Fusion Of Multivesicular Bodies Research Articles

The Ether Lipid Precursor Hexadecylglycerol Stimulates the Release and Changes the Composition of Exosomes Derived from PC-3 Cells

Exosomes are vesicles released by cells after fusion of multivesicular bodies with the plasma membrane. In this study, we have investigated whether ether lipids affect the release of exosomes in PC-3 cells. To increase the cellular levels of ether lipids, the ether lipid precursor hexadecylglycerol was added to cells. Lipidomic analysis showed that this compound was in fact able to double the cellular levels of ether lipids in these cells. Furthermore, increased levels of ether lipids were also found in exosomes released by cells containing high levels of these lipids. Interestingly, as measured by nanoparticle tracking analysis, cells containing high levels of ether lipids released more exosomes than control cells, and these exosomes were similar in size to control exosomes. Moreover, silver staining and Western blot analyses showed that the protein composition of exosomes released in the presence of hexadecylglycerol was changed; the levels of some proteins were increased, and the levels of others were reduced. In conclusion, this study clearly shows that an increase in cellular ether lipids is associated with changes in the release and composition of exosomes.

Exosomes in neurological disease, neuroprotection, repair and therapeutics: problems and perspectives.

Exosomes: Exosomes are a sub-population of micro-vesicles ranging from 40–100 nm that were earlier thought as artefacts under electron microscope. They recently came into attention for their storage of biological information, cell-to-cell communication, serving as biomarkers and potential use in neural protection and regeneration (Kalani et al., 2013, 2014a). Exosomes are secreted by different brain cells including neurons, microglia, astrocytes, oligodendrocytes, and neural stem cells and their presence in cerebro-spinal fluid has also been confirmed. Exosomes represent cup-shaped morphology with a buoyant density of 1.13–1.19 g/cm2. They originate through the endocytic pathway after the fusion of multivesicular bodies (MVBs) with the plasma membrane and then release into the extracellular environment. Exosomes contain different proteins (membrane transport and fusion proteins, tetraspannins, heat-shock proteins, MVB biogenesis proteins), lipids, saccharides, and genetic materials (miRNA and other non-coding RNAs) which are collectively termed as ‘cargo’ contents (Kalani et al., 2014a). Recent findings suggest that exosomes contain unique molecular conservatories and therefore represent pathophysiological state of the cellular source from where they originate. On functional aspects, exosomes have been confirmed to perform roles as transporters, bridging the communication gap between cells and shuttling material from their cells of origin to the other distantly located cells. Because of their nano-size and non-complexed structure, exosomes efficiently cross blood-brain barrier (BBB) and therefore present novel ways to design therapeutic strategies against different cerebral diseases.

Serotonin stimulates secretion of exosomes from microglia cells.

Microglia are resident immune cells in the brain and exert important functions in the regulation of inflammatory processes during infection or cellular damage. Upon activation, microglia undergo complex morphological and functional transitions, including increased motility, phagocytosis and cytokine secretion. Recent findings indicate that exosomes, small vesicles that derive from fusion of multivesicular bodies with the plasma membrane, are involved in secretion of certain cytokines. The presence of specific receptors on the surface of microglia suggests communication with neurons by neurotransmitters. Here, we demonstrate expression of serotonin receptors, including 5-HT2a,b and 5-HT4 in microglial cells and their functional involvement in the modulation of exosome release by serotonin. Our data demonstrate the involvement of cAMP and Ca(2+) dependent signaling pathways in the regulation of exosome secretion. Co-culture of microglia with embryonic stem cell-derived serotonergic neurons further demonstrated functional signaling between neurons and microglia. Together, these data provide evidence for neurotransmitter-dependent signaling pathways in microglial cells that regulate exosome release.

