Intro summary-The cytoskeleon is built of long, non-covalent protein polymers that self-assemble in the cytoplasm

- Some of these polymers are polar (actin, microtubules), others are non-polar (intermediate filaments); polarity is central to the biology of polar filaments

- Eukaryotic cytoskeleton polymers are evolutionary cousins of prokaryotic homologs:- Actin <>ParM, MreB; Tubulin <> FtsZ

CB201.2Intermediate filaments and Polymerization dynamics

- Dynamic vs. non-dynamic filaments- Intermediate filaments and nuclear lamins- Measuring polymerization dynamics- NTP hydrolysis during polymerization: treadmilling and dynamic instability- Proteins and drugs that modulate polymerization dynamics

Polymerization dynamics

Actin, ParM, MreB, Tubulin, FtsZ Weak affinity of polymer for monomer (~M)Fast, unidirectional turnover cycle powered by NTP hydrolysis during polymerization

NTP NDP + Pi

Polymerization dynamics

Actin, ParM, MreB, Tubulin, FtsZ Weak affinity of polymer for monomer (~M)Fast, unidirectional turnover cycle powered by NTP hydrolysis during polymerization

NTP NDP + Pi

Key conceptSubunits only come on and off at ends. This allows nm- and msec-scale biochemistry at ends to control the behavior of polymers that exhibit m- and sec- or min-scale biology

Are all protein polymers controlled by reactions at their ends?

Is all the polymerization/depolymerization biochemistry of microtubules and actin confined to their ends?

Polymerization dynamics

Actin, ParM, MreB, Tubulin, FtsZ Weak affinity of polymer for monomer (~M)Fast, unidirectional turnover cycle powered by NTP hydrolysis during polymerization

Intermediate filamentsHigh affinity of polymer for monomer (~nM-pM) Polymerization ~irreversible, may occur co-translationallySubsequent dynamics requires protein modification (phosphorylation, proteolysis)

NTP NDP + Pi

Intermediate filaments; keratin, vimentin, neurofilaments, other cell-type specific filaments, nuclear lamins

Mechanical integrity. Nuclear organization (nuclear lamins)

Keratin filaments in an epithelial cell monolayer cultured on glass

Intermediate filament structure

IF polypeptide forms an -helix

2 -helices dimerize into a coiled-coil

2 coiled-coils assemble into an anti-parallel tetramer

Tetrameric subunits assemble into a non-polar polymer

Polymers bundle to give rope-like intermediate filaments

What kind of interaction provides the main driving force that makes two alpha helices interact to form a coiled-coil?

1) Van der Waals interactions2) Electrostatic interactions3) Hydrogen bonds4) Hydrophobic interactions5) All the above

The two polypeptides in a coiled coils can run:

1) Parallel2) Anti-parallel3) Either is possible

Keratin mutations compromise the physical integrity of skin

Keratin filaments are abundant in skin keratinocytes, where they provide mechanical integrity to the epidermis.

Point mutations in skin keratin subunits cause inherited skin disease in humans and mouse models. Severity of the disease correlates with the degree to which polymerization of the mutant keratin subunit into intermediate filaments is inhibited.

In these conditions, called epidermolysis bullosa, the epidermis can separate from the dermis, causing severe blistering.

Blistering in the mouse models is first evident at sites where the skin experiences the most mechanical stress.

Fuchs and Cleveland 1998 Science. 279:514-9

Tissue specific expression of IFs

- One of the most striking aspects of IF biology- Keratins in epithelia, GFAP in Glia, Desmin in muscle etc.- ~20 different keratins, always co-expressed as pairs- Useful for histopathology- Keratins in tumor diagnostics- Nestin as a neuronal stem cell marker

- What are possible significances of tissue specific expression of IF genes?

Nuclear lamins

Nuclear lamina

- Lamins are a special type of intermediate filament protein that polymerizes into the nuclear lamina that underlies the nuclear envelope membranes.- Lamin polypeptides contain a nuclear localization sequence (NLS) that makes them enter the nucleus through nuclear pores.

Nuclear lamins depolymerize during mitosis

-The lamina breaks down during mitosis in higher animal cells after phosphorylation by Cdc2.CyclinB kinase, and other kinases- It reforms in the daughter cells through the action of phosphatases

Cdc2.CyclinB kinase

phosphatases

IF dynamics driven by reversible phosphorylation?

