Coupling optogenetic stimulation with NanoLuc-based luminescence (BRET) Ca++ sensing

Abstract

Optogenetic techniques allow intracellular manipulation of Ca++ by illumination of light-absorbing probe molecules such as channelrhodopsins and melanopsins. The consequences of optogenetic stimulation would optimally be recorded by non-invasive optical methods. However, most current optical methods for monitoring Ca++ levels are based on fluorescence excitation that can cause unwanted stimulation of the optogenetic probe and other undesirable effects such as tissue autofluorescence. Luminescence is an alternate optical technology that avoids the problems associated with fluorescence. Using a new bright luciferase, we here develop a genetically encoded Ca++ sensor that is ratiometric by virtue of bioluminescence resonance energy transfer (BRET). This sensor has a large dynamic range and partners optimally with optogenetic probes. Ca++ fluxes that are elicited by brief pulses of light to cultured cells expressing melanopsin and to neurons-expressing channelrhodopsin are quantified and imaged with the BRET Ca++ sensor in darkness, thereby avoiding undesirable consequences of fluorescence irradiation.

Dopamine Receptor DAMB Signals via Gq to Mediate Forgetting in Drosophila

“…Receptors were reconstituted into HEK293T/17 cells together with firefly luciferase-based GloSensor-22F cAMP sensor (Binkowski et al, 2011) or CalfluxVTN Ca 2+ sensor (Yang et al, 2016) that report real time fluctuations in second messenger levels by changes in luminescence and BRET signal, respectively. Consistent with previous reports (Sugamori et al, 1995; Han et al, 1996), we observed that application of DA to cells expressing either dDA1 or Damb stimulated cAMP production (Figure 1B).…”

Buffer NaCl concentration regulates Renilla luciferase activity and ligand-induced conformational changes in the BRET-based PDE5 sensor

“…Ligand bindinginduced structural changes in both the GAFa and the catalytic domains in the full-length PDE5 have been extensively explored by developing a Bioluminescence Resonance Energy Transfer (BRET)-based intramolecular conformational sensor [12]. BRET is being increasingly utilized for monitoring activation of proteins and signaling pathways in live cells [13] [14] [15]. The underlying mechanism behind BRET is the non-radiative transfer of energy from a bioluminescent donor such as Renilla luciferase (Rluc) to a fluorescent acceptor such as GFP 2 (a variant of GFP optimized for excitation by Rluc emission) [16].…”

Advances in bioluminescence imaging: new probes from old recipes

“…CalfluxVTN enabled sensitive imaging of calcium flux in response to stimulation of a rhodopsin photoreceptor. [48] Such measurements were refractory to FRET, as external light interfered with receptor activation. Johnsson and coworkers further developed a universal BRET sensor platform for analyte detection.…”

“…Cellular images were reproduced with permission from ref. 48. (B) Caged luciferins and dioxetanes can report on cellular activities.…”