ORCID Profile
0000-0001-7994-5151
Current Organisation
Baker IDI Heart and Diabetes Institute
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Publisher: Elsevier BV
Date: 10-2015
DOI: 10.1016/J.YMETH.2015.04.008
Abstract: Secretion and exchange of extracellular vesicles (EVs) by most cell types is emerging as a fundamental biological process. Although much is known about EVs, there is still a lack of definition as to how many naturally occurring EV subtypes there are and how their properties and functionalities might differ. This vexing issue is critical if EVs are to be fully harnessed for therapeutic applications. To address this question we have developed and describe here a sequential centrifugal ultrafiltration (SCUF) method to examine, in an unbiased manner, what EV subtypes are released in vitro into cell culture medium using the human colon carcinoma cell line LIM1863 as a model system. Using the culture medium from ∼7.2×10(9) LIM1863 cells, SCUF was performed using hydrophilic PVDF membranes with low protein binding properties (Millipore Durapore™ Ultrafree-CL filters with 0.1, 0.22, 0.45 and 0.65 μm pore size). EV particle sizing was measured using both dynamic light scattering and cryo-electron microscopy. Comparative proteome profiling was performed by GeLC-MS/MS and qualitative protein differences between EV subtypes determined by label-free spectral counting. The results showed essentially two EV subtypes one subtype (fraction Fn1) comprised heterogeneous EVs with particle diameters of 30-1300 nm, the other (fraction Fn5) being homogeneous EVs of 30-100 nm diameter based on cryo-EM both EV subtypes were round shaped. Western blot analysis showed Fn5 (SCUF-Exos) contained traditional exosome marker proteins (Alix(+), TSG101(+), CD81(+), CD63(+)), while Fn1 (SCUF-sMVs) lacked these protein markers. These findings were consistent with sMVs isolated by differential centrifugation (10,000 g, DC-sMVs) and exosomes (100,000 g EVs depleted of 10,000 g material). The buoyant density of sMVs determined by OptiPrep™ density gradient centrifugation was 1.18-1.19 g/mL and exosomes 1.10-1.11 g/mL. Comparative protein profiling of SCUF-Exos/-sMVs revealed 354 and 606 unambiguous protein identifications, respectively, with 256 proteins in common. A salient finding was the first report of 350 proteins uniquely identified in sMVs may of which have the potential to enable discrimination of this EV subtype from exosomes (notably, members of the septin family, kinesin-like protein (KIF23), exportin-2/chromosome segregation like-1 protein (CSE1L), and Rac GTPase-activating protein 1 (RACGAP1)). We report for the first time that both SCUF-Exos and SCUF-sMVs isolated from LIM1863 colon cancer cells induce invasion of recipient NIH3T3 cells. Interestingly, the SCUF-sMVs promote invasion to a significantly greater extent (3-fold) than SCUF-Exos. This analytical SCUF method for fractionating EVs is potentially scalable using tangential flow filtration, thereby providing a solid foundation for future in-depth functional studies of EV subtypes using erse cell types and functional assays.
Publisher: Frontiers Media SA
Date: 22-12-2022
DOI: 10.3389/FCELL.2022.1078096
Abstract: A series of cyclical events within the uterus are crucial for pregnancy establishment. These include endometrial regeneration following menses, under the influence of estrogen (proliferative phase), then endometrial differentiation driven by estrogen rogesterone (secretory phase), to provide a microenvironment enabling attachment of embryo (as a hatched blastocyst) to the endometrial epithelium. This is followed by invasion of trophectodermal cells (the outer layer of the blastocyst) into the endometrium tissue to facilitate intrauterine development. Small extracellular vesicles (sEVs) released by endometrial epithelial cells during the secretory phase have been shown to facilitate trophoblast invasion however, the molecular mechanisms that underline this process remain poorly understood. Here, we show that density gradient purified sEVs (1.06–1.11 g/ml, Alix + and TSG101 + , ∼180 nm) from human endometrial epithelial cells (hormonally primed with estrogen and progesterone vs. estrogen alone) are readily internalized by a human trophectodermal stem cell line and promote their invasion into Matrigel matrix. Mass spectrometry-based proteome analysis revealed that sEVs reprogrammed trophectoderm cell proteome and their cell surface proteome (surfaceome) to support this invasive phenotype through upregulation of pro-invasive regulators associated with focal adhesions (NRP1, PTPRK, ROCK2, TEK), embryo implantation (FBLN1, NIBAN2, BSG), and kinase receptors (EPHB4/B2, ERBB2, STRAP). Kinase substrate prediction highlighted a central role of MAPK3 as an upstream kinase regulating target cell proteome reprogramming. Phosphoproteome analysis pinpointed upregulation of MAPK3 T204/T202 phosphosites in hTSCs following sEV delivery, and that their pharmacological inhibition significantly abrogated invasion. This study provides novel molecular insights into endometrial sEVs orchestrating trophoblast invasion, highlighting the microenvironmental regulation of hTSCs during embryo implantation.
