Under the Microscope: Single-Domain Antibodies for Live-Cell Imaging and Super-Resolution Microscopy

Single-domain antibodies (sdAbs) have substantially expanded the possibilities of advanced cellular imaging such as live-cell or super-resolution microscopy to visualize cellular antigens and their dynamics. In addition to their unique properties including small size, high stability, and solubility in many environments, sdAbs can be efficiently functionalized according to the needs of the respective imaging approach. Genetically encoded intrabodies fused to fluorescent proteins (chromobodies) have become versatile tools to study dynamics of endogenous proteins in living cells. Additionally, sdAbs conjugated to organic dyes were shown to label cellular structures with high density and minimal fluorophore displacement making them highly attractive probes for super-resolution microscopy. Here, we review recent advances of the chromobody technology to visualize localization and dynamics of cellular targets and the application of chromobody-based cell models for compound screening. Acknowledging the emerging importance of super-resolution microscopy in cell biology, we further discuss advantages and challenges of sdAbs for this technology.

[1]  Michelle E. Hung,et al.  Designed Ankyrin Repeat Proteins ( DARPins ) : Binding Proteins for Research , Diagnostics , and Therapy , 2015 .

[2]  J. Reindl,et al.  A New Nanobody-Based Biosensor to Study Endogenous PARP1 In Vitro and in Live Human Cells , 2016, PloS one.

[3]  T. Holak,et al.  Lifeact: a versatile marker to visualize F-actin , 2008, Nature Methods.

[4]  S. Rasmussen,et al.  Crystal Structure of the β2Adrenergic Receptor-Gs protein complex , 2011, Nature.

[5]  R. Insall,et al.  Toxoplasma gondii F-actin forms an extensive filamentous network required for material exchange and parasite maturation , 2017, eLife.

[6]  Bernardo L. Sabatini,et al.  Recombinant Probes for Visualizing Endogenous Synaptic Proteins in Living Neurons , 2013, Neuron.

[7]  B. Ducommun,et al.  Chromatibody, a novel non-invasive molecular tool to explore and manipulate chromatin in living cells , 2016, Journal of Cell Science.

[8]  J. Lippincott-Schwartz,et al.  Imaging Intracellular Fluorescent Proteins at Nanometer Resolution , 2006, Science.

[9]  Andrew D Ellington,et al.  Aptamers as potential tools for super-resolution microscopy , 2012, Nature Methods.

[10]  M. Cristina Cardoso,et al.  A Versatile Nanotrap for Biochemical and Functional Studies with Fluorescent Fusion Proteins*S , 2008, Molecular & Cellular Proteomics.

[11]  Heinrich Leonhardt,et al.  Targeting and tracing antigens in live cells with fluorescent nanobodies , 2006, Nature Methods.

[12]  Emma Lundberg,et al.  Immunofluorescence and fluorescent-protein tagging show high correlation for protein localization in mammalian cells , 2013, Nature Methods.

[13]  H. Ewers,et al.  A simple, versatile method for GFP-based super-resolution microscopy via nanobodies , 2012, Nature Methods.

[14]  Thilo Stehle,et al.  Peptides in headlock – a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy , 2016, Scientific Reports.

[15]  J. Wagner,et al.  Generation of an intrabody‐based reagent suitable for imaging endogenous proliferating cell nuclear antigen in living cancer cells , 2014, Journal of molecular recognition : JMR.

[16]  S. Muyldermans,et al.  A bacterial-two-hybrid selection system for one-step isolation of intracellularly functional Nanobodies. , 2012, Archives of biochemistry and biophysics.

[17]  Felipe Opazo,et al.  Resolving bundled microtubules using anti-tubulin nanobodies , 2015, Nature Communications.

[18]  S. Schornack,et al.  Protein mislocalization in plant cells using a GFP-binding chromobody. , 2009, The Plant journal : for cell and molecular biology.

[19]  Michael J Rust,et al.  Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) , 2006, Nature Methods.

[20]  H. Vogel,et al.  A general method for the covalent labeling of fusion proteins with small molecules in vivo , 2003, Nature Biotechnology.

