INVOLVEMENT OF MAP KINASE IN INSULIN SIGNALING REVEALED BY NONINVASIVE IMAGING OF LUCIFERASE GENE-EXPRESSION IN SINGLE LIVING CELLS

Background: Studies of the mechanisms by which signals are transmitted from receptor tyrosine kinases would be facilitated by a way of monit oring events at the single-cell level. We have explored how luciferase imaging can be used to examine the role of specific signalling pathwa ys in insulin-stimulated gene expression. The analysis of luciferase e xpression in single cells has previously been hampered by the insensit ivity of existing methodologies and the lack of a way of monitoring qu antitatively, and independently, more than one promoter within the sam e cell. We have developed a technique for examining the dynamics of in sulin-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 wit h a high-sensitivity photon-counting camera. The technique involved th e co-microinjection of luciferase- and aequorin-based reporter plasmid s directly into the cell nucleus, and the subsequent analysis of lumin escence in the presence of luciferin and coelenterazine, respectively. The method is quantitative and allows insulin-stimulated gene express ion to be monitored in real time. We found that insulin promoted a sub stantial increase in the expression of a luciferase gene under the con trol of the AP-1-binding site from the collagenase gene promoter. Aequ orin 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-activ ated protein (MAP) kinase phosphatase CL100, or the dominant-negative mutant MAP kinase kinase, MEKS(S217/221A). Conclusions: Microinjection coupled with luciferase imaging allows hormone-regulated gene express ion from relatively weak promoters to be monitored in single living ce lls. We have used this method to demonstrate that MAP kinase plays a c entral role in the ability of insulin to stimulate AP-1-dependent gene transcription.