Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling

Verweij, Frederik J.; Bebelman, Maarten P.; Jiménez, Connie R.; García-Vallejo, Juan J.; Janssen, Hans; Neefjes, Jacques; Knol, Jaco C.; Haas, Richard de Goeij-de; Piersma, Sander R.; Baglio, S. Rubina; Verhage, Matthijs; Middeldorp, Jaap M.; Zomer, Anoek; Rheenen, Jacco van; Coppolino, Marc G.; Hurbain, Ilse; Raposo, Graça; Smit, Martine J.; Toonen, Ruud F.; Niel, Guillaume van; Pegtel, D. Michiel

doi:10.1083/jcb.201703206

Cited by 250 publications

(361 citation statements)

References 45 publications

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“…The release of exosomes is a multistep processes. Though several regulators including nSMase2, phosphorylated SNAP23 and RAB27A/RAB27B are involved in cancer exosome secretion, mechanism of exosome secretion in CSCs is poorly described. Here, we found RAB27B (Fig.…”

Section: Discussionmentioning

confidence: 99%

RAB27B‐activated secretion of stem‐like tumor exosomes delivers the biomarker microRNA‐146a‐5p, which promotes tumorigenesis and associates with an immunosuppressive tumor microenvironment in colorectal cancer

Cheng

Liao

Lin

et al. 2019

Intl Journal of Cancer

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The dynamic cell–cell communication is essential for tissue homeostasis in normal physiological circumstances and contributes to a diversified tumor microenvironment. Although exosomes are extracellular vesicles that actively participate in cell–cell interaction by shutting cellular components, impacts of tumor exosomes in the context of cancer stemness remain elusive. Here, we expand colorectal cancer stem cells (CRCSCs) as cancer spheroids and demonstrate that the β‐catenin/Tcf‐4‐activated RAB27B expression is required for the secretion of CRCSC exosomes. In an exosomal RNA sequencing analysis, a switch of exosomal RNA species from retrotransposons to microRNAs (miRNAs) is identified upon expanding CRCSCs. miRNA‐146a‐5p (miR‐146a) is the major miRNA in CRCSC exosomes and exosomal miR‐146a promotes stem‐like properties and tumorigenicity by targeting Numb in recipient CRC cells. Among 53 CRC patients, those with abundant exosomal miR‐146a expression in serum exhibits higher miR‐146aHigh/NumbLow CRCSC traits, an increased number of tumor‐filtrating CD66(+) neutrophils and a decreased number of tumor‐infiltrating CD8(+) T cells. Our study elucidates a unique mechanism of tumor exosome‐mediated stemness expansion.

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Section: Discussionmentioning

confidence: 99%

RAB27B‐activated secretion of stem‐like tumor exosomes delivers the biomarker microRNA‐146a‐5p, which promotes tumorigenesis and associates with an immunosuppressive tumor microenvironment in colorectal cancer

Cheng

Liao

Lin

et al. 2019

Intl Journal of Cancer

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“…IPMCs are virtually indistinguishable from multivesicular bodies (MVBs) but are not endosomes at all, as their lumens are contiguous with the extracellular space and their membranes are continuous with the cell surface. These different sites of origin can also impart a temporal heterogeneity in exosome release, as vesicles retained within MVBs or IPMCs can be released in a delayed and pulsatile manner via endosome fusion with the plasma membrane (Sung et al, 2015;Verweij et al, 2018) and IPMC opening(Pelchen-Matthews et al, 2012), respectively.…”

