Research from the past few years has overturned our understanding of the field of extracellular vesicles (EVs) and nanoparticles (NPs) by demonstrating a much greater diversity and complexity of ...extracellular carriers of proteins and nucleic acids.Cells release not only lipid-bilayer EVs, such as exosomes, but also non-vesicular extracellular NPs (NVEPs), such as exomeres and supermeres, both of which contain distinguishing proteins and nucleic acids.Supermeres are a newly discovered type of 15–25-nm amembranous extracellular NP that contain RNA and many proteins previously associated with exosomes and other EVs.Technical improvements in the tools used for EV and NVEP research will continue to drive future insights in the cell biology of these distinct modes of intercellular communication.
The study of extracellular vesicles (EVs) and nanoparticles (NPs) is rapidly expanding because recent discoveries have revealed a much greater complexity and diversity than was appreciated only a few years ago. New types of EVs and NPs have recently been described. Proteins and nucleic acids previously thought to be packaged in exosomes appear to be more enriched in different types of EVs and in two recently identified amembranous NPs, exomeres and supermeres. Thus, our understanding of the cell biology and intercellular communication facilitated by the release of EVs and NPs is in a state of flux. In this review, we describe the different types of EVs and NPs, highlight recent advances, and present major outstanding questions.
Accurate identification of risk factors for calcific uremic arteriolopathy (CUA) is necessary to develop preventive strategies for this morbid disease. We investigated whether baseline factors ...recorded at hemodialysis initiation would identify patients at risk for future CUA in a matched case-control study using data from a large dialysis organization. Hemodialysis patients with newly diagnosed CUA (n=1030) between January 1, 2010, and December 31, 2014, were matched by age, sex, and race in a 1:2 ratio to hemodialysis patients without CUA (n=2060). Mean ages for patients and controls were 54 and 55 years, respectively; 67% of participants were women and 49% were white. Median duration between hemodialysis initiation and subsequent CUA development was 925 days (interquartile range, 273-2185 days). In multivariable conditional logistic regression analyses, diabetes mellitus; higher body mass index; higher levels of serum calcium, phosphorous, and parathyroid hormone; and nutritional vitamin D, cinacalcet, and warfarin treatments were associated with increased odds of subsequent CUA development. Compared with patients with diabetes receiving no insulin injections, those receiving insulin injections had a dose-response increase in the odds of CUA involving lower abdomen and/or upper thigh areas (odds ratio, 1.49; 95% confidence interval, 1.03 to 2.51 for one or two injections per day; odds ratio, 1.88; 95% confidence interval, 1.30 to 3.43 for 3 injections per day; odds ratio, 3.74; 95% confidence interval, 2.28 to 6.25 for more than three injections per day), suggesting a dose-effect relationship between recurrent skin trauma and CUA risk. The presence of risk factors months to years before CUA development observed in this study will direct the design of preventive strategies and inform CUA pathobiology.
Lineage mapping has identified both proliferative and quiescent intestinal stem cells, but the molecular circuitry controlling stem cell quiescence is incompletely understood. By lineage mapping, we ...show Lrig1, a pan-ErbB inhibitor, marks predominately noncycling, long-lived stem cells that are located at the crypt base and that, upon injury, proliferate and divide to replenish damaged crypts. Transcriptome profiling of Lrig1+ colonic stem cells differs markedly from the profiling of highly proliferative, Lgr5+ colonic stem cells; genes upregulated in the Lrig1+ population include those involved in cell cycle repression and response to oxidative damage. Loss of Apc in Lrig1+ cells leads to intestinal adenomas, and genetic ablation of Lrig1 results in heightened ErbB1-3 expression and duodenal adenomas. These results shed light on the relationship between proliferative and quiescent intestinal stem cells and support a model in which intestinal stem cell quiescence is maintained by calibrated ErbB signaling with loss of a negative regulator predisposing to neoplasia.
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► Lrig1, an ErbB negative regulator, marks quiescent intestinal stem cells ► Lrig1 and Lgr5 transcriptome profiles are distinct ► Loss of APC in Lrig1+ cells results in intestinal adenomas ► Functionally null Lrig1 mice develop duodenal adenomas
Loss of calibrated EGFR/ErbB signaling in quiescent intestinal stem cells has neoplastic consequences, generating advanced colon adenomas. Although Lrig1+ cells are distinct from proliferating Lgr5+ cells, they are located at the base of the crypt.
Transfer of Functional Cargo in Exomeres Zhang, Qin; Higginbotham, James N.; Jeppesen, Dennis K. ...
