Exosome release is regulated by mTORC1 via a Rab27A-dependent mechanism
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ABSTRACT:
PROVIDER: PRJNA470637 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7138585.fastq.gz | Fastqsanger.gz | |||
| SRR7138586.fastq.gz | Fastqsanger.gz |
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Project description:Exosomes are small membrane-bound vesicles released into extracellular spaces by many types of cells. These nanovesicles carry proteins, mRNA and miRNA and are involved in cell waste management and intercellular communication. In the present study we show that exosome release, which leads to net loss of cellular membrane and protein content, is negatively regulated by mechanistic target of rapamycin complex 1 (mTORC1). We find that in cells and animal models exosome release is inhibited by sustained activation of mTORC1, leading to intracellular accumulation of CD63-positive exosome precursors. Inhibition of mTORC1 by rapamycin or nutrient and growth factor deprivation stimulates exosome release, which occurs concomitantly with autophagy. The drug-stimulated release is blocked by siRNA-mediated downregulation of small GTPase Rab27A. Analysis of the cargo content in exosomes released from rapamycin treated cells reveals that inhibition of mTORC1 does not alter its protein and miRNA profiles. These observations demonstrate that exosome release, like autophagy, is negatively regulated by mTORC1 in response to changes in nutrient and growth factor conditions
2019-03-05 | GSE114235 | GEO
Project description:Exosomes are small membrane-bound vesicles released into extracellular spaces by many types of cells. These nanovesicles carry proteins, mRNA, and miRNA, and are involved in cell waste management and intercellular communication. In the present study, it is shown that exosome release, which leads to net loss of cellular membrane and protein content, is negatively regulated by mechanistic target of rapamycin complex 1 (mTORC1). It is found that in cells and animal models exosome release is inhibited by sustained activation of mTORC1, leading to intracellular accumulation of CD63-positive exosome precursors. Inhibition of mTORC1 by rapamycin or nutrient and growth factor deprivation stimulates exosome release, which occurs concomitantly with autophagy. The drug-stimulated release is blocked by siRNA-mediated downregulation of small GTPase Rab27A. Analysis of the cargo content in exosomes released from rapamycin-treated cells reveals that inhibition of mTORC1 does not significantly alter its majority protein and miRNA profiles. These observations demonstrate that exosome release, like autophagy, is negatively regulated by mTORC1 in response to changes in nutrient and growth factor conditions.
| S-EPMC6364500 | biostudies-literature
Project description:Cells release intraluminal vesicles (ILVs) in multivesicular bodies as exosomes to communicate with other cells. Although recent studies suggest an intimate link between exosome biogenesis and autophagy, the detailed mechanism is not fully understood. Here we employed comprehensive RNAi screening for autophagy-related factors and discovered that Rubicon, a negative regulator of autophagy, is essential for exosome release. Rubicon recruits WIPI2d to endosomes to promote exosome biogenesis. Interactome analysis of WIPI2d identified the ESCRT components that are required for ILV formation. Notably, we found that Rubicon is required for an age-dependent increase of exosome release in mice. In addition, small RNA sequencing of serum exosomes revealed that Rubicon determines the fate of exosomal microRNAs associated with aging and longevity pathways. Taken together, our current results suggest that the Rubicon-WIPI axis functions as a key regulator of exosome biogenesis and is responsible for the age-dependent changes in exosome quantity and quality.
2024-06-25 | GSE262794 | GEO
Project description:To screen miRNAs specifically regulated by mTORC1 or mTORC2, a global miRNA expression profile in MCF-7 cells treated with rapamycin or PP242 (mTORC1/2 kinase inhibitor) was developed using microarray. control, rapamycin or PP242 treated human MCF-7 cells were harvested 48h post-treatment and subjected to total RNA extraction.
2014-09-11 | E-GEOD-61289 | biostudies-arrayexpress
Project description:Extracellular vesicles (EVs) are small membrane-derived vesicles that shuttle proteins or nucleic acids between glia and neurons, thereby promoting neuronal survival and plasticity in the CNS. Exosomes are small EVs (40-150 nm) that are derived from multi-vesicular bodies (MVBs) of the endo-lysosomal pathway, formed by inward budding of the limiting membrane into the MVB lumen and released into the extracellular space upon fusion of the MVB with the plasma membrane (PM). Previous research revealed certain effector molecules to be required for exosome release. For instance, RAB GTPases have been shown to control exosome release in a cell- and tissue-specific manner. In addition, exosome release appears to be evoked by membrane depolarization in conjunction with calcium influx and to depend on members of the SNARE family of proteins. However, the specific cellular and molecular factors that regulate neuronal exosome release and segregate it from the release of neurotransmitter vesicles are currently unknown. Here, we used a combination of molecular biology, patch-clamp electrophysiology and pH-sensitive dye imaging to examine the effect of the neuronal growth and differentiation factors basic fibroblast growth factor (bFGF), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) on neuronal EV release. We found that bFGF-treatment of cultured hippocampal neurons increased the abundance of EVs in the culture medium as measured by Western blot and nanoparticle tracking analysis (NTA), without affecting the number or size of neuronal MVBs. The effect of bFGF depended on calcium and on receptor tyrosine kinase (RTK) activity as the calcium chelating agent BAPTA and the tyrosine kinase inhibitor genistein both abolished the bFGF-induced increase in EV release. In accord with these results, untreated neurons had a low rate of spontaneous and stimulus-evoked MVB-PM fusion events as measured by pH-sensitive dye imaging in conjunction with patch-clamp electrophysiology, but treatment with bFGF significantly increased the number of neurons exhibiting MVB-PM fusion events in response to a high-frequency stimulus. Proteomic analysis of neuronal EVs by Liquid Chromatography Mass Spectrometry (LC-MS) demonstrated bFGF to increase the abundance of the v-SNARE vesicle-associated membrane protein 3 (VAMP3, cellubrevin) on EVs, whereas VAMP2 was decreased. Conversely, knocking-down VAMP3 in cultured neurons abolished the effect of bFGF on EV release. Similar to bFGF, the classical neurotrophins BDNF and NGF increased neuronal EV release in a VAMP3-dependent manner. In summary, our results thus reveal a new function for neurotrophic factor signalling in controlling neuronal exosome release and support the investigation of growth factor-mediated signal transduction via EVs in the healthy and diseased CNS.
