TY - JOUR
T1 - Identification of plasma membrane macro- and microdomains from wavelet analysis of FRET microscopy
AU - Kobrinsky, Evgeny
AU - Mager, Donald E.
AU - Bentil, Sarah A.
AU - Murata, Shin Ichi
AU - Abernethy, Darrell R.
AU - Soldatov, Nikolai M.
N1 - Funding Information:
This study was supported by the Intramural Research Program of the National Institute on Aging, National Institutes of Health. None of the authors declare any competing financial interests that could be perceived as influencing this research.
PY - 2005/5
Y1 - 2005/5
N2 - 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.
AB - 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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U2 - 10.1529/biophysj.104.054056
DO - 10.1529/biophysj.104.054056
M3 - Article
C2 - 15722423
AN - SCOPUS:17844368351
SN - 0006-3495
VL - 88
SP - 3625
EP - 3634
JO - Biophysical journal
JF - Biophysical journal
IS - 5
ER -