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Control of Protein Activity and Cell Fate Specification via Light-Mediated Nuclear Translocation.

Yumerefendi H, Dickinson DJ, Wang H, Zimmerman SP, Bear JE, Goldstein B, Hahn K, Kuhlman B - PLoS ONE (2015)

Bottom Line: Here, we extend this result by characterizing the binding properties of a LOV/NLS switch and demonstrating that it can be used to control gene transcription in yeast.By inserting LANS into the C. elegans lin-1 locus using Cas9-triggered homologous recombination, we demonstrated control of cell fate via light-dependent manipulation of a native transcription factor.We conclude that LANS can be a valuable experimental method for spatial and temporal control of nuclear localization in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina, United States of America.

ABSTRACT
Light-activatable proteins allow precise spatial and temporal control of biological processes in living cells and animals. Several approaches have been developed for controlling protein localization with light, including the conditional inhibition of a nuclear localization signal (NLS) with the Light Oxygen Voltage (AsLOV2) domain of phototropin 1 from Avena sativa. In the dark, the switch adopts a closed conformation that sterically blocks the NLS motif. Upon activation with blue light the C-terminus of the protein unfolds, freeing the NLS to direct the protein to the nucleus. A previous study showed that this approach can be used to control the localization and activity of proteins in mammalian tissue culture cells. Here, we extend this result by characterizing the binding properties of a LOV/NLS switch and demonstrating that it can be used to control gene transcription in yeast. Additionally, we show that the switch, referred to as LANS (light-activated nuclear shuttle), functions in the C. elegans embryo and allows for control of nuclear localization in individual cells. By inserting LANS into the C. elegans lin-1 locus using Cas9-triggered homologous recombination, we demonstrated control of cell fate via light-dependent manipulation of a native transcription factor. We conclude that LANS can be a valuable experimental method for spatial and temporal control of nuclear localization in vivo.

No MeSH data available.


Design and biophysical characterization of light conditioned nuclear localization signal.(A) Schematic of the Light Activated Nuclear Shuttle (LANS) design for light activated nuclear import (B) Sequence alignment of the wild type AsLOV2 and the designed AsLOV2cNLS (sequence identity and homology is marked according to CLUSTALW scheme). (C) Fluorescence polarization competitive binding assay of AsLOV2cNLS against human importin α5 and importin α7.
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pone.0128443.g001: Design and biophysical characterization of light conditioned nuclear localization signal.(A) Schematic of the Light Activated Nuclear Shuttle (LANS) design for light activated nuclear import (B) Sequence alignment of the wild type AsLOV2 and the designed AsLOV2cNLS (sequence identity and homology is marked according to CLUSTALW scheme). (C) Fluorescence polarization competitive binding assay of AsLOV2cNLS against human importin α5 and importin α7.

Mentions: To control nuclear import with light we engineered a conditional Nuclear Localization Signal (cNLS) that would be allosterically blocked in the dark but available for binding to importin in the light (Fig 1A). Previously, the AsLOV2 domain from Avena sativa has been successfully used to control the binding of short, linear sequence epitopes [14–16] and does not contain an endogenous nuclear localization signal. Therefore, to generate an allosterically caged NLS, we first attached the human Myc NLS at the end of the AsLOV2 domain after residue 546, aligning the proline residue from the NLS sequence to the proline residue of AsLOV2. This fusion protein (AsLOV2cMyc) bound importin α5 with low nanomolar affinity and showed no light-dependence (supplemental). Next, we decided to embed the Myc NLS further into the Jα helix, aligning the alanine residues present in both sequences (Fig 1B). To eliminate the conserved proline residue at the beginning of the NLS, which could disrupt the helicity of the Jα, we performed design simulations with the modeling program Rosetta using the karyopherin-Myc NLS complex structure (PDB: 1EE4). We allowed only the proline residue of the Myc NLS to vary and identified a favorable methionine substitution (Fig 1B and S1 Fig). Rosetta was then used to build a model of the designed NLS sequence, grafted onto the AsLOV2 structure (PDB: 2v0u) creating AsLOV2cNLS. The designed methionine pointed towards solvent, not clashing with any residues from the AsLOV2 domain, and the remaining hydrophobic residues present in the NLS were well packed against the core PAS domain (S1 Fig).


