Development and validation of Arc nanobodies: new tools for probing Arc dynamics and function

Abstract

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-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 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 analysis of capsid domain specific functions.