Spatial and temporal relationships between actin-filament nucleation, capping, and disassembly

Area of Science:

• Cell biology
• Cytoskeleton dynamics
• Molecular motors

Background:

• The leading actin network in motile cells consists of the lamellipod and lamellum.
• Lamellipod construction involves a conserved protein biochemical cycle.
• The precise timing and roles of these proteins in actin network architecture are unclear.

Purpose of the Study:

• To investigate the roles of key actin-binding proteins in lamellipod formation and actin network architecture.
• To understand the spatiotemporal dynamics of the Arp2/3 complex and capping protein during cell spreading.
• To determine the contribution of actin filament-stabilizing proteins to lamellipod and lamellum balance.

Main Methods:

• Fluorescent speckle microscopy (FSM) was used on spreading Drosophila S2 cells.
• Labeled actin, Arp2/3 complex, capping protein, and tropomyosin were utilized.
• RNA interference (RNAi) was employed to deplete specific proteins like capping protein and cofilin.

Main Results:

• Capping protein and Arp2/3 complex incorporate at the cell edge; capping protein dissociates earlier than Arp2/3 complex.
• The Arp2/3 complex debranches well before the lamellipodial network disassembles.
• Depletion of capping protein disrupts Arp2/3 complex localization and causes lamellipod disappearance.
• Depletion of cofilin, twinfilin, and tropomyosin expands the lamellipod at the expense of the lamellum.

Conclusions:

• The Arp2/3 complex is essential for lamellipodial network assembly but dissociates before network disassembly.
• Capping protein is required for lamellipod formation and its dissociation coincides with initial filament disassembly.
• Cofilin, twinfilin, and tropomyosin regulate lamellipod size rather than assembly, limiting its expansion.