Direct Extraction and Evaluation of Intraluminal Vesicles Inside a Single Cell

Area of Science:

• Cell Biology
• Biotechnology
• Microscopy

Background:

• Endogenous extracellular vesicles play crucial roles in physiological functions.
• Current methods for evaluating vesicles within cells are limited.
• A need exists for techniques to directly assess intraluminal vesicle (ILV) contents.

Purpose of the Study:

• To present a novel technique for the direct extraction and evaluation of intraluminal vesicles (ILVs) from living cells.
• To analyze the molecular contents, specifically RNA expression profiles, of ILVs.
• To enable direct assessment of miRNA localization within cells.

Main Methods:

• Combined scanning ion conductance microscopy, electrochemical syringes, and confocal microscopy for high-resolution, femtoliter-scale extraction of intracellular structures.
• Directly collected CD63(+) vesicles from HEK293 CD63-pHluorin-RFP cells.
• Compared RNA expression profiles of ILVs with cytosol and ultracentrifuge-isolated extracellular vesicles.

Main Results:

• Successfully extracted and evaluated intraluminal vesicles (ILVs) directly from living cells.
• ILVs exhibited distinct RNA expression profiles compared to cytosol and extracellular vesicles.
• Identified a subset of ILVs containing hsa-miR-145-5p, enabling direct assessment of miRNA accumulation.

Conclusions:

• The developed technique allows for direct extraction and analysis of intraluminal vesicle (ILV) contents within living cells.
• This method reveals unique molecular signatures of ILVs, differing from extracellular vesicles.
• This approach is a powerful new tool for studying ILV composition and function, particularly miRNA localization.