Rincon-Zachary, Magaly , Valster, Aline , Blancaflor, Elison B .
Use of a Yellow Cameleon (YC 3.60) as an intracellular calcium sensor in plant cells using fluorescence resonance energy transfer (FRET) sensitized emission.
Yellow cameleons are chimeric proteins that fluoresce upon binding Ca2+ and are composed of: (1) a cyan fluorescent protein (CFP); (2) the C terminus of calmodulin (CaM); (3) glycine-glycine linker; (4) the CaM-binding domain of myosin light chain kinase (M13); and (5) a yellow fluorescent protein (YFP). The increased interaction between M13 and CaM upon binding of Ca2+ to CaM shortens the distance between CFP and YFP and as a result the efficiency of fluorescence resonance energy transfer (FRET) is enhanced. We are investigating the suitability of the Yellow Cameleon version 3.60 (YC 3.60) with improved dynamic range  for the detection of intracellular calcium levels in plant cells. The YC 3.60 has been shown to be more resistant to cytoplasmic acidification than previous cameleons. The YC 3.60 construct was sub-cloned into the pCAMBIA 1390 binary vector and was expressed under the 35S promoter in Arabidopsis (ecotype Columbia) seedlings. Expression of YC 3.60 was observed in all the tissues of the transgenic Arabidopsis seedlings, including the cotyledons, hypocotyl, trichomes, root and root hairs. FRET efficiency values of whole frames and specific regions of interest were obtained using the FRET sensitized emission method using a Leica SP2 AOBS confocal microscope. Data are presented showing oscillating intracellular calcium gradients at the tip of growing root hairs. Furthermore, validity of the probe was confirmed by observing calcium spikes after treating roots with cold, exogenously added calcium (1 mM), pH 4.5 and 1 mM glutamic acid. Supported by NSF DBI-0400580.  Nagai et al. (2004) PNAS 101: 10554 – 10559.
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1 - Midwestern State University, Dept of Biology, 3410 Taft Blvd, Wichita Falls, TX, 76308, USA
2 - The Samuel Roberts Noble Foundation, Plant Biology
sensitized emission FRET
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM