Abstract
Genetically encoded fluorescent biosensors are powerful tools for studying complex signaling in the nervous system, and now both Ca2+ and voltage sensors are available to study the signaling behavior of entire neural circuits. There is a pressing need for improved sensors, but improving them is challenging because testing them involves a low throughput, labor-intensive processes. Our goal was to create synthetic, excitable cells that can be activated with brief pulses of blue light and serve as a medium throughput platform for screening the next generation of sensors. In this live cell system, blue light activates an adenylyl cyclase enzyme (bPAC) that increases intracellular cAMP (Stierl M et al. 2011). In turn, the cAMP opens a cAMP-gated ion channel. This produces slow, whole-cell Ca2+ transients and voltage changes. To increase the speed of these transients, we add the inwardly rectifying potassium channel Kir2.1, the bacterial voltage-gated sodium channel NAVROSD, and Connexin-43. The result is a highly reproducible, medium-throughput, live cell system that can be used to screen voltage and Ca2+ sensors.
Highlights
Why are biosensors important?Genetically encoded, fluorescent biosensors are powerful tools for studying cell signaling in real-time [1, 2]
The bPAC enzyme is activated with 480 nm light and converts ATP into cyclic adenosine monophosphate
The following day, we stimulated the cells with 20 milliseconds of blue light and collected images continuously with 561 nm excitation light that does not activate bPAC (Fig 1A)
Summary
Encoded, fluorescent biosensors are powerful tools for studying cell signaling in real-time [1, 2]. They are minimally invasive, and since they are genetically encoded their expression can be targeted to specific cell types and tissues [3,4,5]. The short wavelengths of light needed to image green biosensors can heat and damage the brain [6]. There is a need for better biosensors that emit red [7] or near-infrared [8] light because these longer wavelengths enable investigators to image deeper into thick tissues. Higher levels of Ca2+ sensor expression levels can lead to epileptiform activity [11]
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