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Lecture 1
Introduction to the cytoskeleton
Outline:Major cytoskeletal elementsPure polymer dynamicsPolymer dynamics in cells
Paper: Bacterial cytoskeleton
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1) Structural scaffold - cell shape, spatial organization
Roles of cytoskeleton:
2) Dynamic assemblies - movement and force production:
Cell migration Cell divisionIntracellular traffic Contraction
Cytoskeletal functions often involve motor proteins
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3 major elements of the cytoskeleton
1) microtubules – / tubulin dimers - 25 nm diameter relatively stiff – hollow, 13 protofilaments
2) microfilaments = actin filaments - 7 nm diameteractin monomers –more flexible – 2 helicies
3) intermediate filaments – 10 nm diameterfibrous – resistant to shear forces structural, prominent in cells subject to mechanical stress; vimentin, nuclear lamins
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Introduction to polymer dynamics: 3 cases
• simple equilibrium polymers
• polar polymers: asymmetric subunitsundergo conformational change during assembly
• complex polymers: non-equilibrium subunit nucleotide hydrolysis (energy input)
actin andmicrotubules
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Assembles/disassembles by addition/loss of subunits at endsRates = Kon and Koff
KoffKon
Kon depends on concentration of subunit, units of M-1sec-1
Koff does not (unimolecular), units of sec-1
Simple Equilibrium Polymer
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1) lag due to kinetic barrier to nucleation 2) growth 3) equilibrium
[polymer]
lag growth
equilibrium
time
Polymer assembly timecourse
rate of subunit addition = rate of loss
polymer grows, subunit concentration dropsuntil Kon[C] = Koff, when [C] = critical concentration Cc
(M-1sec-1[M] = sec -1)
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Kon[C] = Koff
Kon[C] > Koff
Kon[C] < Koff
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• Equilibrium constant Keq determined by change in free energy
between free subunits and polymer
Keq= Kon/Koff = 1/Cc
Critical Concentration
• Concentration of free subunits at which rate of subunit addition = rate of loss
• Above Cc net growth, below net shrinkage
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Polar Polymer
subunit in polymerfree subunit
plus endminus end
fastslow
Two ends polymerize and depolymerize at different ratesBECAUSEsubunit conformation changes as it incorporates into the polymer
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>The same interactions are broken when a subunit dissociates from either end
>The final state of the subunit is identical
If 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 ends:
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Complex Polymer (non-equilibrium):microtubules, actin filaments
Due to nucleotide hydrolysis upon assembly of subunit into polymer:
Nucleotide hydrolysis reduces binding affinity
D= nucleotidediphosphate
T= nucleotidetriphosphate
T
D D D D
D D D D
T
D
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Internal subunits have different dynamic properties than the ends
T form binds, D form dissociatesKTon>>KDon KDoff>>KToff
T
D D D D
D D D D
T
D
Complex Polymer properties:
Cc = “steady state” concentration:KTon[C]=KDoff
Css=KDoff/KTon
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No longer true equilibrium, rather steady statebecause ATP or GTP subunits must be replenished
D=diphosphate
T=triphosphate
T
D D D D
D D D D
T
D
energy
Steady State Dynamics
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Consequences for polymer dynamics
Treadmilling (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 polymer
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Both 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 polymer
Treadmilling
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D D D D D T
T
D D D D D T
D D D D D T
D
D
D
T
T
Treadmilling
+-
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Dynamic instability (microtubules):
• subunit addition is faster than nucleotide hydrolysis• cap of GTP-tubulin on polymer ends KDoff>>KToff: GTP cap favors growth
GTP Cap present:GrowthGTP Cap lost:rapid disassembly
• stochastic (unpredictable) transitions
• frequency correlates with tubulin concentration
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D D D D D T
D D D D D T
D D D D D T
D D D D D D
D D D D D D
D D D D D D
T
D
T
D
growing
shrinking
Dynamic Instability
catastropherescue
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Classic experiments by Mitchison and Kirschner 1984:Microtubules nucleated from seeds - no lag
5 10 15 20 25 30
Tubulin concentration (M)
[PolymerizedMicrotubules]
1) determine steady state concentration (Css )= 14 M
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2) 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/ml
averagelength:(M)
32
18
32
40
15
22
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non-equilibrium:
• exchange only at the ends • turnover only withdramatic changes insubunit concentration
Summary
• Dynamic instability• Treadmilling • complete and rapid polymer turnover atsteady state• energy required
simple equilibrium:
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Polymer properties regulated in cells:
1) nucleation2) polarity3) dynamics
1) Nucleation: kinetic barrier - slow step
monomer dimertrimer = nucleation site
trimer for actinmore complexfor microtobules
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Actin: protein complexes (Arp2/3)
Microtubules: centrosomes
Nucleating factors in cells
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2) Polarity: due to asymmetry of subunits
structural polarity of polymer lattice
visualized by decoration of actin filaments and microtubules
allows cell to generate asymmetric structures and shapesbasis of motility
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Actin decorated with myosin subfragment 1
actin lattice polarity revealed
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actin structural and dynamic polarity revealed
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Microtubules decoratedand viewed in cross section
microtubule lattice polarity revealed by hook direction
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microtubule dynamic polarity revealed
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Motor proteins recognize polymer asymmetry
mechanochemical enzymeshydrolyze ATP to move along filamentproduce force or carry cargo
polarity generates directionality
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3) dynamics
regulated by many cellular factors
end-cappingsubunit sequesteringpolymer binding proteins
regulators also regulated
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microtubules in interphase
Tournebize and Hyman
QuickTime™ and aMotion JPEG A decompressorare needed to see this picture.
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microtubules in mitosis
Tournebize and Hyman
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