Regulation of exosome release by glycosphingolipids and flotillins

Exosomes are released by cells after fusion of multivesicular bodies with the plasma membrane. The molecular mechanism of this process is still unclear. We investigated the role of sphingolipids and flotillins, which constitute a raft-associated family of proteins, in the release of exosomes. Interestingly, our results show that dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthase, seemed to affect the composition of exosomes released from PC-3 cells. However, the inhibition of ceramide formation from the de novo pathway by fumonisin B1 did not affect exosome secretion. Moreover, in contrast to findings obtained with other cell lines published so far, inhibition of neutral sphingomyelinase 2, an enzyme that catalyzes the formation of ceramide from sphingomyelin, did not inhibit the secretion of exosomes in PC-3 cells. Finally, small interfering RNA-mediated downregulation of flotillin-1 and flotillin-2 did not significantly change the levels of released exosomes as such, but seemed to affect the composition of exosomes. In conclusion, our results reveal the involvement of glycosphingolipids and flotillins in the release of exosomes from PC-3 cells, and indicate that the role of ceramide in exosome formation may be cell-dependent.

C. elegans Ciliated Sensory Neurons Release Extracellular Vesicles that Function in Animal Communication

Cells release extracellular vesicles (ECVs) that play important roles in intercellular communication and may mediate a broad range of physiological and pathological processes. Many fundamental aspects of ECV biogenesis and signaling have yet to be determined, with ECV detection being a challenge and obstacle due to the small size (100 nm) of the ECVs. We developed an in vivo system to visualize the dynamic release of GFP-labeled ECVs. We show here that specific Caenorhabdidits elegans ciliated sensory neurons shed and release ECVs containing GFP-tagged polycystins LOV-1 and PKD-2. These ECVs are also abundant in the lumen surrounding the cilium. Electron tomography and genetic analysis indicate that ECV biogenesis occurs via budding from the plasma membrane at the ciliary base and not via fusion of multivesicular bodies. Intraflagellar transport and kinesin-3 KLP-6 are required for environmental release of PKD-2::GFP-containing ECVs. ECVs isolated from wild-type animals induce male tail-chasing behavior, while ECVs isolated from klp-6 animals and lacking PKD-2::GFP do not. We conclude that environmentally released ECVs play a role in animal communication and mating-related behaviors.

The potential of exosomes in diagnosis and treatment of inborn errors of metabolism

Extracellular vesicles, in particular exosomes, have gained much attention as potent mediators of intercellular signaling. Exosomes are 50-130nm intraluminal vesicles of multivesicular bodies (MVB) that are secreted into the extracellular environment upon fusion of MVB with the plasma membrane. Current research on exosomes focuses on their biogenesis, including specific sorting mechanisms, their potential to transfer proteins and RNA from their cells of origin to target cells, specific methods of vesicle isolation, and their possible application as diagnostic and therapeutic devices. Exosomes are vesicles of endocytic origin that contain a portion of the cytoplasm. Their molecular components represent the composition and thereby the physiological state of the cells from which they originate. In this review, we recapitulate the discovery of exosomes and the subsequent expansion of exosome research into a variety of different areas of interest, with a specific focus on how exosomes could prove to be invaluable for both diagnostic and therapeutic applications within the research field of inborn errors of metabolism.

Proteomic Analysis of C2C12 Myoblast and Myotube Exosome-Like Vesicles: A New Paradigm for Myoblast-Myotube Cross Talk?

Exosomes are nanometer-sized microvesicles formed in multivesicular bodies (MVBs) during endosome maturation. Exosomes are released from cells into the microenvironment following fusion of MVBs with the plasma membrane. During the last decade, skeletal muscle-secreted proteins have been identified with important roles in intercellular communications. To investigate whether muscle-derived exosomes participate in this molecular dialog, we determined and compared the protein contents of the exosome-like vesicles (ELVs) released from C2C12 murine myoblasts during proliferation (ELV-MB), and after differentiation into myotubes (ELV-MT). Using a proteomic approach combined with electron microscopy, western-blot and bioinformatic analyses, we compared the protein repertoires within ELV-MB and ELV-MT. We found that these vesicles displayed the classical properties of exosomes isolated from other cell types containing components of the ESCRT machinery of the MVBs, as well as numerous tetraspanins. Specific muscle proteins were also identified confirming that ELV composition also reflects their muscle origin. Furthermore quantitative analysis revealed stage-preferred expression of 31 and 78 proteins in ELV-MB and ELV-MT respectively. We found that myotube-secreted ELVs, but not ELV-MB, reduced myoblast proliferation and induced differentiation, through, respectively, the down-regulation of Cyclin D1 and the up-regulation of myogenin. We also present evidence that proteins from ELV-MT can be incorporated into myoblasts by using the GFP protein as cargo within ELV-MT. Taken together, our data provide a useful database of proteins from C2C12-released ELVs throughout myogenesis and reveals the importance of exosome-like vesicles in skeletal muscle biology.

