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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

VB6 chromobody visualizes distribution and reorganization of vimentin upon stimulation and knockdown of vimentin.(a,b) A549_VB6-CB cells were left untreated (−TGF-β) or stimulated with TGF-β (5 ng/ml) for 72 h. Cells were either subjected to immunoblot analysis with an α-VIM-IgG and anti-GAPDH antibody (a) or to microscopic analysis (b). (c,d) Knockdown studies of vimentin in A549_VB6-CB cells using three vimentin-specific siRNAs (siVIM1-3) and two control siRNAs (siCTR1-2), followed by immunoblot analysis with an α-VIM-IgG and anti-GAPDH antibody (c) or microscopic analysis (b,d). Shown are representative images from three independent experiments. Scale bars: 20 μm. (e) Time-lapse microscopy of A549_VB6-CB cells. Cells were stimulated with TGF-β (5 ng/ml) for 48 h. Subsequently, TGF-β was removed and cells were cultivated for additional 45 h. Images were taken in 3 h intervals, shown are representative images at indicated time points. Scale bar: 20 μm.
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f4: VB6 chromobody visualizes distribution and reorganization of vimentin upon stimulation and knockdown of vimentin.(a,b) A549_VB6-CB cells were left untreated (−TGF-β) or stimulated with TGF-β (5 ng/ml) for 72 h. Cells were either subjected to immunoblot analysis with an α-VIM-IgG and anti-GAPDH antibody (a) or to microscopic analysis (b). (c,d) Knockdown studies of vimentin in A549_VB6-CB cells using three vimentin-specific siRNAs (siVIM1-3) and two control siRNAs (siCTR1-2), followed by immunoblot analysis with an α-VIM-IgG and anti-GAPDH antibody (c) or microscopic analysis (b,d). Shown are representative images from three independent experiments. Scale bars: 20 μm. (e) Time-lapse microscopy of A549_VB6-CB cells. Cells were stimulated with TGF-β (5 ng/ml) for 48 h. Subsequently, TGF-β was removed and cells were cultivated for additional 45 h. Images were taken in 3 h intervals, shown are representative images at indicated time points. Scale bar: 20 μm.

Mentions: To test whether the chromobody signal is suitable to monitor dynamic modulations of vimentin, we designed two experiments: Firstly, we induced the expression of vimentin by treatment with TGF-β. Secondly, we depleted vimentin by RNA interference. To verify the induction of vimentin, stable A549_VB6-CB cells were either left untreated or incubated with TGF-β for 72 h and corresponding cell lysates were analyzed by immunoblotting. While a minor amount of vimentin was detected in untreated cells, the level was drastically increased upon exposure to TGF-β (Fig. 4a). In parallel, we analyzed the induction of vimentin microscopically. In the absence of TGF-β we observed a weak chromobody signal located mainly around the nucleus. Notably, vimentin filaments extended throughout the entire cell after treatment with TGF-β (Fig. 4b). These findings are in accordance with previous reports describing a TGF-β-induced redistribution of vimentin45. Next, we studied the localization of VB6-CB after knockdown of vimentin. To this end, we transfected A549_VB6-CB cells with vimentin-specific siRNAs (siVIM1-3) or two unrelated siRNAs (siCTR1, siCTR2) as controls. Immunoblot analysis after 72 h showed a highly efficient knockdown with all three vimentin-specific siRNAs (Fig. 4c). Accordingly, cellular imaging revealed a diffuse distribution of the chromobody in the absence of its respective antigen (Fig. 4d).


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

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

VB6 chromobody visualizes distribution and reorganization of vimentin upon stimulation and knockdown of vimentin.(a,b) A549_VB6-CB cells were left untreated (−TGF-β) or stimulated with TGF-β (5 ng/ml) for 72 h. Cells were either subjected to immunoblot analysis with an α-VIM-IgG and anti-GAPDH antibody (a) or to microscopic analysis (b). (c,d) Knockdown studies of vimentin in A549_VB6-CB cells using three vimentin-specific siRNAs (siVIM1-3) and two control siRNAs (siCTR1-2), followed by immunoblot analysis with an α-VIM-IgG and anti-GAPDH antibody (c) or microscopic analysis (b,d). Shown are representative images from three independent experiments. Scale bars: 20 μm. (e) Time-lapse microscopy of A549_VB6-CB cells. Cells were stimulated with TGF-β (5 ng/ml) for 48 h. Subsequently, TGF-β was removed and cells were cultivated for additional 45 h. Images were taken in 3 h intervals, shown are representative images at indicated time points. Scale bar: 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f4: VB6 chromobody visualizes distribution and reorganization of vimentin upon stimulation and knockdown of vimentin.(a,b) A549_VB6-CB cells were left untreated (−TGF-β) or stimulated with TGF-β (5 ng/ml) for 72 h. Cells were either subjected to immunoblot analysis with an α-VIM-IgG and anti-GAPDH antibody (a) or to microscopic analysis (b). (c,d) Knockdown studies of vimentin in A549_VB6-CB cells using three vimentin-specific siRNAs (siVIM1-3) and two control siRNAs (siCTR1-2), followed by immunoblot analysis with an α-VIM-IgG and anti-GAPDH antibody (c) or microscopic analysis (b,d). Shown are representative images from three independent experiments. Scale bars: 20 μm. (e) Time-lapse microscopy of A549_VB6-CB cells. Cells were stimulated with TGF-β (5 ng/ml) for 48 h. Subsequently, TGF-β was removed and cells were cultivated for additional 45 h. Images were taken in 3 h intervals, shown are representative images at indicated time points. Scale bar: 20 μm.
Mentions: To test whether the chromobody signal is suitable to monitor dynamic modulations of vimentin, we designed two experiments: Firstly, we induced the expression of vimentin by treatment with TGF-β. Secondly, we depleted vimentin by RNA interference. To verify the induction of vimentin, stable A549_VB6-CB cells were either left untreated or incubated with TGF-β for 72 h and corresponding cell lysates were analyzed by immunoblotting. While a minor amount of vimentin was detected in untreated cells, the level was drastically increased upon exposure to TGF-β (Fig. 4a). In parallel, we analyzed the induction of vimentin microscopically. In the absence of TGF-β we observed a weak chromobody signal located mainly around the nucleus. Notably, vimentin filaments extended throughout the entire cell after treatment with TGF-β (Fig. 4b). These findings are in accordance with previous reports describing a TGF-β-induced redistribution of vimentin45. Next, we studied the localization of VB6-CB after knockdown of vimentin. To this end, we transfected A549_VB6-CB cells with vimentin-specific siRNAs (siVIM1-3) or two unrelated siRNAs (siCTR1, siCTR2) as controls. Immunoblot analysis after 72 h showed a highly efficient knockdown with all three vimentin-specific siRNAs (Fig. 4c). Accordingly, cellular imaging revealed a diffuse distribution of the chromobody in the absence of its respective antigen (Fig. 4d).

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