CompanyLogowww.themegallery.comBiology Department Drs. H.A.Sofy Permana,MSc.,DSc.CYTOSKELETONCELL BIOLOGYBIOLOGY DEPARTMENTn!"ers!#y of Bra$!%ayaMALANGCompanyLogowww.themegallery.comTo&!cs 'IntroductionStructure ofCytoskeletal filamentsIntermediate Filaments (IFs)Microfilaments (Actin Filament)Microtubules (MTs)CompanyLogowww.themegallery.comThe cytoskeleton is one of several biological elements that define eukaryotic cells (others include the nucleus and mitochondria)IntroductionCompanyLogowww.themegallery.comCytoskeletonMeans cell skeletonInternal framework of cellHas many functionsAnchoring cell organellesProvide cell shapeAids in cell motilityResponse to environmental signalsComprisesMicrotubulesMicrofilaments (Actin filament)ntermediate filamentsCompanyLogowww.themegallery.comIn#er(ce))*)ar Anc+or!n,CompanyLogowww.themegallery.comCytoskeletonfunctionsProvide cell shape !"ow do you thin# about $he structure of these shapes!CompanyLogowww.themegallery.comCytoskeleton"ow do you thin# about $he structure of these shapes!CompanyLogowww.themegallery.com the shape of cells pentagonal hepatocyteCompanyLogowww.themegallery.comIntroductionCytoskeleton (from Greeks):Cyto+skeletonCompanyLogowww.themegallery.comHow do prokaryotic cells Eukaryotic Cells possess CytoskeletonCompanyLogowww.themegallery.comCytoskeleton like-flaments inprokaryotic cellsCompanyLogowww.themegallery.comCompanyLogowww.themegallery.comCytoskeleton like-flaments inprokaryotic cells (CreS)CompanyLogowww.themegallery.comCytoskeleton like-flaments inprokaryotic cells (MreB)CompanyLogowww.themegallery.comCytoskeleton like-flaments inprokaryotic cells (FtsZ)CompanyLogowww.themegallery.comCytoskeleton like-flaments inprokaryotic cellsCompanyLogowww.themegallery.comStructure and FunctionStructures:1. Intermediate Filaments 2. Actins3. MicrotubulesC!"S#$%$!"&Functions:1. Mitosis2. Cell Morp'ology3. Cell MotilityCompanyLogowww.themegallery.comLike our ody! t"e cellsalso"a#e skeletonActin Filament ( Micro)ilament *Actin+Intermediate Filaments *IFs+Microtubules *M!s+CompanyLogowww.themegallery.comEukaryotes Cytoskeleton Components%IntermediateFilaments (IFs)&Microfilaments/Actin Filaments(Actin)'Microtubules(MTs)CompanyLogowww.themegallery.comCytoskeleton structuresdiameter = ! nmdiameter = " nmdiameter = #$ nm%cytomusculatureCell &as fi'ed and stained by Coomassie blueCompanyLogowww.themegallery.comBone CellsOsteoblasts()one formers*. +ormation of bone through ossification or osteogenesis. Collagen produced by ,.R. and -olgi.Released by e.ocytosis.Precursors of hydro.yapetite stored in vesicles/ then released by e.ocytosis. "ssi)ication0formation of bone by osteoblasts.1steoblasts communicate through gap 2unctions.Cells surround themselves by matri..CompanyLogowww.themegallery.comOsteocytesOsteocytes. Mature bone cells. Stellate. Surrounded by matri! but can make small amounts of matri to maintain it.%acunae0 spaces occupied by osteocyte cell bodyCanaliculi0 canals occupied by osteocyte cell processes3utrients diffuse through tiny amount of li4uid surrounding cell and filling lacunae and canaliculi. $hen can transfer nutrients from one cell to the ne.t through gap 2unctions.CompanyLogowww.themegallery.comHyaline CartilageStructure: lar"e amount of colla"en fibers e#enly distributed in proteo"lycan matri. Smoot$ surface in articulations%ocations:+ound in areas for strong support and some fle.ibility0 rib cage/ trachea/ and bronchi/ articular cartilagen embryo forms most of s#eletonnvolved in growth that increases bone lengthCompanyLogowww.themegallery.comElastic CartilageStructure: elastic and colla"en fibers embedded in proteo"lycans. &i"id but elastic properties%ocations: eternal ears and epi"lottisCompanyLogowww.themegallery.comFibrocartilageStructure: t$ick colla"en fibers distributed inproteo"lycan matri' sli"$tly compressible and #ery tou"$%ocations: found in areas of body w$ere a "reat deal of pressure is applied to (oints5iscs of #nee 2oint/ pubic symphysis/ intervertebral discsCompanyLogowww.themegallery.comStructure o$ %ntermediate FilamentsCompanyLogowww.themegallery.comThe IFs are in fully (olymeri)ed state* &ith very little free tetramersCompanyLogowww.themegallery.coman eample of $ow a cell culture is made from tissue.Intermediate filaments as(ectCompanyLogowww.themegallery.comFamily (roteins of Intermediate filamentsCompanyLogowww.themegallery.comCompanyLogowww.themegallery.com+ helical rod domain regions containing he(tad