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Molecular determinants of Arc oligomerization and formation of virus-like capsids

Maria Steene Eriksen, Oleksii Nikolaienko, Erik Ingmar Hallin, Sverre Grødem, Helene J. Bustad, Marte Innselset Flydal, Rory O’Connell, Tomohisa Hosokawa, Daniela Lascu, Shreeram Akerkar, Jorge Cuéllar, James J. Chambers, Gopinath Muruganandam, Remy Loris, Tambudzai Kanhema, Yasunori Hayashi, Margaret M. Stratton, José M. Valpuesta, Petri Kursula, Aurora Martinez, View ORCID ProfileClive R. Bramham
doi: https://doi.org/10.1101/667956
Maria Steene Eriksen
1Department of Biomedicine, University of Bergen, Bergen, Norway
2KG Jebsen Centre for Neuropsychiatric Disorders, University of Bergen, Bergen, Norway
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Oleksii Nikolaienko
1Department of Biomedicine, University of Bergen, Bergen, Norway
2KG Jebsen Centre for Neuropsychiatric Disorders, University of Bergen, Bergen, Norway
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Erik Ingmar Hallin
1Department of Biomedicine, University of Bergen, Bergen, Norway
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Sverre Grødem
1Department of Biomedicine, University of Bergen, Bergen, Norway
2KG Jebsen Centre for Neuropsychiatric Disorders, University of Bergen, Bergen, Norway
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Helene J. Bustad
1Department of Biomedicine, University of Bergen, Bergen, Norway
2KG Jebsen Centre for Neuropsychiatric Disorders, University of Bergen, Bergen, Norway
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Marte Innselset Flydal
1Department of Biomedicine, University of Bergen, Bergen, Norway
2KG Jebsen Centre for Neuropsychiatric Disorders, University of Bergen, Bergen, Norway
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Rory O’Connell
3Department of Biochemistry and Molecular Biology, University of Massachusetts Amherst, Amherst MA, USA
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Tomohisa Hosokawa
4Department of Pharmacology, Kyoto University Graduate School of Medicine, Kyoto, Japan
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Daniela Lascu
1Department of Biomedicine, University of Bergen, Bergen, Norway
2KG Jebsen Centre for Neuropsychiatric Disorders, University of Bergen, Bergen, Norway
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Shreeram Akerkar
1Department of Biomedicine, University of Bergen, Bergen, Norway
2KG Jebsen Centre for Neuropsychiatric Disorders, University of Bergen, Bergen, Norway
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Jorge Cuéllar
5Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, 28049 Madrid, Spain
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James J. Chambers
6Institute for Applied Life Sciences, University of Massachusetts, Amherst, MA, USA
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Gopinath Muruganandam
7VIB-VUB Center for Structural Biology, Vlaams Instituut voor Biotechnologie, 1050 Brussels, Belgium
8Structural Biology Brussels, Department of Bioengineering Sciences, Vrije Universiteit Brussel, 1050 Brussels, Belgium
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Remy Loris
7VIB-VUB Center for Structural Biology, Vlaams Instituut voor Biotechnologie, 1050 Brussels, Belgium
8Structural Biology Brussels, Department of Bioengineering Sciences, Vrije Universiteit Brussel, 1050 Brussels, Belgium
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Tambudzai Kanhema
1Department of Biomedicine, University of Bergen, Bergen, Norway
2KG Jebsen Centre for Neuropsychiatric Disorders, University of Bergen, Bergen, Norway
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Yasunori Hayashi
4Department of Pharmacology, Kyoto University Graduate School of Medicine, Kyoto, Japan
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Margaret M. Stratton
3Department of Biochemistry and Molecular Biology, University of Massachusetts Amherst, Amherst MA, USA
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José M. Valpuesta
5Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, 28049 Madrid, Spain
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Petri Kursula
1Department of Biomedicine, University of Bergen, Bergen, Norway
9Faculty of Biochemistry and Molecular Biology, University of Oulu, Oulu, Finland
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Aurora Martinez
1Department of Biomedicine, University of Bergen, Bergen, Norway
2KG Jebsen Centre for Neuropsychiatric Disorders, University of Bergen, Bergen, Norway
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Clive R. Bramham
1Department of Biomedicine, University of Bergen, Bergen, Norway
2KG Jebsen Centre for Neuropsychiatric Disorders, University of Bergen, Bergen, Norway
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  • ORCID record for Clive R. Bramham
  • For correspondence: clive.bramham@uib.no
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ABSTRACT

