Involvement of MAP kinase in insulin signalling revealed by non-invasive imaging of luciferase gene expression in single living cells.

Abstract

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. Luciferase and aequorin gene expression were examined in single living cells with a high-sensitivity photon-counting camera. The technique involved the comicroinjection 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. 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.