1、Cytoskeleton SystemA.Conception of Cytoskeleton(Narrow sense)A complex network of interconnected microfilaments,microtubules and intermediate filaments that extends throughout the cytosol.Chapter 10MicrobubulesMicrofilamemtsIntermediate filaments1.IntroductionFigure10-2.The three types of protein fi
2、laments that form the cytoskeleton.B.Techniques for studying the cytoskeletonv Fluorescent microscopy and Electron microscopy:Immunofluorescence:fluorescently-labeled antibody Fluorescence:microinject into living cells Video microscopy:in vitro motility assays Electron:Triton X-100,Metal replicav Dr
3、ugs and mutations(about functions)v Biochemical analysis(in vitro)C.The self-assembly and dynamic structure of cytoskeletal filamentsvEach type of cytoskeletal filament is constructed from smaller protein subunits.vThe cytoskeleton is a network of three filamentous structures.vThe cytoskeleton is a
4、dynamic strucrure with many roles.2.Microfilament,MFA.MFs are made of actin and involved in cell motility.vUsing ATP,G-actin polymerizes to form MF(F-actin)Figure16-51The trapping of ADP in an actin filament.B.MF assembly and disassemblyvCharacteristics:(1)Within a MF,all the actin monomers are orie
5、nted in the same direction,so MF has a polarityMyosin 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 filaments plus end and leave from its minus end-“Tread-milling”(3)Dynamic equilibriu
6、m between the G-actin and polymeric forms,which is regulated by ATP hydrolysis and G-actin concentration.(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,al
7、lowing the cell to change its shape and stiffness rapidly in response to changes in its external 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.Figure16-55Lamellipodia and microspikes at the lea
8、ding edge of a human fibroblast migrating in culture.Thearrowinthisscanningelectronmicrographshowsthedirectionofcellmovement.Asthecellmovesforward,lamellipodiaandmicrospikesthatfailtoattachtothetissueculturedishsweepbackwardoveritsdorsalsurface-amovementknownasruffling.(CourtesyofJulianHeath.)C.Spec
9、ific drugs affect polymer dynamicsCytochalasins:Prevent the addition of new monomers to existing MFs,which eventually depolymerize.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 cellsFigu
10、re16-52The effect of cytochalasin on the leading edge of the growth cone of a nerve cell in culture.AlivinggrowthconeisviewedbyNomarskidifferential-interference-contrastmicroscopybothbefore(A)andafter(B)treatmentwithcytochalasin.Thecellin(B)hasthenbeenstainedwithrhodaminephalloidintorevealtheactinfi
11、laments(C).Notehowtheregionbehindtheleadingedgeofthecytochalasin-treatedgrowthconeisdevoidofactinfilaments.CytochalasinB(D).(A,B,andC,courtesyofPaulForscher.)D.Actin-binding proteinsThe structures and functions of cytoskeleton are mainly controlled by its binding proteins(1)Monomer-sequestering prot
12、einsBind with actin monomers and prevent them from polymerizing.thymosin and(profilin)Promoting the assembly of MFFigure16-53Two possible mechanisms by which an actin-monomer-binding protein could inhibit actin polymerization.Itisthoughtthatthymosininhibitsactinpolymerizationinoneoftheseways.(2)MF-b
13、inding proteinsvActin 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 elongated protein.Which can prevent the filament from interacting with other proteins.Another important
14、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 filament,forcing the filament to twist a little more tightly.In addition,cofilin binding cause a large increa
15、se 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 -actinin parallel bundle mediated by fimbrin.Gel-like networkActin filaments are often nucleated at the plasma
16、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 properties of a gel.Loss of filamin causes abnormal cell motilityE.Functions of MFs(1)Maintain cells shape and enforce P
17、MFigure10-75A model for how integrins in the plasma membrane connect intracellular actin filaments to the extracellular matrix at a focal contact.Theformationofafocalcontactoccurswhenthebindingofmatrixglycoproteins(suchasfibronectin)ontheoutsideofthecellcausestheintegrinmoleculestoclusteratthecontac
18、tsite,asillustratedschematicallyin(A).Apossiblearrangementofsomeoftheintracellularattachmentproteinsthatmediatethelinkagebetweenanintegrinandactinfilamentsisshownin(B).(2)Cell migration(Fibroblast et al)vPlatelet activation is a controlled sequence of actin filament severing,uncapping,elongation,rec
19、apping,and cross-linking.(3)Microvillus:Support the projecting membrane of intestinal epithelial cellsFigure16-77Freeze-etch electron micrograph of an intestinal epithelial cell,showing the terminal web beneath the apical plasma membrane.Bundlesofactinfilamentsformingthecoreofmicrovilliextendintothe
20、terminalweb,wheretheyarelinkedtogetherbyacomplexsetofcytoskeletalproteinsthatincludesspectrinandmyosin-II.Beneaththeterminalwebisalayerofintermediatefilaments.(FromN.Hirokawaetal.,J.Cell Biol.91:399-409)(4)Stress fibersComposed of actin filaments and myosin-II Stress FibersFocal contactsFocal contac
21、ts IFsResponse to tensionResponse to tension(5)Contractile ring:For cytokinesis(6)Muscle contractionOrganization of skeletal muscle tissueSarcomereFigure16-84Electron micrographs of an insect flight muscle viewed in cross-section.Themyosinandactinfilamentsarepackedtogetherwithalmostcrystallineregula