Publisher: Wiley
Date: 28-05-2021
Abstract: Doublecortin‐like kinase 1 (DCLK1) is a putative cancer stem cell marker, a promising diagnostic and prognostic maker for malignant tumors and a proposed driver gene for gastric cancer (GC). DCLK1 overexpression in a majority of solid cancers correlates with lymph node metastases, advanced disease and overall poor‐prognosis. In cancer cells, DCLK1 expression has been shown to promote epithelial‐to‐mesenchymal transition (EMT), driving disruption of cell‐cell adhesion, cell migration and invasion. Here, we report that DCLK1 influences small extracellular vesicle (sEV/exosome) biogenesis in a kinase‐dependent manner. sEVs isolated from DCLK1 overexpressing human GC cell line MKN1 (MKN1 OE ‐sEVs), promote the migration of parental (non‐transfected) MKN1 cells (MKN1 PAR ). Quantitative proteome analysis of MKN1 OE ‐sEVs revealed enrichment in migratory and adhesion regulators (STRAP, CORO1B, BCAM, COL3A, CCN1) in comparison to MKN1 PAR ‐sEVs. Moreover, using DCLK1‐IN‐1, a specific small molecule inhibitor of DCLK1, we reversed the increase in sEV size and concentration in contrast to other EV subtypes, as well as kinase‐dependent cargo selection of proteins involved in EV biogenesis (KTN1, CHMP1A, MYO1G) and migration and adhesion processes (STRAP, CCN1). Our findings highlight a specific role of DCLK1‐kinase dependent cargo selection for sEVs and shed new light on its role as a regulator of signaling in gastric tumorigenesis.
Publisher: Springer Science and Business Media LLC
Date: 23-05-2018
DOI: 10.1038/S41571-018-0036-9
Abstract: The sustained growth, invasion, and metastasis of cancer cells depend upon bidirectional cell-cell communication within complex tissue environments. Such communication predominantly involves the secretion of soluble factors by cancer cells and/or stromal cells within the tumour microenvironment (TME), although these cell types have also been shown to export membrane-encapsulated particles containing regulatory molecules that contribute to cell-cell communication. These particles are known as extracellular vesicles (EVs) and include species of exosomes and shed microvesicles. EVs carry molecules such as oncoproteins and oncopeptides, RNA species (for ex le, microRNAs, mRNAs, and long non-coding RNAs), lipids, and DNA fragments from donor to recipient cells, initiating profound phenotypic changes in the TME. Emerging evidence suggests that EVs have crucial roles in cancer development, including pre-metastatic niche formation and metastasis. Cancer cells are now recognized to secrete more EVs than their nonmalignant counterparts, and these particles can be isolated from bodily fluids. Thus, EVs have strong potential as blood-based or urine-based biomarkers for the diagnosis, prognostication, and surveillance of cancer. In this Review, we discuss the biophysical properties and physiological functions of EVs, particularly their pro-metastatic effects, and highlight the utility of EVs for the development of cancer diagnostics and therapeutics.
