Section 9: Actin and microfilaments

Question 1

Describe the structure of actin

Answer 1

• 3 vertebrate isoforms:
  
  • α (muscle) β (cortex) γ (stress fibres)
• Forms G-actin (globular monomer) which polymerises into F-actin (filamentous) microfilaments
  
  • G-actin resembles a 4 leaf clover with 4 domains and an ATP binding cleft
  • ATP cleft gives asymmetrical polarity to the microfilament (+ and - end)

Question 2

Describe the polarity of actin

Answer 2

• The (+) end is the barbed end
• The (-) end is the pointed end
  
  • The ‘arrowheads’ point to the (-) end
  • The arrowheads are S1 myosin (stabilises)

Question 3

How does actin polymerisation work?

Answer 3

• Polymerisation of actin filaments occurs preferably at the (+) end and requires G-actin to be in ATP form
  
  • The monomers also create a Cc like microtubules
  • Also depends on the presence of a nucleus

Question 4

Describe the critical concentrations of actin

Answer 4

• Uniquely, (-) and (+) ends have different critical concentrations
  
  • Cc- = 0.6 μM
  • Cc+ = 0.12 μM
• In between the Cc (Ex., Cc = 0.14), “treadmilling” toward the (-) occurs
  
  • Treadmilling: depolymerisation occurs at the (-), polymerisation occurs at the (+) end

Question 5

The cellular concentration of G-actin is 400 μM! Why isn’t actin being polymerised all the time?

Answer 5

• Because it is regulated by 3 proteins:

1. Thymosin: sequesters (isolates) actin and provides a reservoir
   
   • Binds to G-actin and inactivates it
2. Profilin: promotes actin polymerisation by charging G-ADP into G-ATP actin
   
   • Requires thymosin presence
3. Cofilin: enhances depolymerisation

Question 6

What are the 2 capping proteins for actin?

Answer 6

CapZ: (+) end cap

Tropomodulin: (-) end cap

Question 7

What do these 2 drugs do?

1. Cytochalasin
2. Phalloidin

Answer 7

• Cytochalasin: depolymerises actin filaments
• Phalloidin: stabilises actin filaments (will not shrink or grow)
  
  • Rhodamine-labelled phalloidin stains the actin red for fluorescent imaging

Question 8

How does formin locally regulate actin?

Answer 8

• Formin: a nucleating protein, regulates assembly of unbranched filaments
  
  • Increases the speed of their construction
  • Regulated by Rho-GTPase, which turns it on

Question 9

How does Arp2/3 locally regulate actin?

Answer 9

• Arp2/3: mediates branching off of existing actin filaments
  
  • Regulated by the nucleation promoting factors (NPFs) WASp (activated by Cdc42) and WAVE (activated by Rac)
    
    • They position Arp2/3 on the microfilament, creating the branch

Question 10

How does Listeria move?

Answer 10

• Listeria utilises ActA (an NPF) that activates Arp2/3 causing rapid branching of actin filaments
• These filaments create the shooting star propulsion as actin polymerises at their end

Question 11

How does Arp2/3 function during endocytosis and phagocytosis?

Answer 11

Endocytosis:

• Arp2/3 pulls on the membrane and allows particle entry as it polymerises
  
  • Once the vesicle is formed and brought in, a microtubule is encountered

Phagocytosis:

• Arp2/3 pushes the membrane around the bacterium/pathogen

Question 12

State the function of the 5 actin binding proteins:

1. Fimbrin
2. α-actinin
3. Spectrin
4. Filamin
5. Dystrophin (& ankryin & ezrin)

Answer 12

1. Fimbrin: bundle actin in microvilli
   
   • Microvilli: actin protrusions in intestines (epithelial) that increase surface area
2. α-actinin: bundle actin
3. Spectrin: cross-link actin networks
4. Filamin: cross-link actin networks
5. Dystrophin, ankryin, ezrin: support and link actin to the plasma membrane
   
   • Hold the plasma membrane in place

Question 13

What happens in muscular dystrophy?

Answer 13

• Dystrophin key in muscle function, ezrin and ankryin key in red blood cells
• Muscular dystrophy: dystrophin is ineffective; muscle cannot move despite contraction

Question 14

What is myosin?

Answer 14

• Actin’s motor protein (myosin II is the most abundant, found in muscle)
• Has heavy and light chains
  
  • Head is an ATPase
  • Neck attaches the heavy chain to the light chain
  • Tail bonds the cargo
• 3 main types

Question 15

Describe the 3 classes of myosin

Answer 15

Question 16

What is the ‘myosin thick filament’?

Answer 16

• Myosin II forms a myosin thick filament; many heavy chains bound together in a bipolar structure (like kinesin-5)
  
  • Causes actin sliding

Question 17

Describe the sliding filament assay

Answer 17

• Used to detect myosin-powered movement
  
  1. The S1 head of myosin is bound to glass cover slides
  2. Fluorescently labelled actin is added
  3. ATP is added which causes actin movement (since the myosin is tethered)
• The longer the light chain, the faster the myosin (actin in this experiment) will move
  
  • “Regulatory light chains”

Question 18

Explain how myosin II moves along actin in the muscle

Answer 18

• Starts in a “rigor state” where actin is bound to myosin and no ATP and ADP is present
  
  • But really there’s no start and end since this process is cyclic

1. ATP binding causes myosin to release actin
   
   • The ATP is hydrolysed
2. ATP hydrolysis causes myosin to change shape and bind to actin again, closer to the (+) end
   
   • The myosin head is the only part that is released and moved
3. Pi is released causing a “power stroke”; the relative movement of the actin microfilament
4. ADP release returns the state to rigor

Question 19

What is rigor mortis?

Answer 19

Rigor mortis: myosin never releases actin as there is no ATP present

Question 20

What is the sarcomere?

State the 3 components of it

Answer 20

The sarcomere is a repeating unit found in muscles

• A band: the location of myosin II thick filaments; does not change in size
• Z disk: these come close together during contraction
• I band: the area lacking in myosin; decreases in size during contraction

Contraction occurs as the myosin moves toward the (+) end, pulling the Z disks together

Question 21

Describe the 4 sarcomere proteins

Answer 21

1. CapZ: caps the (+) end of the actin
2. Tropomodulin: caps the (-) end of the actin
   
   • This and CapZ ensures the actin microfilaments are stable
3. Titin: keeps the thick filaments in place in the middle of the sarcomere
4. Nebulin: further coating and stabilising

Question 22

What regulates muscle contraction?

Answer 22

• Muscle contraction is regulated by nerve impulses (Ca²⁺)
• The sarcoplasmic reticulum stores and regulates the level of free Ca2+
  
  • The sarcoplasmic reticulum is found everywhere inside the muscle
• They are near transverse tubules: projections from the plasma membrane that lead into the muscle cell

Question 23

How is Ca²⁺ released from the sarcoplasmic reticulum?

Answer 23

1. The nerve impulse travels down the T tubule and reaches the sarcoplasmic reticulum
2. The impulse causes the calcium release channel on the surface of the SR
3. The calcium release causes the movement of myosin towards the (+) end
4. As soon as the calcium is released, it is immediately pumped back into the ER using an ATP pump
   
   • Important because Ca2+ is dangerous inside the cytoplasm; it forms the precipitate calcium-phosphate

Question 24

What are the 2 key proteins that calcium binds to?

Answer 24

1. Tropomyosin: binds actin in the same place myosin binds in the absence of calcium
   
   • Myosin cannot bind to actin in the presence of tropomyosin
2. Troponin: troponin changes shape upon calcium binding and moves tropomyosin
   
   • When tropomyosin moves, myosin can now bind