@article {70093, title = {iGABASnFR2: Improved genetically encoded protein sensors of GABA}, journal = {eLife}, year = {2026}, month = {2026 Feb 25}, abstract = {

Monitoring GABAergic inhibition in the nervous system has been enabled by development of an intensiometric molecular sensor that directly detects GABA. However, the first generation iGABASnFR exhibits low signal-to-noise and suboptimal kinetics, making in vivo experiments challenging. To improve sensor performance, we targeted several sites in the protein for near-saturation mutagenesis and evaluated the resulting sensor variants in a high throughput screening system using evoked synaptic release in primary cultured neurons. This identified a sensor variant, iGABASnFR2, with 4.2-fold improved sensitivity and 20\% faster kinetics, and binding affinity that remained in a range sensitive to changes in GABA concentration at synapses. We also identified sensors with an inverted response, decreasing fluorescence intensity upon GABA binding. We termed the best such negative-going sensor iGABASnFR2n, which can be used to corroborate observations with the positive-going sensor. These improvements yielded a qualitative enhancement of in vivo performance when compared directly to the original sensor. iGABASnFR2 enabled the first measurements of direction-selective GABA release in the retina. In vivo imaging in somatosensory cortex revealed that iGABASnFR2 can report volume-transmitted GABA release following whisker stimulation. Overall, the improved sensitivity and kinetics of iGABASnFR2 make it a more effective tool for imaging GABAergic transmission in intact neural circuits.

}, doi = {10.7554/eLife.108319.2}, url = {https://doi.org/10.7554/eLife.108319.2}, author = {Kolb, Ilya and Hasseman, Jeremy P and Matsumoto, Akihiro and Jensen, Thomas P and Kopach, Olga and Arthur, Benjamin J and Zhang, Yan and Tsang, Arthur and Reep, Daniel and Tsegaye, Getahun and Zheng, Jihong and Patel, Ronak H and Looger, Loren L and Marvin, Jonathan S and Korff, Wyatt L and Rusakov, Dmitri A and Yonehara, Keisuke and Turner, Glenn C} } @article {70182, title = {iGlucoSnFR2: A genetically encoded fluorescent sensor for measuring intracellular or extracellular glucose in vivo in mouse brain.}, journal = {Sci Adv}, volume = {11}, year = {2025}, month = {2025 Nov 14}, pages = {eadz3889}, abstract = {

Continuous glucose monitors have proven invaluable for monitoring blood glucose levels for diabetics, but they are of limited use for observing glucose dynamics at the cellular (or subcellular) level. We have developed a second generation, genetically encoded intensity-based glucose sensing fluorescent reporter (iGlucoSnFR2). We show that when it is targeted to the cytosol, it reports intracellular glucose consumption and gluconeogenesis in cell culture, along with efflux from the endoplasmic reticulum. It outperforms the original iGlucoSnFR in vivo when observed by fiber photometry in mouse brain and reports transient increase in glucose concentration when stimulated by noradrenaline or electrical stimulation. Last, we demonstrate that membrane localized iGlucoSnFR2 can be calibrated in vivo to indicate absolute changes in extracellular glucose concentration in awake mice. We anticipate iGlucoSnFR2 facilitating previously unobservable measurements of glucose dynamics with high spatial and temporal resolution in living mammals and other experimental organisms.

}, issn = {2375-2548}, doi = {10.1126/sciadv.adz3889}, url = {https://doi.org/10.1126/sciadv.adz3889}, author = {Marvin, Jonathan S and M{\"a}chler, Philipp and Meng, Chengbo and Ates, Tayfun and Patel, Ronak H and Adhikari, Raghabendra and Makurath, Monika A and Ku, Zaneta and Feliciano, Daniel and Atasoy, Deniz and Cui, Guohong and Kleinfeld, David and Brown, Timothy A} } @article {69225, title = {An engineered biosensor enables dynamic aspartate measurements in living cells.}, journal = {Elife}, volume = {12}, year = {2024}, month = {2024 Feb 23}, abstract = {

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.

}, keywords = {Animals, Aspartic Acid, Biosensing Techniques, Cell Line, Green Fluorescent Proteins, Mammals}, issn = {2050-084X}, doi = {10.7554/eLife.90024}, author = {Davidsen, Kristian and Marvin, Jonathan S and Aggarwal, Abhi and Brown, Timothy A and Sullivan, Lucas B} } @article {69250, title = {High-Performance Genetically Encoded Green Fluorescent Biosensors for Intracellular l-Lactate.}, journal = {ACS Central Science}, volume = {10}, year = {2024}, month = {2024 Feb 28}, pages = {402-416}, abstract = {

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.

}, issn = {2374-7943}, doi = {10.1021/acscentsci.3c01250}, author = {Hario, Saaya and Le, Giang N T and Sugimoto, Hikaru and Takahashi-Yamashiro, Kei and Nishinami, Suguru and Toda, Hirofumi and Li, Selene and Marvin, Jonathan S and Kuroda, Shinya and Drobizhev, Mikhail and Terai, Takuya and Nasu, Yusuke and Campbell, Robert E} } @article {69021, title = {Extracellular glutamate and GABA transients at the transition from interictal spiking to seizures.}, journal = {Brain}, year = {2023}, month = {2023 Oct 03}, abstract = {

Focal epilepsy is associated with intermittent brief population discharges (interictal spikes), which resemble sentinel spikes that often occur at the onset of seizures. Why interictal spikes self-terminate whilst seizures persist and propagate is incompletely understood. We used fluorescent glutamate and GABA sensors in an awake rodent model of neocortical seizures to resolve the spatiotemporal evolution of both neurotransmitters in the extracellular space. Interictal spikes were accompanied by brief glutamate transients which were maximal at the initiation site and rapidly propagated centrifugally. GABA transients lasted longer than glutamate transients and were maximal \~{}1.5 mm from the focus where they propagated centripetally. Prior to seizure initiation GABA transients were attenuated, whilst glutamate transients increased, consistent with a progressive failure of local inhibitory restraint. As seizures increased in frequency, there was a gradual increase in the spatial extent of spike-associated glutamate transients associated with interictal spikes. Neurotransmitter imaging thus reveals a progressive collapse of an annulus of feed-forward GABA release, allowing seizures to escape from local inhibitory restraint.

}, issn = {1460-2156}, doi = {10.1093/brain/awad336}, author = {Shimoda, Yoshiteru and Leite, Marco and Graham, Robert T and Marvin, Jonathan S and Hasseman, Jeremy and Kolb, Ilya and Looger, Loren L and Magloire, Vincent and Kullmann, Dimitri M} } @article {68772, title = {Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission.}, journal = {Nature Methods}, volume = {20}, year = {2023}, month = {2023 Jun 01}, abstract = {

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{\textquoteright}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.

}, issn = {1548-7105}, doi = {10.1038/s41592-023-01863-6}, author = {Aggarwal, Abhi and Liu, Rui and Chen, Yang and Ralowicz, Amelia J and Bergerson, Samuel J and Tomaska, Filip and Mohar, Boaz and Hanson, Timothy L and Hasseman, Jeremy P and Reep, Daniel and Tsegaye, Getahun and Yao, Pantong and Ji, Xiang and Kloos, Marinus and Walpita, Deepika and Patel, Ronak and Mohr, Manuel A and Tillberg, Paul W and Looger, Loren L and Marvin, Jonathan S and Hoppa, Michael B and Konnerth, Arthur and Kleinfeld, David and Schreiter, Eric R and Podgorski, Kaspar} } @article {68646, title = {Selective Serotonin Reuptake Inhibitors within Cells: Temporal Resolution in Cytoplasm, Endoplasmic Reticulum, and Membrane.}, journal = {Journal of Neuroscience}, year = {2023}, month = {2023 Mar 02}, abstract = {

