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. 2015 Jun;1850(6):1310-8.
doi: 10.1016/j.bbagen.2015.03.002. Epub 2015 Mar 14.

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

Christopher E Byers  1 , Barbara Barylko  1 , Justin A Ross  2 , Daniel R Southworth  3 , Nicholas G James  2 , Clinton A Taylor 4th  1 , Lei Wang  1 , Katie A Collins  4 , Armando Estrada  1 , Maggie Waung  4 , Tara C Tassin  1 , Kimberly M Huber  4 , David M Jameson  2 , Joseph P Albanesi  5
Affiliations

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

Christopher E Byers et al. Biochim Biophys Acta. 2015 Jun.

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, 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 dynamin 2 and stimulates its GTPase activity under physiologic conditions (37°C and 100mM NaCl). At lower ionic strength Arc also stabilizes pre-formed dynamin 2 polymers against GTP-dependent disassembly, thereby prolonging assembly-dependent GTP hydrolysis catalyzed by dynamin 2. Arc also increases the GTPase activity of dynamin 3, an isoform of implicated in dendrite remodeling, but does not affect the activity of dynamin 1, 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.

Keywords: Arc/Arg3.1; Dynamin; GTPase; Self-assembly.

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Figures

Figure 1
Figure 1. Interaction of Arc with Dyn2 as measured by co-sedimentation and turbidity
A. Binding of Arc to Dyn2 polymers. Polymerization of Dyn2 (final concentration of 1 μM) was induced by lowering the NaCl concentration from 300 mM to 75 mM, and incubating for 15 min at 22°C in the absence or presence of Arc (4 μM). Samples were then centrifuged at 214,000 × g for 15 min at 4°C. Pellets were resuspended in the initial sample volume and equal volumes of pellets (P) and supernatants (S) were subjected to SDS-PAGE and Coomassie blue staining. The right panel shows the results with 4 μM Arc alone. B. Time course of polymerization, measured by the absorbance at 330 nm, of Dyn2 (final concentration of 0.6 μM) upon dilution from 300 mM NaCl to 75 mM NaCl, in the absence of Arc (curve 3) and in the presence of 0.1 μM Arc (curve 2) or 1 μM Arc (curve 1). Curve 4 shows 1 μM Arc alone.
Figure 2
Figure 2. Enhancement of Dyn2 assembly by Arc
A. Absorbance traces showing Dyn2 polymerization upon reduction of NaCl concentration from 300 mM to 50 mM in the absence of Arc or in the presence of 0.5 μM or 1 μM Arc, as designated in the figure. B. Dyn2 polymerization in the presence of 1 μM Arc in buffer containing 75 mM NaCl. C. Dyn2 polymerization in the presence of 1 μM Arc in buffer containing 100 mM NaCl. Arrows designate addition of MgCl2 and GTP to final concentrations of 2 mM and 1 mM, respectively. In all cases, the Dyn2 concentration was 1 μM, the temperature was 37°C, and the absorbance was measured at 330 nm.
Figure 3
Figure 3. Stimulation of the GTPase activity of Dyn2 by Arc
Top panels show the time courses of GTP hydrolysis catalyzed by 1 μM Dyn2 in the absence (●) or presence (○) of 1 μM Arc, measured at 37°C in buffer containing 50 mM NaCl (A), 75 mM NaCl (B), or 100 mM NaCl (C). Bottom panels show the decrease in specific activities at each NaCl concentration over 1 min time courses in the absence (shaded bars) or presence (open bars) of Arc. Each bar represents the activity measured over the 15 seconds prior to the times designated on the abscissa. Less than 10% of substrate (GTP) was depleted in all measurements. Data represent the mean +/− SD of triplicate measurements from two experiments.
Figure 4
Figure 4. Stabilization of the high-activity state of Dyn2 by Arc
A. GTPase activity of Dyn2 (0.1 μM) was measured for various times in 50 mM NaCl at 37°C in the absence (●) or presence (○) of 0.5 μM Arc. Each value corresponds to the percent of maximal specific activity measured over the interval between time points. Under these assay conditions, the activities of Dyn2 in the absence and presence of Arc were ~25 min−1 and ~90 min−1, respectively. Data represent the mean +/− SD of triplicate measurements. B. GTPase activities of Dyn1 (●), Dyn2 (○), and Dyn3 (■) at concentrations of 0.1 μM, were measured for 20 min in 50 mM NaCl at 37°C as a function of Arc concentration.
Figure 5
Figure 5. Size-exclusion chromatography of Arc on a Superdex 200 column
A. Elution profile of untagged Arc (0.5 ml of 45 μM Arc in buffer B). Inset shows the Coomassie blue-stained gel of the loaded sample. Arrows above the profile designate standards: a. β-amylase (Stokes’ radius 5.4 nm), b. catalase (5.2 nm), c. BSA (3.6 nm), d. ovalbumin (3.1 nm), e. carbonic anhydrase (2.4 nm). Void volume and total volume of the column are 40 ml and 110 ml, respectively. B. Elution profile of fractions 44-54 from panel A (shaded), concentrated and then re-chromatographed on the same column. C and D. Elution profiles of 1 μM and 0.25 μM Arc, respectively (4-fold and 16-fold dilutions of the sample chromatographed in panel A).
Figure 6
Figure 6. Sucrose density gradient centrifugation of Arc
His6-Arc (20 μM, 0.4 ml) was layered above a 4 ml 10-40% sucrose gradient and centrifuged at 240,000 × g for 16 h at 4°C. Following centrifugation, fractions were subjected to SDS-PAGE and Coomassie blue staining (top panel). Standard proteins were centrifuged under identical conditions to generate a plot of sedimentation coefficient (S) as a function of sucrose concentration, as measured by refractometry (bottom panel). The data shown are representative of 4 measurements performed using two Arc preparations.
Figure 7
Figure 7. Hydrodynamic analysis of Arc
A. Sedimentation velocity analysis. Experiments were carried out in a Beckman XL-I analytical ultracentrifuge at 20°C, and 35,000 rpm and an Arc concentration of 36 μM. The figure shows UV scans (at 280 nm), taken every four min, from a representative run. B. Fluorescence correlation spectroscopy. Autocorrelation curve of 150 nM Arc-AF488 (grey circles) and fit to data (black line). The results of the fit were D1 = 22 μm2/s (G0 = 0.066), D2 = 430 μm2/s (G0 = 0.15). C. Dynamic Light Scattering. A solution of Arc (16 μM) was introduced into a Wyatt DynoPro DLS instrument and attached temperature Controlled MicroSampler pre-equilibrated to 4°C. The data shown are representative of six measurements, each consisting of thirty 10 second scans at 90% laser intensity. The resulting peak accounts for >99.7% of the mass and >90% of the intensity.
Figure 8
Figure 8. Imaging of Arc by transmission electron microscopy
His6-Arc was negatively stained and imaged at 50,000 x magnification as described in Materials and Methods. Bars (in panels A and B) and box sizes (in panel C) designate dimensions in nm.
Figure 9
Figure 9. Identification of Dyn2 activation determinants in Arc
GTPase assays, performed as described in Figure 4, were carried out in the presence of full-length Arc (residues 1-396), a fragment lacking the putative Dyn2 binding site (Arc-Δ195-214), an N-terminal fragment comprising residues 1- 227 (Arc-N), and a C-terminal fragment comprising residues 228-396 (Arc-C). A scheme of the fragments tested is shown below.
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