Reassessment of Exosome Composition.pdf

Reassessment of Exosome CompositionDennis K.Jeppesen1,Aidan M.Fenix2,Jeffrey L.Franklin1,James N.Higginbotham1,Qin Zhang1,Lisa J.Zimmerman3,Daniel C.Liebler3,Jie Ping4,Qi Liu4,Rachel Evans5,William H.Fissell5,James G.Patton6,Leonard H.Rome7,Dylan T.Burnette2,Robert J.Coffey1,8,*1Department of Medicine,Vanderbilt University Medical Center,Nashville,TN 37232,USA2Department of Cell and Developmental Biology,Vanderbilt University School of Medicine,Nashville,TN 37232,USA3Jim Ayers Institute for Precancer Detection and Diagnosis,Vanderbilt University Medical Center,Nashville,TN 37232,USA4Department of Biostatistics,Vanderbilt University Medical Center,Nashville,TN 37232,USA5Division of Nephrology and Hypertension,Vanderbilt University Medical Center,Nashville,TN 37232,USA6Department of Biological Sciences,Vanderbilt University,Nashville,TN 37235,USA7Department of Biological Chemistry,David Geffen School of Medicine at University at California Los Angeles,Los Angeles,CA 90095,USA8Lead Contact:Robert J.Coffey,MD,Epithelial Biology Center,10415 MRB IV,Vanderbilt University Medical Center,2213 Garland Ave.,Nashville,TN 37232SUMMARYThe heterogeneity of small extracellular vesicles and presence of non-vesicular extracellular matter have led to debate about contents and functional properties of exosomes.Here,we employ high-resolution density gradient fractionation and direct immunoaffinity capture to precisely characterize the RNA,DNA,and protein constituents of exosomes and other non-vesicle material.Extracellular RNA,RNA-binding proteins and other cellular proteins are differentially expressed in exosomes and non-vesicle compartments.Argonaute 14,glycolytic enzymes and cytoskeletal proteins are absent from exosomes.We identify Annexin A1 as a specific marker for microvesicles that are shed directly from the plasma membrane.We further show that small extracellular vesicles*Correspondence:Tel.:615-343-6228;Fax:(615)343-1591,robert.coffeyvumc.org(R.J.C.).AUTHOR CONTRIBUTIONSD.K.J.conceived the study,designed the experimental methodology,performed experiments,analyzed,interpreted and visualized the data,and wrote the manuscript.A.M.F.performed experiments,analyzed and visualized the data.J.L.F.designed experiments and interpreted the data.J.N.H.,Q.Z.,L.J.Z and R.E.performed experiments.J.P.and Q.L.analyzed and interpreted data.D.C.L.and W.H.F.supervised experiments.J.G.P.wrote the manuscript.L.H.R.designed and performed experiments,analyzed the data,and wrote the manuscript.D.C.B.designed and supervised experiments,and interpreted the data.R.J.C.supervised the work.Publishers Disclaimer:This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting,typesetting,and review of the resulting proof before it is published in its final citable form.Please note that during the production process errors may be discovered which could affect the content,and all legal disclaimers that apply to the journal pertain.DECLARATION OF INTERESTSThe authors declare no competing interests.HHS Public AccessAuthor manuscriptCell.Author manuscript;available in PMC 2020 April 04.Published in final edited form as:Cell.2019 April 04;177(2):428445.e18.doi:10.1016/j.cell.2019.02.029.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptare not vehicles of active DNA release.Instead,we propose a new model for active secretion of extracellular DNA through an autophagy-and multivesicular endosome-dependent,but exosome-independent mechanism.This study demonstrates the need for a reassessment of exosome composition and offers a framework for a clearer understanding of extracellular vesicle heterogeneity.eTOC BlurbA reassessment of exosome composition establishes the differential distribution of protein,RNA,and DNA between small extracellular vesicles and non-vesicular extracellular matter and establishes that small extracellular vesicles are not vehicles of active DNA release.Graphical AbstractKeywordsexosomes;microvesicles;exomeres;extracellular vesicles;argonaute;extracellular RNA;extracellular DNA;annexin;autophagy;amphisomesINTRODUCTIONCells release extracellular vesicles(EVs)of different sizes and intracellular origin.The heterogeneity of EVs and presence of non-vesicular extracellular nanoparticles pose major Jeppesen et al.Page 2Cell.Author manuscript;available in PMC 2020 April 04.