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

CB201_12Tim Mitchison Lecture 3

Force generation by polymerization dynamics

Nucleation: controlling where and when polymers form

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



Force from polymerization dynamics

Eukaryotes and prokaryotes

ATPase motor proteins

Only Eukaryotes

Polymerization dynamics can perform mechanical work by pushing or pulling

Pushing by polymerizationLeading edge protrusion (actin)Listeria motility (actin)Plasmid separation in bacteria (ParM)

Pulling by depolymerizationChromosome movement in mitosis(microtubules)

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)

Elementary steps

+

Actin filaments grow by ~2nm per subunit(Actin monomer is ~4nm long, filament has 2 strands)

Kinesin moves 8nm per step

Elementary steps

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Actin filaments grow by ~2nm per subunit


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

Elementary steps



This liberates ~8-12 kilocal per mol(= ~20kT per molecule)

Efficiency = 5pN.8nm/20kT = ~50%

Force distance Chemical energy dissipated

Elementary steps

+

Actin filaments grow by ~2nm per subunit(4nm subunit, 2 stranded polymer)



This liberates ~8-12 kilocal per mol(= ~20kT per molecule)

How do we think about force generation from polymerization or depolymerization?

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 8/13nmSuppose the full energy of GTP hydrolysis is used to promote this reactionGTP -> GDP + G = ~ -50 kJ/mol = 5x10-4 /6x10-23 J/microtubule Force = work/distance = ~ 10-19/0.5x10-9 = ~2x10-10 N = ~200pN


How much force? Simple argument for maximum possible force:For every tubulin added, the microtubules grows 8/13nmSuppose the full energy of GTP hydrolysis is used to promote this reactionGTP -> 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

Actin polymerization force pushes the front of motile cells forward

How do cells control where and when cytoskeleton polymers accumulate?

Bacterium

Neutrophil

Chemotaxis

Phagocytosis

High density of actin filaments

Neutrophil chasing S aureus in a drop of bloodDavid Rogers 1950s

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


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?

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

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

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


- 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 2 bonds. Fast

Break 3 bonds. Slow





Break 3 bonds. Slow




Break 2 bonds. Fast

Break 3 bonds. Slow






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] ~ Kd[monomer]n-1;

nucleation rate ~ Kd[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.

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 + +

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 + +

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.

Leading edge. Contains actin Nucleation + branching factor Arp2/3 complex

Centrosome.Contains microtubule nucleating factor -tubulin ring complex

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+

+

+

+

+ +

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.

Evidence that centrosomes contain microtubule nucleating factors

(cells imaged by fixation and immunofluorescence)

Add nocodazole to depolymerize microtubules

Wash out drug

5 min

20 min

Brinkley BR.(1985). Annu Rev Cell Biol. 1:145-72.

Evidence that centrosomes contain microtubule nucleating factors

(cells imaged by fixation and immunofluorescence)

Add nocodazole to depolymerize microtubules

Wash out drug

5 min

20 min

Permeablize cells with non-ionic detergent

Add tubulin, GTP Incubate at 37o

Brinkley BR.(1985). Annu Rev Cell Biol. 1:145-72.

Microtubule Organizing Centers (MTOCs): Centrosomes, centrioles, basal bodies (animals) and spindle pole bodies

(fungi)

Centrosome = Centriole + Peri-centriolar material (PCM)

Centrioles

PCM (fibrous)

-tubulin ring complex(nucleates MTs)

Discovery of -tubulinAspergillus (a mycelium forming fungus)

-tubulin mutant

Select revertants

-tubulin, -tubulin double mutant

Defects in mitosis, nuclear transport

Oakley and Oakley 1989. Nature 338:662-4.

Discovery of -tubulinAspergillus (a mycelium forming fungus)

-tubulin mutant

Select revertants

-tubulin, -tubulin double mutant

-tubulin knockout: no microtubules

-tubulin localizes to spindle pole bodies by immunofluorescence

Defects in mitosis, nuclear transport

Oakley and Oakley 1989. Nature 338:662-4.

Centrosomes, centrioles, basal bodies and spindle pole bodies

Yeast spindle pole body forms on the nuclear envelope

Wigge et al 1998 J Cell Biol. 141:967-77

Centrosome = Centriole + Peri-centriolar material (PCM)

Animals Fungi

-tubulin ring complex: the template model

Agard 2001 Curr Opin Struct Biol.11:174-81

Agard 2011 Nat Rev Cell Mol Biol.12:709

Note -tubulin has the same fold as tubulin, and the ring complex mimics a plus end

Actin nucleating complexes

Arp2/3 complexNucleates from the pointed (slow growing) endNucleates from the side of a pre-existing filamentGenerates brnached networksLammellipodia, Listeria comet tails, Endocytosis

ForminsNucleate from the barbed (fast growing) endRemain at the growing endGenerate long bundlesYeast actin cables, filopodia?

Formin dimer

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. 109:1597-608.

Listeria moving in cultured cell

Julie Theriot~1992Phase contrast

Listeria provides a system for dissecting the molecular mechanisms underlying leading edge motility

Identification of arp2/3 complex

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. 385:265-9

Listeria provides a system for dissecting the molecular mechanisms underlying leading edge motility

Identification of arp2/3 complex

Listeria moving in cell extract

fractionate cell extract by chromatography

Purify a protein complex that nucleates actin polymerization on the Listeria surface

Listeria movement was later reconstituted using 7 proteins:ActinArp2/3 complex (7 polypeptides)Profilin CofilinCapping proteinVASP+ActA on the bacterium surfaceLoisel et al.(1999). Nature. 401:613-6

Welch et al.(1997) Nature. 385:265-9

Arp2/3 structure

Arp2 and Arp3 subunits have the same fold as actin

Arp2/3 in action

Rhodamine actinTIRF microscopyPollard and Kovar

Arp2/3 mechanism

ActA, WASP etc.

To nucleate, Arp2/3 must:1)bind to the side of a pre-existing filament2)recruiting an activating protein. The activating protein brings in the first subunit of the new polymer

Arp2/3.

Arp2/3 mechanism

This mechanism generates dendritic actin assemblies, as seen in the leading edge of motile cells by EM

Pollard TD, Borisy GG. (2003) Cell. 112:453-65.

ActA, WASP etc.

To nucleate, Arp2/3 must:1)bind to the side of a pre-existing filament2)recruiting an activating protein. The activating protein brings in the first subunit of the new polymer

Arp2/3.


seconds

David Rogers 1950s

Activating proteins make Arp2/3 activity dependent on multiple inputs

NWASP is activated by: Cdc42.GTPPhosphoinositol lipidsTyrosine phosphorylation

WAVE is activated by: Rac.GTPPhosphoinositol lipids

In both cases the WASP homolog acts as an AND gate for multiple biochemical signalsThese signals make Arp2/3 nucleation dependent on multiple signaling pathway inputs at the plasma membrane

fMLP

GPCR G-protein coupled receptor

Different GPCRs for different signals

GDP

Heterotrimeric GTPase (inactive GDP bound state)

Leukocyte chemotactic signals are usually detected by GPCRs

GTP

G G

Signals to the actin cytoskeleton

Bacteria Human cells (eg leukocytes)

Leukotriene B4Chemokine – eg CCL2 etcEtc.

fMLP

GDP

Chemotactic receptors send multiple signals to the actin cytoskeleton

GTP

G G

Actin polymerization at the leading edge

WAVEArp2/3

Myosin-II driven Contraction at the rear of the cell

Rac

The actin cytoskeleton is polarized in motile cells

Actin Myosin-II in a fibroblast cell Actin RhoA in neutrophils

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?

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

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