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Temperature-responsive optogenetic probes of cell signaling.

ABSTRACT: We describe single-component optogenetic probes whose activation dynamics depend on both light and temperature. We used the BcLOV4 photoreceptor to stimulate Ras and phosphatidyl inositol-3-kinase signaling in mammalian cells, allowing activation over a large dynamic range with low basal levels. Surprisingly, we found that BcLOV4 membrane translocation dynamics could be tuned by both light and temperature such that membrane localization spontaneously decayed at elevated temperatures despite constant illumination. Quantitative modeling predicted BcLOV4 activation dynamics across a range of light and temperature inputs and thus provides an experimental roadmap for BcLOV4-based probes. BcLOV4 drove strong and stable signal activation in both zebrafish and fly cells, and thermal inactivation provided a means to multiplex distinct blue-light sensitive tools in individual mammalian cells. BcLOV4 is thus a versatile photosensor with unique light and temperature sensitivity that enables straightforward generation of broadly applicable optogenetic tools.

SUBMITTER: Benman W

PROVIDER: S-EPMC9252025 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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Temperature-responsive optogenetic probes of cell signaling.

Benman William W Berlew Erin E EE Deng Hao H Parker Caitlyn C Kuznetsov Ivan A IA Lim Bomyi B Siekmann Arndt F AF Chow Brian Y BY Bugaj Lukasz J LJ

Nature chemical biology 20211222 2

We describe single-component optogenetic probes whose activation dynamics depend on both light and temperature. We used the BcLOV4 photoreceptor to stimulate Ras and phosphatidyl inositol-3-kinase signaling in mammalian cells, allowing activation over a large dynamic range with low basal levels. Surprisingly, we found that BcLOV4 membrane translocation dynamics could be tuned by both light and temperature such that membrane localization spontaneously decayed at elevated temperatures despite cons ...[more]

PMID: 34937907

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Project description:OBJECTIVE:Optogenetic modulation of neural activity is a ubiquitous tool for basic investigation of brain circuits. While the majority of optogenetic paradigms rely on short light pulses to evoke synchronized activity of optically sensitized cells, many neurobiological processes are associated with slow local field potential (LFP) oscillations. Therefore, we developed a hybrid fiber probe capable of simultaneous electrophysiological recording and optical stimulation and used it to investigate the utility of sinusoidal light stimulation for evoking oscillatory neural activity in vivo across a broad frequency range. APPROACH:We fabricated hybrid fiber probes comprising a hollow cylindrical array of 9 electrodes and a flexible optical waveguide integrated within the core. We implanted these probes in the hippocampus of transgenic Thy1-ChR2-YFP mice that broadly express the blue-light sensitive cation channel channelrhodopsin 2 (ChR2) in excitatory neurons across the brain. The effects of the sinusoidal light stimulation were characterized and contrasted with those corresponding to pulsed stimulation in the frequency range of physiological LFP rhythms (3-128 Hz). MAIN RESULTS:Within hybrid probes, metal electrode surfaces were vertically aligned with the waveguide tip, which minimized optical stimulation artifacts in neurophysiological recordings. Sinusoidal stimulation resulted in reliable and coherent entrainment of LFP oscillations up to 70 Hz, the cutoff frequency of ChR2, with response amplitudes inversely scaling with the stimulation frequencies. Effectiveness of the stimulation was maintained for two months following implantation. SIGNIFICANCE:Alternative stimulation patterns complementing existing pulsed protocols, in particular sinusoidal light stimulation, are a prerequisite for investigating the physiological mechanisms underlying brain rhythms. So far, studies applying sinusoidal stimulation in vivo were limited to single stimulation frequencies. We show the feasibility of sinusoidal stimulation in vivo to induce coherent LFP oscillations across the entire frequency spectrum supported by the gating dynamics of ChR2 and introduce a hybrid fiber probe tailored to continuous light stimulation.

Dataset Information

Temperature-responsive optogenetic probes of cell signaling.

Publications

Temperature-responsive optogenetic probes of cell signaling.

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