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Generation of an alpaca-derived nanobody recognizing γ-H2AX.

Rajan M, Mortusewicz O, Rothbauer U, Hastert FD, Schmidthals K, Rapp A, Leonhardt H, Cardoso MC - FEBS Open Bio (2015)

Bottom Line: In vitro and in vivo characterization showed the specificity of the γ-H2AX nanobody.We found that alternative epitope recognition and masking of the epitope in living cells compromised the chromobody function.These pitfalls should be considered in the future development and screening of intracellular antibody biomarkers.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Technische Universitaet Darmstadt, Germany.

ABSTRACT
Post-translational modifications are difficult to visualize in living cells and are conveniently analyzed using antibodies. Single-chain antibody fragments derived from alpacas and called nanobodies can be expressed and bind to the target antigenic sites in living cells. As a proof of concept, we generated and characterized nanobodies against the commonly used biomarker for DNA double strand breaks γ-H2AX. In vitro and in vivo characterization showed the specificity of the γ-H2AX nanobody. Mammalian cells were transfected with fluorescent fusions called chromobodies and DNA breaks induced by laser microirradiation. We found that alternative epitope recognition and masking of the epitope in living cells compromised the chromobody function. These pitfalls should be considered in the future development and screening of intracellular antibody biomarkers.

No MeSH data available.


Related in: MedlinePlus

γ-H2AX chromobody recruitment to DNA damage sites in living cells. (A) Schematic representation of the experimental strategy. In (B) and (C) HeLa cells were transfected with γ-H2AX-3 chromobody alone or with mRFP-XRCC1 and were microirradiated with a 405 nm laser. Confocal microscopy time series images were acquired 24 h post transfection before and after irradiation. H2AX wild type (E) and knockout (F) MEF cells were transfected with the γ-H2AX-3 chromobody and mRFP-XRCC1. The γ-H2AX-3 chromobody recruitment to microirradiated sites was measured. Scale bar represents 5 μm. (D) and (G) Kinetics of the recruitment of the γ-H2AX-3 chromobody in the presence and absence of XRCC1 is shown for the indicated cell lines. (H) Schematic illustration of FRAP experiments performed on the preselected microirradiated spots. (I-J) Recovery curves of γ-H2AX-3 chromobody in damaged and undamaged sites (± micro IR, respectively) in the presence (J) or absence (I) of XRCC1 overexpression and the corresponding t-half values, as indicated on the left hand side. In both the recruitment and recovery kinetics curves, mean values were plotted and the error bar (shaded and lined) denotes standard deviation.
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f0010: γ-H2AX chromobody recruitment to DNA damage sites in living cells. (A) Schematic representation of the experimental strategy. In (B) and (C) HeLa cells were transfected with γ-H2AX-3 chromobody alone or with mRFP-XRCC1 and were microirradiated with a 405 nm laser. Confocal microscopy time series images were acquired 24 h post transfection before and after irradiation. H2AX wild type (E) and knockout (F) MEF cells were transfected with the γ-H2AX-3 chromobody and mRFP-XRCC1. The γ-H2AX-3 chromobody recruitment to microirradiated sites was measured. Scale bar represents 5 μm. (D) and (G) Kinetics of the recruitment of the γ-H2AX-3 chromobody in the presence and absence of XRCC1 is shown for the indicated cell lines. (H) Schematic illustration of FRAP experiments performed on the preselected microirradiated spots. (I-J) Recovery curves of γ-H2AX-3 chromobody in damaged and undamaged sites (± micro IR, respectively) in the presence (J) or absence (I) of XRCC1 overexpression and the corresponding t-half values, as indicated on the left hand side. In both the recruitment and recovery kinetics curves, mean values were plotted and the error bar (shaded and lined) denotes standard deviation.

Mentions: The γ-H2AX-3 chromobody was next tested for its ability to mark DNA double strand breaks in living cells (Fig. 2). Here, we wanted to mark the phosphorylation event that occurs on the histone H2AX upon DSBs. Initially, cells expressing the γ-H2AX-3 chromobody were irradiated with the 405 nm laser, which has been shown to induce different types of DNA damage including DSBs [21]. No recruitment of γ-H2AX-3 chromobody was observed at the sites of DNA damage (Fig. 2B and D). Hence, XRCC1 (X-ray cross complementing protein 1), a loading protein [22] involved in base excision and single strand break repair in mammalian cells, was co-transfected with the γ-H2AX-3 chromobody to confirm the induction of DNA damage. Recruitment of γ-H2AX-3 chromobody was observed in the presence of the XRCC1 at the microirradiated sites (Fig. 2C and D).


