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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

Quantitative analysis of vimentin upon TGF-β treatment.(a) Live-cell images of A549_VB6-CB cells left untreated (−TGF-β) or stimulated with TGF-β (5 ng/ml) for the indicated time periods. Shown are raw data images (image) and the respective segmentation of vimentin fibers (mask). Scale bars: 20 μm. (b–d) Quantification of vimentin fibers in >100 cells after treatment with TGF-β for 24 h (b), 48 h (c) and 72 h (d). Values represent the means ± s.e.m. of three independent experiments. For statistical analysis student’s t-test was used, **P < 0.01, ***P < 0.001.
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f5: Quantitative analysis of vimentin upon TGF-β treatment.(a) Live-cell images of A549_VB6-CB cells left untreated (−TGF-β) or stimulated with TGF-β (5 ng/ml) for the indicated time periods. Shown are raw data images (image) and the respective segmentation of vimentin fibers (mask). Scale bars: 20 μm. (b–d) Quantification of vimentin fibers in >100 cells after treatment with TGF-β for 24 h (b), 48 h (c) and 72 h (d). Values represent the means ± s.e.m. of three independent experiments. For statistical analysis student’s t-test was used, **P < 0.01, ***P < 0.001.

Mentions: For high-content imaging and phenotypic screening it is necessary to quantify morphological changes in a statistically relevant number of cells. Therefore, we established a phenotypic readout based on the VB6-CB signal. Within raw data images of A549_VB6-CB cells (Supplementary Fig. 7a) the network of vimentin fibers was automatically recognized and segmented (Supplementary Fig. 7b). Branchpoints were detected to separate vimentin fibers into individual fiber segments (Supplementary Fig. 7b’). In order to calculate the number of fiber segments per cell, nuclei were segmented based on the Hoechst signal (Supplementary Fig. 7c). Thereby, spatiotemporal changes of vimentin structures among different treatments can be compared. We examined this readout in a time-lapse experiment following the induction of vimentin by TGF-β (Fig. 5a). Quantitative analysis revealed a seven-fold increase of fiber segments per cell after 24 h compared to the untreated control (Fig. 5b). Notably, we observed a ten-fold elevation after 48 h, which was not further increased upon longer incubation periods (72 h) (Fig. 5c,d). To demonstrate that our vimentin chromobody model is suitable to monitor the effect of vimentin-modulating small compounds, we performed real-time high-content imaging upon Withaferin A (WFA) treatment. WFA is a steroidal lactone of Withaferia somnifera which exhibits anti-tumor and anti-angiogenesis activity in vivo4647. It has been reported to covalently modify vimentin and thereby to induce dominant negative effects20. On molecular level it was shown that WFA promotes phosphorylation and disruption of vimentin48. In addition, WFA has been described to inhibit TGF-β-induced EMT in epithelial breast cancer cells49.


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

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

Quantitative analysis of vimentin upon TGF-β treatment.(a) Live-cell images of A549_VB6-CB cells left untreated (−TGF-β) or stimulated with TGF-β (5 ng/ml) for the indicated time periods. Shown are raw data images (image) and the respective segmentation of vimentin fibers (mask). Scale bars: 20 μm. (b–d) Quantification of vimentin fibers in >100 cells after treatment with TGF-β for 24 h (b), 48 h (c) and 72 h (d). Values represent the means ± s.e.m. of three independent experiments. For statistical analysis student’s t-test was used, **P < 0.01, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4544033&req=5

f5: Quantitative analysis of vimentin upon TGF-β treatment.(a) Live-cell images of A549_VB6-CB cells left untreated (−TGF-β) or stimulated with TGF-β (5 ng/ml) for the indicated time periods. Shown are raw data images (image) and the respective segmentation of vimentin fibers (mask). Scale bars: 20 μm. (b–d) Quantification of vimentin fibers in >100 cells after treatment with TGF-β for 24 h (b), 48 h (c) and 72 h (d). Values represent the means ± s.e.m. of three independent experiments. For statistical analysis student’s t-test was used, **P < 0.01, ***P < 0.001.
Mentions: For high-content imaging and phenotypic screening it is necessary to quantify morphological changes in a statistically relevant number of cells. Therefore, we established a phenotypic readout based on the VB6-CB signal. Within raw data images of A549_VB6-CB cells (Supplementary Fig. 7a) the network of vimentin fibers was automatically recognized and segmented (Supplementary Fig. 7b). Branchpoints were detected to separate vimentin fibers into individual fiber segments (Supplementary Fig. 7b’). In order to calculate the number of fiber segments per cell, nuclei were segmented based on the Hoechst signal (Supplementary Fig. 7c). Thereby, spatiotemporal changes of vimentin structures among different treatments can be compared. We examined this readout in a time-lapse experiment following the induction of vimentin by TGF-β (Fig. 5a). Quantitative analysis revealed a seven-fold increase of fiber segments per cell after 24 h compared to the untreated control (Fig. 5b). Notably, we observed a ten-fold elevation after 48 h, which was not further increased upon longer incubation periods (72 h) (Fig. 5c,d). To demonstrate that our vimentin chromobody model is suitable to monitor the effect of vimentin-modulating small compounds, we performed real-time high-content imaging upon Withaferin A (WFA) treatment. WFA is a steroidal lactone of Withaferia somnifera which exhibits anti-tumor and anti-angiogenesis activity in vivo4647. It has been reported to covalently modify vimentin and thereby to induce dominant negative effects20. On molecular level it was shown that WFA promotes phosphorylation and disruption of vimentin48. In addition, WFA has been described to inhibit TGF-β-induced EMT in epithelial breast cancer cells49.

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