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