2020-02-06 | PXD014401 | Pride
Project description:Background: Docosahexaenoic acid (DHA) is a natural compound with anticancer and anti-angiogenesis activity that is currently under investigation as both a preventative agent and an adjuvant to breast cancer therapy. However, the precise mechanisms of DHA’s anticancer activities are unclear. It is understood that the intercommunication between cancer cells and their microenvironment is essential to tumor angiogenesis. Exosomes are extracellular vesicles that are important mediators of intercellular communication and play a role in promoting angiogenesis. However, very little is known about the contribution of breast cancer exosomes to tumor angiogenesis or whether exosomes can mediate DHA’s anticancer action. Results: Exosomes were collected from MCF7 and MDA-MB-231 breast cancer cells after treatment with DHA. We observed an increase in exosome secretion and exosome microRNA contents from the DHA-treated cells. The expression of 83 microRNAs in the MCF7 exosomes was altered by DHA (>2-fold). The most abundant exosome microRNAs (let-7a, miR-23b, miR-27a/b, miR-21, let-7, and miR-320b) are known to have anti-cancer and/or anti-angiogenic activity. These microRNAs were also increased by DHA treatment in the exosomes from other breast cancer lines (MDA-MB-231, ZR751 and BT20), but not in exosomes from normal breast cells (MCF10A). When DHA-treated MCF7 cells were co-cultured with or their exosomes were directly applied to endothelial cell cultures, we observed an increase in the expression of these microRNAs in the endothelial cells. Furthermore, overexpression of miR-23b and miR-320b in endothelial cells decreased the expression of their pro-angiogenic target genes (PLAU, AMOTL1, NRP1 and ETS2) and significantly inhibited tube formation by endothelial cells, suggesting that the microRNAs transferred by exosomes mediate DHA’s anti-angiogenic action. These effects could be reversed by knockdown of the Rab GTPase, Rab27A, which controls exosome release. Conclusions: We conclude that DHA alters breast cancer exosome secretion and microRNA contents, which leads to the inhibition of angiogenesis. Our data demonstrate that breast cancer exosome signaling can be targeted to inhibit tumor angiogenesis and provide new insight into DHA’s anticancer action, further supporting its use in cancer therapy. Examination of small RNA populations in MCF7 cells and exosomes after DHA treatment.
2015-07-01 | E-GEOD-70432 | biostudies-arrayexpress
Project description:Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a critical regulator of cell growth by integrating multiple signals (nutrients, growth factors, energy and stress) and is frequently deregulated in many types of cancer. We used a robust experimental paradigm involving the combination of two interventions, one genetic and one pharmacologic to identify genes regulated transcriptionally by mTORC1. In Tsc2+/+, but not Tsc2-/- immortalized mouse embryo fibroblasts (MEFs), serum deprivation downregulates mTORC1 activity. In Tsc2-/- cells, abnormal mTORC1 activity can be downregulated by treatment with rapamycin (sirolimus). By contrast, rapamycin has little effect on mTORC1 in Tsc2+/+ cells in which mTORC1 is already inhibited by low serum. Thus, under serum deprived conditions, mTORC1 activity is low in Tsc2+/+ cells (untreated or rapamycin treated), high in Tsc2-/- cells, but lowered by rapamycin; a pattern referred to as a M-^Slow/low/high/lowM-^T or M-^SLLHLM-^T, which allowed the identification of genes regulated by mTORC1 by performing the appropriate comparisons
2011-08-02 | E-GEOD-27982 | biostudies-arrayexpress
Project description:To screen miRNAs specifically regulated by mTORC1 or mTORC2, a global miRNA expression profile in MCF-7 cells treated with rapamycin or PP242 (mTORC1/2 kinase inhibitor) was developed using microarray.
2014-09-11 | GSE61289 | GEO
Project description:Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a critical regulator of cell growth by integrating multiple signals (nutrients, growth factors, energy and stress) and is frequently deregulated in many types of cancer. We used a robust experimental paradigm involving the combination of two interventions, one genetic and one pharmacologic to identify genes regulated transcriptionally by mTORC1. In Tsc2+/+, but not Tsc2-/- immortalized mouse embryo fibroblasts (MEFs), serum deprivation downregulates mTORC1 activity. In Tsc2-/- cells, abnormal mTORC1 activity can be downregulated by treatment with rapamycin (sirolimus). By contrast, rapamycin has little effect on mTORC1 in Tsc2+/+ cells in which mTORC1 is already inhibited by low serum. Thus, under serum deprived conditions, mTORC1 activity is low in Tsc2+/+ cells (untreated or rapamycin treated), high in Tsc2-/- cells, but lowered by rapamycin; a pattern referred to as a “low/low/high/low” or “LLHL”, which allowed the identification of genes regulated by mTORC1 by performing the appropriate comparisons
2011-08-03 | GSE27982 | GEO
Project description:Amiloride ameliorates muscle wasting in cancer cachexia through inhibiting tumor-derived exosome release
| PRJNA724880 | ENA