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Visualizing posttranslational and epigenetic modifications of endogenous proteins in vivo.

Kimura H, Hayashi-Takanaka Y, Stasevich TJ, Sato Y - Histochem. Cell Biol. (2015)

Bottom Line: As a posttranslational protein modification is often associated with a specific function, marking specifically modified protein molecules in living cells is a way to track an important fraction of protein.In the nucleus, histones are subjected to a variety of modifications such as acetylation and methylation that are associated with epigenetic gene regulation.Moreover, these techniques can be applied to any other protein modification, opening up new avenues in broad areas in biology and medicine.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan, hkimura@bio.titech.ac.jp.

ABSTRACT
Protein localization and dynamics can now be visualized in living cells using the fluorescent protein fusion technique, but it is still difficult to selectively detect molecules with a specific function. As a posttranslational protein modification is often associated with a specific function, marking specifically modified protein molecules in living cells is a way to track an important fraction of protein. In the nucleus, histones are subjected to a variety of modifications such as acetylation and methylation that are associated with epigenetic gene regulation. RNA polymerase II, an enzyme that transcribes genes, is also differentially phosphorylated during the initiation and elongation of transcription. To understand the mechanism of gene regulation in vivo, we have developed methods to track histone and RNA polymerase II modifications using probes derived from modification-specific monoclonal antibodies. In Fab-based live endogenous modification labeling (FabLEM), fluorescently labeled antigen-binding fragments (Fabs) are loaded into cells. Fabs bind to target modifications in the nucleus with a binding time of a second to tens of seconds, and so the modification can be tracked without disturbing cell function. For tracking over longer periods of time or in living animals, we have also developed a genetically encoded system to express a modification-specific intracellular antibody (mintbody). Transgenic fruit fly and zebrafish that express histone H3 Lys9 acetylation-specific mintbody developed normally and remain fertile, suggesting that visualizing histone modifications in any tissue in live animals has become possible. These live cell modification tracking techniques will facilitate future studies on epigenetic regulation related to development, differentiation, and disease. Moreover, these techniques can be applied to any other protein modification, opening up new avenues in broad areas in biology and medicine.

No MeSH data available.


Related in: MedlinePlus

Schematic illustration of FabLEM and mintbody methods. Once hybridoma cells that produce specific antibody directed against a modification site are generated, two methods for tracking the modification in living cells are possible. (Top) FabLEM: Modification-specific antibody is purified from hybridoma cell culture supernatant, and fluorescently conjugated Fab fragments are prepared for loading into cells or injecting into mouse embryos. (Bottom) Mintbody: cDNA encoding the variable regions of heavy (VH) and light chains (VL) can be cloned into a vector to be expressed as an scFv fused with the green fluorescent protein. The expression vector can be transfected into cells or embryos to generate transgenic lines
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Fig2: Schematic illustration of FabLEM and mintbody methods. Once hybridoma cells that produce specific antibody directed against a modification site are generated, two methods for tracking the modification in living cells are possible. (Top) FabLEM: Modification-specific antibody is purified from hybridoma cell culture supernatant, and fluorescently conjugated Fab fragments are prepared for loading into cells or injecting into mouse embryos. (Bottom) Mintbody: cDNA encoding the variable regions of heavy (VH) and light chains (VL) can be cloned into a vector to be expressed as an scFv fused with the green fluorescent protein. The expression vector can be transfected into cells or embryos to generate transgenic lines

Mentions: In contrast to the whole IgG, fluorescently labeled antigen-binding fragments (Fabs) were suitable for detecting the endogenous histone modifications (Hayashi-Takanaka et al. 2009, 2011). We prepared Fab fragments from the whole IgG by protease digestion and labeled these with an amine-reactive fluorescent dye, before loading into living cells (Fig. 2, top). After loaded into cells, Fabs diffuse throughout the cytoplasm and enter the nucleus through the pore by diffusion, as their size (~50–60 kD) was just smaller than the pore size. If the target modification is present in the nucleus, Fabs bind to the target and become concentrated in the nucleus (Fig. 3). Fabs could bind to the target modification stably and block the access of cellular factors to the modification; however, Fab binding was transient and did not interfere with cell division. FRAP revealed that the binding time of Fabs to the target modifications in living cell nuclei was in a range from <1 to ~30 s, depending on the binding affinity. This might be unexpected because antibody binding to the epitope is thought to be quite stable. Unlike whole IgG that has a bivalent antigen-binding site, Fabs are monovalent and the binding affinity is much lower. In addition, the high macromolecule concentration (>100 mg/ml) in living cells can cause more frequent molecular collisions to force the dissociation of bound proteins. Indeed, Fabs loaded into cells at ~1 µM did not disturb cell growth. Furthermore, mouse preimplantation embryos injected with Fab developed normally to birth. It is thus concluded that the endogenous modifications can be visualized by using fluorescently labeled Fab without affecting cell function. We named this technique Fab-based live endogenous modification labeling, or FabLEM (Hayashi-Takanaka et al. 2011).Fig. 2


Visualizing posttranslational and epigenetic modifications of endogenous proteins in vivo.

