A light- and calcium-gated transcription factor for imaging and manipulating activated neurons

Abstract

Activity remodels neurons, altering their molecular, structural, and electrical characteristics. To enable the selective characterization and manipulation of these neurons, we present FLARE, an engineered transcription factor that drives expression of fluorescent proteins, opsins, and other genetically-encoded tools only in the subset of neurons that experienced activity during a user-defined time window. FLARE senses the coincidence of elevated cytosolic calcium and externally-applied blue light, which together produce translocation of a membrane-anchored transcription factor to the nucleus to drive expression of any transgene. In cultured rat neurons, FLARE gives a light-to-dark signal ratio of 120 and a high-to-low calcium signal ratio of 10 after 10 minutes of stimulation. Channelrhodopsin expression permitted functional manipulation of FLARE-marked neurons. In adult mice, FLARE also gave light- and motor activity-dependent transcription in the cortex. Due to its modular design, minute-scale temporal resolution, and minimal dark-state leak, FLARE should be useful for the study of activity-dependent processes in neurons and other cells that signal with calcium.

Induction of Signal Transduction by Using Non‐Channelrhodopsin‐Type Optogenetic Tools

“…Currently, several optogenetic tools have been applied both to cultured cells and to in vivo studies, including in Drosophila , in Xenopus embryos, in zebrafish embryos, and in mice . Further improvement of in vivo studies might be facilitated by using red‐ and far‐red‐light‐responsive photoswitches because these light wavelengths can penetrate thick tissues.…”

“…The groups of Kwon and Ting reported back-to-back unique optogenetic tools based on AsLOV2 that label only Ca 2 + -activated neurons during ab lue-light-defined time window. [66] In the Ting group's photoswitch,C aM is fused to aT EV protease, and aC aM-binding peptidei st ethered to an AsLOV2-Ja helixcaged protease sensing motif that is further linked to transcription factor Gal4. When CaM and the CaM-binding peptide associate in aC a 2 + -dependent manner,t he protease sensing motif is exposed from AsLOV2u pon blue light irradiation and then the CaM-fused protease cuts the sensing motif.…”

Directed evolution improves the catalytic efficiency of TEV protease

“…With a kcat of 0.18 s -1 (for its best high-affinity substrate sequence, ENLYFQS [13]), TEV is slower than many other proteases used for biotechnology, such as trypsin (kcat 75 s -1 [14]) and subtilisin (kcat 50 s -1 [15]). This slow turnover fundamentally limits the performance of technologies that rely on TEV, such as FLARE [8]. In vivo, FLARE requires calcium and light stimulation for at least 30 minutes to give TEV sufficient time to release detectable quantities of membrane-anchored transcription factor [8].…”

“…This slow turnover fundamentally limits the performance of technologies that rely on TEV, such as FLARE [8]. In vivo, FLARE requires calcium and light stimulation for at least 30 minutes to give TEV sufficient time to release detectable quantities of membrane-anchored transcription factor [8]. Yet for the neuronal activity integration applications for which FLARE is designed, a temporal resolution of just a few minutes, or even seconds, is desireda goal we have found impossible to achieve using wild-type TEV (vide infra).There have not been systematic efforts to improve the catalytic rate of TEV, apart from optimization of its substrate sequence (TEVcs).…”

“…To devise a platform that could be used to enrich faster proteases over moderately-active ones, we implemented the following: (1) we moved the system into the yeast cytosol, since this reducing environment more closely resembles the eventual context in which evolved TEVs will mostly be used, (2) we coupled TEV activity to the release of a membrane-anchored transcription factor (TF) which in turn drives the expression of a fluorescent protein reporter, in order to increase sensitivity and dynamic range of the protease activity readout, (3) we fused TEV and TEVcs to the photoinducible protein binding pair CRY-CIBN [22] so that our selections can be applied to truncated, low-affinity versions of TEV that are utilized in FLARE and SPARK tools; despite the low affinity, recognition of TEVcs by TEV can be induced by blue light activation of CRY-CIBN. And most importantly, (4), we photocaged the TEVcs substrate sequence with an improved LOV domain (eLOV [8]) in order to exert control over the time window TEV is available to cleave TEVcs.…”