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Nucleotide synthesis is regulated by cytoophidium formation during neurodevelopment and adaptive metabolism.

Aughey GN, Grice SJ, Shen QJ, Xu Y, Chang CC, Azzam G, Wang PY, Freeman-Mills L, Pai LM, Sung LY, Yan J, Liu JL - Biol Open (2014)

Bottom Line: Furthermore, our global metabolic profiling demonstrates that CTPsyn overexpression does not significantly alter CTPsyn-related enzymatic activity, suggesting that cytoophidium formation facilitates metabolic stabilisation.In addition, we show that overexpression of CTPsyn only results in moderate increase of CTP pool in human stable cell lines.Together, our study provides experimental evidence, and a mathematical model, for the hypothesis that inactive CTPsyn is incorporated into cytoophidia.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, United Kingdom.

No MeSH data available.


Related in: MedlinePlus

CTPsyn, cytoophidia and CTP production.(A) Cytoophidia are undetectable in wild-type and GFP vector transfected human 293T cells. (B) The mCTPsyn1-GFP proteins were stably expressed by CTPsyn1 OE#1, CTPsyn1 OE#2 and CTPsyn1 OE#3 cell lines. Almost every cell contains one or more cytoophidia. (C) Quantification of length of cytoophidia expressed by three independent cell lines. (D) Western blot of CTPsyn1 protein in wild-type, and mCTPsyn1-GFP expressing 293T cell lines. (E) Quantification of the relative protein abundance of endogenous CTPsyn1 and exogenous mCTPsyn1-GFP in CTPsyn1 OE#1, CTPsyn1 OE#2 and CTPsyn1 OE#3 cell lines. (F) The relative CTP concentration in cell lysates of wild-type, and three CTPsyn1 overexpression 293T cell lines. Overexpression of CTPsyn moderately increased intracellular CTP concentration in human cells. (G) The fitting of total CTPsyn and CTP concentration using the non-linear relationship derived from our mathematical model (see text). The x axis is total CTPsyn and y axis is CTP concentration. Data points represent experimentally measured values in wild type and CTPsyn overexpressing human 293T cells. (H) Proposed model of cytoophidia assembly in response to nutrient or developmental conditions. Scale bars: 20 µm.
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f06: CTPsyn, cytoophidia and CTP production.(A) Cytoophidia are undetectable in wild-type and GFP vector transfected human 293T cells. (B) The mCTPsyn1-GFP proteins were stably expressed by CTPsyn1 OE#1, CTPsyn1 OE#2 and CTPsyn1 OE#3 cell lines. Almost every cell contains one or more cytoophidia. (C) Quantification of length of cytoophidia expressed by three independent cell lines. (D) Western blot of CTPsyn1 protein in wild-type, and mCTPsyn1-GFP expressing 293T cell lines. (E) Quantification of the relative protein abundance of endogenous CTPsyn1 and exogenous mCTPsyn1-GFP in CTPsyn1 OE#1, CTPsyn1 OE#2 and CTPsyn1 OE#3 cell lines. (F) The relative CTP concentration in cell lysates of wild-type, and three CTPsyn1 overexpression 293T cell lines. Overexpression of CTPsyn moderately increased intracellular CTP concentration in human cells. (G) The fitting of total CTPsyn and CTP concentration using the non-linear relationship derived from our mathematical model (see text). The x axis is total CTPsyn and y axis is CTP concentration. Data points represent experimentally measured values in wild type and CTPsyn overexpressing human 293T cells. (H) Proposed model of cytoophidia assembly in response to nutrient or developmental conditions. Scale bars: 20 µm.

Mentions: According to previous studies, manipulations of certain conditions, such as glutamine deprivation or inhibition of GTP synthesis with drugs, could stimulate cytoophidium formation in some cells (Calise et al., 2014; Gou et al., 2014). To investigate the function of the cytoophidium in mammalian cells, 293T cells were transfected with a construct of mouse CTP synthase 1-GFP fusion protein (mCTPsyn1-GFP) which is driven by the EF1α promoter. Expression of mCTPS1 promoted assembly of cytoophidia in 293T cells in normal culture condition (Fig. 6A,B). Cytoophidia in mCTPS1-GFP expressing cells were significantly longer than those in normal 293T cells with or without DON treatment (Fig. 6B,C; supplementary material Fig. S4), indicating that more CTPsyn proteins accumulated in cytoophidium structure in cells expressing mCTPsyn1. We generated three stable cell lines overexpressing mCTPsyn1-GFP at approximately 6–8-fold higher than endogenous levels. (Fig. 6D,E). However, the intracellular CTP concentration only increased to 2-fold higher than that of wild-type cells indicating that CTPsyn activity does not increase linearly with protein concentration (Fig. 6F).


