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A method to rapidly create protein aggregates in living cells

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      Abstract

      The accumulation of protein aggregates is a common pathological hallmark of many neurodegenerative diseases. However, we do not fully understand how aggregates are formed or the complex network of chaperones, proteasomes and other regulatory factors involved in their clearance. Here, we report a chemically controllable fluorescent protein that enables us to rapidly produce small aggregates inside living cells on the order of seconds, as well as monitor the movement and coalescence of individual aggregates into larger structures. This method can be applied to diverse experimental systems, including live animals, and may prove valuable for understanding cellular responses and diseases associated with protein aggregates.

      Abstract

      Protein aggregates are associated with a wide variety of diseases. Here, in order to address how protein aggregation affects cellular homoeostasis, the authors describe a method to rapidly create protein aggregates in living cells and organisms with precise spatial and temporal control.

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      Most cited references 26

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          Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences.

          We describe a dominant behavioral marker, rol-6(su-1006), and an efficient microinjection procedure which facilitate the recovery of Caenorhabditis elegans transformants. We use these tools to study the mechanism of C.elegans DNA transformation. By injecting mixtures of genetically marked DNA molecules, we show that large extrachromosomal arrays assemble directly from the injected molecules and that homologous recombination drives array assembly. Appropriately placed double-strand breaks stimulated homologous recombination during array formation. Our data indicate that the size of the assembled transgenic structures determines whether or not they will be maintained extrachromosomally or lost. We show that low copy number extrachromosomal transformation can be achieved by adjusting the relative concentration of DNA molecules in the injection mixture. Integration of the injected DNA, though relatively rare, was reproducibly achieved when single-stranded oligonucleotide was co-injected with the double-stranded DNA.
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            Author and article information

            Affiliations
            [1 ]Department of Chemical & Systems Biology Stanford University , Stanford, California 94305, USA
            [2 ]Department of Biology Stanford University , Stanford, California 94305, USA
            [3 ]Cell Sciences Imaging Facility Stanford University , Stanford, California 94305, USA
            Author notes
            [*]

            Present address: Department of Zoology, The University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3

            [†]

            Present address: UCL-MRC Lab for Molecular Cell Biology, London WC1E 6BT, UK.

            Journal
            Nat Commun
            Nat Commun
            Nature Communications
            Nature Publishing Group
            2041-1723
            27 May 2016
            2016
            : 7
            27229621
            4894968
            ncomms11689
            10.1038/ncomms11689
            Copyright © 2016, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

            This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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