Publisher: Wiley
Date: 06-05-2021
Abstract: Embryo implantation into the receptive endometrium is critical in pregnancy establishment, initially requiring reciprocal signalling between outer layer of the blastocyst (trophectoderm cells) and endometrial epithelium however, factors regulating this crosstalk remain poorly understood. Although endometrial extracellular vesicles (EVs) are known to signal to the embryo during implantation, the role of embryo‐derived EVs remains largely unknown. Here, we provide a comprehensive proteomic characterisation of a major class of EVs, termed small EVs (sEVs), released by human trophectoderm cells (Tsc‐sEVs) and their capacity to reprogram protein landscape of endometrial epithelium in vitro. Highly purified Tsc‐sEVs (30–200 nm, ALIX + , TSG101 + , CD9/63/81 + ) were enriched in known players of implantation (LIFR, ICAM1, TAGLN2, WNT5A, FZD7, ROR2, PRICKLE2), antioxidant activity (SOD1, PRDX1/4/6), tissue integrity (EZR, RAC1, RHOA, TNC), and focal adhesions (FAK, ITGA2/V, ITGB1/3). Functionally, Tsc‐sEVs were taken up by endometrial cells, altered transepithelial electrical resistance, and upregulated proteins implicated in embryo attachment (ITGA2/V, ITGB1/3), immune regulation (CD59, CD276, LGALS3), and antioxidant activity (GPX1/3/4, PRDX1/2/4/5/6): processes that are critical for successful implantation. Collectively, we provide critical insights into Tsc‐sEV‐mediated regulation of endometrial function that contributes to our understanding of the molecular basis of implantation.
Publisher: Public Library of Science (PLoS)
Date: 17-10-2014
Publisher: Wiley
Date: 19-03-2021
Abstract: Endometrial extracellular vesicles (EVs) are emerging as important players in reproductive biology. However, how their proteome is regulated throughout the menstrual cycle is not known. Such information can provide novel insights into biological processes critical for embryo development, implantation, and successful pregnancy. Using mass spectrometry‐based quantitative proteomics, we show that small EVs (sEVs) isolated from uterine lavage of fertile women (UL‐sEV), compared to infertile women, are laden with proteins implicated in antioxidant activity (SOD1, GSTO1, MPO, CAT). Functionally, sEVs derived from endometrial cells enhance antioxidant function in trophectoderm cells. Moreover, there was striking enrichment of invasion‐related proteins (LGALS1/3, S100A4/11) in fertile UL‐sEVs in the secretory (estrogen plus progesterone‐driven, EP) versus proliferative (estrogen‐driven, E) phase, with several players downregulated in infertile UL‐sEVs. Consistent with this, sEVs from EP‐ versus E‐primed endometrial epithelial cells promote invasion of trophectoderm cells. Interestingly, UL‐sEVs from fertile versus infertile women carry known players redictors of embryo implantation (PRDX2, IDHC), endometrial receptivity (S100A4, FGB, SERPING1, CLU, ANXA2), and implantation success (CAT, YWHAE, PPIA), highlighting their potential to inform regarding endometrial status regnancy outcomes. Thus, this study provides novel insights into proteome reprograming of sEVs and soluble secretome in uterine fluid, with potential to enhance embryo implantation and hence fertility.
Publisher: Wiley
Date: 12-09-2023
Publisher: Wiley
Date: 11-2021
DOI: 10.1002/JEV2.12164
Abstract: The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gateway by bridging intra‐ and extracellular signalling networks, dictates EVs’ capacity to communicate and interact with their environment, and is a source of potential disease biomarkers and therapeutic targets. However, our understanding of surface protein composition of large EVs (L‐EVs, 100–800 nm, mean 310 nm, ATP5F1A, ATP5F1B, DHX9, GOT2, HSPA5, HSPD1, MDH2, STOML2), a major EV‐subtype that are distinct from small EVs (S‐EVs, 30–150 nm, mean 110 nm, CD44, CD63, CD81, CD82, CD9, PDCD6IP, SDCBP, TSG101) remains limited. Using a membrane impermeant derivative of biotin to capture surface proteins coupled to mass spectrometry analysis, we show that out of 4143 proteins identified in density‐gradient purified L‐EVs (1.07–1.11 g/mL, from multiple cancer cell lines), 961 proteins are surface accessible. The surface molecular ersity of L‐EVs include (i) bona fide plasma membrane anchored proteins (cluster of differentiation, transporters, receptors and GPI anchored proteins implicated in cell‐cell and cell‐ECM interactions) and (ii) membrane surface‐associated proteins (that are released by alent ion chelator EDTA) implicated in actin cytoskeleton regulation, junction organization, glycolysis and platelet activation. Ligand‐receptor analysis of L‐EV surfaceome (e.g., ITGAV/ITGB1) uncovered interactome spanning 172 experimentally verified cognate binding partners (e.g., ANGPTL3, PLG, and VTN) with highest tissue enrichment for liver. Assessment of biotin inaccessible L‐EV proteome revealed enrichment for proteins belonging to COPI/II‐coated ER/Golgi‐derived vesicles and mitochondria. Additionally, despite common surface proteins identified in L‐EVs and S‐EVs, our data reveals surfaceome heterogeneity between the two EV‐subtype. Collectively, our study provides critical insights into erse proteins operating at the interactive platform of L‐EVs and molecular leads for future studies seeking to decipher L‐EV heterogeneity and function.
