Abstracts from the Fourth International Meeting of ISEV, ISEV2. Washington, D. C., USA, 2.
April 2. 01. 5J Extracell Vesicles. Copyright © 2. 01. Journal of Extracellular Vesicles. This is an Open Access article distributed under the terms of the Creative Commons Attribution- Non. Commercial 4. 0 International License, permitting all non- commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Funding statement. Funding for this conference was made possible in part by R1. TR0. 01. 27. 8- 0. PI: Witwer, Kenneth) from the NIH Common Fund, through the Office of Strategic Coordination/Office of the NIH Director and National Center for Advancing Translational Sciences (NCATS).
The views expressed in written conference materials or publications and by speakers and moderators do not necessarily reflect the official policies of the Department of Health and Human Services; nor does mention by trade names, commercial practices, or organizations imply endorsement by the U. S. Government. Funding for this conference was provided in part by a grant from the National Science Foundation (PI: Jay, Steven). Scientific Program ISEV 2. Thursday April 2.
Oral Presentations. Ballroom DSymposium session 1. A - EV biogenesis IChairs: Clotilde Théry and Stephen J. Gould 1. 3: 1. 5- 1. O- 1. A- 1. Apo. E regulates ESCRT- independent sorting on exosomes and endosomal amyloid formation. Guillaume Van Niel.
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Ptissam Bergam. 1, Aurelie Di Cicco. Ilse Hurbain. 1, Alessandra Lo Cicero. Cecile Fort. 1, Florent Dingli. Marie- Claude Potier. Leon Schurgers. 5, Damarys Loew.
Daniel Levy. 2 and Graça Raposo. Department of Cell Biology, Curie Institute, Paris, France; 2. Department of Physical Chemistry, Curie Institute, Paris, France; 3. Department of Mass Spectrometry, Curie Institute, Paris, France; 4.
CNRS UMR7. 22. 5, INSERM U1. UPMC, Institut du Cerveau et de la Moelle, Paris, France; 5. Department of Biochemistry–Vascular Aspects, Faculty of Medicine, Health & Life Science, Maastricht University, Maastricht, The Netherlands Introduction: Exosomes are generated within secretory multivesicular endosomes (MVEs) as intraluminal vesicles (ILVs). To serve specific cellular functions, notably once secreted, ILVs are enriched with defined sets of proteins by various and still elusive sorting mechanisms within MVEs. Pigment cells have tuned their MVEs to produce amyloid fibrils derived from the protein PMEL.
For this purpose, PMEL – the first protein reported as an ESCRT- independent cargo – is sorted in a CD6. ILVs that likely serve as potential seeding platforms for PMEL amyloidogenesis. Contrary to amyloids such as those associated with Alzheimer's disease, PMEL amyloids are non- toxic and are functional as they serve as a scaffolding structure for the synthesis of melanin. To better understand the mechanisms exploited on ILVs to avoid potential toxicity during PMEL amyloidogenesis, we have used exosomes as reporters of these endosomal processes.
Methods: For this purpose, we have characterized exosomes derived from pigment cells by cryo- electron microscopy, mass spectrometry and western blot. We then investigated the role of the intracellular counterparts of exosomes, ILVS in cell lines and in vivo using si. RNA, western blotting and morphological analysis by electron microscopy. Results: Characterization of exosomes derived from pigment cells revealed the association of exosomes and ILVs with apolipoprotein E (Apo. E) and lipoparticles. We could show that Apo. E is targeted to endosomes in a CD6.
ESCRT- independent manner and facilitates the ESCRT- independent sorting of PMEL amyloidogenic fragments onto ILVs. At the surface of ILVs, Apo.
E regulates the formation of mature fibrils in melanocytic cell lines and in pigment cells in vivo. Summary/conclusion: These results established a clear molecular mechanism for ESCRT- independent sorting of PMEL. Moreover, the novel evidence that lipoparticles are associated to exosomes provides a breakthrough that might be exploited to reconsider the respective roles of each extracellular particle in pathologies. Finally our study establishes a paradigm for the mechanism by which Apo. E, the first genetic risk for early onset Alzheimer's disease, regulates the assembly of mature amyloid fibrils under benign and pathological conditions. O- 1. A- 2. Prion protein regulates autophagy and endocytic trafficking with essential roles for the biogenesis of extracellular vesicles. Marcos Dias, Bianca Teixeira, Bruna Rodrigues, Camila Arantes.