Expression of activity-regulated cytoskeleton-associated protein (Arc) is critical for long-term synaptic plasticity, memory formation, and cognitive flexibility. The ability of Arc to self-associate and form virus-like capsid structures implies functionally distinct oligomeric states. However, the molecular mechanism of Arc oligomerization is unknown. Here, we identified a 28-amino-acid region necessary and sufficient for Arc oligomerization. This oligomerization region is located within the second coil of a predicted anti-parallel coiled-coil in the N-terminal domain (NTD). Using alanine scanning mutagenesis, we found a 7-amino-acid motif critical for oligomerization and Arc-mediated transferrin endocytosis in HEK cells. Intermolecular fluorescence lifetime imaging in hippocampal neurons confirmed self-association mediated by the motif. To quantify oligomeric size, we performed a single-molecule photobleaching analysis of purified Arc wild-type and mutant. This analysis revealed a critical role for the NTD motif in the formation of higher-order Arc oligomers (30-170 molecules). Moreover, assembly of higher-order wild-type Arc oligomers was significantly enhanced by addition of GFP RNA. Purified wild-type Arc formed virus-like capsids, as visualized by negative-stain EM, and was estimated by light scattering analysis to contain 40-55 Arc units. In contrast, mutant Arc formed a homogenous dimer population as demonstrated by single-molecule TIRF imaging, size-exclusion chromatography with multi-angle light scattering analysis, small-angle X-ray scattering analysis, and single-particle 3D EM reconstruction. Thus, the dimer appears to be the basic building block for assembly. Herein, we show that the NTD motif is essential for higher-order Arc oligomerization, assembly of virus-like capsid particles, and facilitation of oligomerization by exogenous RNA.

SIGNIFICANCE Arc protein is rapidly expressed in neurons in response to synaptic activity and plays critical roles in synaptic plasticity, postnatal cortical developmental, and memory. Arc has diverse molecular functions, which may be related to distinct oligomeric states of the protein. Arc has homology to retroviral Gag protein and self-assembles into retrovirus-like capsid structures that are capable of intercellular transfer of RNA. Here, we identified a motif in the N-terminal coiled-coil domain of mammalian Arc that mediates higher-order oligomerization and formation of virus-like capsids. The basic building block is the Arc dimer and exogenous RNA facilitates further assembly. The identified molecular determinants of Arc oligomerization will help to elucidate the functional modalities of Arc in the mammalian brain.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted June 12, 2019.
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Molecular determinants of Arc oligomerization and formation of virus-like capsids
Maria Steene Eriksen, Oleksii Nikolaienko, Erik Ingmar Hallin, Sverre Grødem, Helene J. Bustad, Marte Innselset Flydal, Rory O’Connell, Tomohisa Hosokawa, Daniela Lascu, Shreeram Akerkar, Jorge Cuéllar, James J. Chambers, Gopinath Muruganandam, Remy Loris, Tambudzai Kanhema, Yasunori Hayashi, Margaret M. Stratton, José M. Valpuesta, Petri Kursula, Aurora Martinez, Clive R. Bramham
bioRxiv 667956; doi: https://doi.org/10.1101/667956
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Molecular determinants of Arc oligomerization and formation of virus-like capsids
Maria Steene Eriksen, Oleksii Nikolaienko, Erik Ingmar Hallin, Sverre Grødem, Helene J. Bustad, Marte Innselset Flydal, Rory O’Connell, Tomohisa Hosokawa, Daniela Lascu, Shreeram Akerkar, Jorge Cuéllar, James J. Chambers, Gopinath Muruganandam, Remy Loris, Tambudzai Kanhema, Yasunori Hayashi, Margaret M. Stratton, José M. Valpuesta, Petri Kursula, Aurora Martinez, Clive R. Bramham
bioRxiv 667956; doi: https://doi.org/10.1101/667956

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