Selective serotonin reuptake inhibitors (SSRIs) are the most prescribed treatment for individuals experiencing major depressive disorder (MDD). The therapeutic mechanisms that take place before, during, or after SSRIs bind the serotonin transporter (SERT) are poorly understood, partially because no studies exist of the cellular and subcellular pharmacokinetic properties of SSRIs in living cells. We studied escitalopram and fluoxetine using new intensity-based drug-sensing fluorescent reporters ("iDrugSnFRs") targeted to the plasma membrane (PM), cytoplasm, or endoplasmic reticulum (ER) of cultured neurons and mammalian cell lines. We also employed chemical detection of drug within cells and phospholipid membranes. The drugs attain equilibrium in neuronal cytoplasm and ER, at approximately the same concentration as the externally applied solution, with time constants of a few s (escitalopram) or 200-300 s (fluoxetine). Simultaneously, the drugs accumulate within lipid membranes by >= 18-fold (escitalopram) or 180-fold (fluoxetine), and possibly by much larger factors. Both drugs leave cytoplasm, lumen, and membranes just as quickly during washout. We synthesized membrane-impermeant quaternary amine derivatives of the two SSRIs. The quaternary derivatives are substantially excluded from membrane, cytoplasm, and ER for \> 2.4 h. They inhibit SERT transport-associated currents 6- or 11-fold less potently than the SSRIs (escitalopram or fluoxetine derivative, respectively), providing useful probes for distinguishing compartmentalized SSRI effects. Although our measurements are orders of magnitude faster than the "therapeutic lag" of SSRIs, these data suggest that SSRI-SERT interactions within organelles or membranes may play roles during either the therapeutic effects or the "antidepressant discontinuation syndrome".Selective serotonin reuptake inhibitors stabilize mood in several disorders. In general, these drugs bind to the serotonin (5-hydroxytryptamine) transporter (SERT), which clears serotonin from CNS and peripheral tissues. SERT ligands are effective and relatively safe; primary care practitioners often prescribe them. However, they have several side effects and require 2 to 6 weeks of continuous administration until they act effectively. How they work remains perplexing, contrasting with earlier assumptions that the therapeutic mechanism involves SERT inhibition followed by increased extracellular serotonin levels. This study establishes that two SERT ligands, fluoxetine and escitalopram, enter neurons within minutes, while simultaneously accumulating in many membranes. Such knowledge will motivate future research, hopefully revealing where and how SERT ligands "engage" their therapeutic target(s).

}, issn = {1529-2401}, doi = {10.1523/JNEUROSCI.1519-22.2022}, author = {Nichols, Aaron L and Blumenfeld, Zack and Luebbert, Laura and Knox, Hailey J and Muthusamy, Anand K and Marvin, Jonathan S and Kim, Charlene H and Grant, Stephen N and Walton, David P and Cohen, Bruce N and Hammar, Rebekkah and Looger, Loren and Artursson, Per and Dougherty, Dennis A and Lester, Henry A} } @article {67848, title = {Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands.}, journal = {eLife}, volume = {11}, year = {2022}, month = {2022 Jan 04}, abstract = {

Nicotinic partial agonists provide an accepted aid for smoking cessation and thus contribute to decreasing tobacco-related disease. Improved drugs constitute a continued area of study. However, there remains no reductionist method to examine the cellular and subcellular pharmacokinetic properties of these compounds in living cells. Here, we developed new intensity-based drug sensing fluorescent reporters ({\textquoteright}iDrugSnFRs{\textquoteright}) for the nicotinic partial agonists dianicline, cytisine, and two cytisine derivatives - 10-fluorocytisine and 9-bromo-10-ethylcytisine. We report the first atomic-scale structures of liganded periplasmic binding protein-based biosensors, accelerating development of iDrugSnFRs and also explaining the activation mechanism. The nicotinic iDrugSnFRs detect their drug partners in solution, as well as at the plasma membrane (PM) and in the endoplasmic reticulum (ER) of cell lines and mouse hippocampal neurons. At the PM, the speed of solution changes limits the growth and decay rates of the fluorescence response in almost all cases. In contrast, we found that rates of membrane crossing differ among these nicotinic drugs by \> 30 fold. The new nicotinic iDrugSnFRs provide insight into the real-time pharmacokinetic properties of nicotinic agonists and provide a methodology whereby iDrugSnFRs can inform both pharmaceutical neuroscience and addiction neuroscience.

}, issn = {2050-084X}, doi = {10.7554/eLife.74648}, author = {Nichols, Aaron L and Blumenfeld, Zack and Fan, ChengCheng and Luebbert, Laura and Blom, Annet Em and Cohen, Bruce N and Marvin, Jonathan S and Borden, Philip M and Kim, Charlene H and Muthusamy, Anand K and Shivange, Amol V and Knox, Hailey J and Campello, Hugo Rego and Wang, Jonathan H and Dougherty, Dennis A and Looger, Loren L and Gallagher, Timothy and Rees, Douglas C and Lester, Henry A} } @article {67311, title = {The functional organization of excitatory synaptic input to place cells.}, journal = {Nature Communications}, volume = {12}, year = {2021}, month = {2021 Jun 11}, pages = {3558}, abstract = {

Hippocampal place cells contribute to mammalian spatial navigation and memory formation. Numerous models have been proposed to explain the location-specific firing of this cognitive representation, but the pattern of excitatory synaptic input leading to place firing is unknown, leaving no synaptic-scale explanation of place coding. Here we used resonant scanning two-photon microscopy to establish the pattern of synaptic glutamate input received by CA1 place cells in behaving mice. During traversals of the somatic place field, we found increased excitatory dendritic input, mainly arising from inputs with spatial tuning overlapping the somatic field, and functional clustering of this input along the dendrites over ~10 {\textmu}m. These results implicate increases in total excitatory input and co-activation of anatomically clustered synaptic input in place firing. Since they largely inherit their fields from upstream synaptic partners with similar fields, many CA1 place cells appear to be part of multi-brain-region cell assemblies forming representations of specific locations.

}, issn = {2041-1723}, doi = {10.1038/s41467-021-23829-y}, author = {Adoff, Michael D and Climer, Jason R and Davoudi, Heydar and Marvin, Jonathan S and Looger, Loren L and Dombeck, Daniel A} } @article {67627, title = {In~vivo glucose imaging in multiple model organisms with an engineered single-wavelength sensor.}, journal = {Cell Reports}, volume = {35}, year = {2021}, month = {2021 Jun 22}, pages = {109284}, abstract = {

Glucose is arguably the most important molecule in metabolism, and its dysregulation underlies diabetes. We describe a family of single-wavelength genetically encoded glucose sensors with a high signal-to-noise ratio, fast kinetics, and affinities varying over four orders of magnitude (1\ μM to 10\ mM). The sensors allow mechanistic characterization of glucose transporters expressed in cultured cells with high spatial and temporal resolution. Imaging of neuron/glia co-cultures revealed \~{}3-fold faster glucose changes in astrocytes. In larval Drosophila central nervous system explants, intracellular neuronal glucose fluxes suggested a rostro-caudal transport pathway in the ventral nerve cord neuropil. In zebrafish, expected glucose-related physiological sequelae of insulin and epinephrine treatments were directly visualized. Additionally, spontaneous muscle twitches induced glucose uptake in muscle, and sensory and pharmacological perturbations produced large changes in the brain. These sensors will enable rapid, high-resolution imaging of glucose influx, efflux, and metabolism in behaving animals.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2021.109284}, author = {Keller, Jacob P and Marvin, Jonathan S and Lacin, Haluk and Lemon, William C and Shea, Jamien and Kim, Soomin and Lee, Richard T and Koyama, Minoru and Keller, Philipp J and Looger, Loren L} } @article {65902, title = {Directed Evolution of a Selective and Sensitive Serotonin Sensor via Machine Learning.}, journal = {Cell}, volume = {183}, year = {2020}, month = {2020 Dec 23}, pages = {1986-2002.e26}, abstract = {