re(eatsC+terminus,+terminuskeratinsvimentinneurofilament (roteinsnuclear laminseucaryotic cellsmesenchymal originneuronse(ithelium - derivatesFamily (roteins of Intermediate filaments.eratin in rat kangaroo e(ithelial cells (/tk cells)0undles glial filaments (green)in cultured astrocytesCompanyLogowww.themegallery.comFamily (roteins of Intermediate filaments,uclear lamina in frog oocyte1lial filaments in glial cell,eurofilaments in a'onCompanyLogowww.themegallery.comAc#!n Ac#!n -)amen#-)amen#s#r*c#*res#r*c#*reCompanyLogowww.themegallery.com Actin filament structureActin filament structureCompanyLogowww.themegallery.com&ctin in muscle-&ctin in muscle-cellscells('as"-ack)('as"-ack)CompanyLogowww.themegallery.com&ctin in muscle-cells('as"-&ctin in muscle-cells('as"-ack)ack)CompanyLogowww.themegallery.comActin filament formationActin filament formationCompanyLogowww.themegallery.comActin filament formationActin filament formationCompanyLogowww.themegallery.comCompanyLogowww.themegallery.comCompanyLogowww.themegallery.comCompanyLogowww.themegallery.comCompanyLogowww.themegallery.com&ctin! myosin and cell &ctin! myosin and cell mo#ementsmo#ements2o& does a cell move 3CompanyLogowww.themegallery.com Cell movements MecanismText1.xtension!"ase2.Ad'esion,'ase3.!ranslocation,'ase-.De.ad'esion,'aseCell movementsCompanyLogowww.themegallery.comCompanyLogowww.themegallery.comActin mesh-work in filapodiaActin mesh-work in filapodiaCompanyLogowww.themegallery.com&ctin! myosin and cell &ctin! myosin and cell mo#ementsmo#ementsCell movements model(( ackack to to $uncti $unction onCompanyLogowww.themegallery.comMyosin, the actin Myosin, the actin motormotor-protein-proteinCompanyLogowww.themegallery.comMyosin - Actin Myosin - Actin modelmodelCompanyLogowww.themegallery.comMyosin - actin Myosin - actin experimentexperimentCompanyLogowww.themegallery.comMyosin, the actin Myosin, the actin motormotor-protein-proteinCompanyLogowww.themegallery.comMicrotubule structureMicrotubule structureCompanyLogowww.themegallery.comMicrotubule structure PROTOFILAMENT tubulinCompanyLogowww.themegallery.comSchemes of microtubulebstructureSchemes of microtubulebstructure8 nmTubulin dimerprotofilamentplus endminus end25 nmCompanyLogowww.themegallery.com!ubulin " Microtubule structureCompanyLogowww.themegallery.comT"e Microtubule Or#ani$in# Centre (MtOC) in centriolsCompanyLogowww.themegallery.com%amma&tubulin rin# com'lexCompanyLogowww.themegallery.com)olymeri*ation! t"e key process o$ cytoskeleton+s sel$-assemlyCompanyLogowww.themegallery.comanimation by ASA2I Miho (,agoya 4niv56(n)Microtubule formationMicrotubule formationCompanyLogowww.themegallery.comMicrotubule and Microtubule and motormotor proteinproteinCompanyLogowww.themegallery.comM!s # M!s # motormotor protein e$perimentprotein e$perimentCompanyLogowww.themegallery.comM!s M!s motormotor protein and Cargoprotein and CargoCompanyLogowww.themegallery.comM!s # M!s # motormotor protein modelprotein modelCompanyLogowww.themegallery.comCompanyLogowww.themegallery.com%ynamic &nstability of M!sis (in#le MTis (in#le MT be"a)ior be"a)ior ( (in vivo in vivo andand in vitro in vitro) )is (in#le MTis (in#le MT be"a)ior be"a)ior ( (in vivo in vivo andand in vitro in vitro) )repeat / randomly c'angeIn lengt'by undergoing0ro1ing 0ro1ingp'ase p'ase!ransition !ransitionp'ase: p'ase:S'rin2ing S'rin2ingp'ase p'ase.catastrop'e.rescueCompanyLogowww.themegallery.comVarious Regulation factors for microtubule dynamicsVarious Regulation factors for microtubule dynamicsPolymerization factorsNucleation enhancerTuRCTubulin activatorCRMP2 etc.Polymerization acceleratorchTOGp/XMAP215 etc.Depolymerization factorsSevering factor, p56, p48(EF-1) etc.Kinesin that accelerate catastropheKin 13 (KIF2A, XKCM1, MCAK etc. Tubulin inactivator (sequestering factor)stathmin family, HPC-1/syntaxin 1A etc.Rescue acceleratorheat-stable MAP such as MAP22ataninCompanyLogowww.themegallery.com%ynamic instability in vivo %ynamic instability in vivo'in te cell e$tract( 'in te cell e$tract(CompanyLogowww.themegallery.com%ynamic instability in vivo %ynamic instability in vivo'in te cell system( 'in te cell system(CompanyLogowww.themegallery.com%ynamic instability in vivo %ynamic instability in vivo'in te cell system( 'in te cell system(CompanyLogowww.themegallery.comDynamic instability in vitro (in the free cell system)(in the free cell system)10 mby dark+fieldmicrosco(yCompanyLogowww.themegallery.comDynamic instability ProfileCompanyLogowww.themegallery.comHow