Actin and Myosins

Question 1

Actin filaments are found in various architectures to perform different functions.

Describe them.

Answer 1

Cortex - branched and cross-linked networks.

Stress fibres - antiparallel contractile structures, extend into cell tail.
Each end of a stress fibre is a mixture of + and - ends.

Lamellipodium - branched and crosslinked networks.

Filopodium - parallel bundles.

Question 2

Actin assembly and disassembly can be regulated by actin binding proteins.

Name 4 ways in which these proteins could regulate actin organisation.

Answer 2

1. Inhibition of spontaneous self-assembly of G-actin -> F-actin.
2. Nucleation of new actin filaments.
3. Control of actin filament length.
4. Elongation/Shortening of pre-existing actin filaments.

Question 3

Thymosin beta-4 is a G-actin monomer binding protein. Explain its role in actin polymerisation.

Answer 3

Thymosin beta-4 is a small peptide that binds to monomeric actin, that sequesters ADP-G actin which prevents the nucleotide exchange causing an accumulation of ADP-bound G-actin. This prevents polymerisation due to sub-threshold ATP-bound G-actin concentrations.

Question 4

Profilin is a G-actin monomer binding protein. Explain its role in regulating actin polymerisation.

Answer 4

Profilin binds to ADP G-actin from depolymerisation F-actin. It binds specifically opposite the nucleotide binding cleft, which is facing toward the - end.

This prevents polymerisation at the - end, and causes F-actin to extend preferentially at the + end.

Question 5

What does “ARP” stand for in the “ARP2/3 complex”?

Answer 5

Actin Related Protein(s)

Question 6

What is the common role between ARP2/3 complex and Formins in actin polymerisation?

Answer 6

They both aid in nucleation - formation of the actin trimer.

Question 7

What are Filament Binding Proteins?

Answer 7

Proteins that bind to actin in its F-actin state as opposed to G-actin binding proteins which bind to monomeric actin.

Question 8

The ARP2/3 complex is an example of a monomer binding protein and a filament binding protein. True or False?

Answer 8

True.
In addition to its nucleation function, ARP2/3 initiates branching of microfilaments by binding to F-actin. It’s nucleation function results in polymerisation of a new branch, leading to the development of cross links.

Question 9

What are the two ways that Filament Binding Proteins can regulate actin filament length?

Answer 9

1. Capping - An actin filament capped at both ends will never grow nor shrink. It is said to be stabilised.
2. Severing - Breaking up filaments into shorter filaments.

Question 10

Filament Binding Proteins are obligate in the ways they can modify filament length. True or False?

Answer 10

False.

Various proteins serve both capping at either end and severing of the filament that can respond to different signals, allowing multiple layers of regulation. E.G. Gelsolin

Question 11

Describe the capping and severing roles of Gelsolin.

Answer 11

Gelsolin is a Filament Binding Protein
It caps the + end of F-actin preventing further growth and also severs the filament, dissolving meshes of actin filaments.

Actin meshes make the cytosol into a gel-like consistency hence the name Gelsolin

Question 12

State a factor which stimulates and a factor which inhibits Gelsolin activity.

Answer 12

Elevated Ca2+ levels stimulate Gelsolin

PIP2 inhibits.

Question 13

Name three examples of actin binding proteins that are able to bind both monomeric and filamentous actin

Answer 13

1. ARP2/3 Complex
2. Gelsolin
3. ADF/Cofilin

Question 14

What is the function of ADF/Cofilin?

Answer 14

Binds ADP-actin in both monomeric and F-actin.
Enhances depolymerisation if bound at - end, where the depolymerisation occurs.
Also prevents nucleotide exchange, preventing ADP-actin -> ATP-actin, preventing polymerisation at the + end.
Has high overall action of filament disassembly.

Question 15

Compare and contrast the activity of ADF/Cofilin and Profilin

Answer 15

• Profilin and Cofilin both bind to ADP-actin.
• Profilin binds to monomeric actin, whereas Cofilin can bind to monomeric and filamentous actin.
• Both effect the rate of nucleotide exchange, Profilin increases rate while Cofilin decreases.
• Profilin increases polymerisation of actin at the + end and prevents polymerisation at the - end.
• Cofilin increases depolymerisation at the - end while preventing polymerisation at the + end.
• Cofilin exhibits F-actin severing activity, whereas Profilin does not.
• Both accelerate actin dynamics by providing more opportunities for other Actin Binding Proteins to interact with actin.

Question 16

What are stress fibres?

Answer 16

Antiparallel, contractile actin bundles in non-muscle cells that extend into the cell tail.

Each end of a stress fibre is a mixture of + and - ends.

Question 17

Name an agent used to visualise actin filaments.

Answer 17

Phallodin

Question 18

Describe how Phallodin can act as a visualising agent for actin molecules.

State one advantage and one disadvantage of this method.

Answer 18

Phallodin binds selectively and tightly to F-actin. Can be labelled with fluorescent tags and localisation be visualised in light microscopy.

