Actin Filaments (Microfilaments)

What experiment reveals Actin Filament Polarity?

Decoration with myosin S1 Fragments showing a pointed (-) end and barbed (+) end

True or False: Do Actin Filaments have structural polarity?

True

Which end of the Actin Filaments grows FASTER?

The barbed (+) end

What is the approximate diameter of Microfilamets?

7-9nm

True or False: Do actin monomers need to associate with ATP or ADP?

True

What does critical concentration (Cc) represent in actin asesmbly?

The concentration of G-actin above which polymerization can take place to form F-actin.

What does reaching critical concentration (Cc) do to the levels of actin monomers?

Before reaching Cc, there will be more free-monomers (G-actin), but once critical concentration is reached the amount of monomers remains constant. 

Is G-actin (Gobular Actin): 

• Monomer of Actin

• Polymer of Actin

Monomer of Actin

Is F-actin (Filament): 

• Monomer of Actin

• Polymer of Actin

Polymer of Actin

On what end does ATP bind to G-actin?

pointed (-) end

What are the 3 steps in Actin Polymerization? 

1. Nucleation (Lag Phase)

2. Elongation (Growth Phase)

3. Steady State (Equilibrium Phase)

What is Nucleation (Lag Phase)? 

First and Rate-Limiting Step in Actin Polymerization, where actin monomers (G-actin) assemble into a cluster of three monomers (trimer) known as the “nucleus”.

What is the Nucleation Protein? 

ARP 2/3

ARP 2/3

Nucleates Actin Filaments from the sides of existing filaments.

What happens during Elongation Phase (growth)?

Rapid addition of actin monomers (G-actin) to both ends of actin filament.

Profilin

Binds to Actin Monomers & delivers them to growing ends of filaments. Since Profilin causes subunits bound to ADP to release ADP, and leads to the subunits binding to ATP. 

What protein promotes the elongation phase through the Polymerization of actin monomers? 

Profilin

What happens during Equilibrium Phase (steady-state)?

The Actin Filament is no longer growing or shrinking, since there is a balance of polymerization at (+) end and depolymerization at (-) end.

Treadmilling

Actin monomers, which are bound to ATP, add to the more rapidly growing plus end of the filament. As the monomers are incorporated, the ATP is gradually hydrolyzed to ADP. The ADP-bound actin monomers dissociate from the pointed, or minus, end of the filament. Thus, the length of the actin filament is constant. 

What is a “CAP” protein?

A protein that binds to the end of an actin filament to regulate its growth by blocking the addition or removal of actin subunits.

The binding of what protein to a specific end of the filament allows us to determine the Cc for the opposite end? 

CAP Protein

What Toxins alter the Actin monomer?

1. Phalloidin

2. Cytochalasin

3. Latrunculin

What toxin binds alongside actin filaments & stabilizes them? 

Phalloidin

What does cytochalasin do?

Binds to the (+) end of actin filament and prevents addition of subunits.

Thymosin

Binds to actin-ATP monomers & prevents them from polymerizing (monomer-sequestering)

CapZ

Regulates the length of actin filaments through capping the (+) end of actin filaments. Which prevents the addition and loss of actin monomers and consequently stops filament elongation and depolymerization at that end. 

Cofilin

Protein that binds to actin-ADP subunits to disassemble the filaments & regulate actin’s dynamic structure. (Actin-filament depolymerization)

Gelsolin

Protein that binds to and severs actin filaments and decrease cytoplasmic viscosity (filament-severing)

• Ca++(activated by calcium)

• PIP₂ (inactive, it is sequestered by PIP₂, when it is hydrolyzed gelsolin released)

What motor protein walks toward microtubule plus ends (Anterograde)?

Kinesin

What motor protein walks toward microtubule minus ends (Retrograde)?

Dynein

What is Myosin?

The actin motor protein

Is myosin a (+) end or (-) end directed motor?

(+) end directed motors

What part of the myosin has actin and ATP binding sites? 

Myosin Heads 

Myosin II 

Interacts with actin filaments to generate contractile forces, pulling on the filaments to cause them to slide past each other.

Myosin 5

Walks "hand-over-hand" along actin filaments to transport cargo like organelles within cells (organelle or vesicular transport).