Serotonin plays a central role in cognition and is the target of most pharmaceuticals for psychiatric disorders. Existing drugs have limited efficacy; creation of improved versions will require better understanding of serotonergic circuitry, which has been hampered by our inability to monitor serotonin release and transport with high spatial and temporal resolution. We developed and applied a binding-pocket redesign strategy, guided by machine learning, to create a high-performance, soluble, fluorescent serotonin sensor (iSeroSnFR), enabling optical detection of millisecond-scale serotonin transients. We demonstrate that iSeroSnFR can be used to detect serotonin release in freely behaving mice during fear conditioning, social interaction, and sleep/wake transitions. We also developed a robust assay of serotonin transporter function and modulation by drugs. We expect that both machine-learning-guided binding-pocket redesign and iSeroSnFR will have broad utility for the development of other sensors and in\ vitro and in\ vivo serotonin detection, respectively.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2020.11.040}, author = {Unger, Elizabeth K and Keller, Jacob P and Altermatt, Michael and Liang, Ruqiang and Matsui, Aya and Dong, Chunyang and Hon, Olivia J and Yao, Zi and Sun, Junqing and Banala, Samba and Flanigan, Meghan E and Jaffe, David A and Hartanto, Samantha and Carlen, Jane and Mizuno, Grace O and Borden, Phillip M and Shivange, Amol V and Cameron, Lindsay P and Sinning, Steffen and Underhill, Suzanne M and Olson, David E and Amara, Susan G and Temple Lang, Duncan and Rudnick, Gary and Marvin, Jonathan S and Lavis, Luke D and Lester, Henry A and Alvarez, Veronica A and Fisher, Andrew J and Prescher, Jennifer A and Kash, Thomas L and Yarov-Yarovoy, Vladimir and Gradinaru, Viviana and Looger, Loren L and Tian, Lin} } @article {65300, title = {A fast genetically encoded fluorescent sensor for faithful in vivo acetylcholine detection in mice, fish, worms and flies.}, journal = {bioRxiv}, year = {2020}, month = {2020 Feb 8}, abstract = {

Here we design and optimize a genetically encoded fluorescent indicator, iAChSnFR, for the ubiquitous neurotransmitter acetylcholine, based on a bacterial periplasmic binding protein. iAChSnFR shows large fluorescence changes, rapid rise and decay kinetics, and insensitivity to most cholinergic drugs. iAChSnFR revealed large transients in a variety of slice and\ in vivo\ preparations in mouse, fish, fly and worm. iAChSnFR will be useful for the study of acetylcholine in all animals.

}, doi = {https://doi.org/10.1101/2020.02.07.939504}, author = {Borden, Philip and Zhang, Peng and Shivange, Amol V and Marvin, Jonathan S and Cichon, Joseph and Dan, Chuntao and Podgorski, Kaspar and Figueiredo, Antonio and Novak, Ondrej and Tanimoto, Masashi and Shigetomi, Eiji and Lobas, Mark A and Kim, Hyuntae and Zhu, Paula and Zhang, Yajun and Zheng, W. Sharon and Fan, ChengCheng and Wang, Guangfu and Xiang, Bowen and Gan, Li and Zhang, Guang-Xian and Guo, Kaiming and Lin, Li and Cai, Yuan and Yee, Andrew G. and Aggarwal, Aman and Ford, Christopher P. and Rees, Douglas C. and Dietrich, Dirk and Khakh, Baljit S. and Dittman, Jeremy S. and Gan, Wen-Biao and Koyama, Minoru and Jayaraman, Vivek and Cheer, Joseph F. and Lester, Henry A. and Zhu, J. Julius and Looger, Loren L.} } @article {65618, title = {jYCaMP: an optimized calcium indicator for two-photon imaging at fiber laser wavelengths.}, journal = {Nature Methods}, volume = {17}, year = {2020}, month = {2020 May 25}, pages = {694-97}, abstract = {

Femtosecond lasers at fixed wavelengths above 1,000 nm are powerful, stable and inexpensive, making them promising sources for two-photon microscopy. Biosensors optimized for these wavelengths are needed for both next-generation microscopes and affordable turn-key systems. Here we report jYCaMP1, a yellow variant of the calcium indicator jGCaMP7 that outperforms its parent in mice and flies at excitation wavelengths above 1,000 nm and enables improved two-color calcium imaging with red fluorescent protein-based indicators.

}, issn = {1548-7105}, doi = {10.1038/s41592-020-0835-7}, author = {Mohr, Manuel Alexander and Bushey, Daniel and Abhi Aggarwal and Marvin, Jonathan S and Kim, Jeong Jun and Marquez, Emiliano Jimenez and Liang, Yajie and Patel, Ronak and Macklin, John J and Lee, Chi-Yu and Tsang, Arthur and Tsegaye, Getahun and Ahrens, Allison M and Chen, Jerry L and Kim, Douglas S and Wong, Allan M and Looger, Loren L and Schreiter, Eric R and Podgorski, Kaspar} } @article {65604, title = {Nanoscopic visualization of restricted nonvolume cholinergic and monoaminergic transmission with genetically encoded sensors.}, journal = {Nano Letters}, volume = {20}, year = {2020}, month = {2020 Jun}, pages = {4073-83}, abstract = {

How neuromodulatory transmitters diffuse into the extracellular space remains an unsolved fundamental biological question, despite wide acceptance of the volume transmission model. Here, we report development of a method combining genetically encoded fluorescent sensors with high-resolution imaging and analysis algorithms which permits the first direct visualization of neuromodulatory transmitter diffusion at various neuronal and non-neuronal cells. Our analysis reveals that acetylcholine and monoamines diffuse at individual release sites with a spread length constant of \~{}0.75 μm. These transmitters employ varied numbers of release sites, and when spatially close-packed release sites coactivate they can spillover into larger subcellular areas. Our data indicate spatially restricted (i.e., nonvolume) neuromodulatory transmission to be a prominent intercellular communication mode, reshaping current thinking of control and precision of neuromodulation crucial for understanding behaviors and diseases.

}, issn = {1530-6992}, doi = {10.1021/acs.nanolett.9b04877}, author = {Zhu, Paula K and Zheng, W Sharon and Zhang, Peng and Jing, Miao and Borden, Philip M and Ali, Farhan and Guo, Kaiming and Feng, Jiesi and Marvin, Jonathan S and Wang, Yali and Wan, Jinxia and Gan, Li and Kwan, Alex C and Lin, Li and Looger, Loren L and Li, Yulong and Zhang, Yajun} } @article {65827, title = {Optimized Vivid-derived Magnets photodimerizers for subcellular optogenetics in mammalian cells.}, journal = {Elife}, volume = {9}, year = {2020}, month = {2020 Nov 11}, abstract = {

Light-inducible dimerization protein modules enable precise temporal and spatial control of biological processes in non-invasive fashion. Among them, Magnets are small modules engineered from the photoreceptor Vivid by orthogonalizing the homodimerization interface into complementary heterodimers. Both Magnets components, which are well-tolerated as protein fusion partners, are photoreceptors requiring simultaneous photoactivation to interact, enabling high spatiotemporal confinement of dimerization with a single-excitation wavelength. However, Magnets require concatemerization for efficient responses and cell preincubation at 28C to be functional. Here we overcome these limitations by engineering an optimized Magnets pair requiring neither concatemerization nor low temperature preincubation. We validated these {\textquoteright}enhanced{\textquoteright} Magnets (eMags) by using them to rapidly and reversibly recruit proteins to subcellular organelles, to induce organelle contacts, and to reconstitute OSBP-VAP ER-Golgi tethering implicated in phosphatidylinositol-4-phosphate transport and metabolism. eMags represent a very effective tool to optogenetically manipulate physiological processes over whole cells or in small subcellular volumes.

}, issn = {2050-084X}, doi = {10.7554/eLife.63230}, author = {Benedetti, Lorena and Marvin, Jonathan S and Falahati, Hanieh and Guill{\'e}n-Samander, Andres and Looger, Loren L and De Camilli, Pietro} } @article {65242, title = {Biosensors show the pharmacokinetics of S-Ketamine in the endoplasmic reticulum.}, journal = {Frontiers in Cellular Neuroscience}, volume = {13}, year = {2019}, month = {2019 Nov 12}, pages = {499}, abstract = {

The target for the "rapid" (\<24 h) antidepressant effects of S-ketamine is unknown, vitiating programs to rationally develop more effective rapid antidepressants. To describe a drug{\textquoteright}s target, one must first understand the compartments entered by the drug, at all levels-the organ, the cell, and the organelle. We have, therefore, developed molecular tools to measure the subcellular, organellar pharmacokinetics of S-ketamine. The tools are genetically encoded intensity-based S-ketamine-sensing fluorescent reporters, iSKetSnFR1 and iSKetSnFR2. In solution, these biosensors respond to S-ketamine with a sensitivity, S-slope = delta(F/F)/(delta[S-ketamine]) of 0.23 and 1.9/μM, respectively. The iSKetSnFR2 construct allows measurements at \<0.3 μM S-ketamine. The iSKetSnFR1 and iSKetSnFR2 biosensors display \>100-fold selectivity over other ligands tested, including R-ketamine. We targeted each of the sensors to either the plasma membrane (PM) or the endoplasmic reticulum (ER). Measurements on these biosensors expressed in Neuro2a cells and in human dopaminergic neurons differentiated from induced pluripotent stem cells (iPSCs) show that S-ketamine enters the ER within a few seconds after appearing in the external solution near the PM, then leaves as rapidly after S-ketamine is removed from the extracellular solution. In cells, S-slopes for the ER and PM-targeted sensors differ by \<2-fold, indicating that the ER [S-ketamine] is less than 2-fold different from the extracellular [S-ketamine]. Organelles represent potential compartments for the engagement of S-ketamine with its antidepressant target, and potential S-ketamine targets include organellar ion channels, receptors, and transporters.

