Varsity Sci - Prof Randy Schekman - "Selective protein sorting into exosomes" скачать в хорошем качестве

Varsity Sci - Prof Randy Schekman - "Selective protein sorting into exosomes"

Title: "Selective protein sorting into exosomes: A role in cell differentiation and a possible tool in genome editing" Sign up: https://forms.gle/pzFvgxEauhrTiAyQ9 We are honoured to introduce our Keynote Speaker for the Biology Session, Professor Randy Schekman! Prof. Schekman is an investigator of the Howard Hughes Medical Institute and a Professor of Cell and Developmental Biology in the Department of Molecular and Cell Biology at the University of California at Berkeley. His work has attracted many accolades, including election as a Member of the National Academy of Sciences in 1992, the Albert Lasker Award for Basic Medical Research in 2002 and, most notably, the 2013 Nobel Prize in Physiology or Medicine "for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells." Here is the abstract for the keynote talk: "Extracellular vesicles (EVs) are thought to mediate the transfer of cytoplasmic proteins and RNA between cells to inform the processes of differentiation, cell motility and possibly malignant transformation. We have found that undifferentiated or embryonic stem cells (ESCs) which are induced to differentiate into neural progenitor cells secrete EVs that selectively capture proteins implicated in G1/S cell cycle progression. Further, we have found that these EVs promote the differentiation of ESCs to a neural progenitor fate and that cyclin D1 plays a rate- limiting role in that EV-mediated progression. A challenge in genome editing in vivo is to devise an efficient means of delivering editing functions, preferably by a vehicle that evades immune detection. We sought a means to deliver Cas9 and a gRNA enclosed within exosomes, a subclass of EVs, as a vehicle for efficient and targeted gene editing. Cas9 was expressed in a donor cell tethered noncovalently to an integral membrane protein, CD63, enriched in exosomes. Exosomes highly enriched in Cas9 and a gRNA were isolated by buoyant density sedimentation. Isolated exosomes were incubated with reporter cells containing an integrated copy of N-luciferase behind a site which when edited would allow the expression of luciferase. In a control experiment, expression of the Cas9/gRNA construct directly in the reporter cell elicited a 60-70 fold increase in luciferase expression. Exosomes containing a similar level of Cas9 elicited no more than a 50% increase above the background of luciferase. The same was true of conditioned medium containing Cas9-exosomes and even of donor and acceptor cells incubated together separated by a vesicle-permeable membrane in a transwell chamber. Thus, for these EVs, the functional uptake to promote gene expression was not observed as we found for those isolated from differentiating neurons. In contrast, donor and acceptor cells cocultured to near confluence showed a 60-fold increase in luciferase expression. Transfer of Cas9 appears to be mediated by open-end membrane tubular connections, likely dependent on membrane fusion at the point of junction between a tubule from one cell and the target. A molecular dissection for the requirements for this transfer may permit the development of an efficient means for targeted delivery of Cas9/gRNA."