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Biophys. J. BioFAST: First Published February 18, 2005. doi:10.1529/biophysj.104.054056
© 2005 by the Biophysical Society.

A more recent version of this article appeared on May 1, 2005.
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SPECTROSCOPY, IMAGING, OTHER TECHNIQUES

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

Evgeny Kobrinsky 1, Donald E. Mager 2, Sarah A. Bentil 1, Shin-ichi Murata 3, Darrell R. Abernethy 1 and Nikolai M. Soldatov 1*

1 National Institute on Aging, National Institutes of Health
2 University at Buffalo, SUNY
3 Department of Pathology, University of Yamanashi

* To whom correspondence should be addressed. E-mail: soldatovn{at}grc.nia.nih.gov.

Submitted on October 5, 2004
Revised on November 19, 2004
Accepted on 11 February 2005


  Abstract
In this study, we sought to characterize functional signaling domains by applying the multi-resolution properties of the continuous wavelet transform to fluorescence resonance energy transfer (FRET) microscopic images of plasma membranes (PMs). 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 PM 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 PMs. The proposed wavelet based image analysis for the first time provides bases and 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.

Key Words: COS1 cells, Continuous wavelet transform, FRET image analysis, protein kinase C, texture image analysis




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D. E. Mager, E. Kobrinsky, A. Masoudieh, A. Maltsev, D. R. Abernethy, and N. M. Soldatov
Analysis of Functional Signaling Domains from Fluorescence Imaging and the Two-Dimensional Continuous Wavelet Transform
Biophys. J., October 15, 2007; 93(8): 2900 - 2910.
[Abstract] [Full Text] [PDF]


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Copyright © 2005 by the Biophysical Society.