Exosome‐associated polysialic acid modulates membrane potentials, membrane thermotropic properties, and raft‐dependent interactions between vesicles

Sapoń

2020

IJMS

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Intraluminal vesicles (ILVs) are released into the extracellular space as exosomes after the fusion of multivesicular bodies (MVBs) with the plasma membrane. miRNAs are delivered to the raft-like region of MVB by RNA-binding proteins (RBPs). RNA loading into exosomes can be either through direct interaction between RNA and the raft-like region of the MVB membrane, or through interaction between an RBP–RNA complex with this raft-like region. Selection of RNA aptamers that bind to lipid raft region of liposomal membranes was performed using the selection-amplification (SELEX) method. The pool of RNA aptamers was isolated, and the binding of this pool to lipid-raft regions was demonstrated. Sequencing of clones from rafted liposome-eluted RNAs showed sequences apparently of independent origin. Bioinformatics analysis revealed the most frequent raft-motifs present within these sequences. Four raft RNA motifs, one of them an EXO motif, have been identified. These motifs appear to be most frequent both in the case of raft RNA aptamers and in the case of exosomal pro-tumoral miRNAs transferred from cancer cells to macrophages, natural killer cells and dendritic cells, thus suggesting that the selection for incorporation of these miRNAs into ILVs is based on their affinity to the raft-like region of the MVB membrane.

Section: Introductionmentioning

confidence: 99%

“…Surface glycoconjugates, including polysialic acid, play important roles in the interaction of exosomes with cells and between exosomes [4,5]. Interestingly, in calf bovine serum, polysialylated exosomes originate mostly from immune cells [4].…”

Section: Introductionmentioning

confidence: 99%

Binding of RNA Aptamers to Membrane Lipid Rafts: Implications for Exosomal miRNAs Transfer from Cancer to Immune Cells

Sapoń

2020

IJMS

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“…Differential ultracentrifugation, size-exclusion chromatography, and ultrafiltration have been used in consecutive three steps for exosome purification from FBS, as described [ 12 ]. Differential ultracentrifugation: 20 min centrifugation of FBS (total volume 30 mL) at 2000× g , then 18 h centrifugation of the supernatant at 126,000× g (Type 45 Ti rotor, Beckman Coulter, Pasadena, CA, USA), washing the pellet with PBS and resuspending with PBS, re-pelleting it by 70 min ultracentrifugation at 126,000× g , finally resuspending in 200 μL PBS.…”

Section: Methodsmentioning

confidence: 99%

“…Ultrafiltration: 100-nm pore size ultrafiltration (final exosome purification) using Millipore Ultrafree-MC Centrifugal Filter Device (centrifugation at 12,000× g, 1 min). The size distribution and homogeneity of exosomes have been determined by using Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, Worcestershire, UK), the diameter distribution was 50-110 nm [12]. Exosomes from neuroblastoma IMR-32 cell culture supernatants were purified as described [12].…”

Section: Isolation Of Exosomesmentioning

confidence: 99%

Cholera Toxin Subunit B for Sensitive and Rapid Determination of Exosomes by Gel Filtration

et al. 2020

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We developed a sensitive fluorescence-based assay for determination of exosome concentration. In our assay, Cholera toxin subunit B (CTB) conjugated to a fluorescence probe and a gel filtration technique (size-exclusion chromatography) are used. Exosomal membranes are particularly enriched in raft-forming lipids (cholesterol, sphingolipids, and saturated phospholipids) and in GM1 ganglioside. CTB binds specifically and with high affinity to exosomal GM1 ganglioside residing in rafts only, and it has long been the probe of choice for membrane rafts. The CTB-gel filtration assay allows for detection of as little as 3 × 108 isolated exosomes/mL in a standard fluorometer, which has a sensitivity comparable to other methods using advanced instrumentation. The linear quantitation range for CTB-gel filtration assay extends over one order of magnitude in exosome concentration. Using 80 nM fluorescence-labeled CTB, we quantitated 3 × 108 to 6 × 109 exosomes/mL. The assay ranges exhibited linear fluorescence increases versus exosome concentration (r2 = 0.987). The assay was verified for exosomal liposomes. The assay is easy to use, rapid, and does not require any expensive or sophisticated instrumentation.

“…Exosomes were isolated from calf bovine serum (CBS) as described [85,86]. Briefly, CBS was subjected first to 20-min centrifugation at 2000× g, and the supernatant was subjected to 18 h centrifugation at 126,000× g. The obtained pellet was washed with PBS and dissolved by gentle agitation, before re-pelleting it with ultracentrifugation at 126,000× g for 70 min, and next re-suspending in 120 µL PBS.…”

Section: Isolation and Labelling Of Exosomesmentioning

confidence: 99%

Role of RNA Motifs in RNA Interaction with Membrane Lipid Rafts: Implications for Therapeutic Applications of Exosomal RNAs

Mańka

Sapoń

et al. 2021

IJMS

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RNA motifs may promote interactions with exosomes (EXO-motifs) and lipid rafts (RAFT-motifs) that are enriched in exosomal membranes. These interactions can promote selective RNA loading into exosomes. We quantified the affinity between RNA aptamers containing various EXO- and RAFT-motifs and membrane lipid rafts in a liposome model of exosomes by determining the dissociation constants. Analysis of the secondary structure of RNA molecules provided data about the possible location of EXO- and RAFT-motifs within the RNA structure. The affinity of RNAs containing RAFT-motifs (UUGU, UCCC, CUCC, CCCU) and some EXO-motifs (CCCU, UCCU) to rafted liposomes is higher in comparison to aptamers without these motifs, suggesting direct RNA-exosome interaction. We have confirmed these results through the determination of the dissociation constant values of exosome-RNA aptamer complexes. RNAs containing EXO-motifs GGAG or UGAG have substantially lower affinity to lipid rafts, suggesting indirect RNA-exosome interaction via RNA binding proteins. Bioinformatics analysis revealed RNA aptamers containing both raft- and miRNA-binding motifs and involvement of raft-binding motifs UCCCU and CUCCC. A strategy is proposed for using functional RNA aptamers (fRNAa) containing both RAFT-motif and a therapeutic motif (e.g., miRNA inhibitor) to selectively introduce RNAs into exosomes for fRNAa delivery to target cells for personalized therapy.