[21]  M. Neuberger,et al.  Expression and targeting of intracellular antibodies in mammalian cells. , 1990, The EMBO journal.

[22]  Thomas A. Blanpied,et al.  A transsynaptic nanocolumn aligns neurotransmitter release to receptors , 2016, Nature.

[23]  R. Mullins,et al.  Comparative analysis of tools for live cell imaging of actin network architecture , 2014, Bioarchitecture.

[24]  H. Leonhardt,et al.  The fluorescent two-hybrid (F2H) assay for direct analysis of protein-protein interactions in living cells. , 2012, Methods in molecular biology.

[25]  C. Vanhove,et al.  Sortase A-mediated site-specific labeling of camelid single-domain antibody-fragments: a versatile strategy for multiple molecular imaging modalities. , 2016, Contrast media & molecular imaging.

[26]  Mark Bates,et al.  Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation , 2015, eLife.

[27]  Marjeta Urh,et al.  HaloTag: a novel protein labeling technology for cell imaging and protein analysis. , 2008, ACS chemical biology.

[28]  Kai Johnsson,et al.  An engineered protein tag for multiprotein labeling in living cells. , 2008, Chemistry & biology.

[29]  H. Leonhardt,et al.  Novel antibody derivatives for proteome and high-content analysis , 2010, Analytical and bioanalytical chemistry.

[30]  R. Hochstrasser,et al.  Wide-field subdiffraction imaging by accumulated binding of diffusing probes , 2006, Proceedings of the National Academy of Sciences.

[31]  Ray H. Gavin,et al.  Expression of GFP-Actin Leads to Failure of Nuclear Elongation and Cytokinesis in Tetrahymena thermophila , 2003, The Journal of eukaryotic microbiology.

[32]  S. Muyldermans,et al.  Naturally occurring antibodies devoid of light chains , 1993, Nature.

[33]  S. De Clercq,et al.  Mapping cytoskeletal protein function in cells by means of nanobodies , 2013, Cytoskeleton.

[34]  S. Hell,et al.  Fluorogenic probes for live-cell imaging of the cytoskeleton , 2014, Nature Methods.

[35]  Thomas Müller-Reichert,et al.  Cortical Constriction During Abscission Involves Helices of ESCRT-III–Dependent Filaments , 2011, Science.

[36]  R. Grosse,et al.  Actin visualization at a glance , 2017, Development.

[37]  U. Rothbauer,et al.  Real-time analysis of epithelial-mesenchymal transition using fluorescent single-domain antibodies , 2015, Scientific Reports.

[38]  H. Leonhardt,et al.  Direct and Dynamic Detection of HIV-1 in Living Cells , 2012, PloS one.

[39]  A. Castro,et al.  Quantitative Live Imaging of Endogenous DNA Replication in Mammalian Cells , 2012, PloS one.

[40]  R. Goldman,et al.  Vimentin induces changes in cell shape, motility, and adhesion during the epithelial to mesenchymal transition , 2010, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[41]  M. Cristina Cardoso,et al.  Nanobodies and recombinant binders in cell biology , 2015, The Journal of cell biology.

[42]  Guðjón Ólafsson,et al.  Synthetic protein interactions reveal a functional map of the cell , 2016, eLife.

[43]  M. Chalfie.  GREEN FLUORESCENT PROTEIN , 1995, Photochemistry and photobiology.

[44]  Jonathan R. Tomshine,et al.  Conformational biosensors reveal GPCR signalling from endosomes , 2013, Nature.

[45]  R. Grosse,et al.  Nuclear F-actin Formation and Reorganization upon Cell Spreading*♦ , 2015, The Journal of Biological Chemistry.

[46]  U. Rothbauer,et al.  A Multiplexed High-Content Screening Approach Using the Chromobody Technology to Identify Cell Cycle Modulators in Living Cells , 2016, Journal of biomolecular screening.

[47]  D. Janzen,et al.  Visualization of the actin cytoskeleton: Different F‐actin‐binding probes tell different stories , 2014, Cytoskeleton.

[48]  U. Rothbauer,et al.  Visualizing Epithelial-Mesenchymal Transition Using the Chromobody Technology. , 2016, Cancer research.