Kinase-XATP

Pi

Pi Pi

PiPi

PiPi

Binding protein

- Phosphorylation promotes lamin depolymerization in mitosis- IF dynamics are still poorly understood- Keratins in skin and hair become covalently cross-linked as the epithelial cell undergoes terminal differentiation- some IFs more dynamic than others?? e.g. vimentin (mesenchymal cells) more dynamic than keratin (epithelial cells); EMT marker

Phosphatase-Y

Nuclear Lamins

The nuclear lamina contains 3 types of lamins, A,B and C. All are homologous to intermediate filament subnits and assemble into coiled-coil oligomers

Lamin B is prenylated and binds directly to the nuclear envelope membrane. Nucleii in early embryos contain only this lamin type

Lamin A and C are generated from the same precursor protein by a complex set of modification at the protein level.

Mutation in human lamin-A cause “laminopathies” (Gruenbaum et al 2005 Nat Rev Cell Biol 6:21, Capell and Collins 2006 Nat Rev Genet. 7:940)

Mutations in the Lamin-A gene cause laminopathies

AD-EMD, AE-AMD, LGMD1B: muscular dystrophiesDCM1A: cardiomyopathyFPLD, GLD: liopdystrophiesAWS, HGPS: progerias (premature aging)

Worman HJ, Ostlund C, Wang Y.Cold Spring Harb Perspect Biol. 2010 2:a000760. Review

How could two different mutations in the same amino acid in lamin A cause two

very different diseases?

Polymerization dynamics Actin, ParM, tubulin, FtsZ. Weak affinity of polymer for monomer (~M)Polymerization-depolymerization coupled to energy transductionSpontaneous dynamics powered by NTP hydrolysis

Intermediate filamentsHigh affinity of polymer for monomer (~nM) Polymerization ~irreversible, occurs at or near ribosomeSubsequent dynamics requires protein modification (phosphorylation, proteolysis)

NTP NDP + Pi

Polymerization dynamics Experiment. Take a solution of a protein that can polymerize and change the conditions to promote polymerization. Then measure [polymer] over time.

- Tubulin is ~stably dimeric at 0o with GTP present-->Warm to 37o to polymerize

- FtsZ is stably monomeric in GDP-->Add GTP to polymerize

- Actin is stably monomeric at very low ionic strength with ATP present.-->Add physiological Mg++ and K+ to polymerize

Measuring tubulin polymerization by light scattering

When a light beam is passed through a solution of particles, some of the light is scattered. Scattering increases with the molecular weight

Scattering can be quantified by measuring the decrease in light passing through the sample using a spectrophotometer, or by the increase of light emitted at right angles in a fluorimeter.

For long polymers like microtubules, the amount of light scattered is proportional to the polymer mass.

Measuring actin polymerization by fluorescence spectroscopy using pyrene-actin as probe

SHpH 8

Actin

Pyrene-iodoacetate Pyrene- actin

Monomer: pyrene quenched by waterLow fluorescence Polymer: pyrene buried.

High fluorescence

Polymerization dynamics

Polymer mass

Time (seconds-minutes)

Bulk polymerization dynamics

Polymer mass

Time (seconds-minutes)

nucleation“lag phase”

elongation

steady state:Polymerization = depolymerization

Bulk polymerization dynamics

Polymer mass

Time (seconds-minutes)

nucleation“lag phase”

elongation

steady state:Polymerization = depolymerization

What is the difference between “steady-state” and “equilibrium”?How would you tell which applied in a case like the graph above?

Describing elongation dynamics

+

kon c

koff

c = monomer concentration

Assumes:1. monomors are added and lost only at filament ends at single, unique sites for addition and loss

2. kon and koff are constants that do not change with filament length or polymerization/depolymerization rate

Describing elongation dynamics

+

kon c

koff

c = monomer concentration

Assumes:1. monomors are added and lost only at filament ends at single, unique sites for addition and loss

2. kon and koff are constants that do not change with filament length or polymerization/depolymerization rate

kon may be “diffusion limited”, this is, it can occur as fast as monomers can collide with the end of the filament

What is a typcial value for a diffusion-limited rate constant for proteins?