Publisher: Wiley
Date: 03-10-2023
Publisher: Portland Press Ltd.
Date: 22-12-2016
DOI: 10.1042/BCJ20160006
Abstract: Cell–cell communication is critical across an assortment of physiological and pathological processes. Extracellular vesicles (EVs) represent an integral facet of intercellular communication largely through the transfer of functional cargo such as proteins, messenger RNAs (mRNAs), microRNA (miRNAs), DNAs and lipids. EVs, especially exosomes and shed microvesicles, represent an important delivery medium in the tumour micro-environment through the reciprocal dissemination of signals between cancer and resident stromal cells to facilitate tumorigenesis and metastasis. An important step of the metastatic cascade is the reprogramming of cancer cells from an epithelial to mesenchymal phenotype (epithelial–mesenchymal transition, EMT), which is associated with increased aggressiveness, invasiveness and metastatic potential. There is now increasing evidence demonstrating that EVs released by cells undergoing EMT are reprogrammed (protein and RNA content) during this process. This review summarises current knowledge of EV-mediated functional transfer of proteins and RNA species (mRNA, miRNA, long non-coding RNA) between cells in cancer biology and the EMT process. An in-depth understanding of EVs associated with EMT, with emphasis on molecular composition (proteins and RNA species), will provide fundamental insights into cancer biology.
Publisher: Public Library of Science (PLoS)
Date: 04-01-2019
Publisher: Impact Journals, LLC
Date: 23-04-2015
Abstract: Epithelial-mesenchymal transition (EMT) describes a morphogenetic program which confers mesenchymal cell properties, such as reduced cell-cell contact and increased cell migration and invasion, to epithelial cells. Here we investigate the role of the pleiotropic transcription/splicing factor and RNA-binding protein nuclease-sensitive element-binding protein 1 (YBX1/YB-1) in increasing the oncogenic potential of epithelial MDCK cells. Characterization of MDCK cells expressing YBX1 (MDCKYBX1 cells) revealed a partial EMT phenotype, including cytosolic relocalization of E-cadherin, increased cell scattering, and anchorage-independent growth. Subcutaneous injection of parental MDCK cells into NOD/SCID mice did not form tumours. Critically, MDCKYBX1 cells established viable tumour xenografts, and immuno-histochemical staining indicated murine vascularization by CD31+ endothelial cells. We analysed the total secretome (containing soluble and extracellular vesicles) of MDCKYBX1 cells to investigate regulation of the tumour microenvironment. YBX1 expression elevated release of secreted factors known to enhance angiogenesis (TGF-β, CSF-1, NGF, VGF, ADAM9 and ADAM17), compared to MDCK cells. Importantly, treatment with MDCKYBX1 cell-derived secretome increased recipient 2F-2B endothelial cell motility. This defines YBX1 as an oncogenic enhancer that can regulate tumour angiogenesis via release of secreted modulators into the extracellular microenvironment.
