Lecture 3

Summary of Actin Dynamics

• Actin filaments undergo dynamic assembly and disassembly, which is crucial for various cellular processes

Actin at the Leading Edge of a Motile Cell

• Actin filaments are highly dynamic at the leading edge of motile cells

• Electron micrograph shows actin filament branch formation at the leading edge

The Arp2/3 Complex

• Found in all eukaryotes, composed of seven subunits, two of which are related to actin

• Its activity leads to branching of actin filaments, as seen in structures like the leading edge of motile cells

• Electron micrograph shows the branch made by the Arp2/3 complex in an actin filament

Activation of the Arp2/3 Complex

• Arp2/3 activity is enhanced by accessory proteins called Nucleation Promoting Factors (NPFs)

Mechanism of Filament Branching

1. Arp2/3 complex binds to the side of an existing actin filament

2. An activating protein such as WASp binds and induces a conformational change, greatly increasing the complex's ability to bind two actin monomers, forming a (+) end equivalent

3. The new stable seed allows filament formation to follow

Regulation of the Arp2/3 Complex Activating Protein WASp

• WASp exists in an inactive state, with the activation Rho Binding Domain (RBD) hidden

• WASp also has a G-actin binding domain that facilitates actin filament nucleation

• Binding of the signaling molecule Cdc42 to the RBD induces a conformational change that releases the C-terminal acidic activation domain

• Activated WASp:

  • The W domain binds and transfers a G-actin to an activated Arp2/3 complex

  • The acidic A domain and C (central) domain activate the Arp2/3 complex to bind the side of an existing actin filament and initiate a new branch

Actin Branching by Arp2/3 Complex Summary

• Actin branching is used during endocytosis, as endocytosis assembly factors recruit NPFs that activate Arp2/3 complexes

Phagocytosis and Actin Dynamics

1. Opsonization: Bacterium is coated by specific antibodies

2. Leukocyte surface Fc receptor binds the Fc region of the bacterium-bound antibodies

3. Fc receptor-antibody binding signals the cell to activate Arp2/3 complexes, which assemble an actin filament network that moves the cell membrane around the opsonized bacterium

4. Fusion of the membrane projections pinches off a phagosome into the cytoplasm

5. Fusion of lysosomes with the phagosome delivers enzymes that degrade the bacterium

Arp2/3 Mediated Actin Polymerization and Listeria Motility

• The intracellular bacterial parasite Listeria monocytogenes harnesses actin polymerization machinery to propel it through an infected cell (see Listeria movie)

• Listeria actin comet tails can be reconstituted in vitro using just four components: ATP-G-Actin, Arp2/3 complex, CapZ, and Cofilin

Listeria Comet Tail Formation

• The Listeria protein ActA is an Arp2/3 complex activating protein, leading to actin nucleation of branched filaments

• Filaments grow at their (+) ends until capped by CapZ

• Actin is recycled through the action of cofilin, which enhances depolymerization at the (-) end, confining polymerization to the back of the bacterium and propelling it forward

Formins

• Found in all eukaryotes, a diverse family with seven different classes in vertebrates

• They assemble unbranched actin filaments

• All family members have two adjacent domains called Formin Homology Domains, FH1 and FH2, which form the basis of the actin nucleating unit

Regulation of Formins

• Formins contain a Rho Binding Domain (RBD) and two Formin Homology domains (FH1 and FH2)

• The FH1 domain is rich in the amino acid proline, allowing it to bind to profilin and increase the local concentration of ATP-G-actin

• The FH2 domain can then nucleate actin assembly in this favorable ATP-actin rich environment

Mechanism of Formin Nucleation of Actin

• The formin dimer binds two actin filaments

• By rocking back and forth, the dimer can allow the addition of actin monomers to the (+) end of the filament

• Formins protect the (+) end from capping protein activity, promoting filament growth

Organization of Actin-Based Cellular Structures

• Actin filaments in cells are organized into various structures, such as highly organized bundles in microvilli or the meshwork characteristic of the leading edge

• This organization is made possible by the function of actin cross-linking proteins, which have two actin binding sites

Types of Actin Cross-Linkers

• Fimbrin: Arranges filaments with the same polarity in tight bundles

• α-actinin: Holds actin filaments in rigid rods that are more spaced out than those generated by fimbrin

• Spectrin: Exists as a tetramer with two actin binding sites, spanning a greater distance between actin filaments and forming networks under the plasma membrane

• Filamin: Has a highly flexible cross-linker between the actin binding sites, enabling it to make stabilizing cross-links between filaments in a network, as found in the leading edge of motile cells

Summary of Higher Actin Structure Formation

• The Arp2/3 complex and formins work together to organize actin filaments into the diverse structures found in cells, enabling crucial cellular processes like motility, endocytosis, and phagocytosis.