Lecture 15: Properties and Dynamics of Actin

When did electron microscopy define 3 types of filaments?

the 1950s-60s

When were filament subunits purified biochemically?

the late ‘60s - early ‘70s

What are the 3 cytoskeletal polymers?

microfilaments (actin), microtubules (tubulin dimer), and intermediate filaments

True or False: Actin is required for movement; actin assembly can drive movement.

true

What are some examples of actin structures?

microvilli, leading edge filaments, cell cortex, adherens belt, filopodia, lamellipodium, stress fibers, phagocytosis,, moving endocytic vesicles, contractile ring, etc

What are some characteristics of microvilli?

Line the gut to take up nutrients

Are finger-like projections on the surface of the gut

Push the membrane out

Increases the surface area on the cell

Stable filaments (life of 4-5 days)

What are some characteristics of leading edge filaments?

Half-life is 2-4 minutes

Dynamic molecules

True or False: Actin monomers bind to each other to form large filamentous polymer.

true

What is the process for actin regulation?

Signal

Disassembly of filaments and rapid diffusion of subunits

Reassembly of filaments on a different side

True or False: Actin is one of the most abundant intracellular proteins in eukaryotes (10% of muscle protein).

true

Actin is highly what?

conserved through evolution (more than 90% of conservation)

How many different actin genes do humans have?

6 different genes

What is alpha actin involved with?

muscles

What is beta actin involved with?

the leading edge of moving cells

What is gamma actin involved with?

stress fibers

What is the size of actin?

42 kD

What type of protein is actin?

an ATP-binding protein

What is the actin monomer?

G-actin

True or False: There is spontaneous polymeriztion of actin in the presence of Mg, K, and Na, but polyerimization is also reversible.

true

What is polyermization reversibility important for?

cell movement

Actin is a what?

an ATPase

What type of bonds are between actin filaments?

non-covalent bonds

What does the structure of actin resemble?

beads on a string (7-9 nm diameter)

How are subunits arranged in actin?

as a tightly wound helix

What is the short pitch?

1 actin monomer to adjacent neighbor and so forth (every subunit)

What is the long pitch?

a half turn of the helix; the starting monomer is directly facing away, and the other monomer is facing direcly towards you

How large is the full-turn of the helix?

72 nm

What are features shared among cytoskeletal filaments?

They are non-covalent polymers

They are multi-stranded

They grow or shrink by adding or losing monomers from the ends

Why is it so important that filaments are multi-stranded?

single stranded polymers (9-10 subunits) are much shorter than multi-stranded polymers

What part of the myosin attaches to actin?

the S1 domain

How did researchers determine if actin filaments are polar/have an orientation?

Mixed myosin head domains S1 with actin filaments

Allows myosin to bind to actin and process through electron microscopy

Revealed the different morphologies at each end

What are the two ends of actin?

the plus and minus end

What did the experiment involving the addition of myosin-decorated filaments to G-actin reveal?

actin can grow at both ends, but polymerization occurs faster at the plus end

True or False: ATP G-actin can assembled into filaments in the presence of Mg but not in the absence of Mg.

true

How does one study the rate of actin polymerization?

Have a relatively high amount of ATP G-actin in the test tube, and add Mg to trigger F-actin assembly to measure the rate of assembly

What are the phases of actin polymerization?

nucleation, elongation, and steady state

What is occurring at equilibrium?

There is no net F-actin assembly

The ends still exchange monomers

Sme free G-actin

In order to for a complex of stable actin to form, what must happen?

3 monomers must come together

What is a nucleus?

a 3-monomer complex

What type of reaction is nucleation?

a trimolecular reaction

What needs to happen for 3 monomers to come together?

the monomers must collide simultaneously in the correct orientations

What occurs during nucleation?

3 monomers simultaneously collide to form a nucleus

What type of reaction is elongation?

a bimolecular reaction

How many molecules are required to collide during elongation?

2 molecules

How could a researcher test the nucleation idea experimentally?

Bypass nucleation

When a seed is added, the molecular goes right into the elongation phase—skipping nucleation

What type of reaction is actin assembly?

a steady state reaction

What is the critical concentration?

the number of monomers at equilibrium

When does F-actin assembly occur?

when G-actin > critical concentration

When does F-actin disassembly occur?

when G-actin < critical concentration

What happens at the critical concentration?

the rates of actin association and dissociation balance

What 2 different conformations can monomers adopt?

monomers that can add to the plus end and monomers that can add to the minus end

What is important to know about homogenous polymers?

The equilibrium value K = on rate/off rate

Plus ends have a faster on and off rate

Minus ends have a slower on and off rate

All subunits are identical

Polymer products are chemically identical

Addition to the plus or minus end is the same overall reaction

The energy released is identical in both cases

What experiments address whether ATP hydrolysis is required for actin filament assembly?

Use ADP-G-actin in polymerization reaction

Use non-hydrolysable ATP analogues

Is actin a homogenous polymer?

No, it’s not

What is important to know about actin polymers?

Plus ends have ATP actin

Minus ends have ADP actin

Critical concentrations for the two ends are different

ATP hydrolysis results in different end types

The rate of addition at the plus end is faster than ATP hydrolysis

What happens if there’s a positive rate of polymerization?

actin assembly

What happens if there’s a negative rate of polymerization?

actin disassembly

When does treadmilling occur?

when the concentration of G-actin is between the critical concentration of the plus end and critical concentration of the minus end; the plus end is growing and the minus end is shrinking

Treadmilling is a consequence of what?

of ATP hydrolysis and different critical concentrations at the two ends

What are the roles of ATP hydrolysis?

To change the critical concentration value for the plus end vs. the minus end

To make the ends chemically different

What happens after ATP adds onto the ends of actin?

ATP is hydrolyzed to ADP which stays bound to the actin subunits within the polymer

ADP forms dissociate from the polymer more readily than the ATP form

True or False: ADP forms dissociate more readily with the plus end and the ADP form dissociates more readily from the minus end.

true

What type of reaction is treadmilling a part of?

a steady state reaction

What is important to know about equilibrium reactions?

The concentration of the products and reactants remain constant at equilibrium

The rates of the forward and reverse reactions are balanced

Each end loses monomers at the same rate it gains monomers

Monomer gain and loss are balanced at each end

No net gain or loss of monomers at either end

What is important to know about steady state reactions?

At least one parameter of the reaction is not changing over time, but others may vary at steady state

The overall length is constant, but the plus end is growing while the minus end is shrinking

There is a net gain of monomers at the plus end and a net loss of monomers at the minus end

Monomer gain and loss are not balanced at each end

The overall length of each polymer is constant because the rate of gain at the plus end equals the rate of loss at the minus end at steady state

True or False: Equilibrium reactions don’t involve the input of energy; steady state reactions require a continuous input of energy.

true

What happens at steady state?

Treadmilling (as long as ATP is available)

Mass of filaments is constant

Average length of filaments is constant

No net F-actin assembly

Ends are still exchanging monomers

Still some free G-actin

Rate of assembly at the plus end = the rate of disassembly at the minus end

What is important to know about the Speckle microscopy experiment?

Microinject small amounts of fluorescent actin into cell (only a fraction of filaments are labelled)

Image living cells with fluorescence microscopy

Revealed that the plus ends are directed towards the leading edge

What does treadmilling allow a cell to do?

keeps the concentration of monomers higher at the growing edge of the cell

True or False: The rate at which the leading edge is moved forward is the rate at which the actin is assembled.

true