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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

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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

Intro summary


Cb201 2 intermediate filaments and polymerization dynamics

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

NDP + Pi

NTP

Polymerization dynamics

Actin, ParM, MreB, Tubulin, FtsZ

Weak affinity of polymer for monomer (~M)

Fast, unidirectional turnover cycle powered by NTP hydrolysis during polymerization


Polymerization dynamics1

NDP + Pi

NTP

Polymerization dynamics

Actin, ParM, MreB, Tubulin, FtsZ

Weak affinity of polymer for monomer (~M)

Fast, unidirectional turnover cycle powered by NTP hydrolysis during polymerization

Key concept

Subunits only come on and off at ends.

This allows nm- and msec-scale biochemistry at ends to control the behavior of polymers that exhibit mm- and sec- or min-scale biology


Intro summary

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 dynamics2

NDP + Pi

NTP

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 filaments

High affinity of polymer for monomer (~nM-pM)

Polymerization ~irreversible, may occur co-translationally

Subsequent dynamics requires protein modification (phosphorylation, proteolysis)


Intro summary

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

Mechanical integrity. Nuclear organization (nuclear lamins)


Intro summary

Keratin filaments in an epithelial cell monolayer cultured on glass


Intermediate filament structure

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


Intro summary

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

  • Van der Waals interactions

  • Electrostatic interactions

  • Hydrogen bonds

  • Hydrophobic interactions

  • All the above


The two polypeptides in a coiled coils can run

The two polypeptides in a coiled coils can run:

  • Parallel

  • Anti-parallel

  • Either is possible


Keratin mutations compromise the physical integrity of skin

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

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?


Intro summary

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.


Intro summary

Nuclear lamins depolymerize during mitosis

Cdc2.CyclinB

kinase

phosphatases

  • 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


If dynamics driven by reversible phosphorylation

Pi

Pi

Pi

Pi

Pi

Pi

IF dynamics driven by reversible phosphorylation?

Binding protein

Kinase-X

ATP

Pi

Phosphatase-Y

  • 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


Nuclear lamins

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)


Intro summary

Mutations in the Lamin-A gene cause laminopathies

AD-EMD, AE-AMD, LGMD1B: muscular dystrophies

DCM1A: cardiomyopathy

FPLD, GLD: liopdystrophies

AWS, HGPS: progerias (premature aging)

Worman HJ, Ostlund C, Wang Y.

Cold Spring Harb Perspect Biol. 2010 2:a000760. Review


Intro summary

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


Polymerization dynamics3

NDP + Pi

NTP

Polymerization dynamics

Actin, ParM, tubulin, FtsZ.

Weak affinity of polymer for monomer (~M)

Polymerization-depolymerization coupled to energy transduction

Spontaneous dynamics powered by NTP hydrolysis

Intermediate filaments

High affinity of polymer for monomer (~nM)

Polymerization ~irreversible, occurs at or near ribosome

Subsequent dynamics requires protein modification (phosphorylation, proteolysis)


Polymerization dynamics4

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

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

SH

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

pH 8

Actin

Pyrene-iodoacetate

Pyrene- actin

Monomer: pyrene quenched by water

Low fluorescence

Polymer: pyrene buried.

High fluorescence


Polymerization dynamics5

Polymerization dynamics

Polymer mass

Time (seconds-minutes)


Bulk polymerization dynamics

Bulk polymerization dynamics

steady state:

Polymerization = depolymerization

nucleation

“lag phase”

elongation

Polymer mass

Time (seconds-minutes)


Bulk polymerization dynamics1

Bulk polymerization dynamics

steady state:

Polymerization = depolymerization

nucleation

“lag phase”

elongation

Polymer mass

Time (seconds-minutes)

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

Describing elongation dynamics

c = monomer concentration

kon c

+

koff

Assumes:

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

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


Describing elongation dynamics1

Describing elongation dynamics

c = monomer concentration

kon c

+

koff

Assumes:

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

2. kon and koffare 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

koff

kon

Describing polymer dynamics

c = monomer concentration

kon c

+

koff

Growth rate (J) = kon c - koff

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

Cc =

Oosawa and Asakura (1975)

The thermodynamics of protein polymerization. Acadmeic Press


Describing polymer dynamics1

koff

kon

Describing polymer dynamics

c = monomer concentration

kon c

+

koff

Growth rate (J) = kon c - koff

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

kon

J

c

koff

Equilibrium point. Cc = “critical concentration”


A polar polymer without ntp hydrolysis eg bacterial flagellin

A polar polymer without NTP hydrolysis(eg bacterial flagellin)

add from minus end

add from plus end

k+on c

k-on c

+

+

k+off

k-off


A polar polymer without ntp hydrolysis eg bacterial flagellin1

k+on

=

k-on

k-off

k+off

k-on

k+on

=

A polar polymer without NTP hydrolysis(eg bacterial flagellin)

add from minus end

add from plus end

k+on c

k-on c

+

+

k+off

k-off

If there is a conformational change, then in general

But, because DG is the same by either pathway,

Cc+ = Cc-


A polar polymer without ntp hydrolysis eg bacterial flagellin2

k+on

=

k-on c

k-off

k+off

k-on

k+on

=

A polar polymer without NTP hydrolysis(eg bacterial flagellin)

add from minus end

add from plus end

k+on c

k-on c

+

+

k+off

k-off

If there is a conformational change, then in general

But, because DG is the same by either pathway,

plus end

The critical concentration must be the same at each end.

