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

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...

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Published inbioRxiv
Main Authors Maria Steene Eriksen, Nikolaienko, Oleksii, Erik Ingmar Hallin, Grødem, Sverre, Bustad, Helene J, Flydal, Marte Innselset, O'connell, Rory, Hosokawa, Tomohisa, Lascu, Daniela, Akerkar, Shreeram, Cuéllar, Jorge, Chambers, James J, Merski, Ian, Muruganandam, Gopinath, Remy Loris, Kanhema, Tambudzai, Hayashi, Yasunori, Stratton, Margaret M, Valpuesta, José M, Kursula, Petri, Martinez, Aurora, Bramham, Clive R
Format Paper
LanguageEnglish
Published Cold Spring Harbor Cold Spring Harbor Laboratory Press 22.11.2019
Subjects
Alanine
ARC protein
Capsids
Cognitive ability
Cytoskeleton
Endocytosis
Hippocampus
Light scattering
Oligomerization
Photobleaching
Ribonucleic acid
RNA
Scanning mutagenesis
Self-association
Synaptic plasticity
Transferrins
X-ray scattering
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Summary: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. Footnotes * Author list updated
DOI:10.1101/667956