Department of Biomedicine, University of Bergen, Jonas Lies 91, 5009, Bergen, Norway.
Department of Pathophysiology and Metabolism, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama, 701-0192, Japan.
Neurochem Res. 2022 Sep;47(9):2656-2666. doi: 10.1007/s11064-022-03573-5. Epub 2022 Mar 20.
Activity-regulated cytoskeleton-associated (Arc) protein plays key roles in long-term synaptic plasticity, memory, and cognitive flexibility. However, an integral understanding of Arc mechanisms is lacking. Arc is proposed to function as an interaction hub in neuronal dendrites and the nucleus, yet Arc can also form retrovirus-like capsids with proposed roles in intercellular communication. Here, we sought to develop anti-Arc nanobodies (ArcNbs) as new tools for probing Arc dynamics and function. Six ArcNbs representing different clonal lines were selected from immunized alpaca. Immunoblotting with recombinant ArcNbs fused to a small ALFA-epitope tag demonstrated binding to recombinant Arc as well as endogenous Arc from rat cortical tissue. ALFA-tagged ArcNb also provided efficient immunoprecipitation of stimulus-induced Arc after carbachol-treatment of SH-SY5Y neuroblastoma cells and induction of long-term potentiation in the rat dentate gyrus in vivo. Epitope mapping showed that all Nbs recognize the Arc C-terminal region containing the retroviral Gag capsid homology domain, comprised of tandem N- and C-lobes. ArcNbs E5 and H11 selectively bound the N-lobe, which harbors a peptide ligand binding pocket specific to mammals. Four additional ArcNbs bound the region containing the C-lobe and C-terminal tail. For use as genetically encoded fluorescent intrabodies, we show that ArcNbs fused to mScarlet-I are uniformly expressed, without aggregation, in the cytoplasm and nucleus of HEK293FT cells. Finally, mScarlet-I-ArcNb H11 expressed as intrabody selectively bound the N-lobe and enabled co-immunoprecipitation of full-length intracellular Arc. ArcNbs are versatile tools for live-cell labeling and purification of Arc, and interrogation of Arc capsid domain specific functions.
活性调节细胞骨架相关蛋白（Arc）在长时程突触可塑性、记忆和认知灵活性中发挥关键作用。然而，对 Arc 机制的整体理解还很缺乏。Arc 被认为在神经元树突和细胞核中作为相互作用的枢纽发挥作用，但 Arc 也可以形成具有细胞间通讯作用的逆转录病毒样衣壳。在这里，我们试图开发针对 Arc 的纳米抗体（ArcNbs）作为研究 Arc 动力学和功能的新工具。从免疫的羊驼中选择了六个代表不同克隆系的 ArcNbs。用融合了小的 ALFA-表位标签的重组 ArcNbs 进行免疫印迹显示，这些抗体与重组 Arc 以及来自大鼠皮质组织的内源性 Arc 结合。ALFA 标记的 ArcNb 还可以有效地免疫沉淀乙酰胆碱酯酶刺激后的 SH-SY5Y 神经母细胞瘤细胞中的诱导型 Arc，以及体内大鼠齿状回的长时程增强。表位作图显示，所有 Nbs 都识别包含逆转录病毒 Gag 衣壳同源结构域的 Arc C 末端区域，该结构域由串联的 N 端和 C 端叶组成。ArcNbs E5 和 H11 选择性地结合 N 端叶，该叶含有一个特定于哺乳动物的肽配体结合口袋。另外四个 ArcNbs 结合包含 C 端叶和 C 末端尾巴的区域。为了用作遗传编码的荧光内抗体，我们表明，融合到 mScarlet-I 的 ArcNbs 在 HEK293FT 细胞的细胞质和细胞核中均匀表达，没有聚集。最后，作为内抗体表达的 mScarlet-I-ArcNb H11 选择性地结合 N 端叶，并能与全长细胞内 Arc 进行共免疫沉淀。ArcNbs 是活细胞标记和纯化 Arc 的多功能工具，也是研究 Arc 衣壳结构域特定功能的工具。
