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All-optical regulation of gene expression in targeted cells.

Wang Y, He H, Li S, Liu D, Lan B, Hu M, Cao Y, Wang C - Sci Rep (2014)

Bottom Line: Recently, various approaches based on extra-engineered light-sensitive proteins have been developed to provide optogenetic actuators for gene expression.Complicated biomedical techniques including exogenous genes engineering, transfection, and material delivery are needed.Intrinsic or exogenous genes can be activated by a Ca(2+)-sensitive transcription factor nuclear factor of activated T cells (NFAT) driven by a short flash of femtosecond-laser irradiation.

View Article: PubMed Central - PubMed

Affiliation: Ultrafast Laser Laboratory, Key Laboratory of Optoelectronic Information Technology (Ministry of Education), College of Precision Instrument and Optoelectronics Engineering, Tianjin University, Tianjin, 300072, P.R. China.

ABSTRACT
Controllable gene expression is always a challenge and of great significance to biomedical research and clinical applications. Recently, various approaches based on extra-engineered light-sensitive proteins have been developed to provide optogenetic actuators for gene expression. Complicated biomedical techniques including exogenous genes engineering, transfection, and material delivery are needed. Here we present an all-optical method to regulate gene expression in targeted cells. Intrinsic or exogenous genes can be activated by a Ca(2+)-sensitive transcription factor nuclear factor of activated T cells (NFAT) driven by a short flash of femtosecond-laser irradiation. When applied to mesenchymal stem cells, expression of a differentiation regulator Osterix can be activated by this method to potentially induce differentiation of them. A laser-induced "Ca(2+)-comb" (LiCCo) by multi-time laser exposure is further developed to enhance gene expression efficiency. This noninvasive method hence provides an encouraging advance of gene expression regulation, with promising potential of applying in cell biology and stem-cell science.

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Scheme of all-optical regulation of gene expression method.(a). Typical cellular Ca2+ release after exposure of femtosecond laser at different powers (n = 20 cells in each experiment). The power density is around 3 × 105 W/cm2 at the mean power of 10 mW. (b). GFP-tagged NFAT migration into nucleus after the stimulation of femtosecond laser. Bar: 10 μm. (c). Normalized fluorescence intensity of NFAT-GFP in cytosol and nucleus respectively. Significant migration of NFAT appeared at the 12th minute. (d). Proposed mechanism of femtosecond-laser induced gene transcription. The femtosecond laser exposure releases Ca2+ store in ER and provides stress to Ca2+ channel in cytoplasm membrane. Subsequently, NFAT is dephosphorylated and migrate into nucleus regulated by calcineurin. After the activation of corresponding genes, NFAT then moves out and is phosphorylated again. In the activation of gene expression, NFAT may cooperate with other transcriptional partners.
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f1: Scheme of all-optical regulation of gene expression method.(a). Typical cellular Ca2+ release after exposure of femtosecond laser at different powers (n = 20 cells in each experiment). The power density is around 3 × 105 W/cm2 at the mean power of 10 mW. (b). GFP-tagged NFAT migration into nucleus after the stimulation of femtosecond laser. Bar: 10 μm. (c). Normalized fluorescence intensity of NFAT-GFP in cytosol and nucleus respectively. Significant migration of NFAT appeared at the 12th minute. (d). Proposed mechanism of femtosecond-laser induced gene transcription. The femtosecond laser exposure releases Ca2+ store in ER and provides stress to Ca2+ channel in cytoplasm membrane. Subsequently, NFAT is dephosphorylated and migrate into nucleus regulated by calcineurin. After the activation of corresponding genes, NFAT then moves out and is phosphorylated again. In the activation of gene expression, NFAT may cooperate with other transcriptional partners.

Mentions: Femtosecond lasers have been advancing biological researches with significant progresses151617 by their unique advantages including high spatial resolution and good biological safety. It has been found that cellular Ca2+ level can be modulated by tightly focused femtosecond-laser irradiation181920, where Ca2+ store in endoplasmic reticulum (ER) and Ca2+ channels in cytoplasm membrane are both involved21. Since intracellular Ca2+ plays the role of second messenger22, it is reasonable to design a scheme using femtosecond laser to regulate gene expression by taking advantage of a Ca2+-sensitive transcription factor. To this end, parameters of a Ti: Sapphire laser (repetition rate: 80 MHz, pulse duration: 75 fs) coupled in a confocal microscope (Supplementary Fig. S1a) were carefully adjusted to produce appropriate Ca2+ level in the targeted cell as the basic of all following works. As shown in Fig. 1a, a significant Ca2+ rise was excited by 0.3-s exposure of the focused femtosecond laser with a mean power of 40 mW a. It should be noted that high-power and long-duration irradiation of femtosecond laser can induce apoptosis (Supplementary Fig. S1b, c, and d).


All-optical regulation of gene expression in targeted cells.

