Design and Use of AsLOV2-Based Optogenetic Tools for Actin Imaging

Area of Science:

• Cell Biology
• Optogenetics
• Microscopy

Background:

• Lifeact is a commonly used probe for visualizing actin cytoskeleton dynamics.
• High concentrations of Lifeact can lead to concentration-dependent negative side effects.
• There is a need for improved probes to study live-cell actin dynamics with reduced artifacts.

Purpose of the Study:

• To present protocols for using LILAC, a novel optogenetic tool for actin imaging.
• To demonstrate how LILAC, a light-controlled Lifeact variant, overcomes limitations of traditional probes.
• To provide guidance on implementing LILAC for live-cell actin dynamics research.

Main Methods:

• Development of LILAC, a light-controlled probe based on the Avena sativa LOV2 (AsLOV2) domain fused to Lifeact.
• Characterization of LILAC's reduced affinity for the cytoskeleton in the dark.
• Optimization of cell culture, microscopy, and image processing protocols for LILAC usage.

Main Results:

• LILAC exhibits significantly reduced affinity for the cytoskeleton in the dark compared to standard Lifeact.
• This light-dependent modulation minimizes concentration-dependent negative side effects.
• LILAC enables novel image processing strategies for analyzing live-cell actin dynamics.

Conclusions:

• LILAC offers a powerful optogenetic approach for advanced actin imaging in live cells.
• The AsLOV2 domain provides a versatile platform for developing light-controlled biological tools.
• This work paves the way for new imaging and control applications in cell biology.