}, issn = {1662-5102}, doi = {10.3389/fncel.2019.00499}, author = {Bera, Kallol and Kamajaya, Aron and Shivange, Amol V and Muthusamy, Anand K and Nichols, Aaron L and Borden, Philip M and Grant, Stephen and Jeon, Janice and Lin, Elaine and Bishara, Ishak and Chin, Theodore M and Cohen, Bruce N and Kim, Charlene H and Unger, Elizabeth K and Tian, Lin and Marvin, Jonathan S and Looger, Loren L and Lester, Henry A} } @article {65043, title = {Direct wavefront sensing enables functional imaging of infragranular axons and spines.}, journal = {Nature Methods}, volume = {16}, year = {2019}, month = {2019 Jul}, pages = {615-618}, abstract = {

We advance two-photon microscopy for near-diffraction-limited imaging up to 850 {\textmu}m below the pia in awake mice. Our approach combines direct wavefront sensing of light from a guidestar (formed by descanned fluorescence from Cy5.5-conjugated dextran in brain microvessels) with adaptive optics to compensate for tissue-induced aberrations in the wavefront. We achieve high signal-to-noise ratios in recordings of glutamate release from thalamocortical axons and calcium transients in spines of layer 5b basal dendrites during active tactile sensing.

}, issn = {1548-7105}, doi = {10.1038/s41592-019-0434-7}, author = {Liu, Rui and Li, Zengyi and Marvin, Jonathan S and Kleinfeld, David} } @article {65056, title = {A genetically encoded fluorescent sensor for in vivo imaging of GABA.}, journal = {Nature Methods}, volume = {16}, year = {2019}, month = {2019 Jul 15}, pages = {763-770}, abstract = {

Current techniques for monitoring GABA (γ-aminobutyric acid), the primary inhibitory neurotransmitter in vertebrates, cannot follow transients in intact neural circuits. To develop a GABA sensor, we applied the design principles used to create the fluorescent glutamate receptor iGluSnFR. We used a protein derived from a previously unsequenced Pseudomonas fluorescens strain and performed structure-guided mutagenesis and library screening to obtain intensity-based GABA sensing fluorescence reporter (iGABASnFR) variants. iGABASnFR is genetically encoded, detects GABA release evoked by electric stimulation of afferent fibers in acute brain slices and produces readily detectable fluorescence increases in vivo in mice and zebrafish. We applied iGABASnFR to track mitochondrial GABA content and its modulation by an anticonvulsant, swimming-evoked, GABA-mediated transmission in zebrafish cerebellum, GABA release events during interictal spikes and seizures in awake mice, and found that GABA-mediated tone decreases during isoflurane anesthesia.

}, issn = {1548-7105}, doi = {10.1038/s41592-019-0471-2}, author = {Marvin, Jonathan S and Shimoda, Yoshiteru and Magloire, Vincent and Leite, Marco and Kawashima, Takashi and Jensen, Thomas P and Kolb, Ilya and Knott, Erika L and Novak, Ondrej and Podgorski, Kaspar and Leidenheimer, Nancy J and Rusakov, Dmitri A and Ahrens, Misha B and Kullmann, Dimitri M and Looger, Loren L} } @article {64796, title = {A genetically encoded single-wavelength sensor for imaging cytosolic and cell surface ATP.}, journal = {Nature Communications}, volume = {10}, year = {2019}, month = {2019 Feb 12}, pages = {711}, abstract = {

Adenosine 5{\textquoteright} triphosphate (ATP) is a universal intracellular energy source and an evolutionarily ancient, ubiquitous extracellular signal in diverse species. Here, we report the generation and characterization of single-wavelength genetically encoded fluorescent sensors (iATPSnFRs) for imaging extracellular and cytosolic ATP from insertion of circularly permuted superfolder GFP into the epsilon subunit of FF-ATPase from Bacillus PS3. On the cell surface and within the cytosol, iATPSnFR responds to relevant ATP concentrations (30 μM to 3 mM) with fast increases in fluorescence. iATPSnFRs can be genetically targeted to specific cell types and sub-cellular compartments, imaged with standard light microscopes, do not respond to other nucleotides and nucleosides, and when fused with a red fluorescent protein function as ratiometric indicators. After careful consideration of their modest pH sensitivity, iATPSnFRs represent promising reagents for imaging ATP in the extracellular space and within cells during a variety of settings, and for further application-specific refinements.

}, issn = {2041-1723}, doi = {10.1038/s41467-019-08441-5}, author = {Lobas, Mark A and Tao, Rongkun and Nagai, Jun and Kronschl{\"a}ger, Mira T and Borden, Philip M and Marvin, Jonathan S and Looger, Loren L and Khakh, Baljit S} } @article {64866, title = {In vivo glucose imaging in multiple model organisms with an engineered single-wavelength sensor.}, journal = {bioRxiv}, year = {2019}, month = {2019 Mar 8}, abstract = {Glucose is arguably the most important molecule in metabolism, and its mismanagement underlies diseases of vast societal import, most notably diabetes. Although glucose-related metabolism has been the subject of intense study for over a century, tools to track glucose in living organisms with high spatio-temporal resolution are lacking. We describe the engineering of a family of genetically encoded glucose sensors with high signal-to-noise ratio, fast kinetics and affinities varying over four orders of magnitude (1 {\textmu}M to 10 mM). The sensors allow rigorous mechanistic characterization of glucose transporters expressed in cultured cells with high spatial and temporal resolution. Imaging of neuron/glia co-cultures revealed \~{}3-fold higher glucose changes in astrocytes versus neurons. In larval Drosophila central nervous system explants, imaging of intracellular neuronal glucose suggested a novel rostro-caudal transport pathway in the ventral nerve cord neuropil, with paradoxically slower uptake into the peripheral cell bodies and brain lobes. In living zebrafish, expected glucose-related physiological sequelae of insulin and epinephrine treatments were directly visualized in real time. Additionally, spontaneous muscle twitches induced glucose uptake in muscle, and sensory- and pharmacological perturbations gave rise to large but enigmatic changes in the brain. These sensors will enable myriad experiments, most notably rapid, high-resolution imaging of glucose influx, efflux, and metabolism in behaving animals.}, doi = {10.1101/571422}, author = {Keller, Jacob P and Marvin, Jonathan S and Lacin, Haluk and Lemon, William C and Shea, Jamien and Kim, Soomin and Lee, Richard T and Koyama, Minoru and Keller, Philipp J and Looger, Loren L} } @article {49329, title = {Kilohertz frame-rate two-photon tomography.}, journal = {Nature Methods}, volume = {16}, year = {2019}, month = {2019 Jul 29}, pages = {778-86}, abstract = {

Point-scanning two-photon microscopy enables high-resolution imaging within scattering specimens such as the mammalian brain, but sequential acquisition of voxels fundamentally limits imaging speed. We developed a two-photon imaging technique that scans lines of excitation across a focal plane at multiple angles and uses prior information to recover high-resolution images at over 1.4 billion voxels per second. Using a structural image as a prior for recording neural activity, we imaged visually-evoked and spontaneous glutamate release across hundreds of dendritic spines in mice at depths over 250 microns and frame-rates over 1 kHz. Dendritic glutamate transients in anaesthetized mice are synchronized within spatially-contiguous domains spanning tens of microns at frequencies ranging from 1-100 Hz. We demonstrate high-speed recording of acetylcholine and calcium sensors, 3D single-particle tracking, and imaging in densely-labeled cortex. Our method surpasses limits on the speed of raster-scanned imaging imposed by fluorescence lifetime.