Cell reports,
04/2019, Volume:
27, Issue:
3
Journal Article
Peer reviewed
Open access
Exomeres are a recently discovered type of extracellular nanoparticle with no known biological function. Herein, we describe a simple ultracentrifugation-based method for separation of exomeres from ...exosomes. Exomeres are enriched in Argonaute 1–3 and amyloid precursor protein. We identify distinct functions of exomeres mediated by two of their cargo, the β-galactoside α2,6-sialyltransferase 1 (ST6Gal-I) that α2,6- sialylates N-glycans, and the EGFR ligand, amphiregulin (AREG). Functional ST6Gal-I in exomeres can be transferred to cells, resulting in hypersialylation of recipient cell-surface proteins including β1-integrin. AREG-containing exomeres elicit prolonged EGFR and downstream signaling in recipient cells, modulate EGFR trafficking in normal intestinal organoids, and dramatically enhance the growth of colonic tumor organoids. This study provides a simplified method of exomere isolation and demonstrates that exomeres contain and can transfer functional cargo. These findings underscore the heterogeneity of nanoparticles and should accelerate advances in determining the composition and biological functions of exomeres.
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•Exomeres can be isolated using a simplified ultracentrifugation method•Exomeres are enriched for Argonaute proteins and APP•ST6Gal-I in exomeres sialylates recipient cell-surface targets including β1-integrin•AREG in exomeres modulates EGFR trafficking and increases tumor organoid growth
Exomeres are nanoparticles recently identified by asymmetric flow field-flow fractionation. Zhang et al. develop a simplified method for exomere isolation and confirm that exomeres have unique protein and lipid profiles. They show that two exomere cargos, ST6Gal-I and AREG, are biologically active in recipient cells.
Secretion of RNAs in extracellular vesicles is a newly recognized form of intercellular communication. A potential regulatory protein for microRNA (miRNA) secretion is the critical RNA-induced ...silencing complex (RISC) component Argonaute 2 (Ago2). Here, we use isogenic colon cancer cell lines to show that overactivity of KRAS due to mutation inhibits localization of Ago2 to multivesicular endosomes (MVEs) and decreases Ago2 secretion in exosomes. Mechanistically, inhibition of mitogen-activated protein kinase kinases (MEKs) I and II, but not Akt, reverses the effect of the activating KRAS mutation and leads to increased Ago2-MVE association and increased exosomal secretion of Ago2. Analysis of cells expressing mutant Ago2 constructs revealed that phosphorylation of Ago2 on serine 387 prevents Ago2-MVE interactions and reduces Ago2 secretion into exosomes. Furthermore, regulation of Ago2 exosomal sorting controls the levels of three candidate miRNAs in exosomes. These data identify a key regulatory signaling event that controls Ago2 secretion in exosomes.
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•Active KRAS suppresses Ago2 interaction with endosomes and secretion into exosomes•KRAS-MEK-ERK signaling promotes Ago2 phosphorylation on serine 387•S387 phosphorylation inhibits Ago2-endosome association and sorting to exosomes•Ago2 levels and phosphorylation control secretion of candidate miRNAs in exosomes
miRNA secretion in extracellular vesicles is thought to influence cell and tissue phenotypes. In this work, McKenzie et al. show that MEK-ERK signaling downstream of mutant KRAS suppresses Ago2 association with multivesicular endosomes, thereby inhibiting release of Ago2 and several miRNAs in exosomes.
Autocrine, paracrine, and juxtacrine are recognized modes of action for mammalian EGFR ligands including EGF, TGF-α (TGFα), amphiregulin (AREG), heparin-binding EGF-like growth factor (HB-EGF), ...betacellulin, epiregulin, and epigen. We identify a new mode of EGFR ligand signaling via exosomes. Human breast and colorectal cancer cells release exosomes containing full-length, signaling-competent EGFR ligands. Exosomes isolated from MDCK cells expressing individual full-length EGFR ligands displayed differential activities; AREG exosomes increased invasiveness of recipient breast cancer cells 4-fold over TGFα or HB-EGF exosomes and 5-fold over equivalent amounts of recombinant AREG. Exosomal AREG displayed significantly greater membrane stability than TGFα or HB-EGF. An average of 24 AREG molecules are packaged within an individual exosome, and AREG exosomes are rapidly internalized by recipient cells. Whether the composition and behavior of exosomes differ between nontransformed and transformed cells is unknown. Exosomes from DLD-1 colon cancer cells with a mutant KRAS allele exhibited both higher AREG levels and greater invasive potential than exosomes from isogenically matched, nontransformed cells in which mutant KRAS was eliminated by homologous recombination. We speculate that EGFR ligand signaling via exosomes might contribute to diverse cancer phenomena such as field effect and priming of the metastatic niche.
► EGFR ligands are present in exosomes released by human cancer cells ► AREG exosomes increase cancer cell invasion more than HB-EGF or TGFα exosomes ► AREG exosomes are rapidly internalized by recipient cells ► KRAS status correlates with AREG levels in exosomes and their invasive potential
Reassessment of Exosome Composition Jeppesen, Dennis K.; Fenix, Aidan M.; Franklin, Jeffrey L. ...
