Actin Cytoskeleton Dynamics, Rho GTPase Regulation & Cell Motility

Actin Filament Dynamics and Steady-State

• Actin-based “cell contraction” refers to motor-protein (myosin)–actin interactions that shorten cellular regions or move organelles.
• In many cellular regions, actin polymerization and depolymerization reach an equilibrium → no net filament growth.
  • This plateau is called the steady state (analogous to the tubulin treadmilling plateau in microtubules).

Spatial Organization of Actin Networks

• Actin filaments adopt distinct architectures, each suited to a cellular task.
  • Stress fibers: long, contractile bundles—found mostly at the rear of migrating cells; generate tensile force.
  • Lamellipodia (lamellipodia): broad, sheet-like, branched actin networks at the leading edge; drive protrusion.
  • Filopodia (briefly mentioned): thin, spike-like bundles that probe the environment.
• Typical arrangement in a migrating cell:
  • Front edge → Rac/Cdc42 → lamellipodia/filopodia (protrusion).
  • Rear edge → Rho → stress fibers + myosin (contraction).

Major Actin-Binding Proteins (ABPs)

• ABPs either promote or inhibit filament assembly / stability.
• Key ABPs discussed:
  • Profilin: binds G-actin → favors addition at the + end.
  • Formin: +-end processive polymerase; nucleates and elongates unbranched filaments.
  • WASP / N-WASP: receives upstream signals, activates Arp2/3.
  • Arp2/3 complex: nucleates branched filaments at a characteristic 70^{\circ} angle off existing filaments.
  • Cofilin (ADF): severs and depolymerizes ADP-actin regions → generates new barbed ends; over-activation can fully dismantle the network.

Nucleation of Branched Networks (Lamellipodia Mechanism)

1. Extracellular cue → activates WASP.
2. WASP binds and activates Arp2/3.
3. Activated Arp2/3 attaches to the side of an existing (ADP-rich) filament.
4. New daughter filament nucleates → rapid, branched growth → pushes membrane (lamellipodium).

Severing & Recycling (Cofilin Pathway)

• Cofilin preferentially binds ADP-actin in older filament sections.
• Cuts filaments → produces shorter fragments:
  • Creates additional barbed ends for formin-driven regrowth.
  • Excessive cofilin activity → many fragments → complete disassembly.

The Rho-Family GTPases: Master Regulators

• Family name: Rho GTPases; three key members:
  • Rho (RhoA): stress fibers & contraction (rear).
  • Rac: lamellipodia (broad, branched networks).
  • Cdc42: filopodia (thin spikes) + cell polarity.
• Experimental microinjection:
  • RhoA → bright stress fiber staining.
  • Rac → extensive lamellipodial actin.
  • Cdc42 → distinct filopodial arrays.
• Regulation cycle:
  • GEFs (guanine-nucleotide exchange factors) activate (GDP→GTP).
  • GAPs enhance GTP hydrolysis (inactivate).
  • GDIs sequester inactive GTPase in cytosol.

Three Mechanical Steps of Cell Migration

1. Protrusion
   • Lamellipodium & filopodium extend at the front (Rac/Cdc42 driven; Arp2/3 & formin involved).
2. Attachment
   • Nascent adhesions form when integrins bind extracellular-matrix (ECM) ligands.
   • Integrins connect ECM → focal-adhesion complexes → actin cytoskeleton.
   • Integrin signaling also influences differentiation and survival (full topic spans an entire semester; only basic role in motility required here).
3. Contraction / Rear Retraction
   • Stress fibers + non-muscle myosin II contract → pull cell body forward.
   • RhoA signaling coordinates actin assembly, myosin activation, and focal-adhesion maturation at the rear.

Actin–Myosin Interaction Fundamentals

• Motor protein: non-muscle myosin II (NMII).
• Each myosin II head cycles through ATP-dependent states; cycle generates a power stroke that slides actin.

ATPase Cycle (Simplified)

• Experimental questions may ask: “If ATP binding is inhibited, which step fails?” → detachment cannot occur → rigor state persists.

Contractile Array Geometry

• Anti-parallel actin filaments + bipolar NMII filaments → contraction from both sides (heads on opposite ends pull toward their respective + ends).

RhoA Signaling to Stress-Fiber Assembly & Contraction

• RhoA (GTP-bound) activates downstream effectors:
  • ROCK (Rho-associated kinase):
  • Phosphorylates myosin light chain (MLC) → opens NMII into active, filament-forming conformation.
  • Inhibits MLC phosphatase, sustaining phosphorylation.
  • Formin activation: nucleates/elongates linear actin for stress-fiber template.
• Net result: simultaneous generation of long actin bundles and activation of myosin → robust stress-fiber contractility at the cell rear.

Key Take-Home Points

• Steady-state treadmilling keeps filament length constant but allows subunit flux.
• Distinct ABPs sculpt unique actin architectures; Arp2/3 vs. formin determine branch vs. bundle.
• Rho-family GTPases spatially coordinate protrusion (Rac/Cdc42) with contraction (RhoA).
• Cell migration requires protrusion, adhesion, and contraction—each tightly regulated by actin dynamics and integrin signaling.
• ATP-powered NMII–actin interactions generate the mechanical work of rear retraction, governed by RhoA → ROCK → MLC phosphorylation pathway.