}, doi = {10.1101/357269}, author = {Kazemipour, Abbas and Novak, Ondrej and Flickinger, Daniel and Marvin, Jonathan S and Abdelfattah, Ahmed S and King, J. and Borden, PM and Kim, JJ and Al-Abdullatif, SH and Deal, PE and Miller, EW and Schreiter, ER and Druckmann, S and Svoboda, K and Looger, LL and Podgorski, K} } @article {64865, title = {Multiplex imaging relates quantal glutamate release to presynaptic Ca homeostasis at multiple synapses in situ.}, journal = {Nature Communications}, volume = {10}, year = {2019}, month = {2019 03 29}, pages = {1414}, abstract = {

Information processing by brain circuits depends on Ca-dependent, stochastic release of the excitatory neurotransmitter glutamate. Whilst optical glutamate sensors have enabled detection of synaptic discharges, understanding presynaptic machinery requires simultaneous readout of glutamate release and nanomolar presynaptic Ca in situ. Here, we find that the fluorescence lifetime of the red-shifted Ca indicator Cal-590 is Ca-sensitive in the nanomolar range, and employ it in combination with green glutamate sensors to relate quantal neurotransmission to presynaptic Ca kinetics. Multiplexed imaging of individual and multiple synapses in identified axonal circuits reveals that glutamate release efficacy, but not its short-term plasticity, varies with time-dependent fluctuations in presynaptic resting Ca or spike-evoked Ca entry. Within individual presynaptic boutons, we find no nanoscopic co-localisation of evoked presynaptic Ca entry with the prevalent glutamate release site, suggesting loose coupling between the two. The approach enables a better understanding of release machinery at central synapses.

}, issn = {2041-1723}, doi = {10.1038/s41467-019-09216-8}, author = {Jensen, Thomas P and Zheng, Kaiyu and Cole, Nicholas and Marvin, Jonathan S and Looger, Loren L and Rusakov, Dmitri A} } @article {64935, title = {Quantitative in vivo imaging of neuronal glucose concentrations with a genetically encoded fluorescence lifetime sensor.}, journal = {Journal of Neuroscience Research}, volume = {97}, year = {2019}, month = {2019 Aug 01}, pages = {946-60}, abstract = {

Glucose is an essential source of energy for the brain. Recently, the development of genetically encoded fluorescent biosensors has allowed real time visualization of glucose dynamics from individual neurons and astrocytes. A major difficulty for this approach, even for ratiometric sensors, is the lack of a practical method to convert such measurements into actual concentrations in ex vivo brain tissue or in vivo. Fluorescence lifetime imaging provides a strategy to overcome this. In a previous study, we reported the lifetime glucose sensor iGlucoSnFR-TS (then called SweetieTS) for monitoring changes in neuronal glucose levels in response to stimulation. This genetically encoded sensor was generated by combining the Thermus thermophilus glucose-binding protein with a circularly permuted variant of the monomeric fluorescent protein T-Sapphire. Here, we provide more details on iGlucoSnFR-TS design and characterization, as well as pH and temperature sensitivities. For accurate estimation of glucose concentrations, the sensor must be calibrated at the same temperature as the experiments. We find that when the extracellular glucose concentration is in the range 2-10\ mM, the intracellular glucose concentration in hippocampal neurons from acute brain slices is ~20\% of the nominal external glucose concentration (~0.4-2\ mM). We also measured the cytosolic neuronal glucose concentration in vivo, finding a range of ~0.7-2.5\ mM in cortical neurons from awake mice.

}, issn = {1097-4547}, doi = {10.1002/jnr.24433}, author = {D{\'\i}az-Garc{\'\i}a, Carlos Manlio and Lahmann, Carolina and Mart{\'\i}nez-Fran{\c c}ois, Juan Ram{\'o}n and Li, Binsen and Koveal, Dorothy and Nathwani, Nidhi and Rahman, Mahia and Keller, Jacob P and Marvin, Jonathan S and Looger, Loren L and Yellen, Gary} } @article {48984, title = {Stability, affinity and chromatic variants of the glutamate sensor iGluSnFR.}, journal = {Nature Methods}, volume = {15}, year = {2018}, month = {2018 Nov}, pages = {9386-9}, abstract = {

Single-wavelength fluorescent reporters allow visualization of specific neurotransmitters with high spatial and temporal resolution. We report variants of intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) that are functionally brighter; detect submicromolar to millimolar amounts of glutamate; and have blue, cyan, green, or yellow emission profiles. These variants could be imaged in vivo in cases where original iGluSnFR was too dim, resolved glutamate transients in dendritic spines and axonal boutons, and allowed imaging at kilohertz rates.

}, doi = {10.1038/s41592-018-0171-3}, author = {Marvin, Jonathan S and Scholl, Benjamin and Wilson, Daniel E and Podgorski, Kaspar and Kazemipour, Abbas and Mueller, Johannes Alexander and Schoch-McGovern, Susanne and Wang, Samuel S-H and Quiroz, Francisco Jose Urra and Rebola, Nelson and Bao, Huan and Little, Justin P and Tkachuk, Ariana N and Hantman, Adam W and Chapman, Edwin R and Dietrich, Dirk and DiGregorio, David A and Fitzpatrick, David and Looger, Loren L} } @article {48732, title = {Genetically encoded biosensors.}, volume = {B2}, number = {14/350,199}, year = {2017}, month = {2017 Aug 01}, edition = {G01N 33/557 (20060101); G01N 33/58 (20060101); C07K 14/435 (20060101); C07K 14/245 (20060101); C07K 14/195 (20060101); G01N 33/68 (20060101)}, abstract = {

The present disclosure provides, inter alia, genetically encoded recombinant peptide biosensors comprising analyte-binding framework portions and signaling portions, wherein the signaling portions are present within the framework portions at sites or amino acid positions that undergo a conformational change upon interaction of the framework portion with an analyte.

}, url = {http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2\&Sect2=HITOFF\&u=\%2Fnetahtml\%2FPTO\%2Fsearch-adv.htm\&r=2\&p=1\&f=G\&l=50\&d=PTXT\&S1=(\%22Howard+Hughes+medical\%22.ASNM.)\&OS=AN/\%22Howard+Hughes+medical\%22\&RS=AN/\%22Howard+Hughes+medical\%22}, author = {Marvin, Jonathan S and Looger, Loren L and Lee, Richard T and Schreiter, Eric R} } @article {47601, title = {Falling apart.}, journal = {eLife}, volume = {5}, year = {2016}, month = {2016}, abstract = {

Destabilized nanobodies can be used to deliver fluorescent proteins and enzymes to specific targets inside cells.

}, issn = {2050-084X}, doi = {10.7554/eLife.18203}, author = {Marvin, Jonathan S and Looger, Loren L} } @article {6960, title = {Conditions and constraints for astrocyte calcium signaling in the hippocampal mossy fiber pathway.}, journal = {Neuron}, volume = {82}, year = {2014}, month = {2014 Apr 16}, pages = {413-29}, abstract = {

The spatiotemporal activities of astrocyte Ca(2+) signaling in mature neuronal circuits remain unclear. We used genetically encoded Ca(2+) and glutamate indicators as well as pharmacogenetic and electrical control of neurotransmitter release to explore astrocyte activity in the hippocampal mossy fiber pathway. Our data revealed numerous localized, spontaneous Ca(2+) signals in astrocyte branches and territories, but these were not driven by neuronal activity or glutamate. Moreover, evoked astrocyte Ca(2+) signaling changed linearly with the number of mossy fiber action potentials. Under these settings, astrocyte responses were global, suppressed by neurotransmitter clearance, and mediated by glutamate and GABA. Thus, astrocyte engagement in the fully developed mossy fiber pathway was slow and territorial, contrary to that frequently proposed for astrocytes within microcircuits. We show that astrocyte Ca(2+) signaling functionally segregates large volumes of neuropil and that these transients are not suited for responding to, or regulating, single synapses in the mossy fiber pathway.

