Characterization of a dextran-based bifunctional calcium indicator immobilized in cells by the enzymatic addition of isoprenoid lipids.
Cellular processes can be controlled by cell-wide increases in the cytosolic Ca2+ concentration or, alternatively, by localized Ca2+ signals in micro- and nano-domains. The experimental characterization of such localized Ca2+ signals would be facilitated using an immobilized Ca2+ indicator, which could prevent the accelerated spatial spreading of Ca2+ ions that is mediated by binding to diffusible indicators. Here we characterize a dextran-based Ca2+ indicator (CAAX-green) that becomes immobilized in the cytosol by an enzyme-mediated addition of a geranylgeranyl lipid group. CAAX-green consists of a dextran backbone with an attached Ca(2+)-green as well as an 11 residue peptide ending in a C-terminal CAAX-motif. Once introduced into cells by microporation, geranylgeranyl lipid groups are attached to the CAAX peptides by cytosolic enzymes. Measurements in tumor mastcells, myocytes and fibroblasts showed that the indicator becomes membrane attached between 30 min and 1 h following incorporation into the cytoplasm. A time-dependent 10-fold reduction of the diffusion coefficient and a parallel increase in the cytosolic retention after permeabilization indicates that at least 90% of cellular CAAX-green is immobilized. The KD of the indicator in permeabilized cells is 0.65 microM. Overall, these properties make CAAX-green well suited for the investigation of localized Ca2+ signals in a variety of cell types.
Horne
J H
JH
Department of Cell Biology, Duke University, Durham, NC 27710, USA.
Meyer
T
T
D000818:Animals; D002118:Calcium / Q000032:analysis*; D003911:Dextrans; D005456:Fluorescent Dyes; D016257:Fura-2; D008055:Lipids; D008856:Microscopy, Fluorescence / Q000379:methods*; D051381:Rats; D014407:Tumor Cells, Cultured
D003911:Dextrans; D005456:Fluorescent Dyes; D008055:Lipids; D002118:Calcium; D016257:Fura-2