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Identification of Plasma Membrane Macro- and Microdomains from Wavelet Analysis of FRET Microscopy

Evgeny Kobrinsky ^*, Donald E. Mager ^*, Sarah A. Bentil ^*, Shin-ichi Murata , Darrell R. Abernethy ^* and Nikolai M. Soldatov ^*

^* National Institute on Aging, National Institutes of Health, Baltimore, Maryland; and Department of Pathology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Yamanashi, Japan

Correspondence: Address reprint requests to Nikolai M. Soldatov, Tel.: 410-558-8343; E-mail: soldatovn{at}grc.nia.nih.gov.

In this study, we sought to characterize functional signaling domains by applying the multiresolution properties of the continuous wavelet transform to fluorescence resonance energy transfer (FRET) microscopic images of plasma membranes. A genetically encoded FRET reporter of protein kinase C (PKC)-dependent phosphorylation was expressed in COS1 cells. Differences between wavelet coefficient matrices revealed several heterogeneous domains (typically ranging from 1 to 5 µm), reflecting the dynamic balance between PKC and phosphatase activity during stimulation with phorbol-12,13-dibutyrate or acetylcholine. The balance in these domains was not necessarily reflected in the overall plasma membrane changes, and observed heterogeneity was absent when cells were exposed to a phosphatase or PKC inhibitor. Prolonged exposure to phorbol-12,13-dibutyrate and acetylcholine yielded more homogeneous FRET distribution in plasma membranes. The proposed wavelet-based image analysis provides, for the first time, a basis and a means of detecting and quantifying dynamic changes in functional signaling domains, and may find broader application in studying fine aspects of cellular signaling by various imaging reporters.

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