Enhancement of dynamin polymerization and GTPase activity by Arc/Arg3.1

Abstract

BACKGROUND The Activity-regulated cytoskeleton-associated protein, Arc, is an immediate-early gene product implicated in various forms of synaptic plasticity. Arc promotes endocytosis of AMPA type glutamate receptors and regulates cytoskeletal assembly in neuronal dendrites. Its role in endocytosis may be mediated by its reported interaction with dynamin 2 (Dyn2), a 100 kDa GTPase that polymerizes around the necks of budding vesicles and catalyzes membrane scission. METHODS Enzymatic and turbidity assays are used in this study to monitor effects of Arc on dynamin activity and polymerization. Arc oligomerization is measured using a combination of approaches, including size exclusion chromatography, sedimentation analysis, dynamic light scattering, fluorescence correlation spectroscopy, and electron microscopy. RESULTS We present evidence that bacterially-expressed His6-Arc facilitates the polymerization of Dyn2 and stimulates its GTPase activity under physiologic conditions (37°C and 100 mM NaCl). At lower ionic strength Arc also stabilizes pre-formed Dyn2 polymers against GTP-dependent disassembly, thereby prolonging assembly-dependent GTP hydrolysis catalyzed by Dyn2. Arc also increases the GTPase activity of Dyn3, an isoform of implicated in dendrite remodeling, but does not affect the activity of Dyn1, a neuron-specific isoform involved in synaptic vesicle recycling. We further show in this study that Arc (either His6-tagged or untagged) has a tendency to form large soluble oligomers, which may function as a scaffold for dynamin assembly and activation. CONCLUSIONS and GENERAL SIGNIFICANCE The ability of Arc to enhance dynamin polymerization and GTPase activation may provide a mechanism to explain Arc-mediated endocytosis of AMPA receptors and the accompanying effects on synaptic plasticity. This study represents the first detailed characterization of the physical properties of Arc.

Structure of monomeric full-length ARC sheds light on molecular flexibility, protein interactions, and functional modalities

“…In order to facilitate structural studies on full-length ARC and its domains, we set up recombinant expression systems for a number of ARC constructs ( Figure 1A, Table 1, Table S1). Bacterially expressed full-length ARC was insoluble under a variety of conditions, as reported before (Byers et al 2015;Myrum et al 2015). However, high pH made ARC soluble and suitable for structural analyses.…”

“…ARC has been proposed to alternate between monomeric and oligomeric/aggregated forms (Byers et al 2015;Myrum et al 2015). Recently, ARC was shown to build virus-like capsid structures (Ashley et al 2018;Pastuzyn et al 2018), which are packed into membrane vesicles.…”

“…Full-length recombinant ARC is capable of reversible self-oligomerization in water, while in salt-containing buffers, it is prone to aggregation (Byers et al 2015;Myrum et al 2015). Recently, ARC was shown to form virus-like capsid structures; these capsids contain RNA and are released from neurons in extracellular vesicles (Ashley et al 2018;Pastuzyn et al 2018). Alternating oligomeric states may have functional relevance in ARC signalling and capsid formation, but the details of such mechanisms at the molecular level are lacking.…”

c‐Fos and Arc/Arg3.1 expression in auditory and visual cortices after hearing loss: Evidence of sensory crossmodal reorganization in adult rats

“…Arc/Arg3.1, is an immediate early gene related to excitatory glutamatergic neurotransmission through synaptic regulation of AMPA glutamate receptor trafficking and internalization (Ju et al, ; Chowdhury et al, ; Derkach, Oh, Guire, & Soderling, ; Bramham et al, ; Byers et al, ). Arc/Arg3.1‐mediated activity‐dependent endocytosis of AMPA GluR2 subunits, and the subsequent reduction of AMPA receptor mediated currents have been shown in hippocampal slices (Rial Verde, Lee‐Osbourne, Worley, Malinow, & Cline, ).…”

“…c‐Fos is a powerful neuronal functional marker previously used by us to analyze time‐dependent changes in activity in the inferior colliculus after ablation of the AC (Clarkson, Juíz, & Merchán, ). The immediate early gene Arc/Arg3.1 is a key molecular tool for cortical plastic adaptations, which is involved in the turnover of fast excitatory AMPA glutamate receptor subunits, among other functions (Bramham, Worley, Moore, & Guzowski, ; Byers et al, ; Chowdhury et al, ; Derkach et al, ; Ju et al, ). Thus, after performing an immunocytochemical analysis of the activity‐dependent immediate early genes c‐Fos and Arc/Arg3.1, we suggest that spatiotemporal changes in the expression of their protein products in adult rat cortical neurons predict novel, long‐term cellular mechanisms of cross‐modal adaptation to deafness.…”