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Identification of markers for migrasome detection

ABSTRACT: The migrasome is a newly discovered cellular organelle in migrating cells, first described in 20151,2. Migrasomes have been proposed to mediate cell-cell communications and may exert physiological and pathological effects. In zebrafish embryos, generation of migrasomes has been observed during gastrulation, and migrasomes have been shown to be essential for organ morphogenesis during embryonic development (Personal communication). Although migrasome-like structures have been detected by transmission electron microscopy (TEM) in various organs including eye, lung and intestine, definitive evidence for the presence of migrasomes in vivo in mammals is still lacking. Exosomes are defined as 30–100 nm extracellular vesicles derived from the multivesicular body and are secreted by a variety of cell types. Exosomes have attracted great attention in clinical studies since they were detected in solid tissues, such as cartilage3, and in biological fluids, such as blood4. One of the most commonly used methods for detection of extracellular vesicles (EVs) is a biochemical approach using known marker proteins. This strategy allows researchers to identify EVs in complex biological samples such as body fluids and tissues, and thus provides great flexibility for detection of EVs in vivo. So far, there is no satisfactory marker for biochemical detection of migrasomes. TSPAN4 and integrin are known to be enriched on migrasomes1,5. However, both proteins are also present on exosomes6, making it difficult to distinguish the two structures biochemically based only on detection of these proteins. Imaging is the only approach for confirming the presence of migrasomes, but it is not compatible with most clinical samples. Thus, there is an urgent need to identify migrasome-specific markers.

ORGANISM(S): Rattus Rattus

SUBMITTER: Yang Chen

PROVIDER: PXD026960 | iProX | Mon Jun 28 00:00:00 BST 2021

REPOSITORIES: iProX

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Author Correction: Identification of markers for migrasome detection.

Zhao Xiaoxin X Lei Yaxin Y Zheng Jiajia J Peng Junya J Li Ying Y Yu Li L Chen Yang Y

Cell discovery 20220406 1

PMID: 35387977

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Project description:Exosomes are molecular entities derived from membrane vesicles of endocytic origin secreted by most cell types. These vesicles are implicated in cell-to-cell communication, deliver proteins and mRNA molecules between cells. Recent studies have shown that exosomes are found in body fluids such as saliva, blood, urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluids and breast milk. Exosomes secreted through human saliva contain mRNA may potentially be useful for diagnostic purposes. Although the exact protective mechanism of saliva RNA is a topic of debate, the consensus is that the enrichment of mRNAs in these nano-vesicles in one of the features of the biomarker discoveries. Our aim was to determine if exosomes are present in human saliva and to nano-characterize their transcriptomic content. Exosomes were purified by differential ultracentrifugation, identified by immunoelectron microscopy, flow cytometry and western blot using a CD-63 antibody. Atomic force microscopy studies revealed ultra structural analysis of both size and density of exosomes. Microarray analysis revealed the presence of 590 mRNA core transcripts are relatively stable inside the exosomes, which can be of saliva mRNA biomarkers. Exosomal mRNA stability was determined by detergent lyses with treatment of RNase. Under in vitro conditions fluorescent dye labeled saliva exosomes were able to communicate between human oral keratinocytes studied by using fluorescence microscopy. The RNA from saliva exosomes can transfer their genetic information to human oral keratinocytes and alters gene expression in the new location. Together, these results suggest that saliva is involved in mRNA trafficking via exosomes, and provides a mechanism for cargoing passenger mRNAs. Our findings are consistent with proposal that exosomes can shuttle RNAs between cells and mRNA is protected inside these vesicles may be a possible resource for biomarker discovery. Experiment Overall Design: Human saliva exosomes were purified through differential centrifugation followed by RNA extraction and hybridization on Affymetrix microarrays. We were able to obtain normal human subjects saliva which are pooled and subjected to ultracentrifugation. The protocol was approved by UCLA Institutional review board. 1 ml of saliva exosomes were used to extract RNA followed by two rounds of amplification by Actorus Amp kit. The amplified RNA was biotin labled and hybridized with Affymetrix protocol.

Project description:Exosomes are molecular entities derived from membrane vesicles of endocytic origin secreted by most cell types. These vesicles are implicated in cell-to-cell communication, deliver proteins and mRNA molecules between cells. Recent studies have shown that exosomes are found in body fluids such as saliva, blood, urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluids and breast milk. Exosomes secreted through human saliva contain mRNA may potentially be useful for diagnostic purposes. Although the exact protective mechanism of saliva RNA is a topic of debate, the consensus is that the enrichment of mRNAs in these nano-vesicles in one of the features of the biomarker discoveries. Our aim was to determine if exosomes are present in human saliva and to nano-characterize their transcriptomic content. Exosomes were purified by differential ultracentrifugation, identified by immunoelectron microscopy, flow cytometry and western blot using a CD-63 antibody. Atomic force microscopy studies revealed ultra structural analysis of both size and density of exosomes. Microarray analysis revealed the presence of 590 mRNA core transcripts are relatively stable inside the exosomes, which can be of saliva mRNA biomarkers. Exosomal mRNA stability was determined by detergent lyses with treatment of RNase. Under in vitro conditions fluorescent dye labeled saliva exosomes were able to communicate between human oral keratinocytes studied by using fluorescence microscopy. The RNA from saliva exosomes can transfer their genetic information to human oral keratinocytes and alters gene expression in the new location. Together, these results suggest that saliva is involved in mRNA trafficking via exosomes, and provides a mechanism for cargoing passenger mRNAs. Our findings are consistent with proposal that exosomes can shuttle RNAs between cells and mRNA is protected inside these vesicles may be a possible resource for biomarker discovery. Keywords: Human saliva, exosomes, mRNA profiling, gene expression, disease diagnosis

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Identification of markers for migrasome detection

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Author Correction: Identification of markers for migrasome detection.

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