}, issn = {1097-4199}, doi = {10.1016/j.neuron.2014.02.041}, author = {Haustein, Martin D and Kracun, Sebastian and Lu, Xiao-Hong and Shih, Tiffany and Jackson-Weaver, Olan and Tong, Xiaoping and Xu, Ji and Yang, X William and O{\textquoteright}Dell, Thomas J and Marvin, Jonathan S and Ellisman, Mark H and Bushong, Eric A and Looger, Loren L and Khakh, Baljit S.} } @article {45697, title = {Role of adaptor TrfA and ClpPC in controlling levels of SsrA-tagged proteins and antitoxins in staphylococcus aureus.}, journal = {Journal of Bacteriology}, volume = {196}, year = {2014}, month = {2014 Dec 1}, pages = {4140-51}, abstract = {

Staphylococcus aureus responds to changing extracellular environments in part by adjusting its proteome through alterations of transcriptional priorities and selective degradation of the preexisting pool of proteins. In Bacillus subtilis, the proteolytic adaptor protein MecA has been shown to play a role in assisting with the proteolytic degradation of proteins involved in competence and the oxidative stress response. However, the targets of TrfA, the MecA homolog in S. aureus, have not been well characterized. In this work, we investigated how TrfA assists chaperones and proteases to regulate the proteolysis of several classes of proteins in S. aureus. By fusing the last 3 amino acids of the SsrA degradation tag to Venus, a rapidly folding yellow fluorescent protein, we obtained both fluorescence-based and Western blot assay-based evidence that TrfA and ClpCP are the adaptor and protease, respectively, responsible for the degradation of the SsrA-tagged protein in S. aureus. Notably, the impact of TrfA on degradation was most prominent during late log phase and early stationary phase, due in part to a combination of transcriptional regulation and proteolytic degradation of TrfA by ClpCP. We also characterized the temporal transcriptional regulation governing TrfA activity, wherein Spx, a redox-sensitive transcriptional regulator degraded by ClpXP, activates trfA transcription while repressing its own promoter. Finally, the scope of TrfA-mediated proteolysis was expanded by identifying TrfA as the adaptor that works with ClpCP to degrade antitoxins in S. aureus. Together, these results indicate that the adaptor TrfA adds temporal nuance to protein degradation by ClpCP in S. aureus.

}, issn = {1098-5530}, doi = {10.1128/JB.02222-14}, author = {Donegan, Niles P and Marvin, Jonathan S and Cheung, Ambrose L} } @article {6541, title = {Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics.}, journal = {Frontiers in Molecular Neuroscience}, volume = {6}, year = {2013}, month = {2013 Mar 4}, pages = {2}, abstract = {

Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, "RCaMPs," engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca(2+)-bound state of the sensors and the engineered protein domain interfaces of the different indicators. We characterized the biophysical properties and performance of RCaMP sensors in vitro and in vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca(2+)]) and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2) or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca(2+) affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan, and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics.

}, issn = {1662-5099}, doi = {10.3389/fnmol.2013.00002}, author = {Akerboom, Jasper and Carreras Calder{\'o}n, Nicole and Tian, Lin and Wabnig, Sebastian and Prigge, Matthias and Tol{\"o}, Johan and Gordus, Andrew and Orger, Michael B. and Severi, Kristen E and Macklin, John J and Patel, Ronak and Pulver, Stefan R and Wardill, Trevor J. and Fischer, Elisabeth and Sch{\"u}ler, Christina and Chen, Tsai-Wen and Sarkisyan, Karen S and Marvin, Jonathan S and Bargmann, Cornelia I and Kim, Douglas S. and K{\"u}gler, Sebastian and Lagnado, Leon and Hegemann, Peter and Gottschalk, Alexander and Schreiter, Eric R and Looger, Loren L} } @article {6128, title = {An optimized fluorescent probe for visualizing glutamate neurotransmission.}, journal = {Nature Methods}, volume = {10}, year = {2013}, month = {2013 Feb}, pages = {162-70}, abstract = {

We describe an intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) with signal-to-noise ratio and kinetics appropriate for in vivo imaging. We engineered iGluSnFR in vitro to maximize its fluorescence change, and we validated its utility for visualizing glutamate release by neurons and astrocytes in increasingly intact neurological systems. In hippocampal culture, iGluSnFR detected single field stimulus-evoked glutamate release events. In pyramidal neurons in acute brain slices, glutamate uncaging at single spines showed that iGluSnFR responds robustly and specifically to glutamate in situ, and responses correlate with voltage changes. In mouse retina, iGluSnFR-expressing neurons showed intact light-evoked excitatory currents, and the sensor revealed tonic glutamate signaling in response to light stimuli. In worms, glutamate signals preceded and predicted postsynaptic calcium transients. In zebrafish, iGluSnFR revealed spatial organization of direction-selective synaptic activity in the optic tectum. Finally, in mouse forelimb motor cortex, iGluSnFR expression in layer V pyramidal neurons revealed task-dependent single-spine activity during running.

}, keywords = {Animals, Astrocytes, Biosensing Techniques, Caenorhabditis elegans, Calcium Signaling, Escherichia coli Proteins, Excitatory Postsynaptic Potentials, Fluorescent Dyes, Glutamic Acid, Green Fluorescent Proteins, Hippocampus, Mice, Motor Cortex, Neurons, Photic Stimulation, Pyramidal Cells, Recombinant Fusion Proteins, Retina, Signal-To-Noise Ratio, Synaptic Transmission, Zebrafish}, issn = {1548-7105}, doi = {10.1038/nmeth.2333}, author = {Marvin, Jonathan S and Borghuis, Bart G and Tian, Lin and Cichon, Joseph and Harnett, Mark T and Akerboom, Jasper and Gordus, Andrew and Renninger, Sabine L. and Chen, Tsai-Wen and Bargmann, Cornelia I and Orger, Michael B. and Schreiter, Eric R and Demb, Jonathan B and Gan, Wen-Biao and Hires, S Andrew and Looger, Loren L} } @article {6129, title = {Two-photon imaging of nonlinear glutamate release dynamics at bipolar cell synapses in the mouse retina.}, journal = {The Journal of Neuroscience : The Official Journal of the Society for Neuroscience}, volume = {33}, year = {2013}, month = {2013 Jul 3}, pages = {10972-85}, abstract = {

Alpha/Y-type retinal ganglion cells encode visual information with a receptive field composed of nonlinear subunits. This nonlinear subunit structure enhances sensitivity to patterns composed of high spatial frequencies. The Y-cell{\textquoteright}s subunits are the presynaptic bipolar cells, but the mechanism for the nonlinearity remains incompletely understood. We investigated the synaptic basis of the subunit nonlinearity by combining whole-cell recording of mouse Y-type ganglion cells with two-photon fluorescence imaging of a glutamate sensor (iGluSnFR) expressed on their dendrites and throughout the inner plexiform layer. A control experiment designed to assess iGluSnFR{\textquoteright}s dynamic range showed that fluorescence responses from Y-cell dendrites increased proportionally with simultaneously recorded excitatory current. Spatial resolution was sufficient to readily resolve independent release at intermingled ON and OFF bipolar terminals. iGluSnFR responses at Y-cell dendrites showed strong surround inhibition, reflecting receptive field properties of presynaptic release sites. Responses to spatial patterns located the origin of the Y-cell nonlinearity to the bipolar cell output, after the stage of spatial integration. The underlying mechanism differed between OFF and ON pathways: OFF synapses showed transient release and strong rectification, whereas ON synapses showed relatively sustained release and weak rectification. At ON synapses, the combination of fast release onset with slower release offset explained the nonlinear response of the postsynaptic ganglion cell. Imaging throughout the inner plexiform layer, we found transient, rectified release at the central-most levels, with increasingly sustained release near the borders. By visualizing glutamate release in real time, iGluSnFR provides a powerful tool for characterizing glutamate synapses in intact neural circuits.

}, issn = {1529-2401}, doi = {10.1523/JNEUROSCI.1241-13.2013}, author = {Borghuis, Bart G and Marvin, Jonathan S and Looger, Loren L and Demb, Jonathan B} } @article {5164, title = {Optimization of a GCaMP calcium indicator for neural activity imaging.}, journal = {The Journal of Neuroscience}, volume = {32}, year = {2012}, month = {2012 Oct 3}, pages = {13819-40}, chapter = {13819}, abstract = {

Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo . Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of {\textquotedblleft}GCaMP5{\textquotedblright} sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3.GCaMP5allows more sensitive detection of neural activity in vivo andmayfind widespread applications for cellular imaging in general.

