Temporally precise labeling and control of neuromodulatory circuits in the mammalian brain

Lee, Dongmin; Creed, Meaghan C.; Jung, Kanghoon; Stefanelli, Thomas; Wendler, Daniel J.; Oh, Won Chan; Mignocchi, Neymi; Lüscher, Christian; Kwon, Hyung Bae

doi:10.1038/nmeth.4234

Cited by 140 publications

(140 citation statements)

References 38 publications

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“…1B). Note that β2AR is not fused to the tail domain of the vasopressin receptor 9,13 or any other motif that artificially boosts its recruitment of arrestin; only native genes are utilized. HEK 293T cells expressing these FLARE components were stimulated for 5 minutes with both 467 nm light (50 mW/cm 2 ) and isoproterenol.…”

Section: Design and Optimization Of Ppi-flarementioning

confidence: 99%

Time-gated detection of protein-protein interactions with transcriptional readout

Kim

Wang

Sánchez

et al. 2017

Preprint

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Transcriptional assays such as yeast two hybrid, split ubiquitin, and Tango that convert transient protein-protein interactions (PPIs) in cells into stable expression of transgenes are powerful tools for PPI discovery, high-throughput screens, and analysis of large cell populations. However, these assays frequently suffer from high background and they lose all information about PPI dynamics. To address these limitations, we developed a light-gated transcriptional assay for PPI detection called PPI-FLARE (PPI-Fast Light-and Activity-Regulated Expression). PPI-FLARE requires both a PPI to deliver TEV protease proximal to its cleavage peptide, and externally-applied blue light to uncage the cleavage peptide, in order to release a membrane-tethered transcription factor (TF) for translocation to the nucleus. We used PPI-FLARE to detect the ligand-induced association of 12 different PPIs in living mammalian cells, with a temporal resolution of 5 minutes and a ±ligand signal ratio up to 37. By systematically shifting the light irradiation window, we could reconstruct PPI time-courses, distinguishing between GPCRs that engage in transient versus sustained interactions with the cytosolic effector arrestin. When combined with FACS, PPI-FLARE enabled >100-fold enrichment of cells experiencing a specific GPCR-arrestin PPI during a short 10-minute light window over cells missing that PPI during the same time window. Due to its high specificity, sensitivity, and generality, PPI-FLARE should be a broadly useful tool for PPI analysis and discovery.Protein-protein interactions (PPIs) are central to cellular signal transduction. Consequently, many assays have been developed to detect and study them, particularly in the context of living cells, where native PPIs are unperturbed by cell lysis, detergents, fixatives, or dilution. For example, FRET 1 , BRET 2 , fluorescence correlation spectroscopy (FCS) 3 , protein complementation assays (PCAs) 4 , and fluorescence relocalization assays 5,6 have all been used to visualize PPI dynamics in living cells. Though information-rich, these assays have the downside of being labor-intensive, requiring high-content or time-lapse microscopy, and are consequently difficult to perform on a large scale. This makes them non-optimal for PPI discovery, for adaptation to high-throughput screens, or for analysis of large cell populations such as in complex tissue. Instead, real-time assays are more suited for the focused study of a small number of known PPIs under a small set of conditions or in a small number of cells.A separate class of assays detects PPIs by signal integration rather than real-time imaging, and produces gene transcription as the readout. Examples include the yeast two hybrid assay 7 , the split ubiquitin assay 8 , and Tango 9-11 . Benefits of these assays include scalability (because real-time microscopy is not needed), versatility of read out (transcription of a fluorescent protein or an antibiotic resistance gene, for example), and high sensitivity due to signal amplification. Thes...

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Section: Design and Optimization Of Ppi-flarementioning

confidence: 99%

Time-gated detection of protein-protein interactions with transcriptional readout

Kim

Wang

Sánchez

et al. 2017

Preprint

View full text Add to dashboard Cite

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“…Optical approaches, such as injected cell-based systems (CNiFERs) (14) and reporter gene-based iTango (15) can reveal dopamine release with high molecular specificity. However, these systems are limited by poor temporal resolution (seconds to hours), preventing direct detection of DA release events that occur on a sub-second time scale (16, 17).…”

mentioning

confidence: 99%

Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors

Patriarchi

Cho

Merten

et al. 2018

Science

897

892

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Neuromodulatory systems exert profound influences on brain function. Understanding how these systems modify the operating mode of target circuits requires measuring spatiotemporally precise neuromodulator release. We developed dLight1, an intensity-based genetically encoded dopamine indicator, to enable optical recording of dopamine dynamics with high spatiotemporal resolution in behaving mice. We demonstrated the utility of dLight1 by imaging dopamine dynamics simultaneously with pharmacological manipulation, electrophysiological or optogenetic stimulation, and calcium imaging of local neuronal activity. dLight1 enabled chronic tracking of learning-induced changes in millisecond dopamine transients in striatum. Further, we used dLight1 to image spatially distinct, functionally heterogeneous dopamine transients relevant to learning and motor control in cortex. We also validated our sensor design platform for developing norepinephrine, serotonin, melatonin, and opioid neuropeptide indicators.

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“…To achieve this, we introduce two novel peptide elements for controlling the Tet-repressor (TetR). We fuse TetR with a lightresponsive protein domain (LOV2) 27,30,31 , which is further fused to either a Tet-inhibiting peptide (TIP) 32 or a degradation tag consisting of four amino acids 33 . These components remain hidden until illuminated, at which point they ensure light-dependent inhibition or degradation of the TetR protein [32][33][34] .…”

Section: Mainmentioning

confidence: 99%

“…To enable NF in optogenetics, we turned towards the blue-light responsive (450 nm) LOV2 protein domain which has been fused to various proteins previously 27,31 . We fused the LOV2 protein domain with the repressor protein TetR ( Figure 1A) and added the Tet-inhibiting peptide (TIP) 32 capable of inhibiting the TetR protein to the C-terminal end of LOV2.…”

Section: Liter10 Gene Circuits Improve the Precision Of Single-cell mentioning

confidence: 99%

Noise-Reducing Negative-Feedback Optogenetic Circuits in Mammalian Cells

Guinn

Balázsi

2019

Preprint

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Gene autorepression is widely present in nature and is also employed in synthetic biology, partly to reduce gene expression noise in cells. Optogenetic systems have recently been employed for controlling gene expression levels in mammalian cells, but most have utilized activator-based proteins, neglecting negative feedback. Here, we engineer optogenetic negative-feedback gene circuits in mammalian cells to achieve noise-reduction for precise gene expression control. We build a toolset of these noise-reducing Light-Inducible Tuner (LITer) gene circuits using the TetR repressor fused with a Tet-Inhibitory peptide (TIP) or a degradation tag through the light-sensitive LOV2 protein domain. These LITers provide nearly a range of 4-fold gene expression control and up to five-fold noise reduction from existing optogenetic systems. Moreover, we use the LITer gene circuit architecture to control gene expression of the cancer oncogene KRAS(G12V) and study its downstream effects through phospho-ERK levels and cellular proliferation. Overall, these novel LITer optogenetic platforms should enable precise spatiotemporal perturbations for studying multicellular phenotypes in developmental biology, oncology, and other biomedical fields of research.be utilized in single-cell studies of processes as diverse as embryonic development, cancer metastasis, and neuron migration.

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Temporally precise labeling and control of neuromodulatory circuits in the mammalian brain

Cited by 140 publications

References 38 publications

Time-gated detection of protein-protein interactions with transcriptional readout

Time-gated detection of protein-protein interactions with transcriptional readout

Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors

Noise-Reducing Negative-Feedback Optogenetic Circuits in Mammalian Cells

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