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Lecture 1

Lecture 1. Introduction to the cytoskeleton. Outline: Major cytoskeletal elements Pure polymer dynamics Polymer dynamics in cells. Paper: Bacterial cytoskeleton. Roles of cytoskeleton:. 1) Structural scaffold - cell shape, spatial organization.

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Lecture 1

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  1. Lecture 1 Introduction to the cytoskeleton Outline: Major cytoskeletal elements Pure polymer dynamics Polymer dynamics in cells Paper: Bacterial cytoskeleton

  2. Roles of cytoskeleton: 1) Structural scaffold - cell shape, spatial organization 2) Dynamic assemblies - movement and force production: Cell migration Cell division Intracellular traffic Contraction Cytoskeletal functions often involve motor proteins

  3. 3 major elements of the cytoskeleton 1) microtubules – a/b tubulin dimers - 25 nm diameter relatively stiff – hollow, 13 protofilaments 2) microfilaments = actin filaments - 7 nm diameter actin monomers –more flexible – 2 helicies 3) intermediate filaments – 10 nm diameter fibrous – resistant to shear forces structural, prominent in cells subject to mechanical stress; vimentin, nuclear lamins

  4. actin and microtubules Introduction to polymer dynamics: 3 cases • simple equilibrium polymers • polar polymers: asymmetric subunits • undergo conformational change during assembly • complex polymers: non-equilibrium •  subunit nucleotide hydrolysis (energy input)

  5. Kon Koff Simple Equilibrium Polymer Assembles/disassembles by addition/loss of subunits at ends Rates = Kon and Koff Kon depends on concentration of subunit, units of M-1sec-1 Koff does not (unimolecular), units of sec-1

  6. equilibrium [polymer] growth lag time polymer grows, subunit concentration drops until Kon[C] = Koff, when [C] = critical concentration Cc (M-1sec-1[M] = sec -1) Polymer assembly timecourse 1) lag due to kinetic barrier to nucleation 2) growth 3) equilibrium rate of subunit addition = rate of loss

  7. Kon[C] > Koff Kon[C] = Koff Kon[C] < Koff

  8. Critical Concentration • Concentration of free subunits at which • rate of subunit addition = rate of loss • Above Cc net growth, below net shrinkage • Equilibrium constant Keq determined by change in free energy • between free subunits and polymer • Keq= Kon/Koff = 1/Cc

  9. subunit in polymer free subunit minus end plus end slow fast Polar Polymer Two ends polymerize and depolymerize at different rates BECAUSE subunit conformation changes as it incorporates into the polymer

  10. Plus and minus ends: • Different Kon and Koff • But! • Koff/Kon ratio or Cc must be the same for both ends: >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

  11. T D= nucleotide diphosphate T= nucleotide triphosphate D D D D T D D D D D Complex Polymer (non-equilibrium): microtubules, actin filaments Due to nucleotide hydrolysis upon assembly of subunit into polymer: Nucleotide hydrolysis reduces binding affinity

  12. T D D D D D T D D D D Complex Polymer properties:  Internal subunits have different dynamic properties than the ends T form binds, D form dissociates KTon>>KDon KDoff>>KToff Cc = “steady state” concentration: KTon[C]=KDoff Css=KDoff/KTon

  13. T D= diphosphate T= triphosphate D D D D T D D D D D energy Steady State Dynamics No longer true equilibrium, rather steady state because ATP or GTP subunits must be replenished

  14. _ + D D D D T T T D D D D D D Consequences for polymer dynamics Treadmilling (actin and microtubules) • Two different reactions at each end of the polymer • Critical concentration different Cc(- end) > Cc(+ end)

  15. Treadmilling 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

  16. D D D D D T D D D D D T D D D D D T Treadmilling - + T D T D T D

  17. 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: Growth GTP Cap lost: rapid disassembly • stochastic (unpredictable) transitions • frequency correlates with tubulin concentration

  18. D D D D D D D D D D D D D D D T T T catastrophe rescue Dynamic Instability T growing T D D D D D D D shrinking D D D D D D D D D D D D D

  19. [Polymerized Microtubules] 5 10 15 20 25 30 Tubulin concentration (mM) Classic experiments by Mitchison and Kirschner 1984: Microtubules nucleated from seeds - no lag 1) determine steady state concentration (Css ) = 14 mM

  20. Before dilution 15 uM 7.5 uM #concentration: x108/ml 32 32 15 average length: (mM) 18 40 22 2) dilution experiment: Grow microtubule seeds Dilute into tubulin solution above or below Css Wait 10 minutes Measure Mt number-concentration, Mt length (spun onto EM grids)

  21. Summary simple equilibrium: non-equilibrium: • exchange only at the ends • turnover only with • dramatic changes in • subunit concentration • Dynamic instability • Treadmilling • complete and rapid • polymer turnover at • steady state • energy required

  22. trimer = nucleation site monomer dimer Polymer properties regulated in cells: 1) nucleation 2) polarity 3) dynamics 1) Nucleation: kinetic barrier - slow step trimer for actin more complex for microtobules

  23. Nucleating factors in cells Microtubules: centrosomes Actin: protein complexes (Arp2/3)

  24. 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 shapes basis of motility

  25. Actin decorated with myosin subfragment 1 actin lattice polarity revealed

  26. actin structural and dynamic polarity revealed

  27. Microtubules decorated and viewed in cross section microtubule lattice polarity revealed by hook direction

  28. microtubule dynamic polarity revealed

  29. Motor proteins recognize polymer asymmetry mechanochemical enzymes hydrolyze ATP to move along filament produce force or carry cargo polarity generates directionality

  30. 3) dynamics regulated by many cellular factors end-capping subunit sequestering polymer binding proteins regulators also regulated

  31. microtubules in interphase Tournebize and Hyman

  32. microtubules in mitosis Tournebize and Hyman

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