22、rity.(FromJ.Auber,J.de Microsc.8:197-232)Proteins play important roles in muscle contraction Myosin:The actin motor porteinATPaseBinding sitesMyosin II-DimerMainly in muscle cellsThick filamemtsLight-chain phosphorylation and the regulation of the assembly of myosin II into thick filaments Tropomyos
23、in,Tm and Tropnin,TnRopelike 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 cytosolTnTmSliding3.Microtubule,MTTubulin heterodi
24、mersare the protein building blocks of MTsA.Structures:Arrangement of protofilaments in singlet,double,and triplet MTsSingletDoubleTripletABABCIn cilia and flagellaIn centrioles and basal bodiesB.MTs assemble from microtubule-organizing centers(MTOCs)(1)Interphase:CentrosomeDynamic instability(2)Div
25、iding cell:Mitotic spindleDynamic instability(3)Ciliated cell:Basal bodyStabilityBasal body structureC.Characteristics of MT assemblyDynamic instability due to the structural differences between a growing and a shrinking microtubule end.GTP cap;Catastrophe:accidental loss of GTP cap;Rescue:regain of
26、 GTP capv Why the centrosome can act as MTOCStructureNo centrioles in Plant and fungi v Experiments supporting that centrosome is the MTOCTreat cell with colcemidCytosolic MTs depoly,except those in centrosomeRemove colcemidTublin repolyExpla I:MTOC nucleate poly of tubulinsExpla II:MTOC gather MTs
27、in cytosolcentrosome+TubulinsMT+TubulinsNoABvMT are nucleated by a protein complex containing -tubulinThe centrosome is the major MTOC of animal cellsv Drugs affect the assembly of MTs(1)ColchicineBinding to tubulin dimers,prevent MTs polymerization(2)TaxolBinding to MTs,stabilize MTsThese compounds
28、 are called antimitotic drugs,and have application in medical practice as anticancer drugsv Microtuble-associated proteins(MAPs)MAPs modulate MT structure,assembly,and functionKatanin like proteinsMAPsTau:In axon,cause MTs to form tight bundlesMAP2:In dendrites,cause MTs to form looser bundlesMAP1B:
29、In both axons and dendrites to form crossbridge between microtubulesControl organization MAPsMAP1A,MAP1B,MAP1CMAP2,MAP2cMAP3，MAP4TauThe importance of MAPs for neurite formation Organization of MT bundles by MAPs.Spacing of MTs depends on MAPsInsect cell expressing MAP2Insect cell expressing tauFrom
30、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 help to localize Mt in budding yeast cell.5.Functions of MTs1.Maintain cell shapeFig.10-31Microtubule dynami
31、cs in a living cell.Afibroblastwasinjectedwithtubulinthathadbeencovalentlylinkedtorhodamine,sothatapproximately1tubulinsubunitin10inthecellwaslabeledwithafluorescentdye.Note,forexample,thatmicrotubule#1firstgrowsandthenshrinksrapidly,whereasmicrotubule#4growscontinuously.(P.J.Sammaketal.,Nature 332:
32、724-736)细胞内物质运输Motor Protein1.Kinesin Family2.Dynein Family 染色体运动染色体运动Intermediate filaments,IFs IFs are the most abundant and stable components of the cytoskeletonFigure10-13The domain organization of intermediate filament protein monomers.Mostintermediatefilamentproteinsshareasimilarroddomainthati
33、susuallyabout310aminoacidslongandformsanextendedalphahelix.Theamino-terminalandcarboxyl-terminaldomainsarenon-alpha-helicalandvarygreatlyinsizeandsequenceindifferentintermediatefilaments.Figure10-14.A current model of intermediate filament construction.Figure1016.Electron micrographs of two types of
34、 intermediate filaments in cells of the nervous system.(A)Freeze-etchimageofneurofilamentsinanervecellaxon,showingtheextensivecross-linkingthroughproteincross-bridges.(B)Freeze-etchimageofglialfilamentsinglialcellsillustratingthatthesefilamentsaresmoothandhavefewcross-bridges.(C)Conventionalelectron
35、micrographofacross-sectionofanaxonshowingtheregularside-to-sidespacingoftheneurofilaments,whichgreatlyoutnumberthemicrotubules.(AandB,courtesyofNobutakaHirokawa;C,courtesyofJohnHopkins.)3.Function of IFs:Confer mechanical strength on tissuesDisruption of keratin networks causes blisteringFigure10-18
36、.The nuclear lamina.(A)Schematicdrawingshowingthenuclearlaminaincross-sectionintheregionofanuclearpore.Thelaminaisassociatedwithboththechromatinandtheinnernuclearmembrane.(B)Electronmicrographofaportionofthenuclearlaminainafrogoocytepreparedbyfreeze-dryingandmetalshadowing.(C)Electronmicrographofmet
37、al-shadowedisolatedlamindimers(markedL).Theyhaveanoverallformsimilartomusclemyosin(markedM),witharodliketailandtwoglobularheads,buttheyaremuchsmallermolecules.(BandC,courtesyofUeliAebi.)Summary:Cytoskeletal functionsSummary of cytoskeleton1.Three types of cytoskeletal filaments are common to many eu
38、caryotic cells and are fundamental to the spatial organization of these cells.2.The set of accessory proteins is essential for the controlled assembly of the cytoskeletal filaments(includes the motor proteins:myosins,dynein and kinesin)3.Cytoskeletal systems are dynamic and adaptable.Nucleation is rate-limiting step in the formation of a cytoskeletal polymer.4.Regulation of the dynamic behavior and assembly of the cytoskeletal filaments allows eucaryotic cells to build an enormous range of structures from the three basic filaments systems.
