ASGSB 1998 Annual Meeting Abstracts

[21]
USE OF GFP-BASED CA²⁺ AND CA²⁺ -CALMODULIN SENSORS TO INVESTIGATE MECHANICAL STIMULATION. M. Magidin and K. Beckingham. Dept. Biochemistry and Cell Biology, Rice University, Houston, Texas.

Mechanical stimulation elicits an elevation of calcium levels in a wide variety of cell types, ranging from maize coleoptile to mammalian epithelial cells (Gehring et al., 1990; Hansen et al., 1993). The broad evolutionary conservation of this response points to a central role for calcium in mechanical signaling.

Recently calcium and calcium/calmodulin fluorescent indicator proteins have been developed, based on engineered forms of green fluorescent protein (EGFP’s; Romoser et al., 1997). These indicators consist of two different EGFP’s linked together by a region of the calcium/calmodulin target myosin light chain kinase. The overlap of the emission spectrum of one EGFP with the absorption spectrum of the second EGFP allows fluorescent energy transfer (FRET) to occur when these two moeities are close. Binding of calcium-saturated calmodulin to the linking target region moves the two EGFP’s apart and FRET is disrupted. These indicators provide a novel method for analysis of calcium-based responses that avoids the requirement for introduction of indicator dyes into the cells under analysis. As a result, calcium imaging in the tissues of an intact organism is now possible. We are expressing these indicators in the organism Drosophila melanogaster so that the spatial and temporal characteristics of calcium responses can be investigated in detail. Three types of indicators are currently being introduced into flies, two calcium/calmodulin sensors and one calcium indicator. The latter contains a calmodulin coding region in addition to the two EGFP modules so that the sensor is independent of external calmodulin. Progress in calcium and calcium/calmodulin imaging in vivo will be presented.

Supported by the NASA sponsored NSCORT at Rice University.

Gehring et al. 1990. Nature 345, 528-530.
Hansen et al. 1993 J.Cell. Sci. 106, 995-1004.
Romoser et al. 1997 J. Biol. Chem. 272, 13270-13274.

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