[49]  B. Goud,et al.  Recombinant Antibodies Against Subcellular Fractions Used to Track Endogenous Golgi Protein Dynamics in Vivo , 2003, Traffic.

[50]  J. Rain,et al.  NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies , 2016, eLife.

[51]  Le A. Trinh,et al.  Nanobody-targeted E3-ubiquitin ligase complex degrades nuclear proteins , 2015, Scientific Reports.

[52]  S. Jobling,et al.  Immunomodulation of enzyme function in plants by single-domain antibody fragments , 2003, Nature Biotechnology.

[53]  O. Thoumine,et al.  Optimized labeling of membrane proteins for applications to super-resolution imaging in confined cellular environments using monomeric streptavidin , 2017, Nature Protocols.

[54]  C. Hawes,et al.  Fluorescent labelling of the actin cytoskeleton in plants using a cameloid antibody , 2014, Plant Methods.

[55]  U. Rothbauer,et al.  Monitoring Interactions and Dynamics of Endogenous Beta-catenin With Intracellular Nanobodies in Living Cells* , 2015, Molecular & Cellular Proteomics.

[56]  Bacterial two hybrid: a versatile one-step intracellular selection method. , 2012, Methods in molecular biology.

[57]  H. Leonhardt,et al.  Case study on live cell apoptosis-assay using lamin-chromobody cell-lines for high-content analysis. , 2012, Methods in molecular biology.

[58]  E. Snapp,et al.  Fluorescent proteins: a cell biologist's user guide. , 2009, Trends in cell biology.

[59]  Julia Maier,et al.  Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells. , 2014, Biochimica et biophysica acta.

[60]  Suliana Manley,et al.  Putting super-resolution fluorescence microscopy to work , 2008, Nature Methods.

[61]  C. Cepko,et al.  Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies , 2016, eLife.

[62]  M. Affolter,et al.  Fluorescent fusion protein knockout mediated by anti-GFP nanobody , 2011, Nature Structural &Molecular Biology.

[63]  Sung Jun Lim,et al.  Small Quantum Dots Conjugated to Nanobodies as Immunofluorescence Probes for Nanometric Microscopy , 2014, Bioconjugate chemistry.

[64]  J. Gettemans,et al.  A new survivin tracer tracks, delocalizes and captures endogenous survivin at different subcellular locations and in distinct organelles , 2016, Scientific Reports.

[65]  Etienne Weiss,et al.  Extended half‐life upon binding of destabilized intrabodies allows specific detection of antigen in mammalian cells , 2005, The FEBS journal.

[66]  Corella S. Casas-Delucchi,et al.  Modulation of protein properties in living cells using nanobodies , 2010, Nature Structural &Molecular Biology.

[67]  E. Wanker,et al.  A Fluorescent Two-hybrid Assay for Direct Visualization of Protein Interactions in Living Cells*S , 2008, Molecular & Cellular Proteomics.

[68]  S. Hell,et al.  Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. , 1994, Optics letters.

[69]  M. Gustafsson.  Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy , 2000, Journal of microscopy.

[70]  U. Rothbauer,et al.  Live imaging of endogenous protein dynamics in zebrafish using chromobodies , 2015, Development.

[71]  J. Gettemans,et al.  Use of Nanobodies to Localize Endogenous Cytoskeletal Proteins and to Determine Their Contribution to Cancer Cell Invasion by Using an ECM Degradation Assay. , 2016, Methods in molecular biology.

[72]  C. Höbartner,et al.  Aptamers provide superior stainings of cellular receptors studied under super-resolution microscopy , 2017, PloS one.

[73]  Maximilian T. Strauss,et al.  Super-resolution microscopy with DNA-PAINT , 2017, Nature Protocols.

[74]  G. Winter,et al.  Phage-encoded combinatorial chemical libraries based on bicyclic peptides. , 2009, Nature chemical biology.

[75]  H. Leonhardt,et al.  A guide to super-resolution fluorescence microscopy , 2010, The Journal of cell biology.

[76]  R. Tsien,et al.  The Fluorescent Toolbox for Assessing Protein Location and Function , 2006, Science.