Control of Protein Activity and Cell Fate Specification via Light-Mediated Nuclear Translocation.

Yumerefendi H, Dickinson DJ, Wang H, Zimmerman SP, Bear JE, Goldstein B, Hahn K, Kuhlman B - PLoS ONE (2015)

Design and biophysical characterization of light conditioned nuclear localization signal.(A) Schematic of the Light Activated Nuclear Shuttle (LANS) design for light activated nuclear import (B) Sequence alignment of the wild type AsLOV2 and the designed AsLOV2cNLS (sequence identity and homology is marked according to CLUSTALW scheme). (C) Fluorescence polarization competitive binding assay of AsLOV2cNLS against human importin α5 and importin α7.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4471001&req=5

pone.0128443.g001: Design and biophysical characterization of light conditioned nuclear localization signal.(A) Schematic of the Light Activated Nuclear Shuttle (LANS) design for light activated nuclear import (B) Sequence alignment of the wild type AsLOV2 and the designed AsLOV2cNLS (sequence identity and homology is marked according to CLUSTALW scheme). (C) Fluorescence polarization competitive binding assay of AsLOV2cNLS against human importin α5 and importin α7.
Mentions: To control nuclear import with light we engineered a conditional Nuclear Localization Signal (cNLS) that would be allosterically blocked in the dark but available for binding to importin in the light (Fig 1A). Previously, the AsLOV2 domain from Avena sativa has been successfully used to control the binding of short, linear sequence epitopes [14–16] and does not contain an endogenous nuclear localization signal. Therefore, to generate an allosterically caged NLS, we first attached the human Myc NLS at the end of the AsLOV2 domain after residue 546, aligning the proline residue from the NLS sequence to the proline residue of AsLOV2. This fusion protein (AsLOV2cMyc) bound importin α5 with low nanomolar affinity and showed no light-dependence (supplemental). Next, we decided to embed the Myc NLS further into the Jα helix, aligning the alanine residues present in both sequences (Fig 1B). To eliminate the conserved proline residue at the beginning of the NLS, which could disrupt the helicity of the Jα, we performed design simulations with the modeling program Rosetta using the karyopherin-Myc NLS complex structure (PDB: 1EE4). We allowed only the proline residue of the Myc NLS to vary and identified a favorable methionine substitution (Fig 1B and S1 Fig). Rosetta was then used to build a model of the designed NLS sequence, grafted onto the AsLOV2 structure (PDB: 2v0u) creating AsLOV2cNLS. The designed methionine pointed towards solvent, not clashing with any residues from the AsLOV2 domain, and the remaining hydrophobic residues present in the NLS were well packed against the core PAS domain (S1 Fig).

Bottom Line: Here, we extend this result by characterizing the binding properties of a LOV/NLS switch and demonstrating that it can be used to control gene transcription in yeast.By inserting LANS into the C. elegans lin-1 locus using Cas9-triggered homologous recombination, we demonstrated control of cell fate via light-dependent manipulation of a native transcription factor.We conclude that LANS can be a valuable experimental method for spatial and temporal control of nuclear localization in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina, United States of America.

ABSTRACT
Light-activatable proteins allow precise spatial and temporal control of biological processes in living cells and animals. Several approaches have been developed for controlling protein localization with light, including the conditional inhibition of a nuclear localization signal (NLS) with the Light Oxygen Voltage (AsLOV2) domain of phototropin 1 from Avena sativa. In the dark, the switch adopts a closed conformation that sterically blocks the NLS motif. Upon activation with blue light the C-terminus of the protein unfolds, freeing the NLS to direct the protein to the nucleus. A previous study showed that this approach can be used to control the localization and activity of proteins in mammalian tissue culture cells. Here, we extend this result by characterizing the binding properties of a LOV/NLS switch and demonstrating that it can be used to control gene transcription in yeast. Additionally, we show that the switch, referred to as LANS (light-activated nuclear shuttle), functions in the C. elegans embryo and allows for control of nuclear localization in individual cells. By inserting LANS into the C. elegans lin-1 locus using Cas9-triggered homologous recombination, we demonstrated control of cell fate via light-dependent manipulation of a native transcription factor. We conclude that LANS can be a valuable experimental method for spatial and temporal control of nuclear localization in vivo.

No MeSH data available.