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22 Publications

Showing 1-10 of 22 results
03/22/24 | Visualization of Glutamatergic Neurotransmission in Diverse Model Organisms with Genetically Encoded Indicators
Aggarwal A, Chan J, Waring AK, Negrean A, Marvin JS, Podgorski K, Looger LL, Kukley M
New Technologies for Glutamate Interaction: Neurons and Glia;2780:3–34. doi: 10.1007/978-1-0716-3742-5_1

Glutamate is the principal excitatory neurotransmitter, and occasionally subserves inhibitory roles, in the vertebrate nervous system. Glutamatergic synapses are dense in the vertebrate brain, at \textasciitilde1/μm3. Glutamate is released from and onto diverse components of the nervous system, including neurons, glia, and other cells. Methods for glutamate detection are critically important for understanding the function of synapses and neural circuits in normal physiology, development, and disease. Here we describe the development, optimization, and deployment of genetically encoded fluorescent glutamate indicators. We review the theoretical considerations governing glutamate sensor properties from first principles of synapse biology, microscopy, and protein structure-function relationships. We provide case studies of the state-of-the-art iGluSnFR glutamate sensor, encompassing design and optimization, mechanism of action, in vivo imaging, data analysis, and future directions. We include detailed protocols for iGluSnFR imaging in common preparations (bacteria, cell culture, and brain slices) and model organisms (worm, fly, fish, rodent).

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02/28/24 | High-Performance Genetically Encoded Green Fluorescent Biosensors for Intracellular l-Lactate.
Hario S, Le GN, Sugimoto H, Takahashi-Yamashiro K, Nishinami S, Toda H, Li S, Marvin JS, Kuroda S, Drobizhev M, Terai T, Nasu Y, Campbell RE
ACS Central Science. 2024 Feb 28;10(2):402-416. doi: 10.1021/acscentsci.3c01250

l-Lactate is a monocarboxylate produced during the process of cellular glycolysis and has long generally been considered a waste product. However, studies in recent decades have provided new perspectives on the physiological roles of l-lactate as a major energy substrate and a signaling molecule. To enable further investigations of the physiological roles of l-lactate, we have developed a series of high-performance (Δ/ = 15 to 30 ), intensiometric, genetically encoded green fluorescent protein (GFP)-based intracellular l-lactate biosensors with a range of affinities. We evaluated these biosensors in cultured cells and demonstrated their application in an preparation of brain tissue. Using these biosensors, we were able to detect glycolytic oscillations, which we analyzed and mathematically modeled.

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02/24/24 | A series of spontaneously blinking dyes for super-resolution microscopy
Katie L. Holland , Sarah E. Plutkis , Timothy A. Daugird , Abhishek Sau , Jonathan B. Grimm , Brian P. English , Qinsi Zheng , Sandeep Dave , Fariha Rahman , Liangqi Xie , Peng Dong , Ariana N. Tkachuk , Timothy A. Brown , Robert H. Singer , Zhe Liu , Catherine G. Galbraith , Siegfried M. Musser , Wesley R. Legant , Luke D. Lavis
bioRxiv. 2024 Feb 24:. doi: 10.1101/2024.02.23.581625

Spontaneously blinking fluorophores permit the detection and localization of individual molecules without reducing buffers or caging groups, thus simplifying single-molecule localization microscopy (SMLM). The intrinsic blinking properties of such dyes are dictated by molecular structure and modulated by environment, which can limit utility. We report a series of tuned spontaneously blinking dyes with duty cycles that span two orders of magnitude, allowing facile SMLM in cells and dense biomolecular structures.

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02/29/24 | An engineered biosensor enables dynamic aspartate measurements in living cells.
Davidsen K, Marvin JS, Aggarwal A, Brown TA, Sullivan LB
Elife. 2024 Feb 23;12:. doi: 10.7554/eLife.90024