Exosomal miRNAs as potential biomarkers of cardiovascular risk in children.

Intercellular interactions are essential for basic cellular activities and errors in either receiving or transferring these signals have shown to cause pathological conditions. These signals are not only regulated by membrane surface molecules but also by soluble secreted proteins, thereby allowing for an exquisite coordination of cell functions. Exosomes are released by cells upon fusion of multivesicular bodies (MVB) with the plasma membrane. Their envelope reflects their cellular origin and their surface and internal contents include important signaling components. Exosomes contain a wide variety of proteins, lipids, RNAs, non-transcribed RNAs, miRNAs and small RNAs that are representative to their cellular origin and shuttle from donor cells to recipient cells. The exosome formation cargo content and delivery is of immense biological interest because exosomes are believed to play major roles in various pathological conditions, and therefore provide unique opportunities for biomarker discovery and development of non-invasive diagnostics when examined in biological fluids such as urine and blood plasma. For example, circulating miRNAs in exosomes have been applied as functional biomarkers for diagnosis and outcomes prediction, while synthetic miRNAs in polymer-based nanoparticles are applicable for therapeutics. This review provides insights into the composition and functional properties of exosomes, and focuses on their potential value as diagnostic markers in the context of cardiovascular disease risk estimates in children who suffer from conditions associated with heightened prevalence of adverse cardiovascular disease, namely obesity and sleep-disordered-breathing.

Normal prion protein is expressed on exosomes isolated from human plasma

Ritchie, Adam J Crawford, Damian M Ferguson, David J P Burthem, John Roberts, David J England Br J Haematol. 2013 Dec;163(5):678-80. doi: 10.1111/bjh.12543. Epub 2013 Sep 30.

Discarding the haystack to examine the needles: the potential role of urinary exosome analysis

exosomes are tiny vesicles (usually in the order of tens of nanometers), released by several cell types after fusion of multivesicular bodies with the plasma membrane. For many years, exosomes, described for the first time over 30 years ago ([8][1]), were thought to represent but an alternative to

Oncogenic H-Ras Reprograms Madin-Darby Canine Kidney (MDCK) Cell-derived Exosomal Proteins Following Epithelial-Mesenchymal Transition

Epithelial-mesenchymal transition (EMT) is a highly conserved morphogenic process defined by the loss of epithelial characteristics and the acquisition of a mesenchymal phenotype. EMT is associated with increased aggressiveness, invasiveness, and metastatic potential in carcinoma cells. To assess the contribution of extracellular vesicles following EMT, we conducted a proteomic analysis of exosomes released from Madin-Darby canine kidney (MDCK) cells, and MDCK cells transformed with oncogenic H-Ras (21D1 cells). Exosomes are 40-100 nm membranous vesicles originating from the inward budding of late endosomes and multivesicular bodies and are released from cells on fusion of multivesicular bodies with the plasma membrane. Exosomes from MDCK cells (MDCK-Exos) and 21D1 cells (21D1-Exos) were purified from cell culture media using density gradient centrifugation (OptiPrep™), and protein content identified by GeLC-MS/MS proteomic profiling. Both MDCK- and 21D1-Exos populations were morphologically similar by cryo-electron microscopy and contained stereotypical exosome marker proteins such as TSG101, Alix, and CD63. In this study we show that the expression levels of typical EMT hallmark proteins seen in whole cells correlate with those observed in MDCK- and 21D1-Exos, i.e. reduction of characteristic inhibitor of angiogenesis, thrombospondin-1, and epithelial markers E-cadherin, and EpCAM, with a concomitant up-regulation of mesenchymal makers such as vimentin. Further, we reveal that 21D1-Exos are enriched with several proteases (e.g. MMP-1, -14, -19, ADAM-10, and ADAMTS1), and integrins (e.g. ITGB1, ITGA3, and ITGA6) that have been recently implicated in regulating the tumor microenvironment to promote metastatic progression. A salient finding of this study was the unique presence of key transcriptional regulators (e.g. the master transcriptional regulator YBX1) and core splicing complex components (e.g. SF3B1, SF3B3, and SFRS1) in mesenchymal 21D1-Exos. Taken together, our findings reveal that exosomes from Ras-transformed MDCK cells are reprogrammed with factors which may be capable of inducing EMT in recipient cells.

