Analysis of functional signaling domains from fluorescence imaging and the two-dimensional continuous wavelet...

A technique that utilizes the one-dimensional (1D) continuous wavelet transform (CWT) of linearized fluorescence resonance energy transfer (FRET) microscopic images has been extended to identify signaling macro- and microdomains in cell plasma membranes by incorporating the two-dimensional (2D) CWT of time-lapse fluorescence and/or FRET images. Signaling domains were identified from differences in wavelet coefficient matrices, and there was good agreement between the 1D and 2D methods on examining a), static fluorescent images of COS1 cells expressing calmodulin kinase II fused with enhanced yellow fluorescent protein, and b), time lapse FRET images of reporters of protein kinase C (PKC) (PKC activity reporter) and adenylyl cyclase dynamics (cAMP) activity within COS1 plasma membrane confines after stimulation by phorbol-12,13-dibutyrate or forskolin, respectively. The proposed 2D wavelet-based image analysis effectively detected phosphorylation/dephosphorylation signaling microdomains (PKC) as well as those reflective of cAMP without the limitation of requiring linearized signals imposed by the 1D approach. Illustrating successful application to the analysis of intracellular compartments, the 2D CWT was further used to identify signaling domains of cAMP response element-binding (CREB)-induced transcriptional activation in the nuclei of COS1 cells, which could not be achieved with the 1D approach. This technique may be eventually used to characterize complex cellular signaling and protein-protein interactions within localized cytoplasmic domains.