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.
Metrics
-
- 10,583
- views
-
- 1,538
- downloads
-
- 41
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
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
Further reading
-
- Neuroscience
As the global population ages, the prevalence of neurodegenerative disorders is fast increasing. This neurodegeneration as well as other central nervous system (CNS) injuries cause permanent disabilities. Thus, generation of new neurons is the rosetta stone in contemporary neuroscience. Glial cells support CNS homeostasis through evolutionary conserved mechanisms. Upon damage, glial cells activate an immune and inflammatory response to clear the injury site from debris and proliferate to restore cell number. This glial regenerative response (GRR) is mediated by the neuropil-associated glia (NG) in Drosophila, equivalent to vertebrate astrocytes, oligodendrocytes (OL), and oligodendrocyte progenitor cells (OPCs). Here, we examine the contribution of NG lineages and the GRR in response to injury. The results indicate that NG exchanges identities between ensheathing glia (EG) and astrocyte-like glia (ALG). Additionally, we found that NG cells undergo transdifferentiation to yield neurons. Moreover, this transdifferentiation increases in injury conditions. Thus, these data demonstrate that glial cells are able to generate new neurons through direct transdifferentiation. The present work makes a fundamental contribution to the CNS regeneration field and describes a new physiological mechanism to generate new neurons.
-
- Neuroscience
Brain states fluctuate between exploratory and consummatory phases of behavior. These state changes affect both internal computation and the organism’s responses to sensory inputs. Understanding neuronal mechanisms supporting exploratory and consummatory states and their switching requires experimental control of behavioral shifts and collecting sufficient amounts of brain data. To achieve this goal, we developed the ThermoMaze, which exploits the animal’s natural warmth-seeking homeostatic behavior. By decreasing the floor temperature and selectively heating unmarked areas, we observed that mice avoided the aversive state by exploring the maze and finding the warm spot. In its design, the ThermoMaze is analogous to the widely used water maze but without the inconvenience of a wet environment and, therefore, allows the collection of physiological data in many trials. We combined the ThermoMaze with electrophysiology recording, and report that spiking activity of hippocampal CA1 neurons during sharp-wave ripple events encode the position of mice. Thus, place-specific firing is not confined to locomotion and associated theta oscillations but persist during waking immobility and sleep at the same location. The ThermoMaze will allow for detailed studies of brain correlates of immobility, preparatory–consummatory transitions, and open new options for studying behavior-mediated temperature homeostasis.
