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Using LEXY and LINuS Optogenetics Tools and Automated Image Analysis to Quantify Nucleocytoplasmic Transport Dynamics in Live Cells

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A Fast and Reliable Nuclei Extraction Method for Phytochrome Functional Analysis.

Ilse Villmann¹, Akira Nagatani², Mathias Zeidler³

¹Justus-Liebig-University Giessen, Institute for Plant Physiology, Giessen, Germany.

Methods in Molecular Biology (Clifton, N.J.)

|October 1, 2025

Summary

This summary is machine-generated.

Phytochromes, plant light sensors, move between cytoplasm and nucleus to regulate growth. A new protocol isolates these compartments to identify their interaction partners and functions in each location.

Keywords:

Nuclear Nuclear import Phytochrome Protein extraction cytoplasmic partitioning

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Area of Science:

Plant biology
Molecular signaling

Background:

Phytochromes are crucial plant photoreceptors sensing red/far-red light.
Light-dependent nuclear translocation of phytochromes regulates plant development.
Emerging evidence suggests cytoplasmic roles for phytochromes.

Purpose of the Study:

To develop a protocol for isolating intact nuclei and cytoplasmic fractions from Arabidopsis thaliana seedlings.
To enable identification of phytochrome interaction partners in distinct cellular compartments.
To elucidate the differential roles of phytochromes in the cytoplasm versus the nucleus.

Main Methods:

Shearing of Arabidopsis thaliana seedlings.
Purification of intact nuclei using a Percoll gradient.
Separation of cytoplasmic and nuclear fractions for downstream analysis.

Main Results:

A protocol was established for rapid and straightforward isolation of nuclei and cytoplasm.
This method is applicable to both etiolated and light-exposed seedlings.
The isolated fractions facilitate identification of protein interaction partners.

Conclusions:

The developed protocol allows for compartment-specific analysis of phytochrome signaling.
It provides a foundation for understanding the distinct functions of phytochromes in the cytoplasm and nucleus.
This method aids in discovering novel phytochrome interaction partners and signaling pathways.