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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.


Related in: MedlinePlus

Real time light induced nuclear translocation of LANS4 in mammalian tissue culture cells.(A) Representative images for light activation and reversion in HeLa cells and Cos7 (B) (scale bar = 25 μm); (c) Plotting the fold change of nuclear accumulations in HeLa, Cos7 and HEK293 (n = 4 each, mean reported ± SEM with dashed line). See also S1, S2 and S3 Movies. The blue shaded region indicates pulsed blue light activation (see Supplemental experimental procedures). (C) Multple activation reversion cycles in Cos7 (n = 2, mean reported ± SEM with shaded grey area). The blue shaded regions indicate pulsed blue light activation.
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pone.0128443.g003: Real time light induced nuclear translocation of LANS4 in mammalian tissue culture cells.(A) Representative images for light activation and reversion in HeLa cells and Cos7 (B) (scale bar = 25 μm); (c) Plotting the fold change of nuclear accumulations in HeLa, Cos7 and HEK293 (n = 4 each, mean reported ± SEM with dashed line). See also S1, S2 and S3 Movies. The blue shaded region indicates pulsed blue light activation (see Supplemental experimental procedures). (C) Multple activation reversion cycles in Cos7 (n = 2, mean reported ± SEM with shaded grey area). The blue shaded regions indicate pulsed blue light activation.

Mentions: We next sought to characterize the kinetics of nuclear import and export in response to light stimulation. We performed blue light stimulation and measured the rates of nuclear import and dark reversion for LANS4 in three types of mammalian tissue culture cells—HeLa, Cos7 and HEK293 (Fig 3A, 3B and S1–S3 Movies). Nuclear fluorescence upon activation was measured and fold changes of nuclear accumulation were fit by single exponentials with t1/2 = 3.3 ± 0.02 minutes for HeLa (n = 4), t1/2 = 2.7 ± 0.03 minutes for Cos7 (n = 3) and t1/2 = 5.9 ± 0.01 minutes for HEK293 (n = 5) (Fig 3C). Upon stopping the blue light stimulation, the nuclear export kinetics were similarly measured and fit, yielding t1/2 = 2.5 ± 0.01 minutes for HeLa, t1/2 = 2.8 ± 0.02 minutes for Cos7 and t1/2 = 3.2 ± 0.02 minutes for HEK293 (Fig 3C). The differences observed between the cell types may result from differential expression of importins and exportins [27]. Nuclear localization appeared fully reversible, with no significant loss in activation level after multiple cycles of blue light activation and reversion over the course of a few hours (Fig 3D).


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)

Real time light induced nuclear translocation of LANS4 in mammalian tissue culture cells.(A) Representative images for light activation and reversion in HeLa cells and Cos7 (B) (scale bar = 25 μm); (c) Plotting the fold change of nuclear accumulations in HeLa, Cos7 and HEK293 (n = 4 each, mean reported ± SEM with dashed line). See also S1, S2 and S3 Movies. The blue shaded region indicates pulsed blue light activation (see Supplemental experimental procedures). (C) Multple activation reversion cycles in Cos7 (n = 2, mean reported ± SEM with shaded grey area). The blue shaded regions indicate pulsed blue light activation.
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pone.0128443.g003: Real time light induced nuclear translocation of LANS4 in mammalian tissue culture cells.(A) Representative images for light activation and reversion in HeLa cells and Cos7 (B) (scale bar = 25 μm); (c) Plotting the fold change of nuclear accumulations in HeLa, Cos7 and HEK293 (n = 4 each, mean reported ± SEM with dashed line). See also S1, S2 and S3 Movies. The blue shaded region indicates pulsed blue light activation (see Supplemental experimental procedures). (C) Multple activation reversion cycles in Cos7 (n = 2, mean reported ± SEM with shaded grey area). The blue shaded regions indicate pulsed blue light activation.
Mentions: We next sought to characterize the kinetics of nuclear import and export in response to light stimulation. We performed blue light stimulation and measured the rates of nuclear import and dark reversion for LANS4 in three types of mammalian tissue culture cells—HeLa, Cos7 and HEK293 (Fig 3A, 3B and S1–S3 Movies). Nuclear fluorescence upon activation was measured and fold changes of nuclear accumulation were fit by single exponentials with t1/2 = 3.3 ± 0.02 minutes for HeLa (n = 4), t1/2 = 2.7 ± 0.03 minutes for Cos7 (n = 3) and t1/2 = 5.9 ± 0.01 minutes for HEK293 (n = 5) (Fig 3C). Upon stopping the blue light stimulation, the nuclear export kinetics were similarly measured and fit, yielding t1/2 = 2.5 ± 0.01 minutes for HeLa, t1/2 = 2.8 ± 0.02 minutes for Cos7 and t1/2 = 3.2 ± 0.02 minutes for HEK293 (Fig 3C). The differences observed between the cell types may result from differential expression of importins and exportins [27]. Nuclear localization appeared fully reversible, with no significant loss in activation level after multiple cycles of blue light activation and reversion over the course of a few hours (Fig 3D).

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.


Related in: MedlinePlus