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Fluorescence anisotropy imaging microscopy maps calmodulin binding during cellular contraction and locomotion
1Center for Light Microscope Imaging and Biotechnology, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
The Journal of Cell Biology
|June 1, 1993
Summary
Calmodulin (CaM) binding to cellular targets increases significantly with calcium levels in fibroblasts. This binding is spatially correlated with myosin II-based contractions during wound healing, suggesting local CaM activation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biophysics
Background:
- Calmodulin (CaM) acts as a calcium ion (Ca2+) sensor, regulating numerous cellular processes by binding to target proteins.
- Understanding CaM's dynamic binding in living cells is crucial for deciphering calcium signaling pathways.
Purpose of the Study:
- To investigate the temporal and spatial dynamics of CaM binding to cellular targets in fibroblasts.
- To correlate CaM binding with cellular events like serum stimulation and wound healing.
Main Methods:
- Utilized a fluorescent CaM analog with ratio imaging and steady-state fluorescence anisotropy imaging microscopy.
- Studied CaM distribution and binding in serum-deprived fibroblasts and during wound healing in polarized fibroblasts.
Main Results:
- In serum-deprived fibroblasts, CaM binding increased from <10% to ~95% upon serum stimulation, correlating with Ca2+ transients.
- CaM binding was elevated (~50%) in the leading lamellae during wound healing, associated with transverse fiber contraction and myosin II.
- Highest CaM binding occurred in retracting tails and associated with transverse fibers in motile fibroblasts.
Conclusions:
- Local activation of myosin II-based contractions is linked to localized CaM binding to target proteins.
- The study demonstrates a powerful microscopy technique for mapping molecular binding in living cells.