Byers Christopher E, Barylko Barbara, Ross Justin A, Southworth Daniel R, James Nicholas G, Taylor Clinton A, Wang Lei, Collins Katie A, Estrada Armando, Waung Maggie, Tassin Tara C, Huber Kimberly M, Jameson David M, Albanesi Joseph P
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390, United States.
Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 9681, United States.
Biochim Biophys Acta. 2015 Jun;1850(6):1310-8. doi: 10.1016/j.bbagen.2015.03.002. Epub 2015 Mar 14.
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.
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 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.
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.
活性调节细胞骨架相关蛋白Arc是一种即早基因产物，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。其在内吞作用中的作用可能是通过其与发动蛋白2的相互作用介导的，发动蛋白2是一种100 kDa的GTP酶，它围绕出芽囊泡的颈部聚合并催化膜分裂。
本研究使用酶法和浊度测定法来监测Arc对发动蛋白活性和聚合作用的影响。使用多种方法组合测量Arc寡聚化，包括尺寸排阻色谱法、沉降分析、动态光散射、荧光相关光谱法和电子显微镜。
我们提供的证据表明，细菌表达的His6-Arc在生理条件下（37°C和100mM NaCl）促进发动蛋白2的聚合并刺激其GTP酶活性。在较低离子强度下，Arc还能稳定预先形成的发动蛋白2聚合物，防止其依赖GTP的解聚，从而延长发动蛋白2催化的依赖组装的GTP水解。Arc还增加了发动蛋白3的GTP酶活性，发动蛋白3是一种与树突重塑有关的同工型，但不影响发动蛋白1的活性，发动蛋白1是一种参与突触小泡循环的神经元特异性同工型。我们在本研究中进一步表明，Arc（His6标记或未标记）倾向于形成大的可溶性寡聚体，这可能作为发动蛋白组装和激活的支架。
Arc增强发动蛋白聚合和GTP酶激活的能力可能提供一种机制，来解释Arc介导的AMPA受体内吞作用以及对突触可塑性的伴随影响。
