Reassessment of Exosome Composition.

Area of Science:

• Extracellular Vesicle Biology
• Molecular Biology
• Cellular Biology

Background:

• Exosome heterogeneity and contamination with non-vesicular matter complicate understanding of their cargo and function.
• Current methods lack precision in distinguishing exosomes from other extracellular components.

Purpose of the Study:

• To precisely characterize the molecular constituents of exosomes versus non-vesicular extracellular matter.
• To clarify the role of small extracellular vesicles in DNA transport and secretion.
• To propose a refined model for extracellular DNA secretion.

Main Methods:

• High-resolution density gradient fractionation to separate extracellular components.
• Direct immunoaffinity capture for precise molecular analysis.
• Comprehensive analysis of RNA, DNA, and protein content in isolated fractions.

Main Results:

• Differential expression of extracellular RNA, RNA-binding proteins, and cellular proteins between exosomes and non-vesicle compartments.
• Absence of Argonaute 1-4, glycolytic enzymes, and cytoskeletal proteins in exosomes.
• Identification of annexin A1 as a specific marker for plasma membrane-derived microvesicles.
• Demonstration that small extracellular vesicles are not involved in active DNA release.
• Proposal of a novel autophagy- and multivesicular-endosome-dependent, exosome-independent pathway for active extracellular DNA secretion.

Conclusions:

• A reassessment of exosome composition is necessary due to heterogeneity and contamination.
• Annexin A1 serves as a specific marker for microvesicles.
• Small extracellular vesicles are not the primary mechanism for active DNA release.
• A new model for active extracellular DNA secretion via an exosome-independent pathway is proposed.
• This work provides a framework for understanding extracellular vesicle heterogeneity.