Nucleotide synthesis is regulated by cytoophidium formation during neurodevelopment and adaptive metabolism.

Aughey GN, Grice SJ, Shen QJ, Xu Y, Chang CC, Azzam G, Wang PY, Freeman-Mills L, Pai LM, Sung LY, Yan J, Liu JL - Biol Open (2014)

CTPsyn, cytoophidia and CTP production.(A) Cytoophidia are undetectable in wild-type and GFP vector transfected human 293T cells. (B) The mCTPsyn1-GFP proteins were stably expressed by CTPsyn1 OE#1, CTPsyn1 OE#2 and CTPsyn1 OE#3 cell lines. Almost every cell contains one or more cytoophidia. (C) Quantification of length of cytoophidia expressed by three independent cell lines. (D) Western blot of CTPsyn1 protein in wild-type, and mCTPsyn1-GFP expressing 293T cell lines. (E) Quantification of the relative protein abundance of endogenous CTPsyn1 and exogenous mCTPsyn1-GFP in CTPsyn1 OE#1, CTPsyn1 OE#2 and CTPsyn1 OE#3 cell lines. (F) The relative CTP concentration in cell lysates of wild-type, and three CTPsyn1 overexpression 293T cell lines. Overexpression of CTPsyn moderately increased intracellular CTP concentration in human cells. (G) The fitting of total CTPsyn and CTP concentration using the non-linear relationship derived from our mathematical model (see text). The x axis is total CTPsyn and y axis is CTP concentration. Data points represent experimentally measured values in wild type and CTPsyn overexpressing human 293T cells. (H) Proposed model of cytoophidia assembly in response to nutrient or developmental conditions. Scale bars: 20 µm.
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Related In: Results  -  Collection

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f06: CTPsyn, cytoophidia and CTP production.(A) Cytoophidia are undetectable in wild-type and GFP vector transfected human 293T cells. (B) The mCTPsyn1-GFP proteins were stably expressed by CTPsyn1 OE#1, CTPsyn1 OE#2 and CTPsyn1 OE#3 cell lines. Almost every cell contains one or more cytoophidia. (C) Quantification of length of cytoophidia expressed by three independent cell lines. (D) Western blot of CTPsyn1 protein in wild-type, and mCTPsyn1-GFP expressing 293T cell lines. (E) Quantification of the relative protein abundance of endogenous CTPsyn1 and exogenous mCTPsyn1-GFP in CTPsyn1 OE#1, CTPsyn1 OE#2 and CTPsyn1 OE#3 cell lines. (F) The relative CTP concentration in cell lysates of wild-type, and three CTPsyn1 overexpression 293T cell lines. Overexpression of CTPsyn moderately increased intracellular CTP concentration in human cells. (G) The fitting of total CTPsyn and CTP concentration using the non-linear relationship derived from our mathematical model (see text). The x axis is total CTPsyn and y axis is CTP concentration. Data points represent experimentally measured values in wild type and CTPsyn overexpressing human 293T cells. (H) Proposed model of cytoophidia assembly in response to nutrient or developmental conditions. Scale bars: 20 µm.
Mentions: According to previous studies, manipulations of certain conditions, such as glutamine deprivation or inhibition of GTP synthesis with drugs, could stimulate cytoophidium formation in some cells (Calise et al., 2014; Gou et al., 2014). To investigate the function of the cytoophidium in mammalian cells, 293T cells were transfected with a construct of mouse CTP synthase 1-GFP fusion protein (mCTPsyn1-GFP) which is driven by the EF1α promoter. Expression of mCTPS1 promoted assembly of cytoophidia in 293T cells in normal culture condition (Fig. 6A,B). Cytoophidia in mCTPS1-GFP expressing cells were significantly longer than those in normal 293T cells with or without DON treatment (Fig. 6B,C; supplementary material Fig. S4), indicating that more CTPsyn proteins accumulated in cytoophidium structure in cells expressing mCTPsyn1. We generated three stable cell lines overexpressing mCTPsyn1-GFP at approximately 6–8-fold higher than endogenous levels. (Fig. 6D,E). However, the intracellular CTP concentration only increased to 2-fold higher than that of wild-type cells indicating that CTPsyn activity does not increase linearly with protein concentration (Fig. 6F).

Bottom Line: Furthermore, our global metabolic profiling demonstrates that CTPsyn overexpression does not significantly alter CTPsyn-related enzymatic activity, suggesting that cytoophidium formation facilitates metabolic stabilisation.In addition, we show that overexpression of CTPsyn only results in moderate increase of CTP pool in human stable cell lines.Together, our study provides experimental evidence, and a mathematical model, for the hypothesis that inactive CTPsyn is incorporated into cytoophidia.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, United Kingdom.

No MeSH data available.


Related in: MedlinePlus