Browsing by Author "St-Pierre, François"
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Item A synthetic circuit for buffering gene dosage variation between individual mammalian cells(Springer Nature, 2021) Yang, Jin; Lee, Jihwan; Land, Michelle A.; Lai, Shujuan; Igoshin, Oleg A.; St-Pierre, François; Systems, Synthetic, and Physical Biology ProgramPrecise control of gene expression is critical for biological research and biotechnology. However, transient plasmid transfections in mammalian cells produce a wide distribution of copy numbers per cell, and consequently, high expression heterogeneity. Here, we report plasmid-based synthetic circuits – Equalizers – that buffer copy-number variation at the single-cell level. Equalizers couple a transcriptional negative feedback loop with post-transcriptional incoherent feedforward control. Computational modeling suggests that the combination of these two topologies enables Equalizers to operate over a wide range of plasmid copy numbers. We demonstrate experimentally that Equalizers outperform other gene dosage compensation topologies and produce as low cell-to-cell variation as chromosomally integrated genes. We also show that episome-encoded Equalizers enable the rapid generation of extrachromosomal cell lines with stable and uniform expression. Overall, Equalizers are simple and versatile devices for homogeneous gene expression and can facilitate the engineering of synthetic circuits that function reliably in every cell.Item Ancestral circuits for vertebrate color vision emerge at the first retinal synapse(American Association for the Advancement of Science, 2021) Yoshimatsu, Takeshi; Bartel, Philipp; Schröder, Cornelius; Janiak, Filip K.; St-Pierre, François; Berens, Philipp; Baden, Tom; Systems, Synthetic, and Physical Biology ProgramFor color vision, retinal circuits separate information about intensity and wavelength. In vertebrates that use the full complement of four “ancestral” cone types, the nature and implementation of this computation remain poorly understood. Here, we establish the complete circuit architecture of outer retinal circuits underlying color processing in larval zebrafish. We find that the synaptic outputs of red and green cones efficiently rotate the encoding of natural daylight in a principal components analysis–like manner to yield primary achromatic and spectrally opponent axes, respectively. Blue cones are tuned to capture most remaining variance when opposed to green cones, while UV cone present a UV achromatic axis for prey capture. We note that fruitflies use essentially the same strategy. Therefore, rotating color space into primary achromatic and chromatic axes at the eye’s first synapse may thus be a fundamental principle of color vision when using more than two spectrally well-separated photoreceptor types.Item Embargo Developing Genetically Encoded Voltage Indicators for in vivo Neuroimaging(2023-08-09) Lu, Helen; Robinson, Jacob T.; St-Pierre, FrançoisA fundamental goal of neuroscience is to decipher the neural activities underlying behaviors in vivo. Among existing tools for neural recording, genetically encoded voltage indicators (GEVIs) — protein-based fluorescent indicators whose brightness is directly modulated by membrane potential — hold the most promise for large-scale measuring of neural activities with cell-type specificity, subcellular spatial resolution, and sub-millisecond temporal resolution. However, current GEVIs have limited utility in vivo due to their suboptimal performance, especially under two-photon microscopy (2PM), a desired method for deep-tissue imaging. To address these limitations, we started from building a high-throughput screening platform that can evaluate all key metrics of a GEVI under one-photon illumination. Directed evolution on this platform led to JEDI-1P, a green-emitting GEVI optimized for widefield voltage imaging. With improved brightness, kinetics, sensitivity and photostability, JEDI-1P empowered chronic pan-cortical voltage imaging and robust detection of rapid voltage signals in behaving mice. Next, we sought to optimize GEVI for deep tissue imaging by extending the screening platform with a two-photon resonant scanning system. Using this 2P screening platform, we identified JEDI-2P, whose brightness, sensitivity and photostability under 2PM were all significantly improved over its parental indicator. We showed that JEDI-2P can capture voltage responses to visual stimuli in the amacrine cells of isolated mouse retina and the axonal projections of Drosophila interneurons. With excellent 2P photostability, JEDI-2P enabled prolonged continuous recording from individual cortical neurons in awake behaving mice with both resonant-scanning 2PM and ULoVE random-access microscopy. In particular, we highlighted that the improved sensitivity and brightness of JEDI-2P allowed the first high-fidelity voltage recording from mice Layer 5 cortical neurons as well as robust recordings of pairwise voltage correlations during behavior. Taken together, JEDI-2P fills the vacancy of a GEVI optimized for 2P applications and paves the way for long-term studying of deep brain neural activities. Finally, to enable all-optical electrophysiology and multi-spectral imaging under two-photon microscopy, we designed a red-emitting voltage indicator from scratch. After rationally engineering the interface between the chromophore and the voltage-sensing domain, we identified VADER1, the first red-emitting GEVI that has demonstrated the capability to resolve single action potentials in mice under two-photon illumination. We anticipate the expanded GEVI toolbox will enable high-throughput and real-time recording of action potentials from the genetically specified group of neurons in live animals, thereby helping interpret the computation mechanism of neural circuits with unprecedented spatiotemporal resolution.Item Multimodal High-Content Optimization of Neural Activity Biosensors(2022-12-01) Liu, Zhuohe; Veeraraghavan, Ashok; St-Pierre, FrançoisProtein-based biosensors enable optical monitoring of neural activities with cell-type specificity. Genetically encoded voltage indicator (GEVI) is an emerging fluorescent biosensor that reports voltage dynamics. However, the performance of engineered biosensors cannot suffice the applications that require prolonged and faithful recording of rapid activity in deep-tissue