Actin Filament Dynamics and Rho-GTPases

Actin Microfilaments

• Actin is very abundant, comprising 20% of skeletal muscle mass and 5% of total proteins in non-muscle tissues.
• Actin plays crucial roles in cell motility, contractility, shape changes, cytokinesis, cell polarity, phagocytosis, and macropinocytosis.
• Actin assembly is crucial for membrane ruffles, lamellipodia formation, and cell movement in non-muscle cells.
• Actin assembly is initiated and regulated by various factors, including the type and activity of actin binding proteins, and cell signaling.
• Actin proteins are highly conserved, with 375 amino acids and a molecular weight of 43 kDa.
• Actin exists in at least six isoforms in mammals, each encoded by separate genes.
• Actin is categorized into three classes:
  • Class 1: Non-muscle β, γ and smooth muscle γ-actin.
  • Class 2: Skeletal, cardiac and vascular α-actin
  • Class 3: Actin-RPV, centractin, lower eukaryote actins.
• Actin isoforms exhibit specific localization within cells.
  • γ-actin is primarily found at the cell periphery.
  • α-actin is concentrated within stress fibers.
  • β-actin is associated with membrane ruffles, a process involved in cell movement.
• Actin filaments are polarized, with distinct plus (+) and minus (-) ends exhibiting different assembly rates and kinetics.
• The assembly of actin filaments requires ATP hydrolysis which regulates the assembly and disassembly process.
• Cell Signaling alters actin assembly through small GTPases, including Rac, Rho, and Cdc42.
• Actin assembly proceeds through three stages:
  • Nucleation: Initial formation of actin filaments.
  • Polymerization: Elongation of actin filaments.
  • Steady state: Equilibrium between actin filament assembly and disassembly.

Actin Structures in Cells

• Transient/labile structures: Lamellipodia, Filopodia, Ruffles
• Stable structures: Microvilli
• Actin-associated junctions: Focal complexes, focal adhesions
• Actin is crucial for cell movement and shape changes.
• The presence of actin associated proteins contributes to a variety of cellular functions.

Stable Actin Filament Structures

• Microvilli are fingerlike projections on cell surfaces, particularly in epithelia like the intestines.
• Microvilli increase surface area for absorption by forming bundles of 20-30 actin filaments that have their barbed ends facing the tip of the microvilli.
• Actin filaments in microvilli are crosslinked by the bundling proteins villin, fimbrin, and espin.

Actin Filaments - Control and Regulation

• Actin filaments are polarized, with distinct plus (+) and minus (-) ends.
• Actin assembly requires energy from nucleotide (ATP-ADP) hydrolysis in cells.
• Actin binding proteins regulate the ratio of monomer to filament.
• Cell signaling through small GTPases (Rac, Rho, and Cdc42) alters actin assembly to modulate cellular processes.

Actin assembly into filaments

• Pure actin nucleates as trimers and grows by monomer addition.
• The resulting filaments are typically found in bundles or networks.
• Filaments have a diameter of 6-8 nm and are composed of polar subunits arranged in a right-handed double-helical structure.

Actin Filament Polarity

• Actin filaments exhibit polarity with different kinetics at plus (+) and minus (-) ends.
• Myosin decoration reveals a characteristic arrowhead pattern: barbed end (+ end) and pointed end (- end)
• Critical concentration for assembly is lower at the barbed end (plus end).

Actin Assembly

• Subunits add and detach from both ends of filaments, but critical concentrations differ, allowing for treadmilling.
• Plus end has a lower critical concentration (0.1 μM), enabling subunit addition with a higher rate compared to the minus end (0.8 μM)

Formation of New Actin Filaments

• New filaments can be formed by three mechanisms: de novo nucleation, uncapping, and severing existing filaments.

Actin binds ATP

• Actin tightly binds ATP and Mg++.
• ATP is then hydrolyzed to ADP after polymerization.
• ATP binding determines polymerization and depolymerization rates.

Structures of monomeric G-actin and F-actin filaments

• G-actin is a globular monomeric protein with two domains.
• F-actin is a filamentous polymer with a double helix arrangement of G-actin subunits.

Formation of New Actin Filaments (cont.)

• Actin assembly occurs spontaneously from ATP-bound G-actin monomers.
• ATP is hydrolyzed to ADP after polymerization, lagging behind.
• ATP cap marks the end of filament.
• ADP-actin marks the de-polymerization state.

Actin Polymerisation

• Actin polymerization involves the addition of monomers to the plus end (+), with a higher rate of assembly compared to the minus end (-).
• The process has three stages: nucleation, elongation and steady state.

Regulation of filament formation by actin-binding proteins

• Several key actin-binding proteins, including profilin, cofilin, and thymosin-β4, regulate actin filament dynamics and thereby affecting cellular processes.
• These proteins manipulate the assembly and disassembly of actin filaments.
• The interaction sites between actin monomers are affected by the various proteins. (This is often detailed in the diagram)

Actin Binding Proteins

• Various actin-binding proteins affect actin assembly and stability
• Several functions are categorized in the various types of binding proteins.

Actin Ends - Summary

• Actin growth predominantly occurs at the plus (+) end.
• Critical concentration for ATP-actin differs between plus and minus ends (0.1 μM vs 0.6 μM respectively.)
• This difference results in treadmilling, maintaining a constant flux of monomers.

