Methods for Studying the Cell Cyclecell fusion live and fixed imaginggeneticsbiochemistryin vitro systemsinhibitors of cellular processes(transcription, replication, microtubules)DISCUSSION SECTIONS BY STUDENT NUMBERENDING IN ODD NUMBERS 2-3, EVEN 3-4Genetic Screens: Yeast ‘Cell Division Cycle’ (CDC) Mutants‘Dominos’sequential, dependent events‘Oscillator’central controllercan individual protein mutations block steps or whole process ?Cdc Genetic Screens• Lee Hartwell (cerevisiae); Paul Nurse (pombe)• Goal: find mutants unable to transit the cell cycle• Why yeast?– Cell shape --> cell cycle stage– Grow as haploids (easier to find mutants), or diploids (cando genetics)• Problem:– the screen is for cells that can’t grow• Solution:– temperature sensitive mutants– Replica platingGenetic Screens: Yeast ‘Cell Division Cycle’ (CDC) MutantsIn vitro Dissection of the Cell Cycle -Xenopus Egg Extractsuse to isolate proteins present at particular stagesmanipulate proteins-deplete and observe changes to cell cycleDNA TubulinLecture 2Introduction to the CytoskeletonOutline:Composition of the cytoskeleton Polymer Dynamics in theoryPolymer Dynamics in cellsPaper: Identification of pathways regulating cell size and cell-cycle progression by RNAi Paul Nurse “Controlling the Cell Cycle” !!! Thu 4 PM, 100 GPBBStructural scaffold - cell shape, spatial organizationRoles of the CytoskeletonDynamic assemblies - movement and force production:cell migration cell divisionintracellular traffic contractioncytoskeletal functions often involve motor proteins3 major elements of the cytoskeleton microtubulesα/β tubulin dimers 25 nm diameter relatively stiff – hollow, 13 protofilaments3 major elements of the cytoskeleton microfilaments = actin filaments actin monomers7 nm diametermore flexible – 2 helicies3 major elements of the cytoskeleton intermediate filaments – 10 nm diameterfibrous – resistant to shear forces structural – prominent in cells subject to mechanical stress vimentin, nuclear laminsIntroduction to polymer dynamics: 3 cases•1) simple equilibrium polymers•2) polar polymers: asymmetric subunitsundergo conformational change during assembly •3) complex polymers: non-equilibrium ☞ subunit nucleotide hydrolysis (energy input)actin andmicrotubulesassembles & disassembles by addition & loss of subunits at endsrates = Kon and KoffKon depends on concentration of subunit, units of M-1sec-1Koff does not (unimolecular), units of sec-1Simple Equilibrium PolymerKoffKon1) lag due to kinetic barrier to nucleation 2) growth 3) equilibrium[polymer]laggrowthequilibriumtimePolymer assembly timecourserate of subunit addition = rate of losspolymer grows as time proceedssubunit concentration drops until Kon[C] = Koff [C] = critical concentration Cc(M-1sec-1[M] = sec -1)Kon[C] = KoffKon[C] > KoffKon[C] < KoffEquilibrium constant Keq determined by change in free energy between free subunits and polymer Keq= Kon/Koff = 1/CcCritical ConcentrationConcentration of free subunits at which rate of subunit addition = rate of loss Above Cc net growth, below net shrinkagePolar Polymersubunit in polymerfree subunitplus endminus endfastslowTwo ends polymerize and depolymerize at different ratesBECAUSEsubunit conformation changes as it incorporates into the polymer> the same interactions are broken when a subunit dissociates from either end> the final state of the subunit is identicalIf the plus end grows 3 times faster it must also shrink 3 times faster. above Cc both ends grow, below Cc, both shrink• different Kon and Koff• BUT !Koff / Kon ratio or Cc must be the same for both ends:Plus and minus endsComplex Polymer (non-equilibrium):microtubules, actin filamentsdue to nucleotide hydrolysis upon assembly of subunit into polymernucleotide hydrolysis reduces binding affinity TD= nucleotidediphosphateT= nucleotidetriphosphate D D D D D D D D T Dinternal subunits have different dynamic properties than the ends T D D D D D D D D T DComplex Polymer propertiesCss = “steady state” concentrationKTon[C] = KDoffCss = KDoff / KTonKTon>>KDon KDoff>>KToffT form binds, D form dissociatesno longer true equilibrium, rather steady statebecause ATP or GTP subunits must be replenishedD=diphosphateT=triphosphate T D D D D D D D D T DenergySteady State DynamicsConsequences for polymer dynamicstreadmilling (actin and microtubules)• critical concentration different•Cc(- end) > Cc(+ end)_ T D D D D D D D D T D+ T D• two different reactions at each end of the polymerboth ends exposed Steady state occurs at concentration between Cc(- end) and Cc(+ end)net assembly at the plus end net disassembly at the minus end subunits “flux” through the polymerTreadmillingD D D D D T T D D D D D T D D D D D T D D D T TTreadmilling+-Dynamic instability (microtubules)• subunit addition is faster than nucleotide hydrolysis• CAP of GTP-tubulin on polymer ends KDoff >> KToff : GTP CAP favors growthGTP CAP present: growthGTP CAP lost: rapid disassembly• stochastic (unpredictable) transitions• frequency correlates with tubulin concentration5 10 15 20 25 30Classic experiments by Mitchison and Kirschner 1984Tubulin concentration (µM)[PolymerizedMicrotubules]1) determine steady state concentration (Css ) = 14 µMmicrotubules nucleated from seeds - no lag2) dilution experiment:grow microtubule seedsdilute into tubulin solution above or below Csswait 10 minutesmeasure Mt number-concentration, Mt length(spun onto EM grids)before dilution 15 uM 7.5 uM#concentration: x108/mlaverage length: (µM)321832401522size is dependent on the concentration of tubulinDynamic instability in vitroMicrotubules are really....tubes, not simple polymersnon-equilibrium:exchange only at the ends turnover only with dramatic changes in subunit concentration SummaryDynamic instabilityTreadmilling complete and rapid polymer turnover at steady stateenergy requiredsimple equilibrium:Polymer properties regulated in cells1) nucleation2) polarity3) dynamics1) Nucleation: kinetic barrier - slow stepmonomer dimertrimer = nucleation sitetrimer for actinmore complexfor microtobulesActin: protein complexes (Arp2/3) Nucleating factors in cellsMicrotubules: centrosomes2) Polarity: due to asymmetry of subunitsstructural polarity of polymer latticevisualized by decoration of actin filaments and microtubulesallows cell to generate asymmetric structures and shapesbasis of motilityActin decorated with myosin subfragment 1