Chromobodies are nanobodies genetically fused to fluorescent proteins, which were developed to visualize endogenous intracellular antigens. These versatile bioimaging nanotools can also be used to ...detect cell surface epitopes, and we describe here how we use them as an alternative to conjugated antibodies. This way, we routinely test the binding efficiency of nanobodies for their cognate cell surface antigens, before integrating them as sensing domains into complex synthetic receptor architectures.
The identification of diagnostic and therapeutic targets requires a comprehensive understanding of cellular processes, for which advanced technologies in biomedical research are needed. The emergence ...of nanobodies (Nbs) derived from antibody fragments of camelid heavy chain-only antibodies as intracellular research tools offers new possibilities to study and modulate target antigens in living cells. Here we summarize this rapidly changing field, beginning with a brief introduction of Nbs, followed by an overview of how target-specific Nbs can be generated, and introduce the selection of intrabodies as research tools. Intrabodies, by definition, are intracellular functional Nbs that target ectopic or endogenous intracellular antigens within living cells. Such binders can be applied in various formats, e.g. as chromobodies for live cell microscopy or as biosensors to decipher complex intracellular signaling pathways. In addition, protein knockouts can be achieved by target-specific Nbs, while modulating Nbs have the potential as future therapeutics. The development of fine-tunable and switchable Nb-based systems that simultaneously provide spatial and temporal control has recently taken the application of these binders to the next level.
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⁃Nanobodies are versatile recombinant binding molecules for different fields of biomedical research⁃Nanobody derived intrabodies provide unique opportunities to study antigens in living cells and organisms⁃Recent developments and application of intrabodies as imaging probes, biosensors and to modulate antigens are discussed
Single-domain antibodies such as nanobodies (Nbs) have substantially expanded the possibilities of advanced cellular imaging. In comparison to conventional antibodies, Nbs are characterized by small ...size, high stability, and solubility in many environments, including the cytoplasm. Nbs can be efficiently functionalized or modified according to the needs of the imaging approach. Target-specific Nbs can be easily converted into genetically encoded fluorescently labeled intrabodies, also known as chromobodies (CBs), which represent powerful tools to study the dynamics of different proteins of interest within living cells. In this context, CBs specific for a short peptide epitope provide a versatile alternative to bypass the limitations observed with larger fluorescent protein fusions and can be readily used to visualize and monitor peptide-tagged proteins for which specific Nbs are not available. Here, we present our novel detection system comprising a 15 amino acid peptide-tag (PepTag) in combination with a peptide-tag specific CB (PepCB). We provide protocols for adding the PepTag to different proteins of interest, reformatting the peptide-specific Nb (PepNb) into a CB for expression in mammalian cells, and establishment of stable cell lines expressing the PepCB for protein interaction assays and compound screenings.
In biomedical research there is an ongoing demand for new technologies, which help to elucidate disease mechanisms and provide the basis to develop novel therapeutics. In this context a comprehensive ...understanding of cellular processes and their pathophysiology based on reliable information on abundance, localization, posttranslational modifications and dynamic interactions of cellular components is indispensable. Besides their significant impact as therapeutic molecules, antibodies are arguably the most powerful research tools to study endogenous proteins and other cellular components. However, for cellular diagnostics their use is restricted to endpoint assays using fixed and permeabilized cells.
Alternatively, live cell imaging using fluorescent protein-tagged reporters is widely used to study protein localization and dynamics in living cells. However, only artificially introduced chimeric proteins are visualized, whereas the endogenous proteins, their posttranslational modifications as well as non-protein components of the cell remain invisible and cannot be analyzed. To overcome these limitations, traceable intracellular binding molecules provide new opportunities to perform cellular diagnostics in real time. In this review we summarize recent progress in the generation of intracellular and cell penetrating antibodies and their application to target and trace cellular components in living cells. We highlight recent advances in the structural formulation of recombinant antibody formats, reliable screening protocols and sophisticated cellular targeting technologies and propose that such intrabodies will become versatile research tools for real time cell-based diagnostics including target validation and live cell imaging. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.
•Advances in scaffold engineering for intrabodies•Summary on how to select intrabodies•Overview on the application of intrabodies for cellular research•Recent advances in intrabodies for live cell imaging•Highlight chromobody technology
Chromobodies made of nanobodies fused to fluorescent proteins are powerful tools for targeting and tracing intracellular proteins in living cells. Typically, this is achieved by transfecting plasmids ...encoding the chromobodies. However, an excess of unbound chromobody relative to the endogenous antigen can result in high background fluorescence in live cell imaging. Here, we overcome this problem by using mRNA encoding chromobodies. Our approach allows one to precisely control the amount of chromobody expressed inside the cell by adjusting the amount of transfected mRNA. To challenge our method, we evaluate three chromobodies targeting intracellular proteins of different abundance and cellular localization, namely lamin A/C, Dnmt1 and actin. We demonstrate that the expression of chromobodies in living cells by transfection of tuned amounts of the corresponding mRNAs allows the accurate tracking of their cellular targets by time‐lapse fluorescence microscopy.