}, doi = {10.1523/​JNEUROSCI.2601-12.2012}, author = {Akerboom, Jasper and Chen, Tsai-Wen and Wardill, Trevor J. and Marvin, Jonathan S and Mutlu, Sevinc and Carreras Caldero, Nicole and Esposti, Federico and Borghuis, Bart G and Sun, Xiaonan Richard and Gordus, Andrew and Orger, Michael B. and Portugues, Ruben and Engert, Florian and Macklin, John J and Filosa, Alessandro and Aggarwal, Aman and Kerr, Rex and Takagi, Ryousuke and Kracun, Sebastian and Shigetomi, Eiji and Khakh, Baljit S. and Baier, Herwig and Lagnado, Leon and Wang, Samuel S-H and Bargmann, Cornelia and Kimmel, Bruce and Jayaraman, Vivek and Svoboda, Karel and Kim, Douglas S. and Schreiter, Eric R and Looger, Loren L} } @article {4371, title = {A genetically encoded, high-signal-to-noise maltose sensor.}, journal = {Proteins}, volume = {79}, year = {2011}, month = {2011 Nov}, pages = {3025-36}, abstract = {

We describe the generation of a family of high-signal-to-noise single-wavelength genetically encoded indicators for maltose. This was achieved by insertion of circularly permuted fluorescent proteins into a bacterial periplasmic binding protein (PBP), Escherichia coli maltodextrin-binding protein, resulting in a four-color family of maltose indicators. The sensors were iteratively optimized to have sufficient brightness and maltose-dependent fluorescence increases for imaging, under both one- and two-photon illumination. We demonstrate that maltose affinity of the sensors can be tuned in a fashion largely independent of the fluorescent readout mechanism. Using literature mutations, the binding specificity could be altered to moderate sucrose preference, but with a significant loss of affinity. We use the soluble sensors in individual E. coli bacteria to observe rapid maltose transport across the plasma membrane, and membrane fusion versions of the sensors on mammalian cells to visualize the addition of maltose to extracellular media. The PBP superfamily includes scaffolds specific for a number of analytes whose visualization would be critical to the reverse engineering of complex systems such as neural networks, biosynthetic pathways, and signal transduction cascades. We expect the methodology outlined here to be useful in the development of indicators for many such analytes.

}, issn = {1097-0134}, doi = {10.1002/prot.23118}, author = {Marvin, Jonathan S and Schreiter, Eric R and Echevarr{\'\i}a, Ileabett M and Looger, Loren L} } @article {4373, title = {Structure of the escherichia coli phosphonate binding protein PhnD and rationally optimized phosphonate biosensors.}, journal = {Journal of Molecular Biology}, volume = {414}, year = {2011}, month = {2011 Dec 2}, pages = {356-69}, abstract = {

The phnD gene of Escherichia coli encodes the periplasmic binding protein of the phosphonate (Pn) uptake and utilization pathway. We have crystallized and determined structures of E. coli PhnD (EcPhnD) in the absence of ligand and in complex with the environmentally abundant 2-aminoethylphosphonate (2AEP). Similar to other bacterial periplasmic binding proteins, 2AEP binds near the center of mass of EcPhnD in a cleft formed between two lobes. Comparison of the open, unliganded structure with the closed 2AEP-bound structure shows that the two lobes pivot around a hinge by \~{}70{\textdegree} between the two states. Extensive hydrogen bonding and electrostatic interactions stabilize 2AEP, which binds to EcPhnD with low nanomolar affinity. These structures provide insight into Pn uptake by bacteria and facilitated the rational design of high signal-to-noise Pn biosensors based on both coupled small-molecule dyes and autocatalytic fluorescent proteins.

}, issn = {1089-8638}, doi = {10.1016/j.jmb.2011.09.047}, author = {Alicea, Ismael and Marvin, Jonathan S and Miklos, Aleksandr E and Ellington, Andrew D and Looger, Loren L and Schreiter, Eric R} } @article {1555, title = {Crystal structures of the GCaMP calcium sensor reveal the mechanism of fluorescence signal change and aid rational design.}, journal = {The Journal of Biological Chemistry}, volume = {284}, year = {2009}, month = {2009 Mar 6}, pages = {6455-64}, abstract = {

The genetically encoded calcium indicator GCaMP2 shows promise for neural network activity imaging, but is currently limited by low signal-to-noise ratio. We describe x-ray crystal structures as well as solution biophysical and spectroscopic characterization of GCaMP2 in the calcium-free dark state, and in two calcium-bound bright states: a monomeric form that dominates at intracellular concentrations observed during imaging experiments and an unexpected domain-swapped dimer with decreased fluorescence. This series of structures provides insight into the mechanism of Ca2+-induced fluorescence change. Upon calcium binding, the calmodulin (CaM) domain wraps around the M13 peptide, creating a new domain interface between CaM and the circularly permuted enhanced green fluorescent protein domain. Residues from CaM alter the chemical environment of the circularly permuted enhanced green fluorescent protein chromophore and, together with flexible inter-domain linkers, block solvent access to the chromophore. Guided by the crystal structures, we engineered a series of GCaMP2 point mutants to probe the mechanism of GCaMP2 function and characterized one mutant with significantly improved signal-to-noise. The mutation is located at a domain interface and its effect on sensor function could not have been predicted in the absence of structural data.

}, keywords = {Animals, Calcium, Calcium-Binding Proteins, Crystallography, Fluorescent Dyes, Green Fluorescent Proteins, Models, Molecular, Peptides, Protein Binding, Protein Structure, Recombinant Fusion Proteins, Structure-Activity Relationship, Tertiary, X-Ray}, issn = {0021-9258}, doi = {10.1074/jbc.M807657200}, author = {Akerboom, Jasper and Rivera, Jonathan D V{\'e}lez and Guilbe, Mar{\'\i}a M Rodr{\'\i}guez and Malav{\'e}, Elisa C Alfaro and Hernandez, Hector H and Tian, Lin and Hires, S Andrew and Marvin, Jonathan S and Looger, Loren L and Schreiter, Eric R} } @article {1556, title = {Engineering human IgG1 affinity to human neonatal Fc receptor: impact of affinity improvement on pharmacokinetics in primates.}, journal = {Journal of Immunology}, volume = {182}, year = {2009}, month = {2009 Jun 15}, pages = {7663-71}, abstract = {

The pH-dependent binding of Igs to the neonatal FcR (FcRn) plays a critical role in the in vivo homeostasis of IgGs. Modulating the interaction between Fc and FcRn through protein engineering is one method for improving the pharmacokinetics of therapeutic Abs. Recent studies disputed the direct relationship between increasing FcRn affinity and improved pharmacokinetic properties. In this work, we studied the pharmacokinetics of two human IgG1 Fc variants in cynomolgus monkey to further clarify the affinity-pharmacokinetic relationship. First, we report a number of novel Fc point mutations and combination variants, including some with primate-specific FcRn-binding improvements. By studying these variants along with some previously described variants across a wide range of affinities, we discovered a direct correlation of pH 6 affinity improvements with neutral pH improvements, suggesting that all of the tested variants exhibit similar pH dependency in FcRn binding. We then evaluated the pharmacokinetics of variants N434A and N434W, which, respectively, gave approximately 4- and 80-fold improvements in pH 6-binding affinity to both human and nonhuman primate FcRn. Surprisingly, clearance of N434W was similar to that of wild type. N434W is the first variant studied in primates that exhibits significant binding to FcRn at pH 7.4, and its clearance substantiates the principle that too much affinity improvement, i.e., beyond that of N434W, does not yield improved pharmacokinetics. In contrast, N434A exhibited a approximately 2-fold decrease in clearance in cynomolgus monkey, supporting the notion that modest increases in pH 6 FcRn affinity can result in improved pharmacokinetics in primates.

