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.
Highlights
Nanobodies are the recombinant minimal antigen binding fragments derived from the atypical monomeric immunoglobulins present in camelid mammals and cartilaginous fish (HamersCasterman et al, 1993; Muyldermans, 2013; Desmyter et al, 2015; Beghein and Gettemans, 2017; Konning et al, 2017; De Meyer et al, 2014)
We isolated lymphocytes from a single llama immunized with recombinant fragments of these five target proteins and generated nAb phage display cDNA libraries that were subsequently used to isolate target-specific nAbs via phage binding to the individual target proteins
In the case of AMIGO-1, we identified nAbs that colocalized with endogenous AMIGO-1 (Figure 1— figure supplement 3), which is present in large clusters at endoplasmic reticulum-plasma membrane (ER-PM) junctions on the soma and proximal dendrites of cultured rat hippocampal neurons (CHNs) (Bishop et al, 2018)
Summary
Nanobodies (nAbs) are the recombinant minimal antigen binding fragments derived from the atypical monomeric immunoglobulins present in camelid mammals and cartilaginous fish (HamersCasterman et al, 1993; Muyldermans, 2013; Desmyter et al, 2015; Beghein and Gettemans, 2017; Konning et al, 2017; De Meyer et al, 2014). Neuroscience target protein from the 10–20 nm obtained with conventional primary and secondary antibodies down to 2–4 nm (Beghein and Gettemans, 2017; Ries et al, 2012; Szymborska et al, 2013; Pleiner et al, 2015) Their ability to precisely target specific proteins in living cells, and/or label them in post vivo samples with a high degree of efficacy and spatial resolution make nAbs attractive for numerous biomedical research applications, including in neuroscience research (Sudhof, 2018). Integral to the functional complexity of neurons is the diversity of proteins they express (estimated to encompass the products of two-thirds of the genome), a complexity markedly enhanced by compartmentalization of specific proteins at highly restricted sites within the neuron’s complex structure This includes the basic polarized compartments (dendrite, cell body, axon), and distinct subcompartments within these domains (e.g., dendritic spines, the axon initial segment [AIS], nodes of Ranvier, presynaptic terminals, etc.). These targets are the postsynaptic scaffolding proteins Homer (Brandstatter et al, 2004), IRSp53 (Soltau et al, 2002), and SAPAP2 (Takeuchi et al, 1997) that are present at partially overlapping sets of excitatory synapses, Gephyrin (Kneussel et al, 2001) found postsynaptically at most inhibitory synapses, and the Kv2 channel auxiliary subunit AMIGO-1 (36) found in large clusters at endoplasmic reticulum-plasma membrane (ER-PM) junctions present on the soma, proximal dendrites and AIS
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