Development of a Simple and Rapid Method for In Situ Vesicle Detection in Cultured Media

Kawano, Kenichi; Yokoyama, Fumiaki; Kawamoto, Jun; Ogawa, Teiichiro; Kurihara, Tatsuo; Futaki, Shiroh

doi:10.1016/j.jmb.2020.09.009

Cited by 7 publications

(3 citation statements)

References 47 publications

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“…We have developed a simple and rapid method for in situ vesicle detection in cell-cultured media without cell removal and EV purification. 14 In this study, to isolate small EVs using a simple and convenient method, we propose a novel methodology: an EV catch-and-release isolation system (EV-CaRiS) using a net- charge invertible curvature-sensing peptide (NIC). Curvaturesensing peptides recognize vesicles by binding to lipid-packing defects on highly curved membranes, regardless of the expression levels of biomarkers.…”

Section: ■ Introductionmentioning

confidence: 99%

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Extracellular-Vesicle Catch-and-Release Isolation System Using a Net-Charge Invertible Curvature-Sensing Peptide

Kawano,

Kuzuma,

Yoshio

et al. 2024

Anal. Chem.

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Extracellular vesicles (EVs) carry various informative components, including signaling proteins, transcriptional regulators, lipids, and nucleic acids. These components are utilized for cell−cell communication between donor and recipient cells. EVs have shown great promise as pharmaceutical-targeting vesicles and have attracted the attention of researchers in the fields of biological and medical science because of their importance as diagnostic and prognostic markers. However, the isolation and purification of EVs from cell-cultured media remain challenging. Ultracentrifugation is the most widely used method, but it requires specialized and expensive equipment. In the present study, we proposed a novel methodology to isolate EVs using a simple and convenient method, i.e., an EV catch-and-release isolation system (EV-CaRiS) using a net-charge invertible curvature-sensing peptide (NIC). Curvature-sensing peptides recognize vesicles by binding to lipid-packing defects on highly curved membranes regardless of the expression levels of biomarkers. NIC was newly designed to reversibly capture and release EVs in a pH-dependent manner. NIC allowed us to achieve reproducible EV isolation from three human cell lines on resin using a batch method and single-particle imaging of EVs containing the ubiquitous exosome markers CD63 and CD81 by total internal reflection fluorescence microscopy (TIRFM). EV-CaRiS was demonstrated as a simple and convenient methodology for EV isolation, and NIC is promising for applications in the single-particle analysis of EVs.

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Section: ■ Introductionmentioning

confidence: 99%

“…nFAAV5 selectively binds to bacterial EVs with high sensitivity, even in the presence of EV-secretory cells in cultured media. We have developed a simple and rapid method for in situ vesicle detection in cell-cultured media without cell removal and EV purification …”

Section: Introductionmentioning

confidence: 99%

Extracellular-Vesicle Catch-and-Release Isolation System Using a Net-Charge Invertible Curvature-Sensing Peptide

Kawano,

Kuzuma,

Yoshio

et al. 2024

Anal. Chem.

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“…This strain, Shewanella vesiculosa HM13, is a Gram-negative and cold-adapted bacterium that was isolated from horse mackerel intestines (Chen et al, 2020). Vesiculation by this bacterium has two unique features: higher vesicle production than other strains such as Escherichia coli and high purity and productivity of a single specific protein in EMVs; these characteristics make S. vesiculosa HM13 useful for elucidating the mechanisms of bacterial vesiculation and protein transport to EMVs (Chen et al, 2020; Guida et al, 2020; Kamasaka et al, 2020; Kawano et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

Identification of a Putative Sensor Protein Involved in Regulation of Vesicle Production by a Hypervesiculating Bacterium,Shewanella vesiculosaHM13

Yokoyama

Imai

Aoki

et al. 2020

Preprint

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Bacteria secrete and utilize nanoparticles, called extracellular membrane vesicles (EMVs), for survival in their growing environments. Therefore, the amount and components of EMVs should be tuned in response to the environment. However, how bacteria regulate vesiculation in response to the extracellular environment remains largely unknown. In this study, we identified a putative sensor protein, HM1275, involved in the induction of vesicle production in a hypervesiculating Gram-negative bacterium, Shewanella vesiculosa HM13. This protein was predicted to possess typical sensing and signaling domains of sensor proteins, such as methyl-accepting chemotaxis proteins. Comparison of vesicle production between the hm1275-disrupted mutant and the parent strain revealed that HM1275 is involved in lysine-induced hypervesiculation. Moreover, HM1275 has sequence similarity to a biofilm dispersion protein, BdlA, of Pseudomonas aeruginosa PAO1, and hm1275 disruption increased the amount of biofilm. Thus, this study showed that the induction of vesicle production and suppression of biofilm formation in response to lysine concentration are under the control of the same putative sensor protein.

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Structural factors governing binding of curvature-sensing peptides to bacterial extracellular vesicles covered with hydrophilic polysaccharide chains

Kawano

Kamasaka

Yokoyama

et al. 2023

Biophysical Chemistry

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Development of a Simple and Rapid Method for In Situ Vesicle Detection in Cultured Media

Cited by 7 publications

References 47 publications

Extracellular-Vesicle Catch-and-Release Isolation System Using a Net-Charge Invertible Curvature-Sensing Peptide

Extracellular-Vesicle Catch-and-Release Isolation System Using a Net-Charge Invertible Curvature-Sensing Peptide

Identification of a Putative Sensor Protein Involved in Regulation of Vesicle Production by a Hypervesiculating Bacterium,Shewanella vesiculosaHM13

Structural factors governing binding of curvature-sensing peptides to bacterial extracellular vesicles covered with hydrophilic polysaccharide chains

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