A toolbox of nanobodies developed and validated for use as intrabodies and nanoscale immunolabels in brain neurons
- University of California, Davis, United States
- University of Washington, United States
- Stanford School of Medicine, United States
Abstract
Nanobodies (nAbs) are small, minimal antibodies that have distinct attributes that make them uniquely suited for certain biomedical research, diagnostic and therapeutic applications. Prominent uses include as intracellular antibodies or intrabodies to bind and deliver cargo to specific proteins and/or subcellular sites within cells, and as nanoscale immunolabels for enhanced tissue penetration and improved spatial imaging resolution. Here, we report the generation and validation of nAbs against a set of proteins prominently expressed at specific subcellular sites in mammalian brain neurons. We describe a novel hierarchical validation pipeline to systematically evaluate nAbs isolated by phage display for effective and specific use as intrabodies and immunolabels in mammalian cells including brain neurons. These nAbs form part of a robust toolbox for targeting proteins with distinct and highly spatially-restricted subcellular localization in mammalian brain neurons, allowing for visualization and/or modulation of structure and function at those sites.
Data availability
All data generated or analyzed during this study are included in the manuscript and supporting files.
Article and author information
Author details
L Fernando Santana
Funding
National Institutes of Health (U01NS099714)
- James S Trimmer
National Institutes of Health (U24NS109113)
- James S Trimmer
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All procedures involving llamas were performed at Triple J Farms of Kent Laboratories (Bellingham, WA) in strict accordance with the Guide for the Care and Use of Laboratory Animals of the NIH. All procedures involving rats were approved by the University of California, Davis, Institutional Animal Care and Use Committee (IACUC) under protocols 20485 and 21265 and were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals of the NIH. All rats were maintained under standard light-dark cycles and allowed to feed and drink ad libitum. All procedures involving mice were approved by the Stanford University IACUC under protocol 18846 and were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals of the NIH.
Copyright
© 2019, Dong et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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A toolbox of nanobodies developed and validated for use as intrabodies and nanoscale immunolabels in brain neurons
eLife 8:e48750.
https://doi.org/10.7554/eLife.48750
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We present near-atomic-resolution cryoEM structures of the mammalian voltage-gated potassium channel Kv1.2 in open, C-type inactivated, toxin-blocked and sodium-bound states at 3.2 Å, 2.5 Å, 3.2 Å, and 2.9 Å. These structures, all obtained at nominally zero membrane potential in detergent micelles, reveal distinct ion-occupancy patterns in the selectivity filter. The first two structures are very similar to those reported in the related Shaker channel and the much-studied Kv1.2–2.1 chimeric channel. On the other hand, two new structures show unexpected patterns of ion occupancy. First, the toxin α-Dendrotoxin, like Charybdotoxin, is seen to attach to the negatively-charged channel outer mouth, and a lysine residue penetrates into the selectivity filter, with the terminal amine coordinated by carbonyls, partially disrupting the outermost ion-binding site. In the remainder of the filter two densities of bound ions are observed, rather than three as observed with other toxin-blocked Kv channels. Second, a structure of Kv1.2 in Na+ solution does not show collapse or destabilization of the selectivity filter, but instead shows an intact selectivity filter with ion density in each binding site. We also attempted to image the C-type inactivated Kv1.2 W366F channel in Na+ solution, but the protein conformation was seen to be highly variable and only a low-resolution structure could be obtained. These findings present new insights into the stability of the selectivity filter and the mechanism of toxin block of this intensively studied, voltage-gated potassium channel.
