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

2. 1) Structural scaffold - cell shape, spatial organization Roles of cytoskeleton: 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 –  /  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

7. 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) actin and microtubules

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

9. 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 drops until K on [C] = K off, when [C] = critical concentration Cc (M -1 sec -1 [M] = sec -1 )

10. K on [C] = K off K on [C] > K off K on [C] < K off

11. Equilibrium constant K eq determined by change in free energy between free subunits and polymer K eq = K on /K off = 1/Cc Critical Concentration Concentration of free subunits at which rate of subunit addition = rate of loss Above Cc net growth, below net shrinkage

12. Polar Polymer subunit in polymer free subunit plus endminus end fastslow Two ends polymerize and depolymerize at different rates BECAUSE subunit conformation changes as it incorporates into the polymer

13. >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 K on and K off But! K off /K on ratio or Cc must be the same for both ends: Plus and minus ends:

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

15.  Internal subunits have different dynamic properties than the ends T form binds, D form dissociates K Ton >>K Don K Doff >>K Toff T D D D D D D D D T D Complex Polymer properties: Cc = “steady state” concentration: K Ton [C]=K Doff Css=K Doff /K Ton

16. No longer true equilibrium, rather steady state because 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

17. 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

18. 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

19. D D D D D T T D D D D D T D D D D D T D D D T T + -

20. Dynamic instability (microtubules): subunit addition is faster than nucleotide hydrolysis cap of GTP-tubulin on polymer ends K Doff >>K Toff : GTP cap favors growth GTP Cap present: Growth GTP Cap lost: rapid disassembly stochastic (unpredictable) transitions frequency correlates with tubulin concentration

21. 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 catastrophe rescue

22. Classic experiments by Mitchison and Kirschner 1984: Microtubules nucleated from seeds - no lag 51015202530 Tubulin concentration (  M) [Polymerized Microtubules] 1) determine steady state concentration (Css ) = 14  M

23. 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) Before dilution15 uM7.5 uM #concentration: x10 8 /ml average length: (  M) 32 18 32 40 15 22

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

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

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

29. 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

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

31. actin structural and dynamic polarity revealed

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

33. microtubule dynamic polarity revealed

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

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

36. microtubules in interphase Tournebize and Hyman

37. microtubules in mitosis Tournebize and Hyman