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptobstacles to our understanding of the composition and functional properties of distinct secreted components.Greater precision in assigning RNA,DNA and protein to their correct extracellular compartments and identifiying their mechanisms of secretion is crucial for identification of biomarkers and design of future drug interventions.Exosomes are 40150 nm,endosome-derived,small extracellular vesicles(sEVs)secreted by most,if not all,cells.RNA(including mRNA,miRNA and other non-coding RNA),DNA and lipids are reported to be actively and selectively incorporated into intraluminal vesicles(ILVs),which reside within multivesicular endosomes(MVEs)and are the precursor of exosomes(van Niel et al.,2018).In addition to accounting for the presence of membrane proteins in exosomes,inward budding of endosomal membranes is thought to result in the engulfment of cytosolic proteins and other components to the lumen of ILVs(Mathieu et al.,2019;van Niel et al.,2018).Fusion of MVEs with the plasma membrane then releases ILVs into the extracellular space as exosomes.In contrast,microvesicles are 1501000 nm large extracellular vesicles(lEVs)generated by shedding from the plasma membrane(Mathieu et al.,2019;van Niel et al.,2018).However,specific markers that distinguish microvesicles from exosomes are lacking.Much of the recent interest in EVs was triggered by the discovery that exosomes function in the transport of secreted extracellular RNA(exRNA),including extracellular miRNA and mRNA transport(Skog et al.,2008;Valadi et al.,2007).Argonautes(Agos)are important miRNA-processing proteins,but the exosome-mediated secretion of human Ago proteins is an unsettled issue(Arroyo et al.,2011;Melo et al.,2014;Shurtleff et al.,2016).Other RNA-binding proteins(RBPs)have also been reported to be present in exosomes with possible roles for sorting of RNA(Mateescu et al.,2017;Shurtleff et al.,2016;Villarroya-Beltri et al.,2013).However,the heterogeneity of extracellular vesicles and nanoparticles,as well as differences in purification strategies,have confounded analyses.Here,we employ high-resolution density gradient fractionation to separate sEVs from non-vesicular material,and direct immunoaffinity capture(DIC)to specifically isolate exosomes from other types of sEVs.DIC was performed without ultracentrifugation and using capture beads targeting classical exosomal tetraspanins.Comprehensive proteomic and nucleic acid analysis revealed that exRNA and proteins are differentially expressed between sEVs and non-vesicle compartments.Many RBPs linked to inclusion or loading of exRNA in exosomes,including Ago14,are not associated with classical exosomes displaying the exosomal markers CD63,CD81 and CD9(Kowal et al.,2016;van Niel et al.,2018).Exosomes lack cytoskeletal elements and common glycolytic enzymes;the absence of these highly abundant cytosolic proteins suggests that exosome loading must be a highly regulated process.These studies were performed using human colon(DKO-1)and glioblastoma(Gli36)cancer cell lines.The major findings were validated in normal human kidney epithelial cells and human plasma.Exosomes are touted to be vehicles of extracellular DNA secretion,making them attractive targets as liquid biopsies for cancer patients.We provide evidence that double-stranded DNA(dsDNA)and DNA-binding histones are not carried by exosomes or any other type of sEV.Instead,we show that active secretion of extracellular dsDNA and histones can occur through an autophagy-and MVE-dependent,exosome-independent mechanism.Additionally,we identify Annexin A1 as a specific marker for classical microvesicles budding from the plasma membrane.Jeppesen et al.Page 3Cell.Author manuscript;available in PMC 2020 April 04.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor ManuscriptThese findings clarify exosomal constituents and provide sounder footing for exploring their functional properties.RESULTSHigh-Resolution Density Gradient Fractionation Separates Small Extracellular Vesicles from Non-Vesicular ComponentsIt is increasingly clear that“exosomal”samples contain a heterogeneous mixture of sEVs and non-vesicular compartments(operational definitions in Figure 1A and Table S1).Crude lEVs(P15)and crude sEVs(P120)were prepared from DKO-1 and Gli36 cells using conventional differential centrifugation(STAR Methods).These P120 preparations were highly enriched for the classical exosome markers CD63,CD81 and CD9 and devoid of gross contamination(Figure 1B).To further separate the membrane-enclosed sEVs from non-vesicular(NV)components,we employed high-resolution iodixanol gradients(STAR Methods).CD63 and CD81 were present in low-density fractions distinct from those containing the extracellular matrix protein Fibronectin and the ribosomal protein RPS3(Figures 1C and S1AC).Based on the presence of these exosomal markers,low-density fraction pools of purified sEVs and high-density pools of NV components were identified(Figures 1C and S1BC).Subjecting the purified sEVs to a second round of density fractionation did not cause a reappearance of GAPDH and eEF1A1 in high-density fractions,indicating that components in the NV fractions are unlikely to arise de novo from damage to vesicles(Figure S1D).Only the low-density pool containing the gradient-purified sEVs was highly susceptible to detergent-mediated disruption(Figure 1D).The low-density pooled fractions contained cup-shaped vesicles of size and morphology consistent with sEVs/exosomes while