Development and Validation of Arc Nanobodies: New Tools for Probing Arc Dynamics and Function

Area of Science:

• Neuroscience
• Molecular Biology
• Protein Engineering

Background:

• Activity-regulated cytoskeleton-associated (Arc) protein is crucial for synaptic plasticity, memory, and cognitive flexibility.
• Understanding Arc's diverse functions, including its role as an interaction hub and in forming retrovirus-like capsids, remains incomplete.

Purpose of the Study:

• To develop anti-Arc nanobodies (ArcNbs) as novel tools for investigating Arc protein dynamics and functions.
• To characterize the binding specificities and applications of these ArcNbs.

Main Methods:

• Six anti-Arc nanobodies (ArcNbs) were generated from immunized alpacas.
• ArcNbs were tested for binding to recombinant and endogenous Arc via immunoblotting and immunoprecipitation.
• Epitope mapping was performed to identify the Arc regions recognized by the ArcNbs.
• Genetically encoded fluorescent intrabodies were created by fusing ArcNbs to mScarlet-I.

Main Results:

• Developed ArcNbs that successfully bind both recombinant and endogenous Arc protein.
• Demonstrated efficient immunoprecipitation of stimulus-induced Arc in cell culture and in vivo models.
• Epitope mapping revealed that ArcNbs target the Arc C-terminal region, with some specific to the N-lobe.
• Fluorescently tagged ArcNbs (mScarlet-I-ArcNb) showed uniform expression and enabled live-cell labeling and co-immunoprecipitation of intracellular Arc.

Conclusions:

• Anti-Arc nanobodies (ArcNbs) are versatile tools for live-cell labeling, purification, and functional studies of Arc protein.
• ArcNbs targeting specific domains, like the N-lobe, can help elucidate the distinct functions of Arc's capsid domain.
• This work provides new reagents for advancing research into Arc's roles in neuronal function and cognitive processes.