actin形成形成F-actin多聚体。多聚体。影响微丝的稳定性、长度和构型。影响微丝的稳定性、长度和构型。控制微丝的形成、交联、盖帽和截断等。控制微丝的形成、交联、盖帽和截断等。参与细胞移动。参与细胞移动。80 Control pool of unpolymerized actin Bind to a

49、ctin monomer，inhibit actins polymerization Two proteins Profilin（抑制蛋白）（抑制蛋白）: promotes MF Thymosin 4 （胸腺素）（胸腺素）: inhibits MF1. monomer sequestering protein ProfilinlBinds to ATP-G-actin in 1:1 stable complexlProfilin binds to actin monomers and assists actin polymerization.lInhibits addition of mono

50、mers to minus end effectively making the plus end to grow fasterlIt does not block the ATP binding domain.Thymosin 4 Abundant in cytosol-2 x concentration of actin Binds to ATP-G-actin in 1:1 complex preventing polymerization.Role of profilin at the cell membrane Profilin competes Thymosin lHold tog

51、ether actin filaments.lHave two binding sites - one for each filament.Short cross-linking proteins support bundles.Long cross-linking proteins support networks.2. crosss-linking protein and bundling protein Actin network in a spreading platelet.Organization of actin cytoskeleton bundles closely pack

52、ed parallel arrays usually connected by fascin or similar actin cross-linking proteins （肌成束蛋白）（肌成束蛋白） networks loosely packed criss-crossed fibers usually connected by filamin or similar actin cross-linking proteins （细丝蛋白）（细丝蛋白）Mian classes of crosss-linking protein and bundling protein -actinin-act

53、inin filamin fascin （肌成束蛋白）（肌成束蛋白） fimbrin ( (毛缘蛋白毛缘蛋白) )Vinculin （纽蛋白）（纽蛋白）villin ( (绒毛蛋白绒毛蛋白) )(A) Each filamin homodimer is about 160 nm long when fully extended and forms a flexible, high-angle link between two adjacent actin filaments. (B) A set of actin filaments cross-linked by filamin forms

54、a mechanically strong web or gel. filaminlPreferably bind to polymerized actinlPermit growth only at the opposite endlStabilize the filament If capped at both ends, it is effectively stabilizedlbind at the ends of actin filaments. Some of them cap plus end slow the growthSome of them cap minus end s

55、peed up the growth because there is no loss.3. End blocking proteinl Break filaments into shorter fragments.l Remain attached to the plus end.l Regulate the length of “polymer”.l Process is calcium dependent.l Such as gelsolin (凝溶胶蛋白凝溶胶蛋白)4. Filament Severing protein lDestabilizes and breaks the fil

56、ament.lsuch as ADF/cofilin.lCofilin binds the actin filament, forcing the filament to twist a little more tightly. 5. Actin filamentde depolymerizing proteinsPart 3 Assembly of Actin FilamentsnThe filaments also possess a faster growing plus end and a slower growing minus end. nWhen the actin filame

57、nt reaches its desired length, members of a family of small proteins (capping proteins) attach to the plus end, terminating the lengthening of the filament. nThe process of shorting of actin filaments is regulated in the presence of ATP, ADP, and Ca2+ by capping proteins.（lag phase ） Two actin molec

58、ules bind weakly, but addition of a third stabilizes the complex. This trimer then adds additional molecules and forms a nucleation site. This is the slow, or lag phase of the polymerization process. 1. The process of polymerization treadmilling model dynamic instability model 2. dynamic regulation

59、of MF assembly l Assembly of actin filaments (F-actin) is polarized, with G-actin subunits added to the plus (+) end and removed at the minus () end. Even actin filaments of a constant length are highly dynamic structures, balancing G-actin assembly and disassembly at the opposite ends, with a net m

60、ovement or flow along the polymer known as treadmilling.Treadmilling model(2)Dynamic instability modell Free actin is preferably bound to ATP.l The ATP is hydrolysed to ADP soon after assembly.l Rate of subunit addition faster than hydrolysis.l “ATP CAP” cell cortex or actin cortex102 the concentration of mono