Indo-1 Hybrid Biosensors For Calcium Monitoring In Cellular Organelles

Abstract

The central role of calcium in signal transduction depends on the precise spatial and temporal control of its concentration. The existing possibilities to detect fluctuations in Ca2+ concentration with adequate temporal and spatial resolution and in specific cellular organelles, are limited. We have developed a method to measure Ca2+ concentrations in defined subcellular locations that uses derivatives of the dye Indo-1 covalently bound to fusions of “SNAP-tag” (a multiply mutated version of human alkylguanyl DNA alkyl transferase) expressed inside cells. SNAP-Indo-1 conjugates retained the Ca2+−sensing ability of Indo-1 in vitro. One of the derivatives of Indo-1 displayed a four-fold higher fluorescence after coupling to SNAP-tag, which improves specificity of Ca2+ sensing in living cells. In a proof-of-principle experiment, local Ca2+ sensing was demonstrated in muscle cells of mice expressing a SNAP fusion localized to nuclei. [Ca2+] inside nuclei ([Ca2+]n) was evaluated by SEER (shifted excitation and emission ratioing) of confocal microscopic images of fluorescence of the sensor. After permeabilizing the plasma membrane, changes to bathing solutions containing different [Ca2+] induced corresponding changes in [Ca2+]n that were readily detected and used for a preliminary calibration of the technique. Similar hybrid sensors using Indo-1 but targeted to the mitochondrial matrix and the SR were also constructed. In principle, these hybrid sensors should combine the spatial specificity of biosensors with the superior kinetics and dynamic range of small synthetic fluorescent monitors. Factors that tended to limit their performance in initial experiments include targeting specificity of SNAP fusions and unspecific staining by the Indo-1 not reacted with SNAP-tag. Overall, the hybrid biosensor approach is a promising tool for organellar Ca2+ imaging. Support: NIAMS/NIH grants to E.R., MDA to J.Z. and a Marie-Curie Fellowship (EC) to M.B.