Enhancement of dynamin polymerization and GTPase activity by Arc/Arg3.1.
Enhancement of dynamin polymerization and GTPase activity by Arc/Arg3.1.
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DOI：
10.1016/j.bbagen.2015.03.002
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发表时间：
2015-06
期刊：
影响因子：
--
通讯作者：
Albanesi JP
中科院分区：
文献类型：
--
作者：
Byers CE;Barylko B;Ross JA;Southworth DR;James NG;Taylor CA 4th;Wang L;Collins KA;Estrada A;Waung M;Tassin TC;Huber KM;Jameson DM;Albanesi JP
活性调控的细胞骨架相关蛋白Arc是一种即刻早期基因产物，与多种形式的突触可塑性有关。ARC促进AMPA型谷氨酸受体的内吞作用，并调节神经元树突中的细胞骨架组装。它在内吞作用中的作用可能是通过报道它与Dynamin 2(Dy2)的相互作用而介导的，Dynamin 2是一种100 kDa的GTP酶，聚合在萌芽小泡的颈部并催化膜断裂。在这项研究中，使用了酶分析和浊度分析来监测Arc对动力蛋白活性和聚合的影响。使用尺寸排除层析、沉淀物分析、动态光散射、荧光相关光谱和电子显微镜等多种方法来测量弧寡聚反应。我们提供的证据表明，细菌表达的His6-Arc促进了Dy2的聚合，并在生理条件下(37℃和100 mM的氯化钠)刺激了其GTP酶活性。在较低的离子强度下，Arc还可以稳定预先形成的Dy2聚合物，防止GTP依赖的拆解，从而延长Dy2催化的组装依赖的GTP水解。ARC还增加了DYN3的GTP酶活性，但不影响DYN1的活性，DYN3是参与树突重塑的一种亚型，而DY1是参与突触小泡循环的神经元特异性亚型。我们在这项研究中进一步表明，Arc(无论是His6标记的还是非标记的)有形成大的可溶低聚物的趋势，这些低聚物可能作为动力蛋白组装和激活的支架。Arc促进动力蛋白聚合和GTP酶激活的能力可能为解释Arc介导的AMPA受体内吞作用及其对突触可塑性的伴随效应提供了机制。这项研究首次详细描述了Arc的物理性质。
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. 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. 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. 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.
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