Visualization of Protein Interactions in Living Cells

Molecular networks and system-level properties

“…Recently developed techniques, such as high-resolution NMR spectroscopy that allows to detect phosphorylation of proteins in situ (Selenko et al, 2008;Selenko and Wagner, 2007), provide novel and exciting opportunities to obtain the phosphosignature of proteins in physiologically relevant conditions allowing to directly probe their relation with the emerging properties of the network to which they belong. Of course the knowledge of the phosphosignature of a given protein needs to be complemented by that of the kinetic constants of each signature protein with its interaction partners obtained "in vitro" (Jason-Moller et al, 2006;Karlsson, 2004) or better in vivo (Ciruela, 2008;Roy et al, 2008;Zal, 2008).…”

“…Besides, phosphorylation is the preferred biochemical means used by cells to alter protein-protein interactions and to fine tune catalytic activity of enzymes (Lodish et al, 2007). Thus, the study of the molecular phenotypes induced by expression of non-phosphorylatable mutants or of mutants mimicking a constitutive phosphorylation or the determination of the phosphosignature (that is the positional, quantitative estimation of the phosphorylation sites in a protein) of a given interactor (Schmidt et al, 2008) and knowedge from in vitro [i.e., by BIacore (Jason-Moller et al, 2006;Karlsson, 2004)] or in vivo [i.e., by FRET and related techniques (Ciruela, 2008;Roy et al, 2008;Zal, 2008)] analysis of the effects of any given phosphorylation site on its molecular interaction, should substantially contribute to understanding of system-level properties in biochemically relevant networks.…”

Genetically Encodable Fluorescent Biosensors for Tracking Signaling Dynamics in Living Cells

“…402–405 Recently, several insightful reviews have been published that discuss the advantages (and disadvantages) of using these approaches to study protein-protein interactions in living cells. 402–406 Therefore, here we will briefly describe each technique and discuss only a few of the innovative ways in which they have been applied to study protein-protein interactions that occur during signal transduction.…”

“…411;412 Importantly, several FRET detection methods allow the apparent FRET efficiency, E app , to be quantified within a heterogeneous population of donor-acceptor pairs. Using mathematical models generated from measured E app values, important information can be gained about the structure and kinetics of protein-protein interaction networks (reviewed in 406 ). This approach has been particularly useful in defining the steps involved in the assembly and disassembly of signalosomes formed at the immune synapse.…”

“…This approach has been particularly useful in defining the steps involved in the assembly and disassembly of signalosomes formed at the immune synapse. 406 Likewise, FRET-based techniques have provided insights into the assembly of other signaling complexes designed to coordinate the activities of several signaling molecules in space and time. For example, by measuring FRET between CFP- and YFP-tagged versions of CaN and the RII subunit of PKA, respectively, Oliveria et al demonstrated that the molecular scaffold, AKAP79, organizes a kinase-scaffold-phosphatase ternary complex involved in the regulation of synaptic plasticity.…”