An optogenetic method for interrogating YAP1 and TAZ nuclear-cytoplasmic shuttling.

Area of Science:

• Cell Biology
• Molecular Biology
• Biophysics

Background:

• Nuclear shuttling of transcription factors is crucial for biological regulation.
• Existing methods for studying nuclear transport dynamics are limited.

Purpose of the Study:

• To develop and validate a novel optogenetic tool for quantifying nuclear entry and exit rates of transcriptional regulators.
• To investigate the dynamics of YAP1 and TAZ, key regulators of gene expression.

Main Methods:

• Utilized a photo-responsive LOV domain from Avena sativa to sequester fluorescently tagged YAP1 and TAZ on mitochondria.
• Employed blue light to reversibly release these proteins and a custom application to quantify fluorescent signals in cellular compartments.
• Integrated the system with lattice light-sheet microscopy for high-resolution dynamic imaging.

Main Results:

• Demonstrated that phosphorylation of YAP1 enhances its nuclear export rate.
• Showed correlated YAP1 import and export rates within individual cells, despite intercellular variability.
• Revealed correlated nuclear transport dynamics between YAP1 and TAZ when released simultaneously.
• Observed significant heterogeneity in YAP1 dynamics across different cytoplasmic regions.

Conclusions:

• The developed optogenetic tool provides a versatile platform for studying protein nuclear transport dynamics.
• Findings offer new insights into the regulation of YAP1 and TAZ activity and their complex interplay.
• The method highlights the heterogeneous nature of protein dynamics within living cells.