title: Indo-1 derivatives for local calcium sensing.
abstract: The role of calcium in signal transduction relies on the precise spatial and temporal control of its concentration. The existing means to detect fluctuations in Ca2+ concentrations with adequate temporal and spatial resolution are limited. We introduce here a method to measure Ca2+ concentrations in defined locations in living cells that is based on linking the Ca2+-sensitive dye Indo-1 to SNAP-tag fusion proteins. Fluorescence spectroscopy of SNAP-Indo-1 conjugates in vitro showed that the conjugates retained the Ca2+-sensing ability of Indo-1. In a proof-of-principle experiment, local Ca2+ sensing was demonstrated in single cells dissociated from muscle of adult mice expressing a nucleus-localized SNAP-tag fusion. Ca2+ concentrations inside nuclei of resting cells were measured by shifted excitation and emission ratioing of confocal microscopic images of fluorescence. After permeabilizing the plasma membrane, changes in the bathing solution induced corresponding changes in nuclear [Ca2+] that were readily detected and used for a preliminary calibration of the technique. This work thus demonstrates the synthesis and application of SNAP-tag-based Ca2+ indicators that combine the spatial specificity of genetically encoded calcium indicators with the advantageous spectroscopic properties of synthetic indicators.
authors.affiliation: Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015, Lausanne, Switzerland.
authors.forename: Michael
authors.initials: M
authors.lastname: Bannwarth
authors.forename: Ivan R
authors.initials: IR
authors.lastname: Correa
authors.forename: Monika
authors.initials: M
authors.lastname: Sztretye
authors.forename: Sandrine
authors.initials: S
authors.lastname: Pouvreau
authors.forename: Cindy
authors.initials: C
authors.lastname: Fellay
authors.forename: Annina
authors.initials: A
authors.lastname: Aebischer
authors.forename: Leandro
authors.initials: L
authors.lastname: Royer
authors.forename: Eduardo
authors.initials: E
authors.lastname: Rois
authors.forename: Kai
authors.initials: K
authors.lastname: Johnsson
mesh_terms: D000818:Animals; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006147:Guanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D007211:Indoles / Q000737:chemistry*; D051379:Mice; D008856:Microscopy, Fluorescence; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D007211:Indoles; D011993:Recombinant Fusion Proteins; C064976:O(6)-benzylguanine; D006147:Guanine; D019853:O(6)-Methylguanine-DNA Methyltransferase; C048960:indo-1; D002118:Calcium