Nanobody-Based Probes for Subcellular Protein Identification and Visualization

Abstract

Understanding how building blocks of life contribute to physiology is greatly aided by protein identification and cellular localization. The two main labeling approaches developed over the past decades are labeling with antibodies such as immunoglobulin G (IgGs) or use of genetically encoded tags such as fluorescent proteins. However, IgGs are large proteins (150 kDa), which limits penetration depth and uncertainty of target position caused by up to ∼25 nm distance of the label created by the chosen targeting approach. Additionally, IgGs cannot be easily recombinantly modulated and engineered as part of fusion proteins because they consist of multiple independent translated chains. In the last decade single domain antigen binding proteins are being explored in bioscience as a tool in revealing molecular identity and localization to overcome limitations by IgGs. These nanobodies have several potential benefits over routine applications. Because of their small size (15 kDa), nanobodies better penetrate during labeling procedures and improve resolution. Moreover, nanobodies cDNA can easily be fused with other cDNA. Multidomain proteins can thus be easily engineered consisting of domains for targeting (nanobodies) and visualization by fluorescence microscopy (fluorescent proteins) or electron microscopy (based on certain enzymes). Additional modules for e.g., purification are also easily added. These nanobody-based probes can be applied in cells for live-cell endogenous protein detection or may be purified prior to use on molecules, cells or tissues. Here, we present the current state of nanobody-based probes and their implementation in microscopy, including pitfalls and potential future opportunities.

Keywords: chromobody; electron microscopy; fluobody; light microscopy; nanobody; probes; super-resolution microscopy; tagging.

FIGURE 1

Nanobodies improve penetration and detect endogenous proteins. (A) Anti-GFP nanobody labeling (mCherry and peroxidase fused) and IgG labeling in H2B-GFP expressing cells. Cells permeabilized for 5 min with 0.1% Triton before labeling. Nanobodies and primary and secondary antibodies incubated for 1 h each. Note the colozalization between GFP and mCherry (nanobody), most prominently in the low-expressing cells, while Alexa Fluor 594 (IgG) mainly localizes in the cytoplasm. (B) High HER2 expressing cells, SkBr3, labeled with nanobodies targeting HER2. Overlay of nanobody fluoresence and EM image. Note the positive labeling at cell-cell contact sites. Bars: 10 μm. Reproduced from De Beer et al. (2018), http://creativecommons.org/licenses/by/4.0/.

FIGURE 2

Nanobodies delivered for intracellular live-cell imaging. (A) Chromobodies cDNA loaded in e.g., adeno-associated viral particles (AAV). These viruses are used to infect cells in culture or in animals e.g., mouse. (B) Magnetic optical dimerization tool exists of two nMagHigh1 and pMagHigh1. Here, the nMagHigh1 is fused to nanobody fragment containing CDR1/2 and pMagHigh1 is fused to nanobody fragment containing CDR3. Upon stimulation of light, the magnetic tool paired together, resulting in restoring the nanobody. (C) Fluorescent nanobodies in e.g., oligomers can be taken up via endocytosis. Here, when endosomal rupture is induced, the free fluorescent nanobodies can bind to their target. (D) Microfluidic cell squeezing for temporary cell permeabilization. Purified fluorescent nanobodies are present in the medium, and diffuse into the accessible cytoplasm. (E) MoonTag: array of 24 peptide sequence repeat allows visualization of single molecules directly following translation by signal amplification. Chromobodies accumulate at the peptide chain. Major inspiration for this cartoon is from Roder et al. (2017), Klein et al. (2018), Boersma et al. (2019), Yu et al. (2019).

FIGURE 3

Nanobody technology for EM. (A) Cells were expressing only C-terminus YFP or both C- and N-terminus YFP. The cells also expressed a conditionally stable anti-YFP, for proteasomal degradation of unbound probe. The nanobodies were genetically fused with peroxidase, APEX2, for EM detection. Note that only black precipitation is visible in cells expressing C-terminus YFP and N-terminus YFP. This confirmed the degradation of the nanobody with the peroxidase. Bars: 1 μm. (B) Purified nanobodies were used as primary antigen binding protein to reduce the distance between label and antigen. Nanobodies can be detected using an anti-VhH IgG conjugated to gold for EM visualization. Bars: 0.5 μm. (C) Cells express H2B-GFP from Figure 1A, and are permeabilized after fixation followed by labeling with anti-GFP FLIPPER-bodies. Note the colocalization in LM and the dark, positive nucleus in EM versus an unlabeled nucleus. Bars: 10 μm. (D) Neuronal cells expressing pHluorin on the plasma membrane. Added anti-GFP nanobodies bind to the pHluorin, and after a pulse stimulation, synaptic vesicles are formed. In EM, a population of unlabeled and labeled synaptic vesicles is detected. Arrows indicate synaptic vesicles, arrow heads indicate unstained vesicles and open arrow heads indicate residual staining (PM, plasma membrane, SV, Synaptic vesicle). Bar: 0.5 μm. The images in (A–D) have been reproduced from the following studies (Joensuu et al., 2016; Kijanka et al., 2017; Ariotti et al., 2018; De Beer et al., 2018), all of which have been published under a Creative Commons Attribution License.