Section: Implications For Exosome Composition and Heterogeneitymentioning

confidence: 99%

A shared pathway of exosome biogenesis operates at plasma and endosome membranes

Fordjour

Daaboul

Gould

2019

Preprint

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This study of exosome cargo protein budding reveals that cells use a common pathway for budding exosomes from plasma and endosome membranes, providing a new mechanistic explanation for exosome heterogeneity and a rational roadmap for exosome engineering. AbstractEukaryotic cells secrete exosomes, which are small (~30-200 nm dia.), single membranebound organelles that transmit signals and molecules to other cells. Exosome-mediated signaling contributes to diverse physiological and disease processes, rendering their biogenesis of high biomedical importance. The prevailing hypothesis is that exosomes bud exclusively at endosome membranes and are released only upon endosome fusion with the plasma membrane. Here we tested this hypothesis by examining the intracellular sorting and exosomal secretion of the exosome cargo proteins CD63, CD9, and CD81. We report here that CD9 and CD81 are both localized to the plasma membrane and bud >5-fold more efficiently than endosome-localized CD63. Furthermore, we show that redirecting CD63 from endosomes to the plasma membrane by mutating its endocytosis signal (CD63/Y235A) increased its exosomal secretion ~6-fold, whereas redirecting CD9 to endosomes by adding an endosome targeting signal (CD9/YEVM) reduced its exosomal secretion ~5-fold. These data demonstrate that the plasma membrane is a major site of exosome biogenesis, and more importantly, that cells possess a common pathway for exosome protein budding that operates at both plasma and endosome membranes. Using a combination of single-particle interferometry reflectance (SPIR) imaging and immunofluorescence (IF) microscopy, we also show that variations in exosome composition are controlled by differential intracellular protein trafficking rather than by separate mechanisms of exosome biogenesis. This new view of exosome biogenesis offers a simple explanation for the pronounced compositional heterogeneity of exosomes and a validated roadmap for exosome engineering.

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“…In a previous study, correlative light-transmission electron microscopy imaging revealed that fluorescent flashes of pHluorin-CD63 at the plasma membrane indeed correspond to MVB fusion events 21 . To determine whether pHluo_M153R-CD63 likewise reports exosome secretion, we knocked down the MVB docking protein Rab27a 23 with shRNA in pHluo_M153R-CD63-expressing HT1080 cells ( Fig.…”

Section: Resultsmentioning

confidence: 89%

“…We also observed that pHluorin-CD63-positive punctate trails were left behind migrating cells 20 . Subsequently, a similar reporter (pHluorin group placed 7 amino acids away from ours in the first extracellular loop of CD63) was used by the Pegtel group to study GPCR regulation of exosome secretion 21 . pHluorin-CD63 is a powerful tool to track exosome secretion and MVB fusion with the plasma membrane.…”

Section: Introductionmentioning

confidence: 99%

pHluo_M153R-CD63, a bright, versatile live cell reporter of exosome secretion and uptake, reveals pathfinding behavior of migrating cells

Guerrero

et al. 2019

Preprint

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Small extracellular vesicles (EVs) called exosomes affect a variety of autocrine and paracrine cellular phenotypes, including cellular migration, immune activation, and neuronal function. Understanding the function of exosomes in these processes requires a variety of tools, including live cell imaging. We previously constructed a live-cell reporter, pHluorin-CD63, that allowed dynamic subcellular monitoring of exosome secretion in migrating and spreading cells. However, there were some caveats to its use, including relatively low fluorescent expression in cells and the inability to make cell lines that stably express the protein. By incorporating a stabilizing mutation in the pHluorin moiety, M153R, pHluorin-CD63 now exhibits higher and stable expression in cells and superior monitoring of exosome secretion. Using this improved construct, we demonstrate visualization of exosome secretion in 3D culture and identify a role for exosomes in promoting leader-follower behavior in 2D and 3D collective migration. By incorporating a further non-pH-sensitive red fluorescent tag, this reporter allows visualization of the entire exosome lifecycle, including multivesicular body (MVB) trafficking, MVB fusion, exosome uptake and endosome acidification. This new reporter will be a useful tool for understanding both autocrine and paracrine roles of exosomes.

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Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling

Cited by 250 publications

References 45 publications

RAB27B‐activated secretion of stem‐like tumor exosomes delivers the biomarker microRNA‐146a‐5p, which promotes tumorigenesis and associates with an immunosuppressive tumor microenvironment in colorectal cancer

RAB27B‐activated secretion of stem‐like tumor exosomes delivers the biomarker microRNA‐146a‐5p, which promotes tumorigenesis and associates with an immunosuppressive tumor microenvironment in colorectal cancer

A shared pathway of exosome biogenesis operates at plasma and endosome membranes

pHluo_M153R-CD63, a bright, versatile live cell reporter of exosome secretion and uptake, reveals pathfinding behavior of migrating cells

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