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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

Selection of single-domain antibodies (nano-/chromobodies) against vimentin.(a) Amino acid sequence alignment of eight unique vimentin-specific VHH/VH domains (nanobodies, Nbs) identified after two rounds of biopanning followed by phage ELISA. (b) Representative images of HeLa cells expressing selected sdAbs fused to GFP (vimentin chromobodies). Cells stained with an anti-vimentin antibody (α-VIM-IgG) or expressing GFP served as controls. Scale bar: 20 μm.
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f1: Selection of single-domain antibodies (nano-/chromobodies) against vimentin.(a) Amino acid sequence alignment of eight unique vimentin-specific VHH/VH domains (nanobodies, Nbs) identified after two rounds of biopanning followed by phage ELISA. (b) Representative images of HeLa cells expressing selected sdAbs fused to GFP (vimentin chromobodies). Cells stained with an anti-vimentin antibody (α-VIM-IgG) or expressing GFP served as controls. Scale bar: 20 μm.

Mentions: To generate vimentin-specific nanobodies, an alpaca (Vicugna pacos) was immunized with recombinant vimentin. Subsequently, a phagemid library (~2 × 107 clones) was established from peripheral blood mononuclear cells (PBMCs), representing the respective Nb repertoire. After two cycles of biopanning against full-length vimentin, 47 single clones were analyzed in a solid-phase phage ELISA (Supplementary Fig. 1). Sequencing of the positive clones resulted in eight unique Nb sequences (Fig. 1a). Analysis of the hallmark residues (VH/VHH: V37Y; G44Q; L45R; W47L) located in framework 2 revealed that four Nbs (VB3, VE3, VG1 and VF6) are derived from heavy-chain-only antibodies, whereas VB6, VC4, VG4 and VH3 are VH domains derived from conventional IgGs36. To identify suitable candidates for the envisaged intracellular studies, all selected binders were analyzed in living cells. To this end, we generated chromobody constructs by fusing the coding sequences of the selected Nbs to eGFP and transiently expressed them in HeLa cells. The chromobodies VE3-CB and VH3-CB formed intracellular aggregates, whereas VC4-CB, VF6-CB and VG4-CB were diffusely distributed. Notably, VB3-CB, VB6-CB and VG1-CB displayed a filamentous pattern that resembles the distribution of vimentin (Fig. 1b). Additional structures reminiscent of a midbody pattern were observed in the majority of VG1-CB expressing cells. Hence, we selected VB3 nanobody/chromobody and VB6 nanobody/chromobody as best candidates for further biochemical and cell-biological studies.


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

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

Selection of single-domain antibodies (nano-/chromobodies) against vimentin.(a) Amino acid sequence alignment of eight unique vimentin-specific VHH/VH domains (nanobodies, Nbs) identified after two rounds of biopanning followed by phage ELISA. (b) Representative images of HeLa cells expressing selected sdAbs fused to GFP (vimentin chromobodies). Cells stained with an anti-vimentin antibody (α-VIM-IgG) or expressing GFP served as controls. Scale bar: 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Selection of single-domain antibodies (nano-/chromobodies) against vimentin.(a) Amino acid sequence alignment of eight unique vimentin-specific VHH/VH domains (nanobodies, Nbs) identified after two rounds of biopanning followed by phage ELISA. (b) Representative images of HeLa cells expressing selected sdAbs fused to GFP (vimentin chromobodies). Cells stained with an anti-vimentin antibody (α-VIM-IgG) or expressing GFP served as controls. Scale bar: 20 μm.
Mentions: To generate vimentin-specific nanobodies, an alpaca (Vicugna pacos) was immunized with recombinant vimentin. Subsequently, a phagemid library (~2 × 107 clones) was established from peripheral blood mononuclear cells (PBMCs), representing the respective Nb repertoire. After two cycles of biopanning against full-length vimentin, 47 single clones were analyzed in a solid-phase phage ELISA (Supplementary Fig. 1). Sequencing of the positive clones resulted in eight unique Nb sequences (Fig. 1a). Analysis of the hallmark residues (VH/VHH: V37Y; G44Q; L45R; W47L) located in framework 2 revealed that four Nbs (VB3, VE3, VG1 and VF6) are derived from heavy-chain-only antibodies, whereas VB6, VC4, VG4 and VH3 are VH domains derived from conventional IgGs36. To identify suitable candidates for the envisaged intracellular studies, all selected binders were analyzed in living cells. To this end, we generated chromobody constructs by fusing the coding sequences of the selected Nbs to eGFP and transiently expressed them in HeLa cells. The chromobodies VE3-CB and VH3-CB formed intracellular aggregates, whereas VC4-CB, VF6-CB and VG4-CB were diffusely distributed. Notably, VB3-CB, VB6-CB and VG1-CB displayed a filamentous pattern that resembles the distribution of vimentin (Fig. 1b). Additional structures reminiscent of a midbody pattern were observed in the majority of VG1-CB expressing cells. Hence, we selected VB3 nanobody/chromobody and VB6 nanobody/chromobody as best candidates for further biochemical and cell-biological studies.

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