Graphical and Lay Summary
Chromobodies are valuable tools for tracking intracellular proteins in living cells. However, transfection of plasmids encoding chromobodies results in an excess of unbound chromobody relative to the endogenous antigen. Our approach allows fine‐tuning of the amount of chromobody expressed in the cell by adjusting the amount of electroporated IVT mRNA, resulting in an optimal signal‐to‐noise ratio for live cell imaging.
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 immunoglobulins (IgGs) or use of genetically-encoded tags such as fluorescent proteins (FPs). 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 targeting approach. Additionally, IgGs cannot be easily recombinant expressed because they consist of multiple independent translated chains. In the last decade single-chain 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 be fused genetically with FPs cDNA and expressed in cells for live-cell endogenous protein detection. Alternatively, the nanobodies may be purified from cellular system and used on other cells or tissues. Given the option to combine different modular domains for targeting (nanobodies), visualization by fluorescence light microscopy (LM) or electron microscopy (EM; based on enzymes) and purification becomes possible. Here, we present the current state of nanobody-based probes implementation in microscopy, including pitfalls and potential future opportunities.
Understanding cellular processes requires the determination of dynamic changes in the concentration of endogenous proteins. We demonstrate the dependency of the intracellular level of chromobodies ...(CB, fluorescently labeled nanobodies) on the amount of their endogenous antigens and present a broadly applicable strategy how to employ turnover-accelerating CBs, to quantify dynamic changes of endogenous protein levels by quantitative live-cell imaging. This will enable unprecedented insights into the dynamic regulation of proteins, e.g. during cellular signaling, cell differentiation, or upon drug action.
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Highlights
•Chromobodies are stabilized by antigen binding in live cells.•Monitoring changes of endogenous protein levels in living cells with chromobodies.•Broadly applicable system to generate turnover-accelerated chromobodies.•Quantification of time- and dose-dependent compound effects.
Understanding cellular processes requires the determination of dynamic changes in the concentration of genetically nonmodified, endogenous proteins, which, to date, is commonly accomplished by end-point assays in vitro. Molecular probes such as fluorescently labeled nanobodies (chromobodies, CBs) are powerful tools to visualize the dynamic subcellular localization of endogenous proteins in living cells. Here, we employed the dependence of intracellular levels of chromobodies on the amount of their endogenous antigens, a phenomenon, which we termed antigen-mediated CB stabilization (AMCBS), for simultaneous monitoring of time-resolved changes in the concentration and localization of native proteins. To improve the dynamic range of AMCBS we generated turnover-accelerated CBs and demonstrated their application in visualization and quantification of fast reversible changes in antigen concentration upon compound treatment by quantitative live-cell imaging. We expect that this broadly applicable strategy will enable unprecedented insights into the dynamic regulation of proteins, e.g. during cellular signaling, cell differentiation, or upon drug action.
Activity-regulated cytoskeleton-associated (Arc) protein plays key roles in long-term synaptic plasticity, memory, and cognitive flexibility. However, an integral understanding of Arc mechanisms is ...lacking. Arc is proposed to function as an interaction hub in neuronal dendrites and the nucleus, yet Arc can also form retrovirus-like capsids with proposed roles in intercellular communication. Here, we sought to develop anti-Arc nanobodies (ArcNbs) as new tools for probing Arc dynamics and function. Six ArcNbs representing different clonal lines were selected from immunized alpaca. Immunoblotting with recombinant ArcNbs fused to a small ALFA-epitope tag demonstrated binding to recombinant Arc as well as endogenous Arc from rat cortical tissue. ALFA-tagged ArcNb also provided efficient immunoprecipitation of stimulus-induced Arc after carbachol-treatment of SH-SY5Y neuroblastoma cells and induction of long-term potentiation in the rat dentate gyrus in vivo. Epitope mapping showed that all Nbs recognize the Arc C-terminal region containing the retroviral Gag capsid homology domain, comprised of tandem N- and C-lobes. ArcNbs E5 and H11 selectively bound the N-lobe, which harbors a peptide ligand binding pocket specific to mammals. Four additional ArcNbs bound the region containing the C-lobe and C-terminal tail. For use as genetically encoded fluorescent intrabodies, we show that ArcNbs fused to mScarlet-I are uniformly expressed, without aggregation, in the cytoplasm and nucleus of HEK293FT cells. Finally, mScarlet-I-ArcNb H11 expressed as intrabody selectively bound the N-lobe and enabled co-immunoprecipitation of full-length intracellular Arc. ArcNbs are versatile tools for live-cell labeling and purification of Arc, and interrogation of Arc capsid domain specific functions.
Artificially tethering two proteins or protein fragments together is a powerful method to query molecular mechanisms. However, this approach typically relies upon a prior understanding of which two ...proteins, when fused, are most likely to provide a specific function and is therefore not readily amenable to large-scale screening. Here, we describe the Synthetic Physical Interaction (SPI) method to create proteome-wide forced protein associations in the budding yeast Saccharomyces cerevisiae. This method allows thousands of protein-protein associations to be screened for those that affect either normal growth or sensitivity to drugs or specific conditions. The method is amenable to proteins, protein domains, or any genetically encoded peptide sequence.
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