Fluorescent indicators for Ca2+based on green fluorescent proteins and calmodulin

Abstract

Important Ca2+ signals in the cytosol and organelles are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations. We have dubbed these fluorescent indicators 'cameleons'. They consist of tandem fusions of a blue- or cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13, and an enhanced green- or yellow-emitting GFP. Binding of Ca2+ makes calmodulin wrap around the M13 domain, increasing the fluorescence resonance energy transfer (FRET) between the flanking GFPs. Calmodulin mutations can tune the Ca2+ affinities to measure free Ca2+ concentrations in the range 10(-8) to 10(-2) M. We have visualized free Ca2+ dynamics in the cytosol, nucleus and endoplasmic reticulum of single HeLa cells transfected with complementary DNAs encoding chimaeras bearing appropriate localization signals. Ca2+ concentration in the endoplasmic reticulum of individual cells ranged from 60 to 400 microM at rest, and 1 to 50 microM after Ca2+ mobilization. FRET is also an indicator of the reversible intermolecular association of cyan-GFP-labelled calmodulin with yellow-GFP-labelled M13. Thus FRET between GFP mutants can monitor localized Ca2+ signals and protein heterodimerization in individual live cells.

Chemical tags: Applications in live cell fluorescence imaging

“…The particular disadvantage of chemical Ca 2þ -sensors is the difficulty of controlling cellular localization, for example, the targeting to specific organelles. As an alternative to the chemical sensors, the Tsien lab and others developed genetically encoded Ca 2þ -indicators (GECIs) based on fusions of FPs with calcium responsive elements like calmodulin or troponin [75][76][77][78]. These indicators allow for the dissection of the spatial and temporal control of calcium signaling processes in cells because they are genetically encoded and therefore specifically localized.…”

Reporting neural activity with genetically encoded calcium indicators

“…Most GECI recognition elements are based on naturally evolved calcium-binding proteins with large Ca 2+ -dependent conformational changes such as calmodulin (CaM) (Miyawaki et al, 1997;Nakai et al, 2001) or troponin-C (TnC) (Heim and Griesbeck, 2004). Wild-type recognition elements have been engineered to improve binding and response properties Garaschuk et al, 2007).…”

“…GECIs can be delivered in a minimally invasive manner to specific cell types or subcellular compartments (Miyawaki et al, 1997). They are compatible with long-term, repeated, in vivo measurements (Hasan et al, 2004).…”

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

“…signals are found in the cytosol and different organelles, but these signals are often difficult to measure by conventional fluorescent indicators. The yellow cameleon fluorescent calcium indicators are the most suitable reporters for in vivo detection of calcium because they do not require any cofactors and can be targeted to specific intracellular locations (Miyawaki et al 1997(Miyawaki et al , 1999. Several different variants of yellow cameleons have been constructed but all consist of a tandem fusion of ECFP, a calmodulin domain having four calcium binding sites, a calmodulin binding peptide M13 and EYFP (Miyawaki et al 1997;Varadi and Rutter 2002).…”