Cell,
04/2019, Volume:
177, Issue:
2
Journal Article
Peer reviewed
Open access
The 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 1–4, glycolytic enzymes, and cytoskeletal proteins were not detected in 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 are 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.
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•Human cells release Argonaute 1–4 and major vault protein independently of exosomes•Annexin A1 is a specific marker of microvesicles shed from the plasma membrane•Small extracellular vesicles do not contain DNA•Active secretion of cytosolic DNA occurs through an amphisome-dependent mechanism
A 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.
Activating mutations in KRAS occur in 30% to 40% of colorectal cancers. How mutant KRAS alters cancer cell behavior has been studied intensively, but non-cell autonomous effects of mutant KRAS are ...less understood. We recently reported that exosomes isolated from mutant KRAS-expressing colon cancer cells enhanced the invasiveness of recipient cells relative to exosomes purified from wild-type KRAS-expressing cells, leading us to hypothesize mutant KRAS might affect neighboring and distant cells by regulating exosome composition and behavior. Herein, we show the results of a comprehensive proteomic analysis of exosomes from parental DLD-1 cells that contain both wild-type and G13D mutant KRAS alleles and isogenically matched derivative cell lines, DKO-1 (mutant KRAS allele only) and DKs-8 (wild-type KRAS allele only). Mutant KRAS status dramatically affects the composition of the exosome proteome. Exosomes from mutant KRAS cells contain many tumor-promoting proteins, including KRAS, EGFR, SRC family kinases, and integrins. DKs-8 cells internalize DKO-1 exosomes, and, notably, DKO-1 exosomes transfer mutant KRAS to DKs-8 cells, leading to enhanced three-dimensional growth of these wild-type KRAS-expressing non-transformed cells. These results have important implications for non-cell autonomous effects of mutant KRAS, such as field effect and tumor progression.
There is an increasing appreciation for the heterogeneous nature of extracellular vesicles (EVs). In addition, two nonvesicular extracellular nanoparticles (NVEPs), exomeres and supermeres, have been ...discovered recently that are enriched in many cargo previously ascribed to EVs. The EV field has largely focused on EV isolation and characterization, while studies on NVEPs are limited. At this juncture, it is critically important to have robust and reliable methods to separate distinct populations of EVs and NVEPs to assign cargo to their correct carrier. Here, we provide a comprehensive step-by-step protocol for sequential isolation of large and small EVs, nonvesicular fractions, exomeres and supermeres from the same starting material. We describe in detail the use of differential ultracentrifugation, filtration, concentration and high-resolution density-gradient fractionation to obtain purified fractions of distinct populations of EVs and NVEPs. This protocol allows assignment and enrichment of a biomolecule of interest to its specific extracellular compartment. Compared to other isolation methods, our protocol has unique advantages, including high purity and reproducibility, with minimal expertise required. The protocol can be applied to purification of EVs and NVEPs from cell culture medium and human plasma and requires ~72 h to complete. Adoption of this protocol will help translational investigators identify potential circulating biomarkers and therapeutic targets for a host of human diseases and allow basic scientists to better understand EV and NVEP biogenesis and function. Overall, this protocol will allow those interested in isolating EVs and extracellular particles to advance scientific inquiry to answer outstanding questions in the field.
Oligodendrocytes derive from progenitors (OPCs) through the interplay of epigenomic and transcriptional events. By integrating high-resolution methylomics, RNA-sequencing, and multiple transgenic ...lines, this study defines the role of DNMT1 in developmental myelination. We detected hypermethylation of genes related to cell cycle and neurogenesis during differentiation of OPCs, yet genetic ablation of Dnmt1 resulted in inefficient OPC expansion and severe hypomyelination associated with ataxia and tremors in mice. This phenotype was not caused by lineage switch or massive apoptosis but was characterized by a profound defect of differentiation associated with changes in exon-skipping and intron-retention splicing events and by the activation of an endoplasmic reticulum stress response. Therefore, loss of Dnmt1 in OPCs is not sufficient to induce a lineage switch but acts as an important determinant of the coordination between RNA splicing and protein synthesis necessary for myelin formation.
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•Oligodendrocyte-lineage-specific analysis of DNA methylome and transcriptome•No fate switch induced by Dnmt1 ablation in early oligodendrocyte progenitors•DNMT1-dependent regulation of differentiation-specific alternative splicing events•Impaired myelination in Dnmt1 mutants characterized by aberrant ER stress response
Moyon et al. combine high-resolution methylomics, RNA sequencing, and analysis of multiple transgenic lines to define a critical role for DNA methylation in developmental myelination. Ablation of Dnmt1 decreases proliferation yet minimally impacts progenitors while impairing the late stages of differentiation, which require alternative splicing.
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