Publisher: Wiley
Date: 06-05-2021
Abstract: Cardiac intercellular communication is critical for heart function and often dysregulated in cardiovascular diseases. While cardiac extracellular vesicles (cEVs) are emerging mediators of signalling, their isolation remains a technical challenge hindering our understanding of cEV protein composition. Here, we utilised Langendorff‐collagenase‐based enzymatic perfusion and differential centrifugation to isolate cEVs from mouse heart (yield 3–6 μg/heart). cEVs are ∼200 nm, express classical EV markers (Cd63/81/9 + , Tsg101 + , Pdcd6ip/Alix + ), and are depleted of blood (Alb/Fga/Hba) and cardiac damage markers (Mb, Tnnt2, Ldhb). Comparison with mechanically‐derived EVs revealed greater detection of EV markers and decreased cardiac damage contaminants. Mass spectrometry‐based proteomic profiling revealed 1721 proteins in cEVs, implicated in proteasomal and autophagic proteostasis, glycolysis, and fatty acid metabolism essential functions often disrupted in cardiac pathologies. There was striking enrichment of 942 proteins in cEVs compared to mouse heart tissue ‐ implicated in EV biogenesis, antioxidant activity, and lipid transport, suggesting active cargo selection and specialised function. Interestingly, cEVs contain marker proteins for cardiomyocytes, cardiac progenitors, B‐cells, T‐cells, macrophages, smooth muscle cells, endothelial cells, and cardiac fibroblasts, suggesting erse cellular origin. We present a method of cEV isolation and provide insight into potential functions, enabling future studies into EV roles in cardiac physiology and disease.
Publisher: Springer US
Date: 2023
Publisher: Springer Science and Business Media LLC
Date: 13-03-2023
DOI: 10.1038/S42003-023-04641-4
Abstract: Atherosclerotic plaque rupture leading to myocardial infarction is a major global health burden. Applying the tandem stenosis (TS) mouse model, which distinctively exhibits the characteristics of human plaque instability/rupture, we use quantitative proteomics to understand and directly compare unstable and stable atherosclerosis. Our data highlight the disparate natures and define unique protein signatures of unstable and stable atherosclerosis. Key proteins and pathway networks are identified such as the innate immune system, and neutrophil degranulation. The latter includes calprotectin S100A8/A9, which we validate in mouse and human unstable plaques, and we demonstrate the plaque-stabilizing effects of its inhibition. Overall, we provide critical insights into the unique proteomic landscape of unstable atherosclerosis (as distinct from stable atherosclerosis and vascular tissue). We further establish the TS model as a reliable preclinical tool for the discovery and testing of plaque-stabilizing drugs. Finally, we provide a knowledge resource defining unstable atherosclerosis that will facilitate the identification and validation of long-sought-after therapeutic targets and drugs for plaque stabilization.
Publisher: Informa UK Limited
Date: 28-11-2016
DOI: 10.1080/14789450.2017.1260450
Abstract: Extracellular vesicles (EVs) are critical mediators of intercellular communication, capable of regulating the transcriptional landscape of target cells through horizontal transmission of biological information, such as proteins, lipids, and RNA species. This capability highlights their potential as novel targets for disease intervention. Areas covered: This review focuses on the emerging importance of discovery proteomics (high-throughput, unbiased quantitative protein identification) and targeted proteomics (hypothesis-driven quantitative protein subset analysis) mass spectrometry (MS)-based strategies in EV biology, especially exosomes and shed microvesicles. Expert commentary: Recent advances in MS hardware, workflows, and informatics provide comprehensive, quantitative protein profiling of EVs and EV-treated target cells. This information is seminal to understanding the role of EV subtypes in cellular crosstalk, especially when integrated with other 'omics disciplines, such as RNA analysis (e.g., mRNA, ncRNA). Moreover, high-throughput MS-based proteomics promises to provide new avenues in identifying novel markers for detection, monitoring, and therapeutic intervention of disease.
