Applications of cell resealing to reconstitute microRNA loading to extracellular vesicles

Yuki Sonoda,Fumi Kano,Masayuki Murata

doi:10.1038/s41598-021-82452-5

Abstract

MicroRNAs (miRNAs) are cargo carried by extracellular vesicles (EVs) and are associated with cell–cell interactions. The response to the cellular environment, such as disease states, genetic/metabolic changes, or differences in cell type, highly regulates cargo sorting to EVs. However, morphological features during EV formation and secretion involving miRNA loading are unknown. This study developed a new method of EV loading using cell resealing and reconstituted the elementary miRNA-loading processes. Morphology, secretory response, and cellular uptake ability of EVs obtained from intact and resealed HeLa cells were comparable. Exogenously added soluble factors were introduced into multivesicular endosomes (MVEs) and their subsequent secretion to the extracellular region occurred in resealed HeLa cells. In addition, miRNA transport to MVEs and miRNA encapsulation to EVs followed a distinct pathway regulated by RNA-binding proteins, such as Argonaute and Y-box binding protein 1, depending on miRNA types. Our cell-resealing system can analyze disease-specific EVs derived from disease model cells, where pathological cytosol is introduced into cells. Thus, EV formation in resealed cells can be used not only to create a reconstitution system to give mechanistic insight into EV encapsulation but also for applications such as loading various molecules into EVs and identifying disease-specific EV markers.

Highlights

MicroRNAs are cargo carried by extracellular vesicles (EVs) and are associated with cell–cell interactions
These results indicated that at least 60 min of further incubation of resealed HeLa cells after resealing is likely enough to restore the possible effects of the stress-induced by cell resealing on EV formation in resealed HeLa cells
Via morphological and biochemical characterization of EVs derived from resealed cells, the system will be useful for elucidating the regulatory mechanisms underlying EV formation in living cells and introducing various soluble biomaterials, such as proteins, miRNAs, and membrane-impermeable biopharmaceuticals, into EVs

Summary

Introduction

MicroRNAs (miRNAs) are cargo carried by extracellular vesicles (EVs) and are associated with cell–cell interactions. The response to the cellular environment, such as disease states, genetic/metabolic changes, or differences in cell type, highly regulates cargo sorting to EVs. morphological features during EV formation and secretion involving miRNA loading are unknown. MVEs fuse with the plasma membrane, releasing ILVs into the extracellular region These ILVs with a diameter of ~ 100 nm are referred to as “exosomes” in this study. Many studies have reported the molecular mechanisms underlying EV formation, including specific miRNA in/from c ells[10] Various molecules, such as Rab families, the endosomal sorting complexes required for transport (ESCRT) machinery, RNA-binding proteins (RBPs), and the soluble NSF attachment protein (SNAP) receptor (SNARE), are involved in EV formation, sorting, and secretion. The component of the RISC and trans-activation-responsive RNA-binding protein (TARPB) that interacts with Argonaute 2 (Ago2)[11] is involved in miRNA-loaded EV formation. The cells were further incubated in a 5% C O2 incubator for 48 h at 37 °C, and EVs were prepared, as described in (a), and subjected to negative-stained electron microscopy (EM)

Methods

Results

Conclusion