Generation of an alpaca-derived nanobody recognizing γ-H2AX.

Rajan M, Mortusewicz O, Rothbauer U, Hastert FD, Schmidthals K, Rapp A, Leonhardt H, Cardoso MC - FEBS Open Bio (2015)

γ-H2AX chromobody recruitment to DNA damage sites in living cells. (A) Schematic representation of the experimental strategy. In (B) and (C) HeLa cells were transfected with γ-H2AX-3 chromobody alone or with mRFP-XRCC1 and were microirradiated with a 405 nm laser. Confocal microscopy time series images were acquired 24 h post transfection before and after irradiation. H2AX wild type (E) and knockout (F) MEF cells were transfected with the γ-H2AX-3 chromobody and mRFP-XRCC1. The γ-H2AX-3 chromobody recruitment to microirradiated sites was measured. Scale bar represents 5 μm. (D) and (G) Kinetics of the recruitment of the γ-H2AX-3 chromobody in the presence and absence of XRCC1 is shown for the indicated cell lines. (H) Schematic illustration of FRAP experiments performed on the preselected microirradiated spots. (I-J) Recovery curves of γ-H2AX-3 chromobody in damaged and undamaged sites (± micro IR, respectively) in the presence (J) or absence (I) of XRCC1 overexpression and the corresponding t-half values, as indicated on the left hand side. In both the recruitment and recovery kinetics curves, mean values were plotted and the error bar (shaded and lined) denotes standard deviation.
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f0010: γ-H2AX chromobody recruitment to DNA damage sites in living cells. (A) Schematic representation of the experimental strategy. In (B) and (C) HeLa cells were transfected with γ-H2AX-3 chromobody alone or with mRFP-XRCC1 and were microirradiated with a 405 nm laser. Confocal microscopy time series images were acquired 24 h post transfection before and after irradiation. H2AX wild type (E) and knockout (F) MEF cells were transfected with the γ-H2AX-3 chromobody and mRFP-XRCC1. The γ-H2AX-3 chromobody recruitment to microirradiated sites was measured. Scale bar represents 5 μm. (D) and (G) Kinetics of the recruitment of the γ-H2AX-3 chromobody in the presence and absence of XRCC1 is shown for the indicated cell lines. (H) Schematic illustration of FRAP experiments performed on the preselected microirradiated spots. (I-J) Recovery curves of γ-H2AX-3 chromobody in damaged and undamaged sites (± micro IR, respectively) in the presence (J) or absence (I) of XRCC1 overexpression and the corresponding t-half values, as indicated on the left hand side. In both the recruitment and recovery kinetics curves, mean values were plotted and the error bar (shaded and lined) denotes standard deviation.
Mentions: The γ-H2AX-3 chromobody was next tested for its ability to mark DNA double strand breaks in living cells (Fig. 2). Here, we wanted to mark the phosphorylation event that occurs on the histone H2AX upon DSBs. Initially, cells expressing the γ-H2AX-3 chromobody were irradiated with the 405 nm laser, which has been shown to induce different types of DNA damage including DSBs [21]. No recruitment of γ-H2AX-3 chromobody was observed at the sites of DNA damage (Fig. 2B and D). Hence, XRCC1 (X-ray cross complementing protein 1), a loading protein [22] involved in base excision and single strand break repair in mammalian cells, was co-transfected with the γ-H2AX-3 chromobody to confirm the induction of DNA damage. Recruitment of γ-H2AX-3 chromobody was observed in the presence of the XRCC1 at the microirradiated sites (Fig. 2C and D).

Bottom Line: In vitro and in vivo characterization showed the specificity of the γ-H2AX nanobody.We found that alternative epitope recognition and masking of the epitope in living cells compromised the chromobody function.These pitfalls should be considered in the future development and screening of intracellular antibody biomarkers.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Technische Universitaet Darmstadt, Germany.

ABSTRACT
Post-translational modifications are difficult to visualize in living cells and are conveniently analyzed using antibodies. Single-chain antibody fragments derived from alpacas and called nanobodies can be expressed and bind to the target antigenic sites in living cells. As a proof of concept, we generated and characterized nanobodies against the commonly used biomarker for DNA double strand breaks γ-H2AX. In vitro and in vivo characterization showed the specificity of the γ-H2AX nanobody. Mammalian cells were transfected with fluorescent fusions called chromobodies and DNA breaks induced by laser microirradiation. We found that alternative epitope recognition and masking of the epitope in living cells compromised the chromobody function. These pitfalls should be considered in the future development and screening of intracellular antibody biomarkers.

No MeSH data available.


Related in: MedlinePlus