Intracellular levels of the amino acid aspartate are responsive to changes in metabolism in mammalian cells and can correspondingly alter cell function, highlighting the need for robust tools to measure aspartate abundance. However, comprehensive understanding of aspartate metabolism has been limited by the throughput, cost, and static nature of the mass spectrometry (MS)-based measurements that are typically employed to measure aspartate levels. To address these issues, we have developed a green fluorescent protein (GFP)-based sensor of aspartate (jAspSnFR3), where the fluorescence intensity corresponds to aspartate concentration. As a purified protein, the sensor has a 20-fold increase in fluorescence upon aspartate saturation, with dose-dependent fluorescence changes covering a physiologically relevant aspartate concentration range and no significant off target binding. Expressed in mammalian cell lines, sensor intensity correlated with aspartate levels measured by MS and could resolve temporal changes in intracellular aspartate from genetic, pharmacological, and nutritional manipulations. These data demonstrate the utility of jAspSnFR3 and highlight the opportunities it provides for temporally resolved and high-throughput applications of variables that affect aspartate levels.

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01/09/24 | Direct measurement of dynamic attractant gradients reveals breakdown of the Patlak-Keller-Segel chemotaxis model
Trung V. Phan , Henry H. Mattingly , Lam Vo , Jonathan S. Marvin , Loren L. Looger , Thierry Emonet
Proceedings of the National Academy of Sciences. 2024 Jan 09:. doi: 10.1073/pnas.230925112

Chemotactic bacteria not only navigate chemical gradients, but also shape their environments by consuming and secreting attractants. Investigating how these processes influence the dynamics of bacterial populations has been challenging because of a lack of experimental methods for measuring spatial profiles of chemoattractants in real time. Here, we use a fluorescent sensor for aspartate to directly measure bacterially generated chemoattractant gradients during collective migration. Our measurements show that the standard Patlak-Keller-Segel model for collective chemotactic bacterial migration breaks down at high cell densities. To address this, we propose modifications to the model that consider the impact of cell density on bacterial chemotaxis and attractant consumption. With these changes, the model explains our experimental data across all cell densities, offering new insight into chemotactic dynamics. Our findings highlight the significance of considering cell density effects on bacterial behavior, and the potential for fluorescent metabolite sensors to shed light on the complex emergent dynamics of bacterial communities.

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01/01/24 | Transforming chemigenetic bimolecular fluorescence complementation systems into chemical dimerizers using chemistry.
Pratik Kumar , Alina Gutu , Amelia Waring , Timothy A. Brown , Luke D. Lavis , Alison G. Tebo
bioRxiv. 2024 Jan 01:. doi: 10.1101/2023.12.30.573644

Chemigenetic tags are versatile labels for fluorescence microscopy that combine some of the advantages of genetically encoded tags with small molecule fluorophores. The Fluorescence Activating and absorbance Shifting Tags (FASTs) bind a series of highly fluorogenic and cell-permeable chromophores. Furthermore, FASTs can be used in complementation-based systems for detecting or inducing protein-protein interactions, depending on the exact FAST protein variant chosen. In this study, we systematically explore substitution patterns on FAST fluorogens and generate a series of fluorogens that bind to FAST variants, thereby activating their fluorescence. This effort led to the discovery of a novel fluorogen with superior properties, as well as a fluorogen that transforms splitFAST systems into a fluorogenic dimerizer, eliminating the need for additional protein engineering.

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10/16/23 | Optimized Red-Absorbing Dyes for Imaging and Sensing
Grimm JB, Tkachuk AN, Patel R, Hennigan ST, Gutu A, Dong P, Gandin V, Osowski AM, Holland KL, Liu ZJ, Brown TA, Lavis LD
Journal of the American Chemical Society. 2023 Oct 16:. doi: 10.1021/jacs.3c0527310.1021/jacs.3c05273

Rhodamine dyes are excellent scaffolds for developing a broad range of fluorescent probes. A key property of rhodamines is their equilibrium between a colorless lactone and fluorescent zwitterion. Tuning the lactone–zwitterion equilibrium constant (KL–Z) can optimize dye properties for specific biological applications. Here, we use known and novel organic chemistry to prepare a comprehensive collection of rhodamine dyes to elucidate the structure–activity relationships that govern KL–Z. We discovered that the auxochrome substituent strongly affects the lactone–zwitterion equilibrium, providing a roadmap for the rational design of improved rhodamine dyes. Electron-donating auxochromes, such as julolidine, work in tandem with fluorinated pendant phenyl rings to yield bright, red-shifted fluorophores for live-cell single-particle tracking (SPT) and multicolor imaging. The N-aryl auxochrome combined with fluorination yields red-shifted Förster resonance energy transfer (FRET) quencher dyes useful for creating a new semisynthetic indicator to sense cAMP using fluorescence lifetime imaging microscopy (FLIM). Together, this work expands the synthetic methods available for rhodamine synthesis, generates new reagents for advanced fluorescence imaging experiments, and describes structure–activity relationships that will guide the design of future probes.

