Monitoring Interactions and Dynamics of Endogenous Beta-catenin With Intracellular Nanobodies in Living Cells*

Traenkle, Bjoern; Emele, Felix Emele; Anton, Roman; Poetz, Oliver; Haeussler, Ragna S.; Maier, Julia; Kaiser, Philipp; Scholz, Armin M.; Nueske, Stefan; Buchfellner, Andrea; Romer, Tina; Rothbauer, Ulrich

doi:10.1074/mcp.m114.044016

Cited by 76 publications

(97 citation statements)

References 72 publications

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“…However, the subcellular distribution of transiently overexpressed fluorescent β-catenin is different than the endogenous protein, since the overexpressed protein is found throughout the whole cell including the nucleus (even without a signal), membrane staining is lacking, and cytoskeletal organization is disrupted (Krieghoff et al, 2006; Jamieson et al, 2011; Ligon et al, 2001). Even the use of nanobodies targeting endogenous β-catenin in living cells did not mimic the membrane localization of non-induced cells (Traenkle et al, 2015). Hence, using our cell system in which YFP-β-catenin was stably expressed at relatively low levels (80% over the endogenous protein) and was distributed similarly to the endogenous protein, we were able to follow the subcellular dynamics of β-catenin in real-time.…”

Section: Discussionmentioning

confidence: 99%

Quantifying β-catenin subcellular dynamics and cyclin D1 mRNA transcription during Wnt signaling in single living cells

Kafri

Hasenson

Kanter

et al. 2016

eLife

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Signal propagation from the cell membrane to a promoter can induce gene expression. To examine signal transmission through sub-cellular compartments and its effect on transcription levels in individual cells within a population, we used the Wnt/β-catenin signaling pathway as a model system. Wnt signaling orchestrates a response through nuclear accumulation of β-catenin in the cell population. However, quantitative live-cell measurements in individual cells showed variability in nuclear β-catenin accumulation, which could occur in two waves, followed by slow clearance. Nuclear accumulation dynamics were initially rapid, cell cycle independent and differed substantially from LiCl stimulation, presumed to mimic Wnt signaling. β-catenin levels increased simultaneously at adherens junctions and the centrosome, and a membrane-centrosome transport system was revealed. Correlating β-catenin nuclear dynamics to cyclin D1 transcriptional activation showed that the nuclear accumulation rate of change of the signaling factor, and not actual protein levels, correlated with the transcriptional output of the pathway.DOI: http://dx.doi.org/10.7554/eLife.16748.001

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Section: Discussionmentioning

confidence: 99%

Quantifying β-catenin subcellular dynamics and cyclin D1 mRNA transcription during Wnt signaling in single living cells

Kafri

Hasenson

Kanter

et al. 2016

eLife

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“…Studying protein folding using NMR [54] -I mmunization and grafting CDRs to stable nanobodies 460 [54] Proclacitonin High-throughput assay [55b] Chitosane-graphene nanocomposite, randomly attached (glutaraldehyde) Immunization 6.2-24.5 [55b] PCNA ImagingDNA replication [27,60] FPs, genetic fusion Immunization N/A b-catenin Imagingofb-catenin [61] FPs, genetic fusion Immunization 1.9-44 [61] HIV-1 capsid protein ImagingHIV-1 [62] FPs, genetic fusion Immunization 0.16 [62] Nuclear lamina…”

Section: Genetically Encoded Nanobodies In Cellular Biology and Imagingmentioning

confidence: 99%

Nanobodies: Chemical Functionalization Strategies and Intracellular Applications

et al. 2018

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Nanobodies can be seen as next‐generation tools for the recognition and modulation of antigens that are inaccessible to conventional antibodies. Due to their compact structure and high stability, nanobodies see frequent usage in basic research, and their chemical functionalization opens the way towards promising diagnostic and therapeutic applications. In this Review, central aspects of nanobody functionalization are presented, together with selected applications. While early conjugation strategies relied on the random modification of natural amino acids, more recent studies have focused on the site‐specific attachment of functional moieties. Such techniques include chemoenzymatic approaches, expressed protein ligation, and amber suppression in combination with bioorthogonal modification strategies. Recent applications range from sophisticated imaging and mass spectrometry to the delivery of nanobodies into living cells for the visualization and manipulation of intracellular antigens.

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“…Surviving colonies were further characterized and the sequences of the VHHs were determined. After this screen, 19 VHHs that specifically block infection of cells with IAV or VSV were isolated. This screening approach should be applicable to identify inhibitors of any pathogen or biological pathway.…”

Section: Selection Of Cytosolic/nuclear Sdabsmentioning

confidence: 99%

“…As an analytical tool camelid antibodies have been used to illuminate in vivo trafficking of target molecules, to detect conformational states and post‐translational modification of proteins. In addition, they have been employed as chaperons for crystallization in cases where unstable proteins are sensitive to degradation, unfolding or aggregation …”

Section: Introductionmentioning

confidence: 99%

“…In addition, they have been employed as chaperons for crystallization in cases where unstable proteins are sensitive to degradation, unfolding or aggregation. [19][20][21][22][23] In general nanobodies are selected from na€ ıve, immune, synthetic camelid VHH single domain antibody repertoires [24][25][26][27] whereas shark sdAbs and human VH and VL sdAbs are mainly selected from synthetic antibody libraries. 12-1828-34 Antibody fragments are displayed on filamentous phages or yeast.…”

Section: Introductionmentioning

confidence: 99%

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Single domain antibodies for the knockdown of cytosolic and nuclear proteins

Böldicke

2017

Protein Science

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Single domain antibodies (sdAbs) from camels or sharks comprise only the variable heavy chain domain. Human sdAbs comprise the variable domain of the heavy chain (VH) or light chain (VL) and can be selected from human antibodies. SdAbs are stable, nonaggregating molecules in vitro and in vivo compared to complete antibodies and scFv fragments. They are excellent novel inhibitors of cytosolic/nuclear proteins because they are correctly folded inside the cytosol in contrast to scFv fragments. SdAbs are unique because of their excellent specificity and possibility to target posttranslational modifications such as phosphorylation sites, conformers or interaction regions of proteins that cannot be targeted with genetic knockout techniques and are impossible to knockdown with RNAi. The number of inhibiting cytosolic/nuclear sdAbs is increasing and usage of synthetic single pot single domain antibody libraries will boost the generation of these fascinating molecules without the need of immunization. The most frequently selected antigenic epitopes belong to viral and oncogenic proteins, followed by toxins, proteins of the nervous system as well as plant- and drosophila proteins. It is now possible to select functional sdAbs against virtually every cytosolic/nuclear protein and desired epitope. The development of new endosomal escape protein domains and cell-penetrating peptides for efficient transfection broaden the application of inhibiting sdAbs. Last but not least, the generation of relatively new cell-specific nanoparticles such as polymersomes and polyplexes carrying cytosolic/nuclear sdAb-DNA or -protein will pave the way to apply cytosolic/nuclear sdAbs for inhibition of viral infection and cancer in the clinic.

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Monitoring Interactions and Dynamics of Endogenous Beta-catenin With Intracellular Nanobodies in Living Cells*

Cited by 76 publications

References 72 publications

Quantifying β-catenin subcellular dynamics and cyclin D1 mRNA transcription during Wnt signaling in single living cells

Quantifying β-catenin subcellular dynamics and cyclin D1 mRNA transcription during Wnt signaling in single living cells

Nanobodies: Chemical Functionalization Strategies and Intracellular Applications

Single domain antibodies for the knockdown of cytosolic and nuclear proteins

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