Parallel Proteomic and Transcriptomic Microenvironment Mapping (μMap) of Nuclear Condensates in Living Cells

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Cellular functions are spatially organized within organelles, but many organelles' roles remain unclear.
• Understanding organelle function requires profiling biomolecular composition, which is challenging for small, dynamic structures.
• Photoproximity labeling is valuable for mapping molecular interactions but faces challenges in live-cell applications regarding catalyst localization and toxicity.

Purpose of the Study:

• To develop a novel intracellular photocatalyst with reduced cytotoxicity and off-target binding for live-cell proximity labeling.
• To establish a HaloTag-based microenvironment-mapping (μMap) technique for spatially cataloging subnuclear condensates in living cells.
• To create a specialized RNA-focused workflow (μMap-seq) for parallel transcriptomic and proteomic profiling of cellular structures.

Main Methods:

• Development of a novel, low-cytotoxicity intracellular photocatalyst.
• Application of HaloTag-based microenvironment-mapping (μMap) for spatial cataloging of subnuclear condensates.
• Implementation of μMap-seq for parallel RNA and protein profiling of specific cellular structures.

Main Results:

• A new photocatalyst was successfully utilized for live-cell proximity labeling with minimal cytotoxicity.
• The μMap technique enabled spatial cataloging of subnuclear condensates, including the nucleolus, nuclear lamina, Cajal bodies, paraspeckles, and PML bodies.
• The μMap-seq workflow demonstrated accurate parallel transcriptomic and proteomic profiling of these structures.

Conclusions:

• The developed photocatalyst and μMap platform significantly advance live-cell proximity labeling capabilities.
• The study provides a spatial map of subnuclear condensates, offering potential new insights into RNA metabolism and gene regulation.
• This work expands the utility of the μMap platform for comprehensive live-cell biological system analysis.