these were absent from high-density fraction pools(Figures 1E and S1EF).In summary,a high-resolution iodixanol density gradient separates small extracellular membrane vesicles bearing the hallmarks of sEVs/exosomes from non-vesicular components.Proteomic Profiling of Small Extracellular Vesicles and Non-Vesicular FractionsTo assess the protein composition of density gradient-purified sEVs and NVs,LC-MS/MS was performed(Figure S2A and Tables S24).Despite considerable overlap between the fractions in terms of identifiable proteins it became strikingly clear that gradient-purified sEVs are highly distinct from NVs(Figures 2AC and S2BC).Syntenin-1(gene SDCBP)and ALIX(PDCD6IP)were among the most abundant proteins identified in the density gradient-purified sEV samples,and are two of the most highly expressed exosomal proteins found with more sophisticated purification methods(Kowal et al.,2016;Zhang et al.,2018).The most abundant proteins in the NV fractions were metabolic enzymes like GAPDH,PKM and ENO1,and cytosolic proteins including HSP90 and tubulins(Table S5).As expected,common exosomal markers were relatively overexpressed in gradient-purified sEVs(Figures 2D and S2D).Histones are frequently identified and among the most abundant proteins in proteomic analysis of sEVs/exosomes(Zhang et al.,2018).However,Histones H2A and H3 were highly enriched in NV fractions.LC-MS/MS results were validated,including the distinct association of histones with NV fractions(Figures 2E and S2E).Of the 25 most frequently reported proteins in the ExoCarta exosome database Jeppesen et al.Page 4Cell.Author manuscript;available in PMC 2020 April 04.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscript(Keerthikumar et al.,2016),many were more associated with NV fractions than purified sEV fractions,including GAPDH,PKM and HSP90(Figures 2F and S2F).In summary,proteomic profiling reveals that after high-resolution iodixanol density gradient purification,some presumed exosomal proteins were more associated with NV fractions than with fractions containing sEVs/exosomes.Differential Expression of RNA in Small Extracellular Vesicles and Non-Vesicular FractionsExtracellular RNA(exRNA)was extracted from density gradient-purified sEV and NV pooled fractions.Distinct ribosomal RNA(rRNA)peaks(18S and 28S)were diminished in the extracellular lEV,sEV and NV samples,while small RNA species were enriched(Figure S2G).Short RNA sequencing(STAR Methods)revealed enrichment of rRNA fragments and other specific small RNAs or RNA fragments in the extracellular samples(Figure 2G).The overall pattern of miRNA expression was distinctly different not only between cellular and secreted miRNAs,but also between sEV and NV fractions(Figure 2H).Numerous miRNAs displayed significant differential distribution between purified sEV and NV fractions and between purified sEVs and their parental cells(Figure S2H and Tables S67).Many of the most abundant miRNAs were more associated with extracellular NV fractions than with either parental cells or sEV fractions(Figure 2I).Strong enrichment for extracellular transfer RNA(tRNA)fragments was also observed,particularly for lEV and sEV samples(Figure 2G).Y-box protein 1(YBX1)is an RBP reported to be present in exosomes and responsible for sorting miRNA and tRNA into exosomes(Shurtleff et al.,2016;Shurtleff et al.,2017).However,YBX1 was not detected in either sEV or NV samples released from DKO-1 cells and was more abundant in Gli36 NV fractions than sEV fractions after gradient purification(Figure S2I).YBX1 is responsible for sorting miR-223 and miR-144 into HEK293T-derived exosomes(Shurtleff et al.,2016);however,both were detected with very low abundance in our short RNA-seq of DKO-1 and Gli36 cells and extracellular samples(Figure S2J).The 3 termini of cellular miRNAs and purified sEV miRNAs were broadly similar,but the NV fraction miRNAs displayed marked trimming(Figure S2K).YRNAs and vault RNAs(VTRNA)were enriched in extracellular samples,with VTRNA in particular associated with NV fractions(Figure 2G).Long RNA sequencing revealed that the distribution of transcripts among lEVs,sEVs and NV fractions were similar,while protein-coding transcripts comprised a larger percentage of cellular samples compared to extracellular samples(Figure S2L).Strikingly,the overwhelming majority of cellular long RNA reads mapped to exonic regions,while the majority of extracellular reads mapped to intronic regions(Figure S2M).The abundance of lincRNA was greater for all extracellular samples compared to parental cells(Figure S2N)and the patterns of lincRNA expression were clearly distinct(Figure S2O).YRNA,and fragments or processed versions of YRNA,have repeatedly been reported to be present and enriched in crude samples of sEVs compared to cells(Chakrabortty et al.,2015;Nolte-t Hoen et al.,2012).By short RNA-seq,we observed that YRNA was also enriched in lEVs and gradient-purified NV fra