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

J

minus end

k-off

c

Cc+ = Cc-

k+off


What happens if a microtubule can elongate at multiple sites

What happens if a microtubule can elongate at multiple sites?

kon c

+

Growth rate (J) = kon c – koff

koff

?

+

  • kon is likely to change

  • koff likely to change

  • Nothing will change


What happens if a microtubule can elongate at multiple sites1

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

ATP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

Nucleotide hydrolysis during polymerization

Actin binds ATP and hydrolyzes it during polymerization

Phosphate

Subunit addition

Hydrolysis


Nucleotide hydrolysis during polymerization1

ATP

ATP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

Nucleotide hydrolysis during polymerization

Actin binds ATP and hydrolyzes it during polymerization

Phosphate

Subunit addition

Hydrolysis

ParM, MreB are similar

Tubulin binds GTP and hydrolyzes it during polymerization

GDP-tubulin

GTP-tubulin

-

+

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


Intro summary

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

Tubulin heterodimers never dissociate after folding

-tubulin

monomer; rapid GTP exchange, no hydrolysis

polymer; No exchange, rapid hydrolysis

-tubulin

No exchange, no hydrolysis; GTP has purely structural role

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


Intro summary

ATP hydrolysis and actin polymerization

100 means 20mol

actin monomer has polymerized

*

Carlier, Pantaloni and Korn, JCB 1984

  • Note the kinetic lag between hydrolysis and polymerization.

  • From this we can infer:

  • There can be at most a single molecule of ATP-actin at the tip of a polymerizing filament

  • There can be many ATP actin molecules at the tip


Intro summary

ATP hydrolysis and actin polymerization

100 light scattering units means 20mol

actin monomer has polymerized

*

Carlier, Pantaloni and Korn, JCB 1984

  • Note the kinetic lag between hydrolysis and polymerization.

  • From this we can infer:

  • Subunit addition to a polymerizing tip requires ATP hydrolysis

  • Subunit addition does not require ATP hydrolysis


Nucleotide hydrolysis during polymerization2

ATP

ATP

ATP

ATP

ATP

ATP

ATP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

Nucleotide hydrolysis during polymerization

Slow polymerization

Fast polymerization

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


Nucleotide hydrolysis weakens the polymer

ATP

ATP

ATP

ATP

ATP

ATP

ATP

ATP

ATP

ATP

ATP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

Nucleotide hydrolysis “weakens” the polymer

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

Slow

Fast


Nucleotide hydrolysis weakens the polymer1

ATP

ATP

ATP

ATP

ATP

ATP

ATP

ATP

ATP

ATP

ATP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

Nucleotide hydrolysis “weakens” the polymer

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

Slow

Fast

ATP

AMP-PNP

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


Treadmilling of pure actin driven by atp hydrolysis

ATP

ATP

ATP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

Treadmilling of pure actin driven by ATP hydrolysis

Pi

Slow

ADP

ATP

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


Intro summary

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

Barbed end

J

Cc+

Cc-

Pointed end

k-off

c

k+off

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


Two small proteins accelerate actin dynamics in cells

ADP.Pi

ADP.Pi

ADP.Pi

ADP.Pi

ADP.Pi

ATP

ATP

ATP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

Two small proteins accelerate actin dynamics in cells

Pi

Cofilin

Profilin

ADP

ATP

  • - 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


Microtubules dynamic instability

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

Dynamic Instability is driven by GTP hydrolysis

Pi

Growing microtubule

GTP-tubulin

GDP

Nucleotide exchange

Catastrophe

Rescue

GTP

Shrinking microtubule

GDP-tubulin

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)


How long is the gtp cap

How long is the GTP cap?

Pi

Growing microtubule

GTP-tubulin

Short cap?

Long cap?

How would you ask this question experimentally?


Cellular factors regulate dynamic instability

Cellular factors regulate Dynamic Instability

MAPs stabilize microtubules

Kinesin-13 promotes catastrophes

Pi

GDP

GTP


Drugs that stabilize and destabilize actin filaments

ATP

ATP

ATP

ADP

ADP

ADP

ADP

ADP

ADP

ADP

Cell stained with fluorescent phallodin

Drugs that stabilize and destabilize actin filaments

Phalloidin

Binds

Blocks

Blocks

Binds

Latrunculin

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


Drugs that stabilize and destabilize microtubules

Drugs that stabilize and destabilize microtubules

Taxol

Binds

Blocks

Blocks

Binds

Colchicine

Drugs that bind to tubulin dimer and block polymerization:

Colchicine; know to the ancients as a drug. Still used to treat gout

Nocodazole; 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

Why rapid polymerization dynamics?Remodeling of the cytoskeleon

Neutrophil detects a bacterium

Actin depolymerizes as the bacterium is internalized and killed

assemble phagocytic cup

2 minutes

2 minutes


Why rapid polymerization dynamics remodeling of the cytoskeleon1

Interphase

Mitosis

10 minutes

Why rapid polymerization dynamics?Remodeling of the cytoskeleon

Neutrophil detects a bacterium

Actin depolymerizes as the bacterium is internalized and killed

assemble phagocytic cup

2 minutes

2 minutes


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