cb201_12 tim mitchison lecture 3 force generation by polymerization dynamics nucleation: controlling...

Download CB201_12 Tim Mitchison Lecture 3 Force generation by polymerization dynamics Nucleation: controlling where and when polymers form

Post on 23-Dec-2015

215 views

Category:

Documents

2 download

Embed Size (px)

TRANSCRIPT

  • Slide 1
  • CB201_12 Tim Mitchison Lecture 3 Force generation by polymerization dynamics Nucleation: controlling where and when polymers form
  • Slide 2
  • Force generation by the cytoskeleton One of the main functions of the actin and microtubule cytoskeletons, and their prokaryotic counterparts, is to generate force for cell motility in a spatially and temporally controlled manner
  • Slide 3
  • Force generation by the cytoskeleton One of the main functions of the actin and microtubule cytoskeletons, and their prokaryotic counterparts, is to generate force for cell motility in a spatially and temporally controlled manner Force from polymerization dynamics Eukaryotes and prokaryotes
  • Slide 4
  • Force generation by the cytoskeleton One of the main functions of the actin and microtubule cytoskeletons, and their prokaryotic counterparts, is to generate force for cell motility in a spatially and temporally controlled manner Force from polymerization dynamics Eukaryotes and prokaryotes ATPase motor proteins Only Eukaryotes
  • Slide 5
  • Polymerization dynamics can perform mechanical work by pushing or pulling Pushing by polymerization Leading edge protrusion (actin) Listeria motility (actin) Plasmid separation in bacteria (ParM) Pulling by depolymerization Chromosome movement in mitosis (microtubules)
  • Slide 6
  • Mechanical work requires enery dissipation Mechanical work performed = force x distance Total energy dissipated = G per elementary step x number of steps taken Efficiency = work done/energy dissipated In general, the efficiency of converting chemical energy into mechanical work must be less than 100% if the process that does the work is to proceed unidirectionally ie some heat must be dissipated to make the process irreversible. This law of thermodynamics was developed for steam engines but applies equally to biology The efficiency of biological motors can be quite high. Food human rowing Total efficiency = ~ 20% Food ATP efficiency = ~40% Therefore, effecience of ATP mechanical work in muscle = ~50% (Wikipedia)
  • Slide 7
  • Elementary steps + Actin filaments grow by ~2nm per subunit (Actin monomer is ~4nm long, filament has 2 strands) Kinesin moves 8nm per step
  • Slide 8
  • Elementary steps + Actin filaments grow by ~2nm per subunit Kinesin moves 8nm per step Each step is coupled to hydrolysis of 1 molecule of ATP to ADP + Pi This liberates ~8-12 kilocal per mol (= ~20kT per molecule) Bolzman constant ~4pN.nm
  • Slide 9
  • Elementary steps Kinesin moves 8nm per step Each step is coupled to hydrolysis of 1 molecule of ATP to ADP + Pi This liberates ~8-12 kilocal per mol (= ~20kT per molecule) Efficiency = 5pN.8nm/20kT = ~50% Forcedistance Chemical energy dissipated
  • Slide 10
  • Elementary steps + Actin filaments grow by ~2nm per subunit (4nm subunit, 2 stranded polymer) Kinesin moves 8nm per step Each step is coupled to hydrolysis of 1 molecule of ATP to ADP + Pi This liberates ~8-12 kilocal per mol (= ~20kT per molecule) How do we think about force generation from polymerization or depolymerization?
  • Slide 11
  • Microtubule polymerizing in a microfabricated box. The force from polymerization causes the microtubule to buckle. Polymerization slows as the force on the ends increases. Eventually a catastrophe occurs. M. Dogterom and coworkers Science 278:856(1997), J Cell Biol 161:1029(2003)
  • Slide 12
  • Microtubule polymerizing in a microfabricated box. The force from polymerization causes the microtubule to buckle. Polymerization slows as the force on the ends increases. Eventually a catastrophe occurs. M. Dogterom and coworkers Science 278:856(1997), J Cell Biol 161:1029(2003) How much force? Simple argument for maximum possible force: For every tubulin added, the microtubules grows 8/13nm Suppose the full energy of GTP hydrolysis is used to promote this reaction GTP -> GDP + G = ~ -50 kJ/mol = 5x10 -4 /6x10 -23 J/microtubule Force = work/distance = ~ 10 -19 /0.5x10 -9 = ~2x10 -10 N = ~200pN
  • Slide 13
