Nucleation is slow, elongation is fast

1
CB201_12 Tim Mitchison Lecture 3 Force
generation by polymerization dynamics Nucleation
controlling where and when polymers form
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
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
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
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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)
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Mechanical work requires enery dissipation
Mechanical work performed force x
distance Total energy dissipated DG 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)
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Elementary steps

Actin filaments grow by 2nm per subunit (Actin
monomer is 4nm long, filament has 2 strands)
Kinesin moves 8nm per step
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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
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Elementary steps
Chemical energy dissipated
Force
distance
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
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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?
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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 278856(1997), J Cell Biol 1611029(2003)

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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 278856(1997), J Cell Biol 1611029(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 -gt GDP DG -50 kJ/mol 5x10-4
/6x10-23 J/microtubule Force work/distance
10-19/0.5x10-9 2x10-10 N 200pN
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 278856(1997), J Cell Biol 1611029(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 -gt GDP DG -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
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Actin polymerization force pushes the front of
motile cells forward
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How do cells control where and when cytoskeleton
polymers accumulate?
Bacterium
Phagocytosis
Neutrophil
High density of actin filaments
Chemotaxis
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Neutrophil chasing S aureus in a drop of
blood David Rogers 1950s
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How might cells control where and when
cytoskeleton polymers accumulate?
Neutrophil detects a bacterium
Signal (bacterial cell wall)
Receptor in plasma membrane
seconds
Signaling pathway
Cytoskeleton reorganization
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How might cells control where and when
cytoskeleton polymers accumulate?
Neutrophil detects a bacterium
Signal (bacterial cell wall)
Receptor in plasma membrane
seconds
Signaling pathway
Cytoskeleton reorganization
What kind of processes might work for this at the
level of cytoskeleton filaments?
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Many proteins binds to cytoskeleton filaments and
control their behavior in cells
Capping
Cross-linking
Bundling
Nucleating
Gel-forming
Moving
Depolymerizing, Severing
Monomer binding, Monomer sequestering
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Many proteins binds to cytoskeleton filaments and
control their behavior in cells
Capping
Cross-linking
Bundling
Nucleating
Gel-forming
Moving
Depolymerizing, Severing
Monomer binding, Monomer sequestering
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Nucleation is slow, elongation is fast
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
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.
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Origin of the kinetic barrier to nucleation. 1)
Condensation models (Oosawa-type models)
Diffusion controlled
Diffusion controlled
Diffusion controlled
Diffusion controlled
Break one bond. Fast
Break 2 bonds. Fast
Break 3 bonds. Slow
Break 3 bonds. Slow
minimal seed with n subunits
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Origin of the kinetic barrier to nucleation. 1)
Condensation models (Oosawa-type models)
Diffusion controlled
Diffusion controlled
Diffusion controlled
Diffusion controlled
Break one bond. Fast
Break 2 bonds. Fast
Break 3 bonds. Slow
Break 3 bonds. Slow
minimal seed with n subunits
- 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.
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Origin of the kinetic barrier to nucleation. 1)
Condensation models (Oosawa-type models)
Diffusion controlled
Diffusion controlled
Diffusion controlled
Diffusion controlled
Break one bond. Fast
Break 2 bonds. Fast
Break 3 bonds. Slow
Break 3 bonds. Slow
minimal seed with n subunits
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
Kdmonomern-1 nucleation rate
Kdmonomern-1 x kmonomer Kmonomern
N 3-4 for actin Tobacman LS, Korn ED. J Biol
Chem. 1983 2583207-14.
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Origin of the kinetic barrier to nucleation. 2)
Conformational switch models
Seed catalyzes conformational change
Slow, spontaneous conformational change
Non-polymerizing conformation (normal form of
subunit after folding)
Polymerizing conformation (rare form of subunit)
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Origin of the kinetic barrier to nucleation. 2)
Conformational switch models
Seed catalyzes conformational change
Slow, spontaneous conformational change
Non-polymerizing conformation (normal form of
subunit after folding)
Polymerizing conformation (rare form of subunit)

• - 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.
  26157-85 DePace et al 1998 Cell. 931241-52
• More relevant to viral coat proteins and amyloid
  fibers

