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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

Nanobodies specifically recognize vimentin.(a) Immunofluorescence staining of endogenous vimentin in fixed MDCK cells with VB3ATTO488, VB6ATTO488 and VB6-VB6ATTO488 in comparison to α-VIM-IgG. Shown are respresentative images from three independent experiments. Scale bar: 20 μm. (b) Immunoblot analysis of protein lysates derived from HeLa, HEK293T, MDCK or A549 cells using VB6-VB6ATTO488 for direct detection or an α-VIM-IgG as control. (c) Immunoprecipitation of endogenous vimentin with immobilized Nbs. Input and bound fractions of indicated cell lysates were analyzed by immunoblot with an α-VIM-IgG. ctr: pulldown with non-related nanobody. (d,e) VB3 and VB6 bind to the rod domain of vimentin (d) Schematic overview of vimentin domains fused to GFP. (e) HEK293T cells were transfected with the indicated constructs and subjected to immunoprecipitation with immobilized Nbs followed by immunoblot analysis of input and bound fractions with an anti-GFP antibody. ctr: pulldown with non-related nanobody.
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f2: Nanobodies specifically recognize vimentin.(a) Immunofluorescence staining of endogenous vimentin in fixed MDCK cells with VB3ATTO488, VB6ATTO488 and VB6-VB6ATTO488 in comparison to α-VIM-IgG. Shown are respresentative images from three independent experiments. Scale bar: 20 μm. (b) Immunoblot analysis of protein lysates derived from HeLa, HEK293T, MDCK or A549 cells using VB6-VB6ATTO488 for direct detection or an α-VIM-IgG as control. (c) Immunoprecipitation of endogenous vimentin with immobilized Nbs. Input and bound fractions of indicated cell lysates were analyzed by immunoblot with an α-VIM-IgG. ctr: pulldown with non-related nanobody. (d,e) VB3 and VB6 bind to the rod domain of vimentin (d) Schematic overview of vimentin domains fused to GFP. (e) HEK293T cells were transfected with the indicated constructs and subjected to immunoprecipitation with immobilized Nbs followed by immunoblot analysis of input and bound fractions with an anti-GFP antibody. ctr: pulldown with non-related nanobody.

Mentions: For in vitro analyses, the Nbs VB3 and VB6 were recombinantly expressed and purified from Escherichia coli (E.coli). To detect vimentin by direct immunofluorescence, we chemically coupled an organic dye (ATTO488) to the purified Nbs resulting in a DOL (degree of labeling) of 1 for VB3 and 0.8 for VB6. Staining of fixed MDCK cells with ATTO488-labeled Nbs displayed considerable differences. While VB3ATTO488 was primarily localized in the nuclear compartment, VB6ATTO488 showed a similar pattern as obtained with a conventional anti-vimentin antibody (α-VIM-IgG) (Fig. 2a upper panel). Since we detected a residual unspecific signal of VB6ATTO488, we generated a bivalent VB6 dimer (VB6-VB6). The latter consists of two identical VB6 domains connected by a flexible peptide linker. Cellular imaging with VB6-VB6ATTO488 revealed an increase in signal intensity at filamentous structures and a concomitant decrease of unspecific background (Fig. 2a lower panel). In addition, co-staining with VB6-VB6ATTO488 and an α-VIM-IgG showed a broad signal overlap at vimentin filaments in HeLa, MDA-MB-231 and MDCK cells while VB3ATTO488 and VB6ATTO488 only partially colocalized with α-VIM-IgG (Supplementary Fig. 2a–c). Next, we tested whether VB6-VB6ATTO488 is also functional in immunoblotting. To this end, 20 μg of soluble protein derived from four cell lines were subjected to SDS-PAGE and immunoblotting. Subsequently, the western blots were stained either with VB6-VB6ATTO488 or α-VIM-IgG (Fig. 2b). Direct detection using VB6-VB6ATTO488 revealed a high specificity for endogenous vimentin indicated by a comparable band pattern as obtained with α-VIM-IgG detected with a fluorescently labeled secondary antibody (Fig. 2b).