}, keywords = {Amino Acid Sequence, Animals, Antibody Affinity, Fc, Histocompatibility Antigens Class I, Humans, Hydrogen-Ion Concentration, Immunoglobulin G, Macaca fascicularis, Mice, Models, Molecular, Mutation, Protein Structure, Quaternary, Receptors, Recombinant Proteins}, issn = {1550-6606}, doi = {10.4049/jimmunol.0804182}, author = {Yeung, Yik Andy and Leabman, Maya K and Marvin, Jonathan S and Qiu, Julia and Adams, Camellia W and Lien, Samantha and Starovasnik, Melissa A and Lowman, Henry B} } @article {1564, title = {Modulating protein interactions by rational and computational design.}, journal = {Protein Engineering and Design}, year = {2009}, month = {2009}, pages = {343-66}, author = {Marvin, Jonathan S and Looger, Loren L} } @article {1580, title = {Crystallization and preliminary x-ray characterization of the genetically encoded fluorescent calcium indicator protein GCaMP2.}, journal = {Acta Crystallographica. Section F, Structural Biology and Crystallization Communications}, volume = {64}, year = {2008}, month = {2008 Jul 1}, pages = {629-31}, abstract = {

Fluorescent proteins and their engineered variants have played an important role in the study of biology. The genetically encoded calcium-indicator protein GCaMP2 comprises a circularly permuted fluorescent protein coupled to the calcium-binding protein calmodulin and a calmodulin target peptide, M13, derived from the intracellular calmodulin target myosin light-chain kinase and has been used to image calcium transients in vivo. To aid rational efforts to engineer improved variants of GCaMP2, this protein was crystallized in the calcium-saturated form. X-ray diffraction data were collected to 2.0 A resolution. The crystals belong to space group C2, with unit-cell parameters a = 126.1.

}, keywords = {Amino Acid Sequence, Calcium-Calmodulin-Dependent Protein Kinase Type 2, Crystallization, Green Fluorescent Proteins, Indicators and Reagents, Molecular Sequence Data, X-Ray Diffraction}, issn = {1744-3091}, doi = {10.1107/S1744309108016059}, author = {Rodr{\'\i}guez Guilbe, Mar{\'\i}a M and Alfaro Malav{\'e}, Elisa C and Akerboom, Jasper and Marvin, Jonathan S and Looger, Loren L and Schreiter, Eric R} } @article {1557, title = {Bispecific antibodies for dual-modality cancer therapy: killing two signaling cascades with one stone.}, journal = {Current Opinion in Drug Discovery \& Development}, volume = {9}, year = {2006}, month = {2006 Mar}, pages = {184-93}, abstract = {

The additive and synergistic therapeutic effects derived from combinations of chemotherapeutic drugs and radiation have established an indispensable paradigm: cancer must be attacked on multiple fronts. However, the increased antitumor efficacy of such combinational regimens is also associated with severe systemic toxicity, as these drugs cannot selectively target tumor cells. Monoclonal antibodies (mAbs), which have exquisite specificity for their antigens, are becoming an increasingly important class of antitumor agents, as they enhance the efficacy of various therapeutic regimens without significantly increasing systemic toxicity. Furthermore, preclinical and early clinical evidence indicate that combinations of antibody-based drugs provide even greater efficacy with minimal side effects. Unfortunately, the research, manufacturing and regulatory costs of mAb development pose a significant barrier to the use of antibody-based combination therapies. An emerging alternative is the use of dual-targeting bispecific antibodies (BsAbs). BsAbs are derived from the recombination of variable domains of two antibodies with different specificities; BsAbs are thus capable of binding both antigens of their parental antibodies. With the recent progress that has been made in antibody engineering technology, BsAbs that simultaneously target two tumor-associated molecules (eg, growth factor receptors) are being heralded for their potential to deliver two therapeutic moieties in a single molecule.

}, keywords = {Animals, Antibodies, Antibody Specificity, Antineoplastic Agents, Combined Modality Therapy, Humans, Immunotherapy, Neoplasms, Signal Transduction}, issn = {1367-6733}, author = {Marvin, Jonathan S and Zhu, Zhenping} } @article {1566, title = {Antibody humanization and affinity maturation using phage display.}, journal = {Phage Display in Biotechnology and Drug Discovery}, year = {2005}, month = {2005}, pages = {493-528}, author = {Marvin, Jonathan S and Lowman, Henry B} } @article {1565, title = {Computation-based engineering of protein and antibody therapeutics.}, journal = {Drug Design Reviews Online}, volume = {2}, year = {2005}, month = {2005}, pages = {419-25}, abstract = {

Recent advances in computation-based protein engineering offer opportunities to introduce or modify the biophysical characteristics of proteins at will. The power of computational design comes from the ability to surpass the combinatorial and physical limitations inherent to laboratory-based high-throughput or trial-and-error methods. As a result, modifications that require significant changes to the amino acid sequence of a protein are now accessible to the protein engineering community. Hydrophobic cores of proteins have been repacked to increase their thermostability. Binding sites in proteins have been modified to increase affinity or alter specificity for proteins, peptides, and small molecules. Enzymes have been designed de novo. Non-natural protein folds have been created. For the most part, these achievements have been applied to proteins that make good model systems in academic settings. How can these computational methods be applied to therapeutically relevant proteins? This review will focus on the ground-breaking achievements of computation-based protein engineering and on recent applications of rational design to improve therapeutic proteins. - See more at: http://www.eurekaselect.com/90585/article/computation-based-design-and-engineering-protein-and-antibody-therapeutics$\#$sthash.kSt3UaNE.dpuf

}, author = {Marvin, Jonathan S and Zhu, Zhenping} } @article {1558, title = {Recombinant approaches to IgG-like bispecific antibodies.}, journal = {Acta Pharmacologica Sinica}, volume = {26}, year = {2005}, month = {2005 Jun}, pages = {649-58}, abstract = {

One of the major obstacles in the development of bispecific antibodies (BsAb) has been the difficulty of producing the materials in sufficient quality and quantity by traditional technologies, such as the hybrid hybridoma and chemical conjugation methods. In contrast to the rapid and significant progress in the development of recombinant BsAb fragments (such as diabody and tandem single chain Fv), the successful design and production of full length IgG-like BsAb has been limited. Compared to smaller fragments, IgG-like BsAb have long serum half-life and are capable of supporting secondary immune functions, such as antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity. The development of IgG-like BsAb as therapeutic agents will depend heavily on our research progress in the design of recombinant BsAb constructs (or formats) and production efficiency. This review will focus on recent advances in various recombinant approaches to the engineering and production of IgG-like BsAb.

}, keywords = {Animals, Antibodies, Bispecific, Cytotoxicity, Humans, Immunoglobulin Fc Fragments, Immunoglobulin Fragments, Immunoglobulin G, Immunoglobulin Variable Region, Immunologic, Recombinant Fusion Proteins}, issn = {1671-4083}, doi = {10.1111/j.1745-7254.2005.00119.x}, author = {Marvin, Jonathan S and Zhu, Zhenping} } @article {1559, title = {Redesigning an antibody fragment for faster association with its antigen.}, journal = {Biochemistry}, volume = {42}, year = {2003}, month = {2003 Jun 17}, pages = {7077-83}, abstract = {

Traditional approaches for increasing the affinity of protein-protein complexes focus on constructing highly complementary binding surfaces. Recent theoretical simulations and experimental results suggest that electrostatic steering forces can also be manipulated to increase association rates while leaving dissociation rates unchanged, thus increasing affinity. Here we demonstrate that electrostatic attraction can be enhanced between an antibody fragment and its cognate antigen through application of a few simple rules to identify potential on-rate amplification sites that lie at the periphery of the antigen-antibody interface.

}, keywords = {Amino Acid Substitution, Antibody Affinity, Antigen-Antibody Complex, Antigen-Antibody Reactions, Antigens, Computer Simulation, Drug Design, Endothelial Growth Factors, Fluorescence, Humans, Immunoglobulin Fragments, Intercellular Signaling Peptides and Proteins, Kinetics, Lymphokines, Models, Molecular, Osmolar Concentration, Protein Binding, Recombinant Proteins, Spectrometry, Static Electricity, Thermodynamics, Vascular Endothelial Growth Factor A, Vascular Endothelial Growth Factors}, issn = {0006-2960}, doi = {10.1021/bi026947q}, author = {Marvin, Jonathan S and Lowman, Henry B} } @article {1567, title = {Introduction of fluorescent allosteric signal transduction elements into glucose-binding protein: construction of a novel glucose sensor.}, journal = {Journal of the American Chemical Society}, year = {1998}, month = {1998}, pages = {7-11}, author = {Marvin, Jonathan S and Hellinga, H W} }