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.
Ruthenium-catalyzed activation of DNA-binding compounds in aqueous buffers and in cellular environments.
Transcriptional assays, such as yeast two-hybrid and TANGO, that convert transient protein-protein interactions (PPIs) into stable expression of transgenes are powerful tools for PPI discovery, screens, and analysis of cell populations. However, such assays often have high background and lose information about PPI dynamics. We have developed SPARK (Specific Protein Association tool giving transcriptional Readout with rapid Kinetics), in which proteolytic release of a membrane-tethered transcription factor (TF) requires both a PPI to deliver a protease proximal to its cleavage peptide and blue light to uncage the cleavage site. SPARK was used to detect 12 different PPIs in mammalian cells, with 5 min temporal resolution and signal ratios up to 37. By shifting the light window, we could reconstruct PPI time-courses. Combined with FACS, SPARK enabled 51 fold enrichment of PPI-positive over PPI-negative cells. Due to its high specificity and sensitivity, SPARK has the potential to advance PPI analysis and discovery.
Tobacco etch virus protease (TEV) is one of the most widely-used proteases in biotechnology because of its exquisite sequence-specificity. A limitation, however, is its slow catalytic rate. We developed a generalizable yeast-based platform for directed evolution of protease catalytic properties. Protease activity is read out via proteolytic release of a membrane-anchored transcription factor, and we temporally regulate access to TEV's cleavage substrate using a photosensory LOV domain. By gradually decreasing light exposure time, we enriched faster variants of TEV over multiple rounds of selection. Our S153N mutant (uTEV1Δ), when incorporated into the calcium integrator FLARE, improved the signal/background ratio by 27-fold, and enabled recording of neuronal activity in culture with 60-second temporal resolution. Given the widespread use of TEV in biotechnology, both our evolved TEV mutants and the directed evolution platform used to generate them, could be beneficial across a wide range of applications. Proteases are ubiquitous in biology, frequently initiating or terminating endogenous signaling cascades. Their peptide bond cleavage activities have been harnessed for a wide range of biotechnological applications, including bottom-up mass spectrometry (MS)-based proteomics, affinity purification, neuronal silencing (e.g., botulinum protease [1]), lightregulated apoptosis [2], tagging of newly synthesized proteins [3], assembly of protein droplets [4], protease-based synthetic circuits [5,6], regulation of TALENs [7], transcriptional readout of calcium [8,9] and protein-protein interactions [10,11,12]. One of the most frequently-used proteases in biotechnology is TEV, the 27 kD cysteine protease from tobacco etch virus. TEV is appealing because it is active in the mammalian cytosol, has no required cofactors, recognizes a 7-amino acid consensus peptide substrate, and, most importantly, is highly sequence-specific, exhibiting negligible activity towards endogenous mammalian proteomes. Consequently, TEV has been harnessed for sequencespecific transcription factor release in response to calcium and light in FLARE [8], GPCR Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
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