Publisher: Springer Science and Business Media LLC
Date: 05-12-2016
DOI: 10.1038/SREP38397
Abstract: Previously we reported that LIM1863 colorectal cancer (CRC) cells secrete three distinct extracellular vesicle subtypes – two subpopulations of exosomes (apical EpCAM-Exos and basolateral A33-Exos) and shed microvesicles (sMVs) – with distinct protein and miRNA signatures. Here, we extend our omics approach to understand the fundamental role of LIM1863-derived EVs by performing a comprehensive analysis of their mRNAs and long non-coding RNAs (lncRNAs) using RNA-Seq. We show that 2,389 mRNAs, 317 pseudogene transcripts, 1,028 lncRNAs and 206 short non-coding RNAs selectively distributed to (i.e., are enriched in) LIM1863 EVs, relative to the parent cell. An Ensembl/UniProtKB analysis revealed 1,937 mRNAs encode canonical proteins, 348 isoforms (including splice-variant proteins), and 119 ‘missing proteins’ (i.e., annotated in Ensembl but not UniProtKB). Further dissection of our protein/RNA data revealed that 6/151 observed RNA binding proteins have the potential to interact with ~75% of EV-enriched RNAs. Intriguingly, the co-existence of U1 and U2 ribonucleoproteins and their cognate snRNAs in LIM1863 EVs suggests a possible association of CRC EVs with recipient cell splicing events. Our data reveal several potential lncRNA CRC biomarkers and novel splicing/fusion genes that, collectively, will advance our understanding of EV biology in CRC and accelerate the development of EV-based diagnostics and therapeutics.
Publisher: Wiley
Date: 31-01-2019
Abstract: Cancer-associated fibroblasts (CAFs) are a heterogeneous population of activated fibroblasts that constitute a dominant cellular component of the tumor microenvironment (TME) performing distinct functions. Here, the role of tumor-derived exosomes (Exos) in activating quiescent fibroblasts into distinct functional subtypes is investigated. Proteomic profiling and functional dissection reveal that early- (SW480) and late-stage (SW620) colorectal cancer (CRC) cell-derived Exos both activated normal quiescent fibroblasts (α-SMA
Publisher: Wiley
Date: 30-10-2019
Abstract: Embryo implantation into maternal endometrium is critical for initiation and establishment of pregnancy, requiring developmental synchrony between endometrium and blastocyst. However, factors regulating human endometrial-embryo cross talk and facilitate implantation remain largely unknown. Extracellular vesicles (EVs) are emerging as important mediators of this process. Here, a trophectoderm spheroid-based in vitro model mimicking the pre-implantation human embryo is used to recapitulate important functional aspects of blastocyst implantation. Functionally, human endometrial EVs, derived from hormonally treated cells synchronous with implantation, are readily internalized by trophectoderm cells, regulating adhesive and invasive capacity of human trophectoderm spheroids. To gain molecular insights into mechanisms underpinning endometrial EV-mediated enhancement of implantation, quantitative proteomics reveal critical alterations in trophectoderm cellular adhesion networks (cell adhesion molecule binding, cell-cell adhesion mediator activity, and cell adherens junctions) and metabolic and gene expression networks, and the soluble secretome from human trophectodermal spheroids. Importantly, transfer of endometrial EV cargo proteins to trophectoderm to mediate changes in trophectoderm function is demonstrated. This is highlighted by correlation among endometrial EVs, the trophectodermal proteome following EV uptake, and EV-mediated trophectodermal cellular proteome, important for implantation. This work provides an understanding into molecular mechanisms of endometrial EV-mediated regulation of human trophectoderm functions-fundamental in understanding human endometrium-embryo signaling during implantation.
Publisher: Wiley
Date: 05-2013
Abstract: Exosomes are small extracellular 40-100 nm diameter membrane vesicles of late endosomal origin that can mediate intercellular transfer of RNAs and proteins to assist premetastatic niche formation. Using primary (SW480) and metastatic (SW620) human isogenic colorectal cancer cell lines we compared exosome protein profiles to yield valuable insights into metastatic factors and signaling molecules fundamental to tumor progression. Exosomes purified using OptiPrep™ density gradient fractionation were 40-100 nm in diameter, were of a buoyant density ~1.09 g/mL, and displayed stereotypic exosomal markers TSG101, Alix, and CD63. A major finding was the selective enrichment of metastatic factors (MET, S100A8, S100A9, TNC), signal transduction molecules (EFNB2, JAG1, SRC, TNIK), and lipid raft and lipid raft-associated components (CAV1, FLOT1, FLOT2, PROM1) in exosomes derived from metastatic SW620 cells. Additionally, using cryo-electron microscopy, ultrastructural components in exosomes were identified. A key finding of this study was the detection and colocalization of protein complexes EPCAM-CLDN7 and TNIK-RAP2A in colorectal cancer cell exosomes. The selective enrichment of metastatic factors and signaling pathway components in metastatic colon cancer cell-derived exosomes contributes to our understanding of the cross-talk between tumor and stromal cells in the tumor microenvironment.