ARA7(Q69L) expression in transgenic Arabidopsis cells induces the formation of enlarged multivesicular bodies

Arabidopsis thaliana ARA7 (AtRabF2b), a member of the plant Rab5 small GTPases functioning in the vacuolar transport pathway, localizes to pre-vacuolar compartments (PVCs), known as multivesicular bodies (MVBs) in plant cells. Overexpression of the constitutively active GTP-bound mutant of ARA7, ARA7(Q69L), induces the formation of large ring-like structures (1–2 µm in diameter). To better understand the biology of these ARA7(Q69L)-induced ring-like structures, transgenic Arabidopsis cell lines expressing ARA7(Q69L) tagged with green fluorescent protein (GFP) under the control of a heat shock-inducible promoter were generated. In these transgenic cells, robust ring-like structures were formed after 4 h of heat shock induction. Transient co-expression, confocal imaging, and immunogold electron microscopy (immunogold-EM) experiments demonstrated that these GFP–ARA7(Q69L)-labelled ring-like structures were distinct from the Golgi apparatus and trans-Golgi network, but were labelled with an antibody against an MVB marker protein. In addition, live cell imaging and detailed EM analysis showed that the GFP–ARA7(Q69L)-induced spherical structures originated from the homotypic fusion of MVBs. In summary, it was demonstrated that GFP–ARA7(Q69L) expression is an efficient tool for studying PVC/MVB-mediated protein trafficking and vacuolar degradation in plant cells.

Abstract 3085: Expression profiling of exosomal miRNAs derived from human esophageal cancer cells by Solexa high-through sequencing.

Abstract Background: Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment on fusion of multivesicular bodies (MVB) with the plasma membrane. Studies showed that inside exosomes, cellular gene products including proteins, mRNAs, and microRNAs (miRNAs) are packaged and these molecules can be transferred to recipient cells to deliver their molecular signaling. Objective: To explore the miRNA expression profile and distribution characteristics of exosomes derived from human esophageal cancer cells by Solexa high-throughput sequencing. Methods: Exosomes were extracted from cell culture supernatants by ultracentrifugation. Exosomes were identified by transmission electronic microscopy and western blot assay with anti-CD63 antibody. The types and abundance of known and novel miRNAs in cells and exosomes were identified by Solexa high-throughput sequencing. Results:There were 56421(2.94%) unique sequences in cells and 7727(0.63%) in exosomes matched the known miRNAs(Table 1). 64 in cells and 32 in exosomes novel miRNAs were predicted. Further analysis revealed extensive sequence variations (isomiRs) of cell and exosome miRNAs. The known miRNAs showed an increased expression in cells compared with exosomes. The expression level of hsa-miR-chr15_4885 and hsa-miR-chr18_7463 in exosomes were significantly higher than cells, with an average fold-change of 10 while hsa-miR-chr7_20613 and hsa-miR-chr19_8205 in cells were significantly higher than exosomes, with an average fold-change of thousands. Conclusion: The miRNA expression profile in exosomes is different from that in cells. It suggested that the significantly enriched miRNAs in exosomes may be the result of an active uptaken by exosomes. These specific miRNAs expressed in exosomes may play an important role in intercellular communication. Table 1. Distribution of sequence counts for the different classes of small RNAs in cells and exosomes Classification Unique RNA in cells Percentage (%) in cells Unique RNA in exosomes Percentage (%) in exosomes miRNA 56,421 2.94 7,727 0.63 mRNA 59,655 3.11 5,988 0.49 Repbase 31,421 1.64 5,066 0.41 rRNA 260,307 13.56 85,303 6.95 tRNA 80,635 4.20 119,500 9.74 snRNA 27,935 1.45 1,823 0.15 Unknown RNA 1,403,576 73.10 1,001,499 81.63 Total 1,919,950 100.00 1,226,905 100.00 Citation Format: Juan Liao, Ran Liu, Lihong Yin. Expression profiling of exosomal miRNAs derived from human esophageal cancer cells by Solexa high-through sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3085. doi:10.1158/1538-7445.AM2013-3085 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