Kimura H, Hayashi-Takanaka Y, Stasevich TJ, Sato Y - Histochem. Cell Biol. (2015)

Schematic illustration of FabLEM and mintbody methods. Once hybridoma cells that produce specific antibody directed against a modification site are generated, two methods for tracking the modification in living cells are possible. (Top) FabLEM: Modification-specific antibody is purified from hybridoma cell culture supernatant, and fluorescently conjugated Fab fragments are prepared for loading into cells or injecting into mouse embryos. (Bottom) Mintbody: cDNA encoding the variable regions of heavy (VH) and light chains (VL) can be cloned into a vector to be expressed as an scFv fused with the green fluorescent protein. The expression vector can be transfected into cells or embryos to generate transgenic lines
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4522274&req=5

Fig2: Schematic illustration of FabLEM and mintbody methods. Once hybridoma cells that produce specific antibody directed against a modification site are generated, two methods for tracking the modification in living cells are possible. (Top) FabLEM: Modification-specific antibody is purified from hybridoma cell culture supernatant, and fluorescently conjugated Fab fragments are prepared for loading into cells or injecting into mouse embryos. (Bottom) Mintbody: cDNA encoding the variable regions of heavy (VH) and light chains (VL) can be cloned into a vector to be expressed as an scFv fused with the green fluorescent protein. The expression vector can be transfected into cells or embryos to generate transgenic lines
Mentions: In contrast to the whole IgG, fluorescently labeled antigen-binding fragments (Fabs) were suitable for detecting the endogenous histone modifications (Hayashi-Takanaka et al. 2009, 2011). We prepared Fab fragments from the whole IgG by protease digestion and labeled these with an amine-reactive fluorescent dye, before loading into living cells (Fig. 2, top). After loaded into cells, Fabs diffuse throughout the cytoplasm and enter the nucleus through the pore by diffusion, as their size (~50–60 kD) was just smaller than the pore size. If the target modification is present in the nucleus, Fabs bind to the target and become concentrated in the nucleus (Fig. 3). Fabs could bind to the target modification stably and block the access of cellular factors to the modification; however, Fab binding was transient and did not interfere with cell division. FRAP revealed that the binding time of Fabs to the target modifications in living cell nuclei was in a range from <1 to ~30 s, depending on the binding affinity. This might be unexpected because antibody binding to the epitope is thought to be quite stable. Unlike whole IgG that has a bivalent antigen-binding site, Fabs are monovalent and the binding affinity is much lower. In addition, the high macromolecule concentration (>100 mg/ml) in living cells can cause more frequent molecular collisions to force the dissociation of bound proteins. Indeed, Fabs loaded into cells at ~1 µM did not disturb cell growth. Furthermore, mouse preimplantation embryos injected with Fab developed normally to birth. It is thus concluded that the endogenous modifications can be visualized by using fluorescently labeled Fab without affecting cell function. We named this technique Fab-based live endogenous modification labeling, or FabLEM (Hayashi-Takanaka et al. 2011).Fig. 2

Bottom Line: As a posttranslational protein modification is often associated with a specific function, marking specifically modified protein molecules in living cells is a way to track an important fraction of protein.In the nucleus, histones are subjected to a variety of modifications such as acetylation and methylation that are associated with epigenetic gene regulation.Moreover, these techniques can be applied to any other protein modification, opening up new avenues in broad areas in biology and medicine.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan, hkimura@bio.titech.ac.jp.

ABSTRACT
Protein localization and dynamics can now be visualized in living cells using the fluorescent protein fusion technique, but it is still difficult to selectively detect molecules with a specific function. As a posttranslational protein modification is often associated with a specific function, marking specifically modified protein molecules in living cells is a way to track an important fraction of protein. In the nucleus, histones are subjected to a variety of modifications such as acetylation and methylation that are associated with epigenetic gene regulation. RNA polymerase II, an enzyme that transcribes genes, is also differentially phosphorylated during the initiation and elongation of transcription. To understand the mechanism of gene regulation in vivo, we have developed methods to track histone and RNA polymerase II modifications using probes derived from modification-specific monoclonal antibodies. In Fab-based live endogenous modification labeling (FabLEM), fluorescently labeled antigen-binding fragments (Fabs) are loaded into cells. Fabs bind to target modifications in the nucleus with a binding time of a second to tens of seconds, and so the modification can be tracked without disturbing cell function. For tracking over longer periods of time or in living animals, we have also developed a genetically encoded system to express a modification-specific intracellular antibody (mintbody). Transgenic fruit fly and zebrafish that express histone H3 Lys9 acetylation-specific mintbody developed normally and remain fertile, suggesting that visualizing histone modifications in any tissue in live animals has become possible. These live cell modification tracking techniques will facilitate future studies on epigenetic regulation related to development, differentiation, and disease. Moreover, these techniques can be applied to any other protein modification, opening up new avenues in broad areas in biology and medicine.

No MeSH data available.


Related in: MedlinePlus