An Expanded Palette of Genetically Encoded Ca ²⁺ Indicators

Abstract

Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in response to changes in calcium ion (Ca(2+)) concentration are powerful tools for visualizing intracellular signaling activity. However, despite a decade of availability, the palette of single FP-based Ca(2+) indicators has remained limited to a single green hue. We have expanded this palette by developing blue, improved green, and red intensiometric indicators, as well as an emission ratiometric indicator with an 11,000% ratio change. This series enables improved single-color Ca(2+) imaging in neurons and transgenic Caenorhabditis elegans. In HeLa cells, Ca(2+) was imaged in three subcellular compartments, and, in conjunction with a cyan FP-yellow FP-based indicator, Ca(2+) and adenosine 5'-triphosphate were simultaneously imaged. This palette of indicators paints the way to a colorful new era of Ca(2+) imaging.

Reciprocal inhibition between sensory ASH and ASI neurons modulates nociception and avoidance in Caenorhabditis elegans

“…We first monitored Ca 2 þ transients in ASHs using the genetically encoded Ca 2 þ sensor R-GECO1 (Fig. 2a), which has properties of high Ca 2 þ affinity, a large dynamic range and slow photobleaching 31 . A transparent polydimethylsiloxane microfluidic device was used to trap and expose worms to the Cu 2 þ stimulus 32,33 .…”

“…Neuronal calcium responses in the soma were measured by detecting changes in the fluorescence intensity of R-GECO1, a sensitive, rapid kinetic calcium indicator of weak photobleaching 31 . A home-made microfluidic NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6655 ARTICLE device was used for calcium imaging as previously described 32,33 .…”

Excited-state structural dynamics of a dual-emission calmodulin-green fluorescent protein sensor for calcium ion imaging

“…Among the engineered GECO proteins, GEM-GECO1 is an intriguing case with the serine-tyrosine-glycine (SYG) derived chromophore (4). Upon ultraviolet (UV) excitation it fluoresces green in the absence of Ca 2+ but blue upon Ca 2+ binding with a K d of 340 nM.…”

“…The advanced imaging capabilities of the GECO series have been explored (4,5,7), but few spectroscopic studies exist for these unique Ca 2+ sensors. In contrast, spectroscopy on wildtype (wt)GFP included infrared pump probe (16), time-resolved fluorescence (17)(18)(19), transient infrared (20,21), and femtosecond Raman spectroscopy (22), as well as computational studies (23)(24)(25), providing a fairly complete picture of the photophysical and photochemical steps leading to green fluorescence (26,27).…”

“…Recently, the color palette of genetically encoded Ca 2+ sensors for optical imaging (the GECO series) has been expanded to include blue, improved green, red intensiometric, and emission ratiometric sensors (4)(5)(6)(7). The GECO proteins belong to the GCaMP family of Ca 2+ sensors that are chimeras of a circularly permutated (cp)GFP, calmodulin (CaM), and a peptide derived from myosin light chain kinase (M13) (8).…”