do the catastrophe and rescue in MT dynamics occur ?catastro(herescueCompanyLogowww.themegallery.com[Itoh and Hotani, 1994]Bar= 5 mDetailed dynamic instability observation of single microtubuleminus end(lus endminus end(lus endCompanyLogowww.themegallery.comMicrotubule Associated Proteins Microtubule Associated Proteins (MAPs) (MAPs) is MTs is MTs regulatorregulatorCompanyLogowww.themegallery.com*e#ulation of dynamic instability*e#ulation of dynamic instabilityDepolymerization of bulk microtubulesPolymerization of bulk microtubulesSuppression of catastrophe,Enhancement of rescue etc.Enhancement of catastrophe,Severing of microtubule etc.CompanyLogowww.themegallery.comRegulation factors for microtubule dynamics 1Regulation factors for microtubule dynamics 1CRMP2 binds to free tubulin and increases its polymerizing activity, but it is released from microtubule after polymerization. CompanyLogowww.themegallery.comRegulation factors for microtubule dynamics 2Regulation factors for microtubule dynamics 2XMAP215 binds to plus end of microtubule and guides free tubulins to increase polymerization velocity. CompanyLogowww.themegallery.comRegulation factors for microtubule dynamics 3Regulation factors for microtubule dynamics 3Stathmin binds to two free tubulin dimers and sequesters them from the polymerization. CompanyLogowww.themegallery.comRegulation factors for microtubule dynamics 4Regulation factors for microtubule dynamics 4Heat-stable MAPs such as MAP2, tau and MAP4 binds to microtubule lateral surface and increases rescue frequency. ( ack to ( ack to re,ulationre,ulation $actor $actorCompanyLogowww.themegallery.comAXONEMEMTs structure in FlagellaMTs structure in Flagella10 mChlamydomonas sp10 m(( ackack to to $uncti $unction onCompanyLogowww.themegallery.com10 mTriplet MTs in basal-body5 mMTs structure in CiliaMTs structure in Cilia(( ackack to to $uncti $unction on20 mParamaecium spDoublet MTs in ciliaCompanyLogowww.themegallery.comHow do Cilia and Flagella move ?10 m10 m(( ackack to to $uncti $unction onCompanyLogowww.themegallery.comCytoskeleton FunctionsCytoskeleton Functionsin mitosis (drosophyla early embryo)CompanyLogowww.themegallery.comF%3"4$SC$&C$ MIC4"SC",67pecimen is e.cited with a specific wavelength of light/ then fluorescent emissions are observed.Dra1bac2s 5'en to use Fluorescence microscopy 68sed to study specimens/ which can be made to fluoresce.6Certain material emits energy detectable as visible light when irradiated with the light of a specific wavelength. $he sample can either be fluorescing in its natural form li#e chlorophyll and some minerals/ or treated with fluorescing chemicals.6Photo bleaching can significantly cause measurement error CompanyLogowww.themegallery.com67ample you want to study is itself the light source. F%3"4$SC$&C$Main uses:6maging and 4uantification 6Assaying antigens in antigen9antibody reactions6maging and 4uantification of intracellular 53A6Analysis of chromosomal abnormalitiesPrinciple of +luorescence%. ,nergy is absorbed by the atom which becomes e.cited.&. $he electron 2umps to a higher energy level.'. 7oon/ the electron drops bac# to the ground state/ emitting a photon (or a pac#et of light) : the atom is fluorescing CompanyLogowww.themegallery.comCytoskeleton FunctionsCytoskeleton Functionsin mitosis (frog embryo)CompanyLogowww.themegallery.comCytoskeleton FunctionsCytoskeleton Functionsin mitosis (lung cell)CompanyLogowww.themegallery.comDIFF$4$&!IA% I&!$4F$4$&C$ C"&!4AS! MIC4"SC",*D.I.C+6$ransforms minute differences in refraction inde.es of light passing through an unstained specimen/ or optical path differences from the specimen surface shape/ into a monochromatic shadow:cast image enabling observation.6'5:pseudo effect that it gives and also/ unli#e phase contrast there are no halos around the sub2ectDra1bac2s 65C utili;es optical path differences within the specimen (i.e.0 product of refractive inde. and geometric path length) to generate contrast the three:dimensional appearance may not represent reality6)irefringent specimens such as those found in crystals may not be suitable because of their effect upon polari;ed light. 7imilarly/ specimen carriers/ such as culture vessels/ Petri dishes/ etc./ made of plastic may not be suitable5'en to use DIC6 6As with phase contrast microscopy/ 5C microscopy may be used with living specimens. "owever/ it is better suited to thic#er specimens. CompanyLogowww.themegallery.com$he 5C set:up consists of0 A Polari;er/a