Martin Roffe, Glaucia Hajj and Vilma Martins. International Research Center, AC Camargo Cancer Center, Sao Paulo, Brazil Introduction: Prion protein (Pr. PC) plays important roles in neuronal physiology and in tumour biology. Primary cultures from Pr. PC knockout astrocytes (Prnp.
CM) of these cells were reduced when compared to wild- type (WT) astrocytes (Lima et al., J Neurochem. Thus, suggesting that Pr. PC regulates extracellular vesicles (EV) release. Methods: EVs were isolated using ultracentrifugation and their size and concentrations analyzed by NTA. Electron and confocal microscopies and biochemical analysis were performed to evaluate EV and endocytic components. RNA was used to knock down beclin. Results: Prnp. 0/0 astrocytes secrete lower EVs in CM than WT cells whilst the reconstitution of Pr.
PC expression in Prnp. EV secretion. In astrocytes overexpressing Pr. PC, the number of secreted EVs is higher than those secreted by WT cells. The same differences were observed in CM from primary fibroblasts cultures and in the blood circulation of these mice. The absence of Pr. PC causes a delay in caveolin- mediated endocytosis and in the traffic of EGF- EGFR to late endosomes/multivesicular bodies (LE/MVB).
Prnp. 0/0 astrocytes present altered MVB formation, LAMP1- stained lysosomal compartments were enlarged and shown an increased number of autophagosomes when compared with WT cells. Autophagy inhibition by beclin. EVs released by Prnp. Conversely, autophagy induction by serum starvation or rapamycin treatment inhibited exosome release in WT cells. Summary/conclusion: Pr. PC modulates endocytic pathways and impairs autophagy. In the absence of Pr.
PC, endosomes can be directed to fuse with autophagosomes contributing to the impairment in EVs biogenesis/release. The cellular levels of Pr. PC can regulate the amount of secreted vesicles with major roles in heath and disease. O- 1. A- 3. Rabs involved in extracellular vesicle biogenesis each affect multiple different steps in endolysosomal and secretory trafficking in Drosophila secondary cells. Clive Wilson. 1, Siamak Redhai.
Ben Kroeger. 1, Laura Corrigan. Shih- Jung Fan. 1, Mark Wainwright. Ian Dobbie. 2, Aaron Leiblich. Carina Gandy. 1, John Morris. Freddie Hamdy. 3 and Deborah Goberdhan. Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom; 2.
Department of Biochemistry, University of Oxford, Oxford, United Kingdom; 3. Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom Introduction: We have recently characterized a new in vivo system to dissect the role of the endolysosomal and secretory pathways in regulating the biogenesis of extracellular vesicles. It takes advantage of the Drosophila male accessory gland (AG), which shares similarities with the human prostate. A small subset of epithelial cells within the AG called secondary cells (SCs) specifically produces vesicles, which can be marked by a GFP- tagged form of the putative exosome marker, human CD6. CD6. 3- positive vesicles are made inside giant (~5 µm diameter) endolysosomal compartments and secreted into the AG lumen in a process that requires the function of ESCRTs like ALi. X and Hrs, and of different Rabs associated with mammalian exosome secretion, such as Rab.
Rab. 27 and Rab. 35. We investigated how these Rabs alter membrane trafficking within SCs to affect extracellular vesicle secretion.
Methods: We have used molecular genetic approaches in the fly combined with both confocal and super- resolution (3. D- SIM) microscopy to determine how knockdown of specific Rabs alters trafficking and vesicle biogenesis pathways in SCs. Results: Knockdown of Rab. Rab. 27 or Rab. 35 with at least two independent RNAi molecules affects EV secretion.
However, each Rab has very different effects on SC intracellular compartments.