Describing polymer dynamics

+

kon c

koff

c = monomer concentration

Growth rate (J) = kon c - koff

At equilibrium, J = 0. kon c = koff Cc = “critical concentration”

kon

koffCc =

Oosawa and Asakura (1975) The thermodynamics of protein polymerization. Acadmeic Press

Describing polymer dynamics

+

kon c

koff

c = monomer concentration

Growth rate (J) = kon c - koff

At equilibrium, J = 0. kon c = koff Cc =

J

c

kon

koffEquilibrium point. Cc = “critical concentration”

kon

koff

A polar polymer without NTP hydrolysis(eg bacterial flagellin)

+

k+on c

k+off

+

k-on c

k-off

add from plus end

add from minus end

A polar polymer without NTP hydrolysis(eg bacterial flagellin)

+

k+on c

k+off

+

k-on c

k-off

If there is a conformational change, then in general k+on k-

on=

But, because G is the same by either pathway, k+

on

k+off

k-on

k-off=

add from plus end

add from minus end

Cc+ = Cc-

A polar polymer without NTP hydrolysis(eg bacterial flagellin)

J

c

Cc+ = Cc-

+

k+on c

k+off

+

k-on c

k-off

k+off

k-off

If there is a conformational change, then in general k+on k-

on c=

But, because G is the same by either pathway, k+

on

k+off

k-on

k-off=

add from plus end

add from minus end

The critical concentration must be the same at each end.

Depending on c, both ends may either grow, shrink or remain static.

plus end

minus end

What happens if a microtubule can elongate at multiple sites?

+

kon c

koff

Growth rate (J) = kon c – koff

+

1) kon is likely to change2) koff likely to change3) Nothing will change

?

What happens if a microtubule can elongate at multiple sites?

According to a recent paper, multiple independent elongations sites at a microtubule ends cause the off rate increases with tubulin concentration(Gardner, Odde et al 2012 Cell 146:582)

In their model, increased growth rate leads to a more irregular microtubule ends, which have higher average off rates and more fluctuations

Nucleotide hydrolysis during polymerization

ATP

ADP ADP ADP

ADP ADP ADP

ADP ADP ADP

ADP ADP ADP

ATP ADP ADP ADP

ADP ADP ADP

ADP

Phosphate

Subunit addition Hydrolysis

Actin binds ATP and hydrolyzes it during polymerization

Nucleotide hydrolysis during polymerization

ATP

ADP ADP ADP

ADP ADP ADP

ADP ADP ADP

ADP ADP ADP

ATP ADP ADP ADP

ADP ADP ADP

ADP

Phosphate

Subunit addition Hydrolysis

Actin binds ATP and hydrolyzes it during polymerization

- +

GTP-tubulinGDP-tubulin

Tubulin binds GTP and hydrolyzes it during polymerization

ParM, MreB are similar

FtsZ, TubZ are biochemically similar but they do not form tubular polymers

Tubulin is a heterodimer of and polypeptides(FtsZ and other bacterial tubulin subunit are monomers)

Tubulin heterodimers never dissociate after folding

-tubulinmonomer; rapid GTP exchange, no hydrolysispolymer; No exchange, rapid hydrolysis

-tubulinNo exchange, no hydrolysis; GTP has purely structural role

How do you think this was shown – that the GTP on -tubulin is structural?

ATP hydrolysis and actin polymerization

100 means 20mol actin monomer has polymerized*

Carlier, Pantaloni and Korn, JCB 1984

Note the kinetic lag between hydrolysis and polymerization.From this we can infer:1) There can be at most a single molecule of ATP-actin at the

tip of a polymerizing filament2) There can be many ATP actin molecules at the tip

ATP hydrolysis and actin polymerization

100 light scattering units means 20mol actin monomer has polymerized

*

Carlier, Pantaloni and Korn, JCB 1984

Note the kinetic lag between hydrolysis and polymerization.From this we can infer:1) Subunit addition to a polymerizing tip requires ATP hydrolysis2) Subunit addition does not require ATP hydrolysis

Nucleotide hydrolysis during polymerization

ATP

ADP ADP ADP

ADP ADP ADP

ADP ADP ADP

ADP ADP ADP

ATP

Slow polymerization

ATP

ADP ADP ADP

ADP ADP ADP

ADP ADP

ADP

ATP

Fast polymerization

ATP

ATP

ATP

A kinetic lag between subunit addition and ATP hydrolysis can generate a “cap” of unhydrolyzed subunits

Nucleotide hydrolysis “weakens” the polymer

For both actin filaments and microtubules, ATP (GTP) hydrolysis has the effect of increasing the dissociation rate constant

ATP

ADP ADP ADP

ADP ADP ADP

ATP ATP

ATP ATP ATP

AT

P

AT

P

AT

P

ATP

ATP

AD

PADP

ADP

AD

PAD

PSlow

Fast

Nucleotide hydrolysis “weakens” the polymer

For both actin filaments and microtubules, ATP (GTP) hydrolysis has the effect of increasing the dissociation rate constant