RNA Interference: Systemic RNAi SIDes with Endosomes

Systemic RNAi, the intercellular spreading of RNAi silencing, requires SID-1 and SID-3 to import silencing signals in Caenorhabditis elegans. How are these signals exported? SID-5, an endosome-associated protein, is a candidate for the job.

Profiling of microRNAs in exosomes released from PC-3 prostate cancer cells

Exosomes are small extracellular vesicles released to the extracellular milieu through fusion of multivesicular bodies with the plasma membrane. These vesicles contain microRNAs and might therefore be vehicles transferring genetic information between cells. The aim of this study was to investigate whether there was a sorting of microRNAs into exosomes in the prostate cancer cell line PC-3. In addition, microRNAs in PC-3 cells and in the non-cancerous prostate cell line RWPE-1 were compared. Exosomes were isolated from the conditioned media from PC-3 cells by ultracentrifugation and inspected by electron microscopy. Total RNA was isolated and microRNAs were analyzed by microarray analysis and real time RT-PCR. MicroRNA microarray analysis revealed that the microRNA profile of PC-3 released exosomes was similar to the profile of the corresponding parent cells. Nevertheless, a sorting of certain microRNAs into exosomes was observed, and low number microRNAs (microRNAs with a low number in their name) were found to be underrepresented in these vesicles. Moreover, the miRNA profile of PC-3 cells resembled the miRNA profile of RWPE-1 cells, though some miRNAs were found to be differently expressed in these cell lines. These results show that exosomes from PC-3 cells, in agreement with previous reports from other cell types, contain microRNAs. Furthermore, this study supports the idea that there is a sorting of microRNAs into exosomes and adds a new perspective by pointing at the underrepresentation of low number miRNAs in PC-3 released exosomes.

Abstract 176: Kinase activity of tumor-derived exosomes as a potential biomarker for response to treatment

Abstract Introduction Exosomes are nanometer-sized vesicles secreted by tumor cells after fusion of multivesicular bodies with the plasma membrane that reflect tumor cell biology. Exosomes contain distinct classes of molecules including mRNA, miRNA and proteins. Because they can be isolated from blood they are promising candidate biomarkers. Diagnostic biomarkers for selection of patients for treatment with tyrosine kinase inhibitors are urgently warranted. We hypothesize that exosomes contain tumor derived kinases that may serve as biomarkers for treatment with kinase inhibitors. Methods The U87 glioma cell line with or without EGFRvIII (U87ΔEGFR) was used as an in vitro model in this study. Cell viability was studied with the MTT-assay. Exosomes were isolated from 32h conditioned media either serum-free or containing 10% exosome-deprived serum and from 5-9mL of fresh human serum from 3 cancer patients. The isolation procedure consisted of subsequent centrifugation at 500G, 2000G, 10.000G and 100.000G. The exosome pellet was treated with proteases for 1h at 37°C to digest soluble proteins, washed in PBS and lysed. Western blots for phospho-EGFR (y1068), EGFR, phospho-AKT, AKT, phospho-ERK, ERK, Alpha-Tubulin and exosome markers CD63, ALIX and CD81 were performed. Results The sensitivity of U87ΔEGFR to erlotinib (ERL) was significantly higher compared to parental U87 cells with IC50s of 1µM and 5.4µM (p<0.001), respectively. Treatment with 1 μM ERL resulted in reduced phosphorylation of EGFR, AKT and ERK in U87ΔEGFR, but not in U87 cells. The overexpression of constitutively active EGFRvIII mutant increased the phosphorylation of EGFR and downstream AKT and ERK in cells as well as in exosomes. Trypsin treatment only affected membrane proteins, where AKT and ERK levels were not altered. Exosomes from U87ΔEGFR cells treated with 0, 1 and 10µM ERL reflected the phosphorylation patterns of the cells with dose-dependent effects on AKT and ERK. Phosphorylated AKT and ERK could also be detected in exosomes from the cancer patients. Conclusion We here show that kinase activity and treatment response are reflected by exosomes derived from tumor cells in vitro. In addition we found that phosphorylated proteins can be detected in exosomes derived from blood of patients with cancer. Our aim is to further develop exosomal kinase activity analysis as an early biomarker for treatment response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 176. doi:1538-7445.AM2012-176

