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Real-time analysis of epithelial-mesenchymal transition using fluorescent single-domain antibodies.

Maier J, Traenkle B, Rothbauer U - Sci Rep (2015)

Bottom Line: Following chromobody fluorescence in a cancer-relevant cellular model, we were able for the first time to monitor and quantify dynamic changes of endogenous vimentin upon siRNA-mediated knockdown, induction with TGF-β and modification with Withaferin A by high-content imaging.This versatile approach allows detailed studies of the spatiotemporal organization of vimentin in living cells.It enables the identification of vimentin-modulating compounds, thereby providing the basis to screen for novel therapeutics affecting EMT.

View Article: PubMed Central - PubMed

Affiliation: Pharmaceutical Biotechnology, Eberhard Karls University Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany.

ABSTRACT
Vimentin has become an important biomarker for epithelial-mesenchymal transition (EMT), a highly dynamic cellular process involved in the initiation of metastasis and cancer progression. To date there is no approach available to study endogenous vimentin in a physiological context. Here, we describe the selection and targeted modification of novel single-domain antibodies, so-called nanobodies, to trace vimentin in various cellular assays. Most importantly, we generated vimentin chromobodies by combining the binding moieties of the nanobodies with fluorescent proteins. Following chromobody fluorescence in a cancer-relevant cellular model, we were able for the first time to monitor and quantify dynamic changes of endogenous vimentin upon siRNA-mediated knockdown, induction with TGF-β and modification with Withaferin A by high-content imaging. This versatile approach allows detailed studies of the spatiotemporal organization of vimentin in living cells. It enables the identification of vimentin-modulating compounds, thereby providing the basis to screen for novel therapeutics affecting EMT.

No MeSH data available.


Related in: MedlinePlus

VB3 and VB6 chromobodies recognize vimentin in living cells.(a) Intracellular immunoprecipitation (IC-IP) of vimentin. Lysates of HEK293T cells expressing indicated chromobodies (VB3-CB; VB6-CB) or GFP were subjected to immunoprecipitation with the GFP-Trap. Input (I) and bound fractions (B) were analyzed by immunoblot with an α-VIM-IgG (upper panel) and an anti-GFP antibody (lower panel). (b,c) Vimentin chromobodies have a low tendency to aggregate upon intracellular expression. (b) Representative images of HeLa cells expressing GFP-VIM, VB3-CB or VB6-CB from three independent experiments. Scale bar: 20 μm. (c) Quantification of cells with fluorescent aggregates upon expression of GFP-VIM, VB3-CB or VB6-CB. Columns represent the percentage of cells displaying fluorescent granules (total number of analyzed cells > 300). Values represent the means of three independent transfections ± stds. For statistical analysis Chi-squared test was used, ***P < 0.001. (d) Fluorescent recovery after photobleaching (FRAP) analysis of VB3-CB, VB6-CB and GFP-VIM. Shown are representative images of transiently transfected HeLa cells before and after photobleaching of a defined region (white box). Scale bars: 10 μm. (e) Quantitative evaluation of FRAP data showing mean values of fluorescence recovery in photobleached regions. VB6-CB recovered to 84.1 ± 3.1% with a halftime of 3.9 s, the recovery of VB3-CB amounted to 58.4 ± 19.5% with a half time of 4.3 s; n = 10; N = 1. Data are represented as mean ± stds. For statistical analysis students t-test was used, ***P < 0.001.
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f3: VB3 and VB6 chromobodies recognize vimentin in living cells.(a) Intracellular immunoprecipitation (IC-IP) of vimentin. Lysates of HEK293T cells expressing indicated chromobodies (VB3-CB; VB6-CB) or GFP were subjected to immunoprecipitation with the GFP-Trap. Input (I) and bound fractions (B) were analyzed by immunoblot with an α-VIM-IgG (upper panel) and an anti-GFP antibody (lower panel). (b,c) Vimentin chromobodies have a low tendency to aggregate upon intracellular expression. (b) Representative images of HeLa cells expressing GFP-VIM, VB3-CB or VB6-CB from three independent experiments. Scale bar: 20 μm. (c) Quantification of cells with fluorescent aggregates upon expression of GFP-VIM, VB3-CB or VB6-CB. Columns represent the percentage of cells displaying fluorescent granules (total number of analyzed cells > 300). Values represent the means of three independent transfections ± stds. For statistical analysis Chi-squared test was used, ***P < 0.001. (d) Fluorescent recovery after photobleaching (FRAP) analysis of VB3-CB, VB6-CB and GFP-VIM. Shown are representative images of transiently transfected HeLa cells before and after photobleaching of a defined region (white box). Scale bars: 10 μm. (e) Quantitative evaluation of FRAP data showing mean values of fluorescence recovery in photobleached regions. VB6-CB recovered to 84.1 ± 3.1% with a halftime of 3.9 s, the recovery of VB3-CB amounted to 58.4 ± 19.5% with a half time of 4.3 s; n = 10; N = 1. Data are represented as mean ± stds. For statistical analysis students t-test was used, ***P < 0.001.

Mentions: For further analysis of the intracellular binding properties, we performed intracellular immunoprecipitations (IC-IPs). Soluble protein fractions of HEK293T cells expressing VB3-CB, VB6-CB or GFP as a negative control were subjected to pulldown experiments using the fluorescent moiety as an affinity tag. Input and bound fractions were analyzed by immunoblotting with antibodies against GFP and vimentin. While vimentin was not co-precipitated with GFP alone, it was clearly detected in the bound fraction of both chromobodies (Fig. 3a).


