Applications of cell resealing to reconstitute microRNA loading to extracellular vesicles

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.

Home and Away: The Role of Non-Coding RNA in Intracellular and Intercellular DNA Damage Response

“…Additionally, it suggests a mechanism for the active sorting of miRNAs into exosomes upon damage induction [110]. This is supported by findings that the RNA-binding protein YBX1 is involved in the loading of miRNA, such as miR-122, into exosomes [114], as well as in the specific packaging of many other abundant small ncRNA species, including tRNA, Y RNA, and Vault RNA [115]. Although this was not investigated under damage conditions, it is still relevant to the study of RIBE, as it implies active cellular control over the RNA composition of exosomes, which is likely to undergo changes upon alterations to cellular conditions.…”

Targeted protein degradation by Trim-Away using cell resealing coupled with microscopic image-based quantitative analysis

“…In our previous studies, we attempted to apply semi-intact cells or a cell resealing technique to deliver antibodies and inhibit their intracellular targets ( Kano et al, 2000 ; Sonoda et al, 2021 ). However, the standard protocols used in these studies were not thoroughly optimized for antibody delivery, and the actual efficacy or retention of the delivered antibodies has not yet been explored in detail.…”

“…For example, fluorescent-labeled antibodies can be used to visualize endogenous target proteins in live cells ( Teng et al, 2016 ). Additionally, antibodies can be used to inhibit the functions of proteins by binding to their targets ( Sonoda et al, 2021 ). Clift et al (2017) , Clift et al (2018) reported another approach where the intrinsic function of TRIM21, a ubiquitin ligase that binds to the Fc domain of antibodies, was used to target protein degradation via the proteasome.…”

“…To date, cell resealing has been used for the intracellular delivery of various membrane-impermeable molecules, such as chemical compounds, nucleotides, peptides, and soluble proteins ( Murakami et al, 2018 ; Yamaoki et al, 2018 ; Sonoda et al, 2021 ). The technique involves first treating the cell membrane with a cholesterol-dependent pore-forming toxin, such as treatment with streptolysin O (SLO) at 4°C.…”

Endothelial Extracellular Vesicles Enriched in microRNA-34a Predict New-Onset Diabetes in Coronavirus Disease 2019 (COVID-19) Patients: Novel Insights for Long COVID Metabolic Sequelae

“…Quantifying the expression levels of microRNAs (miRNAs) shuttled by extracellular vesicles (EVs) has been shown to be extremely valuable in clinical practice (Cha et al, 2021;Esmaeili et al, 2021;Gu et al, 2021;Liao et al, 2021;Papiewska-Pajak et al, 2021;Silvestro et al, 2021;Sonoda et al, 2021;Wang et al, 2021;Yang and Rhee, 2021;Chen et al, 2022;Fullerton et al, 2022;Pavani et al, 2022). We have recently demonstrated that endothelial EVs (EC-EVs) enriched in miR-24 independently predict cerebrovascular events in COVID-19 .…”