Proteomic analysis of urine exosomes by multidimensional protein identification technology (MudPIT)

Exosomes are membrane vesicles that are secreted by cells upon fusion of multivesicular bodies with the plasma membrane. Exosomal proteomics has emerged as a powerful approach to understand the molecular composition of exosomes and has potential to accelerate biomarker discovery. Different proteomic analysis methods have been previously employed to establish several exosome protein databases. In this study, TFE solution-phase digestion was compared with in-gel digestion and found to yield similar results. Proteomic analysis of urinary exosomes was performed by multidimensional protein identification technology (MudPIT) after TFE digestion. Nearly, 3280 proteins were identified from nine human urine samples with 31% overlap among nine samples. Gene ontology (GO) analysis, coupled with detection of all of the members of ESCRT machinery complex, supports the multivesicular origin of these particles. These results significantly expand the existing database of urinary exosome proteins. Our results also indicate that more than 1000 proteins can be detected from exosomes prepared from as little as 25 mL of urine. This study provides the largest set of proteins present in human urinary exosome proteomes, provides a valuable reference for future studies, and provides methods that can be applied to exosomal proteomic analysis from other tissue sources.

Exosomes and the kidney: prospects for diagnosis and therapy of renal diseases

Exosomes are 40–100 nm membrane vesicles secreted into the extracellular space by numerous cell types. These structures can be isolated from body fluids including urine and plasma. Exosomes contain proteins, mRNAs, miRNAs, and signaling molecules that reflect the physiological state of their cells of origin and consequently provide a rich source of potential biomarker molecules. Aside from diagnostic uses, exosome-mediated transfer of proteins, mRNAs, miRNAs, and signaling molecules offer the promise that they may be used for therapeutic purposes. In this review, we integrate new knowledge about exosomes from outside the field of nephrology with recent progress by renal researchers in order to provide a basis for speculation about how the study of exosomes may affect the fields of nephrology and renal physiology in the next few years.

Exosomes from Plasmodium yoelii-infected reticulocytes protect mice from lethal infections.

Exosomes are 30–100-nm membrane vesicles of endocytic origin that are released after the fusion of multivesicular bodies (MVBs) with the plasma membrane. While initial studies suggested that the role of exosomes was limited to the removal of proteins during the maturation of reticulocytes to erythrocytes, recent studies indicate that they are produced by different types of cells and are involved in promoting inter-cellular communication and antigen presentation. Here, we describe the isolation and characterization of exosomes from peripheral blood of BALB/c mice infected with the reticulocyte-prone non-lethal Plasmodium yoelii 17X strain. Importantly, proteomic analysis revealed the presence of parasite proteins in these vesicles. Moreover, immunization of mice with purified exosomes elicited IgG antibodies capable of recognizing P. yoelii-infected red blood cells. Furthermore, lethal challenge of immunized mice with the normocyte-prone lethal P. yoelii 17XL strain caused a significant attenuation in the course of parasitaemia, increased survival time, and altered the cell tropism to reticulocytes. These results were obtained also when the exosomes were isolated from a P. yoelii-infected reticulocyte culture indicating that reticulocyte-derived exosomes carry antigens and are involved in immune modulation. Moreover, inclusion of CpG ODN 1826 in exosome immunizations elicited IgG2a and IgG2b antibodies and promoted survival, clearance of parasites and subsequent sterile protection of 83% of the animals challenged with P. yoelli 17XL. To our knowledge, this is the first report of immune responses elicited by exosomes derived from reticulocytes opening new avenues for the modulation of anti-malaria responses.

Tumor exosomes: a novel biomarker?

Tumor exosomes: a novel biomarker?