Publisher: Wiley
Date: 04-2019
Abstract: Exosomes are important bidirectional cell-cell communicators in normal and pathological physiology. Although exosomal surface membrane proteins (surfaceome) enable target cell recognition and are an attractive source of disease marker, they are poorly understood. Here, a comprehensive surfaceome analysis of exosomes secreted by the colorectal cancer cell line SW480 is described. Sodium carbonate extraction/Triton X-114 phase separation and mild proteolysis (proteinase K, PK) of intact exosomes is used in combination with label-free quantitative mass spectrometry to identify 1025 exosomal proteins of which 208 are predicted to be integral membrane proteins (IMPs) according to TOPCONS and GRAVY scores. Interrogation of UniProt database-annotated proteins reveals 124 predicted peripherally-associated membrane proteins (PMPs). Surprisingly, 108 RNA-binding proteins (RBPs)/RNA nucleoproteins (RNPs) are found in the carbonate/Triton X-114 insoluble fraction. Mild PK treatment of SW480-GFP labeled exosomes reveal 58 proteolytically cleaved IMPs and 14 exoplasmic PMPs (e.g., CLU/GANAB/LGALS3BP). Interestingly, 18 RBPs/RNPs (e.g., EIF3L/RPL6) appear bound to the outer exosome surface since they are sensitive to PK proteolysis. The finding that outer surface-localized miRNA Let-7a-5p is RNase A-resistant, but degraded by a combination of RNase A/PK treatment suggests exosomal miRNA species also reside on the outer surface of exosomes bound to RBPs/RNPs.
Publisher: Springer US
Date: 2021
Publisher: Wiley
Date: 19-03-2021
Abstract: Epithelial–mesenchymal transition (EMT) describes an evolutionary conserved morphogenic process defined by loss of epithelial characteristics and acquisition of mesenchymal phenotype, and altered patterns of intercellular communication, leading to functional changes in cell migration and invasion. In this regard, we have previously reported that oncogenic H‐Ras induced EMT in Madin‐Darby Canine Kidney (MDCK) cells (21D1 cells) trigger changes in the protein distribution pattern in cells, exosomes, and soluble protein factors (secretome) which modulate the tumor microenvironment. Here, we report that shed microvesicles (also termed microparticles/ectosomes) secreted from MDCK cells following oncogenic H‐Ras‐induced EMT (21D1‐sMVs) are biochemically distinct from exosomes and parental MDCK‐sMVs. The protein spectra of RNA‐binding proteins and mitochondrial proteins in 21D1‐sMVs differ profoundly compared to those of exosomes, likewise proteins associated with suppression of anoikis. We show that 21D1‐sMVs promote cell migration, confer anchorage‐independent growth, and induce EMT in parental MDCK cells. An unexpected and novel finding was the selective sorting of tissue transglutaminase‐2 (TGM2) into 21D1‐sMVs there was no evidence of TGM2 in MDCK‐sMVs. Prior treatment of 21D1‐sMVs with neutralizing anti‐TGM2 or anti‐FN1 antibodies attenuates the invasive capability of fibroblasts. These finding suggest that microvesicle‐associated TGM2 may play an important contributory role in the EMT process and warrants further investigation.
Publisher: Springer Science and Business Media LLC
Date: 25-03-2021
DOI: 10.1038/S42003-021-01882-Z
Abstract: During the final stages of cell ision, newly-formed daughter cells remain connected by a thin intercellular bridge containing the midbody (MB), a microtubule-rich organelle responsible for cytokinetic abscission. Following cell ision the MB is asymmetrically inherited by one daughter cell where it persists as a midbody remnant (MB-R). Accumulating evidence shows MB-Rs are secreted (sMB-Rs) into the extracellular medium and engulfed by neighbouring non-sister cells. While much is known about intracellular MB-Rs, sMB-Rs are poorly understood. Here, we report the large-scale purification and biochemical characterisation of sMB-Rs released from colon cancer cells, including profiling of their proteome using mass spectrometry. We show sMB-Rs are an abundant class of membrane-encapsulated extracellular vesicle (200-600 nm) enriched in core cytokinetic proteins and molecularly distinct from exosomes and microparticles. Functional dissection of sMB-Rs demonstrated that they are engulfed by, and accumulate in, quiescent fibroblasts where they promote cellular transformation and an invasive phenotype.