Real-time analysis of epithelial-mesenchymal transition using fluorescent single-domain antibodies.

Maier J, Traenkle B, Rothbauer U - Sci Rep (2015)

VB3 and VB6 chromobodies recognize vimentin in living cells.(a) Intracellular immunoprecipitation (IC-IP) of vimentin. Lysates of HEK293T cells expressing indicated chromobodies (VB3-CB; VB6-CB) or GFP were subjected to immunoprecipitation with the GFP-Trap. Input (I) and bound fractions (B) were analyzed by immunoblot with an α-VIM-IgG (upper panel) and an anti-GFP antibody (lower panel). (b,c) Vimentin chromobodies have a low tendency to aggregate upon intracellular expression. (b) Representative images of HeLa cells expressing GFP-VIM, VB3-CB or VB6-CB from three independent experiments. Scale bar: 20 μm. (c) Quantification of cells with fluorescent aggregates upon expression of GFP-VIM, VB3-CB or VB6-CB. Columns represent the percentage of cells displaying fluorescent granules (total number of analyzed cells > 300). Values represent the means of three independent transfections ± stds. For statistical analysis Chi-squared test was used, ***P < 0.001. (d) Fluorescent recovery after photobleaching (FRAP) analysis of VB3-CB, VB6-CB and GFP-VIM. Shown are representative images of transiently transfected HeLa cells before and after photobleaching of a defined region (white box). Scale bars: 10 μm. (e) Quantitative evaluation of FRAP data showing mean values of fluorescence recovery in photobleached regions. VB6-CB recovered to 84.1 ± 3.1% with a halftime of 3.9 s, the recovery of VB3-CB amounted to 58.4 ± 19.5% with a half time of 4.3 s; n = 10; N = 1. Data are represented as mean ± stds. For statistical analysis students t-test was used, ***P < 0.001.
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f3: VB3 and VB6 chromobodies recognize vimentin in living cells.(a) Intracellular immunoprecipitation (IC-IP) of vimentin. Lysates of HEK293T cells expressing indicated chromobodies (VB3-CB; VB6-CB) or GFP were subjected to immunoprecipitation with the GFP-Trap. Input (I) and bound fractions (B) were analyzed by immunoblot with an α-VIM-IgG (upper panel) and an anti-GFP antibody (lower panel). (b,c) Vimentin chromobodies have a low tendency to aggregate upon intracellular expression. (b) Representative images of HeLa cells expressing GFP-VIM, VB3-CB or VB6-CB from three independent experiments. Scale bar: 20 μm. (c) Quantification of cells with fluorescent aggregates upon expression of GFP-VIM, VB3-CB or VB6-CB. Columns represent the percentage of cells displaying fluorescent granules (total number of analyzed cells > 300). Values represent the means of three independent transfections ± stds. For statistical analysis Chi-squared test was used, ***P < 0.001. (d) Fluorescent recovery after photobleaching (FRAP) analysis of VB3-CB, VB6-CB and GFP-VIM. Shown are representative images of transiently transfected HeLa cells before and after photobleaching of a defined region (white box). Scale bars: 10 μm. (e) Quantitative evaluation of FRAP data showing mean values of fluorescence recovery in photobleached regions. VB6-CB recovered to 84.1 ± 3.1% with a halftime of 3.9 s, the recovery of VB3-CB amounted to 58.4 ± 19.5% with a half time of 4.3 s; n = 10; N = 1. Data are represented as mean ± stds. For statistical analysis students t-test was used, ***P < 0.001.
Mentions: For further analysis of the intracellular binding properties, we performed intracellular immunoprecipitations (IC-IPs). Soluble protein fractions of HEK293T cells expressing VB3-CB, VB6-CB or GFP as a negative control were subjected to pulldown experiments using the fluorescent moiety as an affinity tag. Input and bound fractions were analyzed by immunoblotting with antibodies against GFP and vimentin. While vimentin was not co-precipitated with GFP alone, it was clearly detected in the bound fraction of both chromobodies (Fig. 3a).

Bottom Line: Following chromobody fluorescence in a cancer-relevant cellular model, we were able for the first time to monitor and quantify dynamic changes of endogenous vimentin upon siRNA-mediated knockdown, induction with TGF-β and modification with Withaferin A by high-content imaging.This versatile approach allows detailed studies of the spatiotemporal organization of vimentin in living cells.It enables the identification of vimentin-modulating compounds, thereby providing the basis to screen for novel therapeutics affecting EMT.

View Article: PubMed Central - PubMed

Affiliation: Pharmaceutical Biotechnology, Eberhard Karls University Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany.

ABSTRACT
Vimentin has become an important biomarker for epithelial-mesenchymal transition (EMT), a highly dynamic cellular process involved in the initiation of metastasis and cancer progression. To date there is no approach available to study endogenous vimentin in a physiological context. Here, we describe the selection and targeted modification of novel single-domain antibodies, so-called nanobodies, to trace vimentin in various cellular assays. Most importantly, we generated vimentin chromobodies by combining the binding moieties of the nanobodies with fluorescent proteins. Following chromobody fluorescence in a cancer-relevant cellular model, we were able for the first time to monitor and quantify dynamic changes of endogenous vimentin upon siRNA-mediated knockdown, induction with TGF-β and modification with Withaferin A by high-content imaging. This versatile approach allows detailed studies of the spatiotemporal organization of vimentin in living cells. It enables the identification of vimentin-modulating compounds, thereby providing the basis to screen for novel therapeutics affecting EMT.

No MeSH data available.


Related in: MedlinePlus