  • Microtubule polymerizing in a microfabricated box. The force from polymerization causes the microtubule to buckle. Polymerization slows as the force on the ends increases. Eventually a catastrophe occurs. M. Dogterom and coworkers Science 278:856(1997), J Cell Biol 161:1029(2003) How much force? Simple argument for maximum possible force: For every tubulin added, the microtubules grows 8/13nm Suppose the full energy of GTP hydrolysis is used to promote this reaction GTP -> GDP + G = ~ -50 kJ/mol = 5x10 -4 /6x10 -23 J/microtubule Force = work/distance = ~ 10 -19 /0.5x10 -9 = ~2x10 -10 N = ~200pN Force can be estimated since we know the bending ridigity of the microtubule, and can thus estimate the force required to buckle it Measured force ~5pN per microtubule (similar to the force exterted by a single motor molecule) Not as efficient as a motor protein, but still substantial force on the molecular scale
  • Slide 14
  • Actin polymerization force pushes the front of motile cells forward
  • Slide 15
  • How do cells control where and when cytoskeleton polymers accumulate? Bacterium Neutrophil Chemotaxis Phagocytosis High density of actin filaments
  • Slide 16
  • Neutrophil chasing S aureus in a drop of blood David Rogers 1950s
  • Slide 17
  • How might cells control where and when cytoskeleton polymers accumulate? Neutrophil detects a bacterium seconds Signal (bacterial cell wall) Receptor in plasma membrane Signaling pathway Cytoskeleton reorganization
  • Slide 18
  • How might cells control where and when cytoskeleton polymers accumulate? Neutrophil detects a bacterium seconds Signal (bacterial cell wall) Receptor in plasma membrane Signaling pathway Cytoskeleton reorganization What kind of processes might work for this at the level of cytoskeleton filaments?
  • Slide 19
  • Many proteins binds to cytoskeleton filaments and control their behavior in cells Bundling Cross- linking Capping Gel-forming Depolymerizing, Severing Nucleating Moving Monomer binding, Monomer sequestering
  • Slide 20
  • Many proteins binds to cytoskeleton filaments and control their behavior in cells Bundling Cross- linking Capping Gel-forming Depolymerizing, Severing Nucleating Moving Monomer binding, Monomer sequestering
  • Slide 21
  • Nucleation is slow, elongation is fast Nucleating a new filament is slow. Each incoming subunit makes only a subset of the favorable bonds Elongating an existing filament is fast. Each incoming subunit makes all favorable bonds The observation that elongating an existing filament is (much) faster than starting a new one is termed the kinetic barrier to nucleation. The physical chemistry of polymer nucleation is similar to crystallization from a saturated solution or freezing of a supercooled liquid. In each case self- assembly can be nucleated by a pre-existing fragment of the polymer/crystal
  • Slide 22
  • Origin of the kinetic barrier to nucleation. 1) Condensation models (Oosawa-type models) Break one bond. Fast Break 2 bonds. Fast Break 3 bonds. Slow Diffusion controlled Break 3 bonds. Slow minimal seed with n subunits
  • Slide 23
  • Origin of the kinetic barrier to nucleation. 1) Condensation models (Oosawa-type models) - Requires multi-stranded polymer - Does not require conformational change of monomer (similar models work for crystallization) - Elongation rate is proportional to the concentration of the subunit. - Nucleation rate depends on concentration of subunit by a power law. Break one bond. Fast Break 2 bonds. Fast Break 3 bonds. Slow Diffusion controlled Break 3 bonds. Slow minimal seed with n subunits
  • Slide 24
  • Origin of the kinetic barrier to nucleation. 1) Condensation models (Oosawa-type models) Break one bond. Fast Break 2 bonds. Fast Break 3 bonds. Slow Diffusion controlled minimal seed with n subunits Break 3 bonds. Slow Assume rapid equilibrium Rate of formation of new filaments = concentration of ( n - 1)mers x rate that they turn into filaments n-1 monomers ( n - 1)mer Assume rapid equilibrium up until minimal seed. Then: [( n - 1)mer] ~ K d [monomer] n-1 ; nucleation rate ~ K d [monomer] n-1 x k[monomer] ~ K[monomer] n N = 3-4 for actin Tobacman LS, Korn ED. J Biol Chem. 1983 258:3207-14.
  • Slide 25
  • Origin of the kinetic barrier to nucleation. 2) Conformational switch models Non-polymerizing conformation (normal form of subunit after folding) Polymerizing conformation (rare form of subunit) Seed catalyzes conformational change Slow, spontaneous conformational change + +
  • Slide 26
  • Origin of the kinetic barrier to nucleation. 2) Conformational switch models - Does not requires multi-stranded polymer (in principle) - Requires conformational change of monomer that is catalyzed by polymer - Nucleation rate is independent of elongation rate and can be very slow. Caspar DL, Namba K. (1990) Adv Biophys. 26:157-85; DePace et al 1998 Cell. 93:1241-52 More relevant to viral coat proteins and amyloid fibers Non-polymerizing conformation (normal form of subunit after folding) Polymerizing conformation (rare form of subunit) Seed catalyzes conformational change Slow, spontaneous conformational change + +
  • Slide 27
  • Nucleation factors in the cell The kinetic barrier to nucleation p

Recommended

View more >