Publisher: Wiley
Date: 07-2020
Publisher: Wiley
Date: 10-01-2023
Abstract: Implantation success relies on intricate interplay between the developing embryo and the maternal endometrium. Extracellular vesicles (EVs) represent an important player of this intercellular signalling through delivery of functional cargo (proteins and RNAs) that reprogram the target cells protein and RNA landscape. Functionally, the signalling reciprocity of endometrial and embryo EVs regulates the site of implantation, preimplantation embryo development and hatching, antioxidative activity, embryo attachment, trophoblast invasion, arterial remodelling, and immune tolerance. Omics technologies including mass spectrometry have been instrumental in dissecting EV cargo that regulate these processes as well as molecular changes in embryo and endometrium to facilitate implantation. This has also led to discovery of potential cargo in EVs in human uterine fluid (UF) and embryo spent media (ESM) of diagnostic and therapeutic value in implantation success, fertility, and pregnancy outcome. This review discusses the contribution of EVs in functional hallmarks of embryo implantation, and how the integration of various omics technologies is enabling design of EV‐based diagnostic and therapeutic platforms in reproductive medicine.
Publisher: MDPI AG
Date: 18-11-2022
Abstract: Extracellular vesicles (EVs) from stem cells have shown significant therapeutic potential to repair injured cardiac tissues and regulate pathological fibrosis. However, scalable generation of stem cells and derived EVs for clinical utility remains a huge technical challenge. Here, we report a rapid size-based extrusion strategy to generate EV-like membranous nanovesicles (NVs) from easily sourced human iPSCs in large quantities (yield 900× natural EVs). NVs isolated using density-gradient separation (buoyant density 1.13 g/mL) are spherical in shape and morphologically intact and readily internalised by human cardiomyocytes, primary cardiac fibroblasts, and endothelial cells. NVs captured the dynamic proteome of parental cells and include pluripotency markers (LIN28A, OCT4) and regulators of cardiac repair processes, including tissue repair (GJA1, HSP20/27/70, HMGB1), wound healing (FLNA, MYH9, ACTC1, ILK), stress response/translation initiation (eIF2S1/S2/S3/B4), hypoxia response (HMOX2, HSP90, GNB1), and extracellular matrix organization (ITGA6, MFGE8, ITGB1). Functionally, NVs significantly promoted tubule formation of endothelial cells (angiogenesis) (p 0.05) and survival of cardiomyocytes exposed to low oxygen conditions (hypoxia) (p 0.0001), as well as attenuated TGF-β mediated activation of cardiac fibroblasts (p 0.0001). Quantitative proteome profiling of target cell proteome following NV treatments revealed upregulation of angiogenic proteins (MFGE8, MYH10, VDAC2) in endothelial cells and pro-survival proteins (CNN2, THBS1, IGF2R) in cardiomyocytes. In contrast, NVs attenuated TGF-β-driven extracellular matrix remodelling capacity in cardiac fibroblasts (ACTN1, COL1A1/2/4A2/12A1, ITGA1/11, THBS1). This study presents a scalable approach to generating functional NVs for cardiac repair.
Publisher: Elsevier BV
Date: 12-2019
DOI: 10.1016/J.BBAPAP.2018.11.008
Abstract: Extracellular vesicles comprise two main classes - exosomes and shed microvesicles (sMVs). Whilst much is known about exosome cargo content and functionality, sMVs are poorly understood. Here, we describe the large-scale purification of sMVs released from primary (SW480) and metastatic (SW620) human isogenic colorectal cancer (CRC) cell lines using a combination of differential ultracentrifugation and isopycnic iodixanol density centrifugation. The yield of SW480-sMVs and SW620-sMVs was 0.75 mg and 0.80 mg, respectively. Both SW480-/SW620-sMVs are heterogeneous in size (100-600 nm diameter) and exhibit identical buoyant densities (1.10 g/mL). In contrast to exosomes, sMVs are ALIX
No related grants have been discovered for Alin Rai.