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06/01/23 | Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission.
Aggarwal A, Liu R, Chen Y, Ralowicz AJ, Bergerson SJ, Tomaska F, Mohar B, Hanson TL, Hasseman JP, Reep D, Tsegaye G, Yao P, Ji X, Kloos M, Walpita D, Patel R, Mohr MA, Tillberg PW, GENIE Project Team , Looger LL, Marvin JS, Hoppa MB, Konnerth A, Kleinfeld D, Schreiter ER, Podgorski K
Nature Methods. 2023 Jun 01;20(6):. doi: 10.1038/s41592-023-01863-6

The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit low in vivo signal-to-noise ratios, saturating activation kinetics and exclusion from postsynaptic densities. Using a multiassay screen in bacteria, soluble protein and cultured neurons, we generated variants with improved signal-to-noise ratios and kinetics. We developed surface display constructs that improve iGluSnFR's nanoscopic localization to postsynapses. The resulting indicator iGluSnFR3 exhibits rapid nonsaturating activation kinetics and reports synaptic glutamate release with decreased saturation and increased specificity versus extrasynaptic signals in cultured neurons. Simultaneous imaging and electrophysiology at individual boutons in mouse visual cortex showed that iGluSnFR3 transients report single action potentials with high specificity. In vibrissal sensory cortex layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines.

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03/15/23 | Fast and sensitive GCaMP calcium indicators for imaging neural populations.
Zhang Y, Rozsa M, Liang Y, Bushey D, Wei Z, Zheng J, Reep D, Broussard GJ, Tsang A, Tsegaye G, Narayan S, Obara CJ, Lim J, Patel R, Zhang R, Ahrens MB, Turner GC, Wang SS, Korff WL, Schreiter ER, Svoboda K, Hasseman JP, Kolb I, Looger LL
Nature. 2023 Mar 15:. doi: 10.1038/s41586-023-05828-9

Calcium imaging with protein-based indicators is widely used to follow neural activity in intact nervous systems, but current protein sensors report neural activity at timescales much slower than electrical signalling and are limited by trade-offs between sensitivity and kinetics. Here we used large-scale screening and structure-guided mutagenesis to develop and optimize several fast and sensitive GCaMP-type indicators. The resulting 'jGCaMP8' sensors, based on the calcium-binding protein calmodulin and a fragment of endothelial nitric oxide synthase, have ultra-fast kinetics (half-rise times of 2 ms) and the highest sensitivity for neural activity reported for a protein-based calcium sensor. jGCaMP8 sensors will allow tracking of large populations of neurons on timescales relevant to neural computation.

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11/13/22 | Brain-wide measurement of protein turnover with high spatial and temporal resolution
Boaz Mohar , Jonathan B. Grimm , Ronak Patel , Timothy A. Brown , Paul Tillberg , Luke D. Lavis , Nelson Spruston , Karel Svoboda
bioRxiv. 2022 Nov 13:. doi: 10.1101/2022.11.12.516226

Cells regulate function by synthesizing and degrading proteins. This turnover ranges from minutes to weeks, as it varies across proteins, cellular compartments, cell types, and tissues. Current methods for tracking protein turnover lack the spatial and temporal resolution needed to investigate these processes, especially in the intact brain, which presents unique challenges. We describe a pulse-chase method (DELTA) for measuring protein turnover with high spatial and temporal resolution throughout the body, including the brain. DELTA relies on rapid covalent capture by HaloTag of fluorophores that were optimized for bioavailability in vivo. The nuclear protein MeCP2 showed brain region- and cell type-specific turnover. The synaptic protein PSD95 was destabilized in specific brain regions by behavioral enrichment. A novel variant of expansion microscopy further facilitated turnover measurements at individual synapses. DELTA enables studies of adaptive and maladaptive plasticity in brain-wide neural circuits.

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