Wang Y, He H, Li S, Liu D, Lan B, Hu M, Cao Y, Wang C - Sci Rep (2014)

Scheme of all-optical regulation of gene expression method.(a). Typical cellular Ca2+ release after exposure of femtosecond laser at different powers (n = 20 cells in each experiment). The power density is around 3 × 105 W/cm2 at the mean power of 10 mW. (b). GFP-tagged NFAT migration into nucleus after the stimulation of femtosecond laser. Bar: 10 μm. (c). Normalized fluorescence intensity of NFAT-GFP in cytosol and nucleus respectively. Significant migration of NFAT appeared at the 12th minute. (d). Proposed mechanism of femtosecond-laser induced gene transcription. The femtosecond laser exposure releases Ca2+ store in ER and provides stress to Ca2+ channel in cytoplasm membrane. Subsequently, NFAT is dephosphorylated and migrate into nucleus regulated by calcineurin. After the activation of corresponding genes, NFAT then moves out and is phosphorylated again. In the activation of gene expression, NFAT may cooperate with other transcriptional partners.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4061554&req=5

f1: Scheme of all-optical regulation of gene expression method.(a). Typical cellular Ca2+ release after exposure of femtosecond laser at different powers (n = 20 cells in each experiment). The power density is around 3 × 105 W/cm2 at the mean power of 10 mW. (b). GFP-tagged NFAT migration into nucleus after the stimulation of femtosecond laser. Bar: 10 μm. (c). Normalized fluorescence intensity of NFAT-GFP in cytosol and nucleus respectively. Significant migration of NFAT appeared at the 12th minute. (d). Proposed mechanism of femtosecond-laser induced gene transcription. The femtosecond laser exposure releases Ca2+ store in ER and provides stress to Ca2+ channel in cytoplasm membrane. Subsequently, NFAT is dephosphorylated and migrate into nucleus regulated by calcineurin. After the activation of corresponding genes, NFAT then moves out and is phosphorylated again. In the activation of gene expression, NFAT may cooperate with other transcriptional partners.
Mentions: Femtosecond lasers have been advancing biological researches with significant progresses151617 by their unique advantages including high spatial resolution and good biological safety. It has been found that cellular Ca2+ level can be modulated by tightly focused femtosecond-laser irradiation181920, where Ca2+ store in endoplasmic reticulum (ER) and Ca2+ channels in cytoplasm membrane are both involved21. Since intracellular Ca2+ plays the role of second messenger22, it is reasonable to design a scheme using femtosecond laser to regulate gene expression by taking advantage of a Ca2+-sensitive transcription factor. To this end, parameters of a Ti: Sapphire laser (repetition rate: 80 MHz, pulse duration: 75 fs) coupled in a confocal microscope (Supplementary Fig. S1a) were carefully adjusted to produce appropriate Ca2+ level in the targeted cell as the basic of all following works. As shown in Fig. 1a, a significant Ca2+ rise was excited by 0.3-s exposure of the focused femtosecond laser with a mean power of 40 mW a. It should be noted that high-power and long-duration irradiation of femtosecond laser can induce apoptosis (Supplementary Fig. S1b, c, and d).

Bottom Line: Recently, various approaches based on extra-engineered light-sensitive proteins have been developed to provide optogenetic actuators for gene expression.Complicated biomedical techniques including exogenous genes engineering, transfection, and material delivery are needed.Intrinsic or exogenous genes can be activated by a Ca(2+)-sensitive transcription factor nuclear factor of activated T cells (NFAT) driven by a short flash of femtosecond-laser irradiation.

View Article: PubMed Central - PubMed

Affiliation: Ultrafast Laser Laboratory, Key Laboratory of Optoelectronic Information Technology (Ministry of Education), College of Precision Instrument and Optoelectronics Engineering, Tianjin University, Tianjin, 300072, P.R. China.

ABSTRACT
Controllable gene expression is always a challenge and of great significance to biomedical research and clinical applications. Recently, various approaches based on extra-engineered light-sensitive proteins have been developed to provide optogenetic actuators for gene expression. Complicated biomedical techniques including exogenous genes engineering, transfection, and material delivery are needed. Here we present an all-optical method to regulate gene expression in targeted cells. Intrinsic or exogenous genes can be activated by a Ca(2+)-sensitive transcription factor nuclear factor of activated T cells (NFAT) driven by a short flash of femtosecond-laser irradiation. When applied to mesenchymal stem cells, expression of a differentiation regulator Osterix can be activated by this method to potentially induce differentiation of them. A laser-induced "Ca(2+)-comb" (LiCCo) by multi-time laser exposure is further developed to enhance gene expression efficiency. This noninvasive method hence provides an encouraging advance of gene expression regulation, with promising potential of applying in cell biology and stem-cell science.

Show MeSH
Related in: MedlinePlus