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• W2044884027 startingPage "890" @default.
• W2044884027 abstract "Background: Studies of the mechanisms by which signals are transmitted from receptor tyrosine kinases would be facilitated by a way of monitoring events at the single-cell level. We have explored how luciferase imaging can be used to examine the role of specific signalling pathways in insulin-stimulated gene expression. The analysis of luciferase expression in single cells has previously been hampered by the insensitivity of existing methodologies and the lack of a way of monitoring quantitatively, and independently, more than one promoter within the same cell. We have developed a technique for examining the dynamics of insulin-stimulated AP-1-dependent transcription in single living cells, and have explored the signalling pathway involved.Results Luciferase and aequorin gene expression were examined in single living cells with a high-sensitivity photon-counting camera. The technique involved the co-microinjection of luciferase- and aequorin-based reporter plasmids directly into the cell nucleus, and the subsequent analysis of luminescence in the presence of luciferin and coelenterazine, respectively. The method is quantitative and allows insulin-stimulated gene expression to be monitored in real time. We found that insulin promoted a substantial increase in the expression of a luciferase gene under the control of the AP-1-binding site from the collagenase gene promoter. Aequorin expression, under the control of a cytomegalovirus promoter, was unaffected by insulin. The effect of insulin on luciferase expression was specifically blocked by overexpression of either the mitogen-activated protein (MAP) kinase phosphatase CL100, or the dominant-negative mutant MAP kinase kinase, MEKS217/221A.Conclusion Microinjection coupled with luciferase imaging allows hormone-regulated gene expression from relatively weak promoters to be monitored in single living cells. We have used this method to demonstrate that MAP kinase plays a central role in the ability of insulin to stimulate AP-1-dependent gene transcription." @default.
• W2044884027 created "2016-06-24" @default.
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• W2044884027 creator A5015737554 @default.
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• W2044884027 date "1995-08-01" @default.
• W2044884027 modified "2023-10-17" @default.
• W2044884027 title "Involvement of MAP kinase in insulin signalling revealed by non-invasive imaging of luciferase gene expression in single living cells" @default.
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• W2044884027 doi "https://doi.org/10.1016/s0960-9822(95)00179-5" @default.
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• W2044884027 hasPublicationYear "1995" @default.
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