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

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

Nanobodies specifically recognize vimentin.(a) Immunofluorescence staining of endogenous vimentin in fixed MDCK cells with VB3ATTO488, VB6ATTO488 and VB6-VB6ATTO488 in comparison to α-VIM-IgG. Shown are respresentative images from three independent experiments. Scale bar: 20 μm. (b) Immunoblot analysis of protein lysates derived from HeLa, HEK293T, MDCK or A549 cells using VB6-VB6ATTO488 for direct detection or an α-VIM-IgG as control. (c) Immunoprecipitation of endogenous vimentin with immobilized Nbs. Input and bound fractions of indicated cell lysates were analyzed by immunoblot with an α-VIM-IgG. ctr: pulldown with non-related nanobody. (d,e) VB3 and VB6 bind to the rod domain of vimentin (d) Schematic overview of vimentin domains fused to GFP. (e) HEK293T cells were transfected with the indicated constructs and subjected to immunoprecipitation with immobilized Nbs followed by immunoblot analysis of input and bound fractions with an anti-GFP antibody. ctr: pulldown with non-related nanobody.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Nanobodies specifically recognize vimentin.(a) Immunofluorescence staining of endogenous vimentin in fixed MDCK cells with VB3ATTO488, VB6ATTO488 and VB6-VB6ATTO488 in comparison to α-VIM-IgG. Shown are respresentative images from three independent experiments. Scale bar: 20 μm. (b) Immunoblot analysis of protein lysates derived from HeLa, HEK293T, MDCK or A549 cells using VB6-VB6ATTO488 for direct detection or an α-VIM-IgG as control. (c) Immunoprecipitation of endogenous vimentin with immobilized Nbs. Input and bound fractions of indicated cell lysates were analyzed by immunoblot with an α-VIM-IgG. ctr: pulldown with non-related nanobody. (d,e) VB3 and VB6 bind to the rod domain of vimentin (d) Schematic overview of vimentin domains fused to GFP. (e) HEK293T cells were transfected with the indicated constructs and subjected to immunoprecipitation with immobilized Nbs followed by immunoblot analysis of input and bound fractions with an anti-GFP antibody. ctr: pulldown with non-related nanobody.
Mentions: For in vitro analyses, the Nbs VB3 and VB6 were recombinantly expressed and purified from Escherichia coli (E.coli). To detect vimentin by direct immunofluorescence, we chemically coupled an organic dye (ATTO488) to the purified Nbs resulting in a DOL (degree of labeling) of 1 for VB3 and 0.8 for VB6. Staining of fixed MDCK cells with ATTO488-labeled Nbs displayed considerable differences. While VB3ATTO488 was primarily localized in the nuclear compartment, VB6ATTO488 showed a similar pattern as obtained with a conventional anti-vimentin antibody (α-VIM-IgG) (Fig. 2a upper panel). Since we detected a residual unspecific signal of VB6ATTO488, we generated a bivalent VB6 dimer (VB6-VB6). The latter consists of two identical VB6 domains connected by a flexible peptide linker. Cellular imaging with VB6-VB6ATTO488 revealed an increase in signal intensity at filamentous structures and a concomitant decrease of unspecific background (Fig. 2a lower panel). In addition, co-staining with VB6-VB6ATTO488 and an α-VIM-IgG showed a broad signal overlap at vimentin filaments in HeLa, MDA-MB-231 and MDCK cells while VB3ATTO488 and VB6ATTO488 only partially colocalized with α-VIM-IgG (Supplementary Fig. 2a–c). Next, we tested whether VB6-VB6ATTO488 is also functional in immunoblotting. To this end, 20 μg of soluble protein derived from four cell lines were subjected to SDS-PAGE and immunoblotting. Subsequently, the western blots were stained either with VB6-VB6ATTO488 or α-VIM-IgG (Fig. 2b). Direct detection using VB6-VB6ATTO488 revealed a high specificity for endogenous vimentin indicated by a comparable band pattern as obtained with α-VIM-IgG detected with a fluorescently labeled secondary antibody (Fig. 2b).

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