targets. The underlying cause is the slow and challenging protein engineering to optimize holistically multiple spatial and temporal properties. To expand the biosensor toolbox, innovations in software, hardware, and wetware are needed in multimodal and automated protein optimization. We first aim to optimize GEVIs by developing a high-throughput platform to screen biosensor variants under two-photon illumination. Electric field stimulation was used to induce transient voltage responses in cells layered onto 96-well plates. Using this platform, we identified an indicator, JEDI-2P, which is faster, brighter, and more sensitive and photostable than its predecessors. JEDI-2P can report voltage response to visual stimuli in the dendrites and somata of amacrine cells of isolated mouse retina and in axonal termini of fruit fly interneurons. In awake behaving mice, JEDI-2P enables optical voltage recording of individual cortical neurons for more than 30 min using both resonant-scanning and ULoVE random-access two-photon microscopy. Especially with ULoVE, JEDI-2P can robustly detect spikes at depths exceeding 400 μm and report voltage correlations in pairs of neurons. To further boost throughput, we developed SPOTlight, a versatile single-cell screening platform. Using a microscope, SPOTlight captures visual phenotypes of individual cells that reflect both spatial and temporal properties. Single cells of interest can be precisely tagged using light because they contain phototransformable proteins or dyes. Tagged cells are retrieved by fluorescence-activated cell sorting to recover genotypes. To demonstrate the platform, we opted to optimize photostability of a yellow fluorescent protein (YFP), a common building block for biosensors, while monitoring brightness. We identified mGold, the most photostable YFP to date after screening ~3 million cells. We anticipate that accelerated and automated screening platforms will empower protein engineering for robust and well-performing biosensors that are crucial for neural activity monitoring, unlocking the secrets of our brains.Item Sustained deep-tissue voltage recording using a fast indicator evolved for two-photon microscopy(Elsevier, 2022) Liu, Zhuohe; Lu, Xiaoyu; Villette, Vincent; Gou, Yueyang; Colbert, Kevin L.; Lai, Shujuan; Guan, Sihui; Land, Michelle A.; Lee, Jihwan; Assefa, Tensae; Zollinger, Daniel R.; Korympidou, Maria M.; Vlasits, Anna L.; Pang, Michelle M.; Su, Sharon; Cai, Changjia; Froudarakis, Emmanouil; Zhou, Na; Patel, Saumil S.; Smith, Cameron L.; Ayon, Annick; Bizouard, Pierre; Bradley, Jonathan; Franke, Katrin; Clandinin, Thomas R.; Giovannucci, Andrea; Tolias, Andreas S.; Reimer, Jacob; Dieudonné, Stéphane; St-Pierre, FrançoisGenetically encoded voltage indicators are emerging tools for monitoring voltage dynamics with cell-type specificity. However, current indicators enable a narrow range of applications due to poor performance under two-photon microscopy, a method of choice for deep-tissue recording. To improve indicators, we developed a multiparameter high-throughput platform to optimize voltage indicators for two-photon microscopy. Using this system, we identified JEDI-2P, an indicator that is faster, brighter, and more sensitive and photostable than its predecessors. We demonstrate that JEDI-2P can report light-evoked responses in axonal termini of Drosophila interneurons and the dendrites and somata of amacrine cells of isolated mouse retina. JEDI-2P can also optically record the voltage dynamics of individual cortical neurons in awake behaving mice for more than 30 min using both resonant-scanning and ULoVE random-access microscopy. Finally, ULoVE recording of JEDI-2P can robustly detect spikes at depths exceeding 400 μm and report voltage correlations in pairs of neurons.Item Versatile phenotype-activated cell sorting(AAAS, 2020) Lee, Jihwan; Liu, Zhuohe; Suzuki, Peter H.; Ahrens, John F.; Lai, Shujuan; Lu, Xiaoyu; Guan, Sihui; St-Pierre, FrançoisUnraveling the genetic and epigenetic determinants of phenotypes is critical for understanding and re-engineering biology and would benefit from improved methods to separate cells based on phenotypes. Here, we report SPOTlight, a versatile high-throughput technique to isolate individual yeast or human cells with unique spatiotemporal profiles from heterogeneous populations. SPOTlight relies on imaging visual phenotypes by microscopy, precise optical tagging of single target cells, and retrieval of tagged cells by fluorescence-activated cell sorting. To illustrate SPOTlight’s ability to screen cells based on temporal properties, we chose to develop a photostable yellow fluorescent protein for extended imaging experiments. We screened 3 million cells expressing mutagenesis libraries and identified a bright new variant, mGold, that is the most photostable yellow fluorescent protein reported to date. We anticipate that the versatility of SPOTlight will facilitate its deployment to decipher the rules of life, understand diseases, and engineer new molecules and cellsItem Widefield imaging of rapid pan-cortical voltage dynamics with an indicator evolved for one-photon microscopy(Springer Nature, 2023) Lu, Xiaoyu; Wang, Yunmiao; Liu, Zhuohe; Gou, Yueyang; Jaeger, Dieter; St-Pierre, FrançoisWidefield imaging with genetically encoded voltage indicators (GEVIs) is a promising approach for understanding the role of large cortical networks in the neural coding of behavior. However, the limited performance of current GEVIs restricts their deployment for single-trial imaging of rapid neuronal voltage dynamics. Here, we developed a high-throughput platform to screen for GEVIs that combine fast kinetics with high brightness, sensitivity, and photostability under widefield one-photon illumination. Rounds of directed evolution produced JEDI-1P, a green-emitting fluorescent indicator with enhanced performance across all metrics. Next, we optimized a neonatal intracerebroventricular delivery method to achieve cost-effective and wide-spread JEDI-1P expression in mice. We also developed an approach to correct optical measurements from hemodynamic and motion artifacts effectively. Finally, we achieved stable brain-wide voltage imaging and successfully tracked gamma-frequency whisker and visual stimulations in awake mice in single trials, opening the door to investigating the role of high-frequency signals in brain computations.