ATP

ADP ADP ADP

ADP ADP ADP

ATP ATP

ATP ATP ATP

AT

P

AT

P

AT

P

ATP

ATP

AD

PADP

ADP

AD

PAD

PSlow

Fast

To do this experiment, you need either a mutation in the actin, or a non-hydrolyzeable ATP analog

ATP AMP-PNP

Treadmilling of pure actin driven by ATP hydrolysis

ADPATP

ADP ADP ADP

ADP ADP ADP

ATPADP ADP ADP

ADP ADP

ATP

ATP ADP

Pi

Note. Treadmilling of pure actin is very slow, and it is not clear if this reaction is relevant inside cells, where turnover is often very fast

Slow

J

k+off

k-off

Cc+Cc-

Steady state monomer concentration, where growth on plus ends is exactly balanced by shrinkage of minus ends

c

Barbed end

Pointed end

Treadmilling occurs at steady state because the critical concentration is lower on the barbed than the pointed end

Two small proteins accelerate actin dynamics in cells

ADP

ADP.PiATP

ATP ADP

- Cofilin only binds ADP-actin. It greatly weakens the filament, promoting faster dissociation of subunits from ends, as well as severing- Profilin helps recycle actin monomer- Cofilin and profilin conserved throughout eukaryotes- The exact pathway of depolymerization in cells is still unknown

Profilin Cofilin

ADP.PiADP.Pi

ADP.PiADP.Pi

Pi

ADP ADP ADP

ADP ADP ADP ATP

ATP

Microtubules: dynamic instability

Microtubules alternate between bouts of polymerization and depolymerization. This is true for pure tubulin in vitro, and also in cytoplasm, though the rates differ.

Rhodamine-tubulin polymerizing from a centrosome in frog egg extract.

Observation by widefield fluorescence microscopy

Dynamic Instability is driven by GTP hydrolysis

Model: GTP subunits like to be straight, while GDP subunits like to be curved. GTP hydrolysis puts the lattice under stress. Growing microtubules are stabilized by a cap of GTP-subunits. When this cap is lost the microtubule depolymerizes rapidly by a kind of tearing or peeling apart. (Nogales and Wang 2006 Curr Opin Struct Biol 16:221-9)

Growing microtubule

Shrinking microtubule

GTP

GDP

Pi

Nucleotide exchange

Catastrophe Rescue

GTP-tubulin

GDP-tubulin

How long is the GTP cap?

Growing microtubulePi

GTP-tubulin

Short cap?

Long cap?

How would you ask this question experimentally?

Cellular factors regulate Dynamic Instability

GTP

GDP

Pi

Kinesin-13 promotes catastrophesMAPs stabilize microtubules

Drugs that stabilize and destabilize actin filaments

Phalloidin

Latrunculin

Binds

BindsBlocks

Blocks

Phalloidin, jasplakinolide bind to actin filaments and block depolymerization. Fluorescent phalloidin is to allow image the actin cytoskeleton in fixed cells. It is not cell permeable. Jasplakinolide is cell permeable and is used for perturbation.

Latrunculin-B binds to unpolymerized actin and blocks polymerization.

Cytochalasin-D binds to the barbed ends of actin filaments and caps them, preventing polymerization. Unlike Latrunculin, it also blocks depolymerization

ADP ADP ADP

ADP ADP ADP

ADP

AT

P

AT

P

ATP

Cell stained with fluorescent phallodin

Drugs that stabilize and destabilize microtubules

Taxol

Colchicine

Binds

BindsBlocks

Blocks

Drugs that bind to tubulin dimer and block polymerization:Colchicine; know to the ancients as a drug. Still used to treat goutNocodazole; similar action, but faster acting and reversible. Preferred for research.

Drugs that bind to microtubules and block depolymerization:Taxol (= paclitaxel). Use in research to stabilize microtubules in cells or with pure tubulin. Widely used anti-cancer drug that triggers the mitotic checkpoint

Why rapid polymerization dynamics? Remodeling of the cytoskeleon

assemble phagocytic cup

Neutrophil detects a bacterium

Actin depolymerizes as the bacterium is internalized and killed

2 minutes 2 minutes

Why rapid polymerization dynamics? Remodeling of the cytoskeleon

Interphase Mitosis

10 minutes

assemble phagocytic cup

Neutrophil detects a bacterium

Actin depolymerizes as the bacterium is internalized and killed

2 minutes 2 minutes