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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: Most importantly, we generated vimentin chromobodies by combining the binding moieties of the nanobodies with fluorescent proteins.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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getmorefigures.php?uid=PMC4544033&req=5

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: Most importantly, we generated vimentin chromobodies by combining the binding moieties of the nanobodies with fluorescent proteins.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