Genetically encoded orientation probes for F-actin for fluorescence polarization microscopy

Area of Science:

• Cell Biology
• Biophysics
• Microscopy

Background:

• Fluorescence polarization microscopy visualizes molecular position and orientation, crucial for studying cytoskeletal protein dynamics in vivo.
• Existing F-actin probes (phalloidin conjugates, SiR-actin) have limited applicability in complex living systems like tissues and embryos.
• Imaging F-actin orientation is vital for understanding cellular architecture and dynamics.

Purpose of the Study:

• To develop novel, genetically encoded F-actin orientation probes for live-cell fluorescence polarization microscopy.
• To overcome limitations of existing probes in introducing them to various living specimens.
• To enable advanced imaging of the 3D architecture of the actin cytoskeleton.

Main Methods:

• Engineered genetically encoded probes by rigidly connecting circular permutated green fluorescent protein (GFP) or mEGFP to actin-binding proteins (Lifeact, UtrCH).
• Evaluated probe performance using ensemble and single-particle fluorescence polarization measurements.
• Utilized the instantaneous FluoPolScope for high-resolution polarization analysis.

Main Results:

• Developed and validated a novel genetically encoded F-actin orientation probe suitable for live-cell imaging.
• The new probe demonstrates properties comparable to conventional GFP-UtrCH F-actin probes.
• Successfully demonstrated the potential for imaging F-actin orientation in various living systems.

Conclusions:

• Genetically encoded F-actin probes offer a powerful tool for advanced fluorescence polarization microscopy.
• These probes facilitate the study of 3D actin cytoskeleton architecture in diverse biological contexts.
• Opens new avenues for single-molecule orientation imaging and network analysis in live systems.