Holistic Methods for the Analysis of cNMP Effects.

Cyclic nucleotide monophosphates (cNMPs) typify the archetype second messenger in living cells and serve as molecular switches with broad functionality. cAMP and cGMP are the best-described cNMPs; however, there is a growing body of evidence indicating that also cCMP and cUMP play a substantial role in signal transduction. Despite research efforts, to date, relatively little is known about the biology of these noncanonical cNMPs, which is due, at least in part, to methodological issues in the past entailing setbacks of the entire field. Only recently, with the use of state-of-the-art techniques, it was possible to revive noncanonical cNMP research. While high-sensitive detection methods disclosed relevant levels of cCMP and cUMP in mammalian cells, knowledge about the biological effectors and their physiological interplay is still incomplete. Holistic biophysical readouts capture cell responses label-free and in an unbiased fashion with the advantage to detect concealed aspects of cell signaling that are arduous to access via traditional biochemical assay approaches. In this chapter, we introduce the dynamic mass redistribution (DMR) technology to explore cell signaling beyond established receptor-controlled mechanisms. Both common and distinctive features in the signaling structure of cCMP and cUMP were identified. Moreover, the integrated response of whole live cells revealed a hitherto undisclosed additional effector of the noncanonical cNMPs. Future studies will show how holistic methods will become integrated into the methodological arsenal of contemporary cNMP research.