Phallodin is smaller than immunofluorescent antibodies typically used in labelling cellular proteins. This allows for denser labelling and higher resolution images.

Phallodin stabilises F-actin, preventing depolymerisation. Phallodin-treated cells therefore have greater actin levels associated with plasma membranes, and cells often die.

Causes distributions to actin organisation, may influence images.

Question 19

Cross-linking proteins organise networks and bundles of actin filaments.

State the cross-linking proteins in stress-fibres.

Answer 19

Alpha-actinin

Question 20

What is the cross-linker protein in the gel-like network of the cell cortex?

Answer 20

Filamin

Question 21

What is the cross linker in the tight, parallel bundles of filopodia?

Answer 21

Fimbrin

Question 22

All cross-linker proteins associate with actin, and therefore contain…?

Answer 22

Actin Binding Domains (ABDs)

Question 23

Out of Fimbrin, Alpha-Actinin and Filamin, which proteins exists as dimers?

Answer 23

Filamin and alpha-actinin

Question 24

Fimbrin is a dimeric protein. True or False?

Answer 24

False. It is a monomer.

Question 25

Describe the structure of alpha-actinin

Answer 25

It has an antiparallel head-to-tail structure such that the actin binding domains are further apart and in opposite directions.

This forms greater spaces between actin filaments for inclusion of motor proteins, and also contributes to the antiparallel structure of stress fibres.

Question 26

How many ABDs does fimbrin have?

Answer 26

2

Question 27

How many ABDs do alpha-actinin and filamen have?

Answer 27

1 each

Question 28

Describe how filamen organises actin into networks.

Answer 28

Filamen forms a dimer with the actin binding domains at the points of a V-type arrangement, so that when the ABD binds actin, they are organised in networks.

Question 29

Filamen, Fimbrin and alpha-actinin are specialised actin filamen cross linkers and are only expressed in certain cell types. True or False?

Answer 29

False. They are all widely expressed in eukaryotic cells.

Question 30

What is the role of spectrin, a specialised actin filament cross linker protein, in erythrocytes/RBCs?

Answer 30

Spectrin forms the meshwork of erythrocytes giving them their biconcave shape. Mutations underlie severe anaemia.

Question 31

What are Myosins?

Answer 31

The most characterised motor proteins - a multi-gene family with actin binding domains.

Question 32

What is Dystrophin and it’s function?

Answer 32

It is an actin filament binding protein with a single ABD.
It is part of a multi-protein complex connecting muscle fibre cytoskeleton to extracellular matrix.
Role in force generation.
Mutations underlie muscular dystrophies.

Question 33

Describe the structure and function of Myosin I

Answer 33

1 heavy chain monomer:
1 head, neck with calmodulin light chains, relatively short tail.

Links the cytoskeleton to membranes, e.g. filopodia and microvilli.

Allows force generation of filopodia to move the cells cytoskeleton in tread-milling.

Question 34

Describe the structure and function of Myosin II

Answer 34

2 heavy chained dimer:
2 heads, neck with essential light chain and regulatory light chain, tail ~130nm.
Used for contractile force, e.g. striated muscle contraction, cytokinesis - tightening contractile ring between daughter cells.

Question 35

Describe the structure and function of Myosin V

Answer 35

2 heavy chained dimer:
2 heads with calmodulin light chains.
Used for cytoskeletal-membrane interactions, e.g. vesicle transport.
Carried transport vesicles through constant association and dissociation with actin filaments and the head regions.

Question 36

What are the key similarities of Myosins?

Answer 36

Head region is the ABD - ATPase activity.
Structure of head/neck region is conserved pocket where ATP binds.
ATP binding site close to ABD.
Light neck chains channels power of ATP hydrolysis to cause conformational change in protein.

Question 37

Which Myosins are the most abundant?

Answer 37

Myosin I and II

Question 38

Described the higher order organisation of Myosin II in the context of muscle fibres.

Answer 38

Individual MII dimers in muscle fibres assembled into thick filaments with tails packed tightly together and the heads - the ABDs - protruding.

Question 39

What are sarcomeres?

Answer 39

Functional unit of striated muscle, comprised of thick myosin II filaments and thin actin filaments, organised into myofibrils which are bundled into myofibres and in turn into muscle tissue.

Question 40

Muscle contraction occurs when…

Answer 40

The thin and thick filaments in sarcomeres slide past each other resulting in a reduction of sarcomere length.

Question 41

Outline the control of the sliding of sarcomere filaments in muscle contraction.

Answer 41

Controlled by interaction of the myosin heads, protruding out the thick filament, with actin in the thin filament.

• Myosin heads bind ATP changing conformational change
• Myosin no longer binds actin filaments
• Ca2+ binds to troponin C on tropomyosin revealing binding site on actin for myosin.
• ATP hydrolysis causes conformational change in neck region
• Myosin head pivots binding new actin subunit
• Pi is released, head pivots back to original position, filament moves in power stroke.

Question 42

What is the name of the structure that anchors actin filaments at either end of the sarcomere?

Answer 42

Z disks