SPLIFF: A Single-Cell Method to Map Protein-Protein Interactions in Time and Space

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Vast amounts of protein-protein interaction data exist without cellular context.
• Tracking protein interactions in real-time within single cells is challenging.
• Need for methods to resolve protein interactions spatially and temporally.

Purpose of the Study:

• To develop a novel method for monitoring protein-protein interactions with spatiotemporal resolution in single cells.
• To utilize a split-ubiquitin system with fluorescent reporters for interaction detection.
• To validate the method in the model organism Saccharomyces cerevisiae.

Main Methods:

• Developed the Split-Ubiquitin-based Interaction Fluorescence assay (SPLIFF).
• Utilized fusion proteins: mCherry-Cub-GFP and a Nub-tagged binding partner.
• Employed two-channel fluorescence time-lapse microscopy in yeast for monitoring interactions post-cell fusion.

Main Results:

• SPLIFF successfully detects protein-protein interactions by monitoring the ratio of green to red fluorescence.
• The method allows for the mapping of spatiotemporal interaction profiles of proteins.
• Demonstrated proof-of-concept in Saccharomyces cerevisiae.

Conclusions:

• SPLIFF provides a robust and simple method for studying protein interactions in a dynamic, single-cell context.
• This technique enhances the understanding of protein function by revealing interaction dynamics.
• Offers a valuable tool for systems biology and molecular interaction studies.