Intracellular calmodulin availability accessed with two-photon cross-correlation

The availability and interactions of signaling proteins are tightly regulated in time and space to produce specific and localized effects. For calmodulin (CaM), a key transducer of intracellular Ca2+ signaling, binding to its variety of targets initiates signaling cascades and regulates its subcellular localization, thereby making it unavailable for subsequent binding interactions. Among CaM's numerous targets, Ca2+ /Ca M-dependent protein kinase 11 is one of the most striking due to its unique ability to increase its affinity for CaM by autophosphorylation and to translocate when bound to Ca2+/CaM. Two-photon fluorescence correlation spectroscopy and cross-correlation spectroscopy were utilized to compare mobility and molecular interactions between CaM and Ca2+/CaM-dependent protein kinase 11 in solution and in living cells. These techniques revealed that CaM availability in cells could be altered by a change in intracellular conditions. Two-photon fluorescence cross-correlation spectroscopy exemplifies a generally applicable approach for studying protein-protein interactions in living cells that allows access to the behavior of signaling molecules within their native environment to probe for heterogeneities in signaling pathways in different cellular compartments.