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Nucleation factors in the cell
The kinetic barrier to nucleation prevents
polymerization of cytoskeleton subunits at random
in the cell. The cell controls where polymers
form using nucleating factors.
Centrosome. Contains microtubule nucleating
factor ?-tubulin ring complex
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Nucleation factors in the cell
The kinetic barrier to nucleation prevents
polymerization of cytoskeleton subunits at random
in the cell. The cell controls where polymers
form using nucleating factors.
Centrosome. Contains microtubule nucleating
factor ?-tubulin ring complex
Leading edge. Contains actin Nucleation
branching factor Arp2/3 complex
These nucleating factors have the same fold as
the filament subunit, suggesting a mechanism
(templating) and an evolutionary origin. We now
know other actin nucleating factors that are
quite different in structure.
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Evidence that centrosomes contain microtubule
nucleating factors (cells imaged by fixation and
immunofluorescence)
Add nocodazole to depolymerize microtubules
Brinkley BR.(1985). Annu Rev Cell Biol. 1145-72.
Wash out drug
5 min
20 min
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Evidence that centrosomes contain microtubule
nucleating factors (cells imaged by fixation and
immunofluorescence)
Add nocodazole to depolymerize microtubules
Brinkley BR.(1985). Annu Rev Cell Biol. 1145-72.
Permeablize cells with non-ionic detergent
Wash out drug
Add tubulin, GTP Incubate at 37o
5 min
20 min
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Microtubule Organizing Centers (MTOCs)
Centrosomes, centrioles, basal bodies (animals)
and spindle pole bodies (fungi)
Centrioles PCM (fibrous) ?-tubulin ring
complex (nucleates MTs)
Centrosome Centriole Peri-centriolar
material (PCM)
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Discovery of ?-tubulin
Aspergillus (a mycelium forming fungus)
?-tubulin mutant
Defects in mitosis, nuclear transport
Select revertants
?-tubulin, ?-tubulin double mutant
Oakley and Oakley 1989. Nature 338662-4.
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Discovery of ?-tubulin
Aspergillus (a mycelium forming fungus)
?-tubulin knockout no microtubules
?-tubulin mutant
Defects in mitosis, nuclear transport
?-tubulin localizes to spindle pole bodies by
immunofluorescence
Select revertants
?-tubulin, ?-tubulin double mutant
Oakley and Oakley 1989. Nature 338662-4.
34
Centrosomes, centrioles, basal bodies and spindle
pole bodies
Fungi
Animals
Yeast spindle pole body forms on the nuclear
envelope
Centrosome Centriole Peri-centriolar
material (PCM)
Wigge et al 1998 J Cell Biol. 141967-77
35
?-tubulin ring complex the template model
Agard 2001 Curr Opin Struct Biol.11174-81
Note g-tubulin has the same fold as tubulin, and
the ring complex mimics a plus end
Agard 2011 Nat Rev Cell Mol Biol.12709
36
Actin nucleating complexes
Arp2/3 complex Nucleates from the pointed (slow
growing) end Nucleates from the side of a
pre-existing filament Generates brnached
networks Lammellipodia, Listeria comet tails,
Endocytosis
Formins Nucleate from the barbed (fast growing)
end Remain at the growing end Generate long
bundles Yeast actin cables, filopodia?
Formin dimer
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A pathogen provides a model for motility driven
by actin polymerization
- Listeria monocytogenes is a gram positive
bacterium that infects us from contaminated
food - Enters the cytoplasm of many cell types by
breaking out of phagosomes - Nucleates actin
filaments and forms a comet tail that propels it
through the cytoplasm and into neighboring
cells - Other pathogens (Shigella, pox virus)
also move using actin comet tails
comet tail of actin filaments
Tilney and Portnoy (1989) J Cell Biol.
1091597-608.
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Listeria moving in cultured cell
Julie Theriot 1992 Phase contrast
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Listeria provides a system for dissecting the
molecular mechanisms underlying leading edge
motility
Listeria moving in cell extract
fractionate cell extract by chromatography

Purify a protein complex that nucleates actin
polymerization on the Listeria surface
Welch et al.(1997) Nature. 385265-9
Identification of arp2/3 complex
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Listeria provides a system for dissecting the
molecular mechanisms underlying leading edge
motility
Listeria moving in cell extract
Listeria movement was later reconstituted using 7
proteins Actin Arp2/3 complex (7
polypeptides) Profilin Cofilin Capping
protein VASP ActA on the bacterium
surface Loisel et al.(1999). Nature. 401613-6
fractionate cell extract by chromatography

Purify a protein complex that nucleates actin
polymerization on the Listeria surface
Welch et al.(1997) Nature. 385265-9
Identification of arp2/3 complex
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Arp2/3 structure
Arp2 and Arp3 subunits have the same fold as actin
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Arp2/3 in action
Rhodamine actin TIRF microscopy Pollard and Kovar
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Arp2/3 mechanism

• To nucleate, Arp2/3 must
• bind to the side of a pre-existing filament
• recruiting an activating protein.
• The activating protein brings in the first
  subunit of the new polymer

ActA, WASP etc.
Arp2/3.
44
Arp2/3 mechanism

• To nucleate, Arp2/3 must
• bind to the side of a pre-existing filament
• recruiting an activating protein.
• The activating protein brings in the first
  subunit of the new polymer

ActA, WASP etc.
Arp2/3.
This mechanism generates dendritic actin
assemblies, as seen in the leading edge of
motile cells by EM
Pollard TD, Borisy GG. (2003) Cell. 112453-65.
45
How might cells control where and when
cytoskeleton polymers accumulate?
Neutrophil detects a bacterium
seconds
David Rogers 1950s
46
Activating proteins make Arp2/3 activity
dependent on multiple inputs
NWASP is activated by Cdc42.GTP Phosphoinositol
lipids Tyrosine phosphorylation
WAVE is activated by Rac.GTP Phosphoinositol
lipids
In both cases the WASP homolog acts as an AND
gate for multiple biochemical signals These
signals make Arp2/3 nucleation dependent on
multiple signaling pathway inputs at the plasma
membrane
47
Leukocyte chemotactic signals are usually
detected by GPCRs
Human cells (eg leukocytes)
Bacteria
Leukotriene B4 Chemokine eg CCL2 etc Etc.
fMLP
GDP
GTP
GPCR G-protein coupled receptor Different GPCRs
for different signals
Ga
Gbg
Heterotrimeric GTPase (inactive GDP bound state)
Signals to the actin cytoskeleton
48
Chemotactic receptors send multiple signals to
the actin cytoskeleton
fMLP
GDP
GTP
Ga
Gbg
WAVE
Rac
Arp2/3
Actin polymerization at the leading edge
Myosin-II driven Contraction at the rear of the
cell
49
The actin cytoskeleton is polarized in motile
cells
Actin Myosin-II in a fibroblast cell
Actin RhoA in neutrophils
50
How does a neutrophil polarize?
How are the multiple signaling outputs from
chemotactic receptors spatially organized to
promote polarization? Do different signals
diffuse away from the receptor to different
extents? Does the front of the cell inhibit
the back (or vice versa) and if so by chemical
signals, or physical signals such as membrane
tension?
?
?