Free Ends and Free Subunits

• Free G-actin monomers are largely present in ATP-bound state making them likely to become incorporated into filaments.
• G-actin complexes with actin-binding proteins regulate actin assembly.
• Capping proteins control the addition of G-actin to the ends of existing filaments, which regulates F-actin growth.

Actin-Binding Proteins: Polymer modifying proteins

• Multiple proteins regulate actin filament formation, stability and function.

Actin Binding Proteins: Over 50 known

• Many proteins impact actin assembly, stability, and function.
• These proteins categorized as severing, capping, crosslinking, and bundling, have diverse roles in cell structure and dynamics.

Actin-binding proteins-regulators of assembly & branching

• Profilin binds G-actin (ATP state).
• Arp2/3 proteins initiate branched actin networks.
• Thymosin-β4 sequesters G-actin (preventing assembly).
• Tropomodulin caps the minus end of actin filaments to inhibit disassembly.

Actin binding proteins-regulators of assembly & cross-linkers

• Several proteins control actin assembly, crosslinking, and attachment.
• Cofilin accelerates disassembly and actin filament severing.
• Gelsolin severs filaments.
• α-actinin bundles filaments, and filamin crosslinks them..
• Tropomyosin stabilizes and modulates filament stability.

Actin associations at a cell edge (and adjacent pages)

• Actin forms complex networks and interactions at the leading edge of migrating cells.
• These contribute to cell protrusion, locomotion, and sensing of the environment.
• Various proteins and complexes affect actin dynamics at cellular edges, leading to changes in protrusion rate and shape.

Actin dynamics and cell protrusions

• Cell surface receptors, Rho family GTPases and signaling proteins such as WASP and SCAR, Arp2/3 complex, capping proteins, and profilin/cofilin are all controllers of actin dynamics that impact cell protrusions like filopodia and lamellipodia

Rho-GTPases

• Rho, Rac and Cdc42 are key signaling molecules in controlling actin dynamics.
• Each is responsible for driving the formation of different actin-based structures such as stress fibers or lamellipodia.

Regulation of the state of actin in cells

• Actin constantly remodels, redistributes, and reassembles. Its dynamics are regulated by signaling pathways and external stimuli.
• Changes to actin configuration occur locally and are controlled by small GTPases such as Rac, Rho, and Cdc42.

How the role of Rac/Rho/cdc42 in actin regulation was discovered

• Experiments with serum-starved cells demonstrated the role of these proteins.
• C3 transferase, a chemical, inactivates Rho and is used as a tool to determine Rho's impact on actin function.

Spatial and temporal regulation in response to external stimuli

• Signaling pathways regulate initiation, branching and growth of actin filaments in response to external stimuli like hormones, growth factors, and cell-to-cell signaling.

Nucleation-promoting factors, rapid filament assembly at the plasma membrane

• Nucleation-promoting factors (NPFs) like WASP and Scar drive actin filament assembly near the plasma membrane.
• The Arp2/3 complex is a crucial component in this process by inducing branching and creating complex actin networks.
• Actin monomers, profilin, formins, and nucleating factors function cooperatively at the leading edge of the cell.

Fish "keratocytes" - model systems for studying movement

• Fish keratocytes are used to study cell movement processes.

The Arp2/3 Complex

• Arp2/3 is a complex of seven proteins that nucleates actin filaments "de novo."
• It promotes actin filament branching and is highly concentrated at the leading edge of the cell in response to external stimuli.
• Arp2/3 proteins are controlled by activation signals through factors like WASP or WAVE.

Arp2 and Arp3 share structural homology to actin

• Arp2 and Arp3 share common structural features with actin, contributing to their function in actin filament nucleation.

Regulating actin structures

• ECM, growth factors, hormones regulate actin structures (filopodia, lamellipodia).
• Cdc42 controls the protrusion through regulated actin interactions with proteins.

Steps in cell movement

• Cell movement is a multi-step process involving extension, adhesion, translocation, and de-adhesion.
• Actin-based structures and interactions are essential components in each step, allowing the cell to extend structures, attach and move across a substrate and then detach and recycle.

Contribution of Cdc42, Rac, and Rho to cell movement

• Cdc42 activation promotes actin assembly and treadmilling at the leading edge.
• Rac activation is needed for the formation and regulation of the lamellipodium.
• Rho activation leads to myosin II activation, promoting stress fiber contraction and pulling the cell body forward

Rac - Lamellipodium formation

• Rac activation is needed for the formation and regulation of lamellipodia through Arp2/3 complex activation, capping protein and cofilin activity.
• During Rac-driven lamellipodia formation, other proteins facilitate a series of branching actin networks.

Dendritic Nucleation Model

• Arp2/3 complex nucleates new filaments at the side of pre-existing filaments forming Y-junctions, promoting branching and anchoring for further elongation in the lamellipodium.

Dendritic Actin Network

• Arp2/3 complex is present in Y-junctions and promotes the dendritic branching patterns seen in lamellipodia.

Hijacked by parasites

• Parasites such as Listeria, Shigella, and Helicobacter hijack actin machinery for their invasion and spread inside host cells

Steps in cell movement (cont.)

• Specific actin-based structures, like lamellipodia and filopodia, are formed to assist cell movement.
• This helps with recognition of the substrate and extension of growth processes.