Recombinant Probes for Visualizing Endogenous Synaptic Proteins in Living Neurons

Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA.
Neuron (Impact Factor: 15.05). 06/2013; 78(6):971-85. DOI: 10.1016/j.neuron.2013.04.017
Source: PubMed


The ability to visualize endogenous proteins in living neurons provides a powerful means to interrogate neuronal structure and function. Here we generate recombinant antibody-like proteins, termed Fibronectin intrabodies generated with mRNA display (FingRs), that bind endogenous neuronal proteins PSD-95 and Gephyrin with high affinity and that, when fused to GFP, allow excitatory and inhibitory synapses to be visualized in living neurons. Design of the FingR incorporates a transcriptional regulation system that ties FingR expression to the level of the target and reduces background fluorescence. In dissociated neurons and brain slices, FingRs generated against PSD-95 and Gephyrin did not affect the expression patterns of their endogenous target proteins or the number or strength of synapses. Together, our data indicate that PSD-95 and Gephyrin FingRs can report the localization and amount of endogenous synaptic proteins in living neurons and thus may be used to study changes in synaptic strength in vivo.

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Available from: Jason Junge, Oct 01, 2015
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    • "Genetic randomization of the CDR-like loops allows the generation of libraries from which to retrieve binders with desired specificities after in vitro display, as was for example demonstrated for a monobody directed against the Abl SH2 domain (Wojcik et al., 2010). In contrast to variable Ig domains, the fibronectin structure does not depend on intramolecular disulphide bridges, thus facilitating functional applications in reducing environments such as the cytoplasm of living cells (Gross et al., 2013). "
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    ABSTRACT: Antibodies are key reagents to investigate cellular processes. The development of recombinant antibodies and binders derived from natural protein scaffolds has expanded traditional applications, such as immunofluorescence, binding arrays, and immunoprecipitation. In addition, their small size and high stability in ectopic environments have enabled their use in all areas of cell research, including structural biology, advanced microscopy, and intracellular expression. Understanding these novel reagents as genetic modules that can be integrated into cellular pathways opens up a broad experimental spectrum to monitor and manipulate cellular processes. © 2015 Helma et al.
    The Journal of Cell Biology 06/2015; 209(5):633-644. DOI:10.1083/jcb.201409074
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    • "Binding events become visible as an overlay of both fluorescent signals. Recently, this has been demonstrated in an assay where the antigen was redirected to the Golgi by fusing the antigen to a Golgi targeting sequence [55] and by the fluorescent-2-hybrid assay (F2H) [54] [56]. The latter is performed in recombinant mammalian cell lines harboring a large copy number of the lac operator array stably integrated into the genome. "
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    ABSTRACT: 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. Beside 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 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.
    Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 01/2014;
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    ABSTRACT: Antibody-like probes report the localization and amount of endogenous proteins in living cells.
    Nature Methods 07/2013; 10(8):696-697. DOI:10.1038/nmeth.2588
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