Structure of the Lifeact-F-actin complex

Area of Science:

• Cell Biology
• Structural Biology
• Biochemistry

Background:

• Lifeact is a widely used peptide for visualizing filamentous actin (F-actin) in live cells.
• Lifeact is known to cause artefacts by altering cell morphology, but the molecular basis is unclear.
• Understanding Lifeact's interaction with F-actin is crucial for accurate live-cell imaging.

Purpose of the Study:

• To determine the high-resolution structure of the Lifeact-F-actin complex.
• To elucidate the molecular mechanism behind Lifeact's effects on cellular morphology.
• To provide a basis for designing improved actin probes.

Main Methods:

• Electron cryo-microscopy (cryo-EM) to determine the structure of the Lifeact-F-actin complex.
• In vitro binding assays to assess competition with other actin-binding proteins.
• Activity measurements to evaluate the functional consequences of Lifeact binding.

Main Results:

• The cryo-EM structure reveals Lifeact binds to a hydrophobic pocket on F-actin, spanning two actin subunits.
• Lifeact stabilizes the DNase I-binding loop (D-loop) of actin in a closed conformation.
• Lifeact competes with known actin-binding proteins (e.g., cofilin, myosin) and toxins for the same binding site.
• Mutations can increase Lifeact's affinity for F-actin.

Conclusions:

• Lifeact's interaction with F-actin explains its artefactual effects on cell morphology.
• The identified binding site is a target for understanding other actin-binding protein interactions.
• This study provides a foundation for developing more precise actin probes for live-cell imaging.