@article {70003, title = {De novo designed bright, hyperstable rhodamine binders for fluorescence microscopy}, journal = {bioRxiv}, year = {2025}, month = {2025 Jun 25}, abstract = {

De novo protein design has emerged as a powerful strategy with the promise to create new tools. The practical performance of designed fluorophore binders, however, has remained far from meeting fluorescence microscopy demands. Here, we design de novo Rhodamine Binder (Rhobin) tags that combine ideal properties including size, brightness, and now adding hyperstability. Rhobin allows live and fixed cell imaging of a wide range of subcellular targets in mammalian cells. Its reversible fluorophore binding further enables live super-resolution STED microscopy with low photobleaching, as well as PAINT-type single-molecule localization microscopy. We showcase Rhobin in the extremophile Sulfolobus acidocaldarius living at 75 degrees Celsius, an application previously inaccessible by existing tags. Rhobin will serve as the basis for a new class of live cell fluorescent tags and biosensors.

}, doi = {10.1101/2025.06.24.661379}, url = {https://www.biorxiv.org/content/early/2025/06/25/2025.06.24.661379}, author = {Chen, Yuda and Yserentant, Klaus and Hong, Kibeom and Kuang, Yiming and Bhowmick, Arghya and Charles-Orszag, Arthur and Lord, Samuel J. and Lu, Lei and Hou, Kaipeng and Mann, Samuel I and Grimm, Jonathan B and Lavis, Luke D and Mullins, R. Dyche and DeGrado, William F and Huang, Bo} } @article {69841, title = {DELTA: a method for brain-wide measurement of synaptic protein turnover reveals localized plasticity during learning.}, journal = {Nat Neurosci}, year = {2025}, month = {2025 Mar 31}, abstract = {

Synaptic plasticity alters neuronal connections in response to experience, which is thought to underlie learning and memory. However, the loci of learning-related synaptic plasticity, and the degree to which plasticity is localized or distributed, remain largely unknown. Here we describe a new method, DELTA, for mapping brain-wide changes in synaptic protein turnover with single-synapse resolution, based on Janelia Fluor dyes and HaloTag knock-in mice. During associative learning, the turnover of the ionotropic glutamate receptor subunit GluA2, an indicator of synaptic plasticity, was enhanced in several brain regions, most markedly hippocampal area CA1. More broadly distributed increases in the turnover of synaptic proteins were observed in response to environmental enrichment. In CA1, GluA2 stability was regulated in an input-specific manner, with more turnover in layers containing input from CA3 compared to entorhinal cortex. DELTA will facilitate exploration of the molecular and circuit basis of learning and memory and other forms of plasticity at scales ranging from single synapses to the entire brain.

}, issn = {1546-1726}, doi = {10.1038/s41593-025-01923-4}, url = {https://doi.org/10.1038/s41593-025-01923-4}, author = {Mohar, Boaz and Michel, Gabriela and Wang, Yi-Zhi and Hernandez, Veronica and Grimm, Jonathan B and Park, Jin-Yong and Patel, Ronak and Clarke, Morgan and Brown, Timothy A and Bergmann, Cornelius and Gebis, Kamil K and Wilen, Anika P and Liu, Bian and Johnson, Richard and Graves, Austin and Tchumatchenko, Tatjana and Savas, Jeffrey N and Fornasiero, Eugenio F and Huganir, Richard L and Tillberg, Paul W and Lavis, Luke D and Svoboda, Karel and Spruston, Nelson} } @article {69840, title = {EPSILON: a method for pulse-chase labeling to probe synaptic AMPAR exocytosis during memory formation.}, journal = {Nat Neurosci}, year = {2025}, month = {2025 Mar 31}, abstract = {

A tool to map changes in synaptic strength during a defined time window could provide powerful insights into the mechanisms of learning and memory. Here we developed a technique, Extracellular Protein Surface Labeling in Neurons (EPSILON), to map α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) exocytosis in vivo by sequential pulse-chase labeling of surface AMPARs with membrane-impermeable dyes. This approach yields synaptic-resolution maps of AMPAR exocytosis, a proxy for synaptic potentiation, in genetically targeted neurons during memory formation. In mice undergoing contextual fear conditioning, we investigated the relationship between synapse-level AMPAR exocytosis in CA1 pyramidal neurons and cell-level expression of the immediate early gene product cFos, a frequently used marker of engram neurons. We observed a strong correlation between AMPAR exocytosis and cFos expression, suggesting a synaptic mechanism for the association of cFos expression with memory engrams. The EPSILON technique is a useful tool for mapping synaptic plasticity and may be extended to investigate trafficking of other transmembrane proteins.

}, issn = {1546-1726}, doi = {10.1038/s41593-025-01922-5}, url = {https://doi.org/10.1038/s41593-025-01922-5}, author = {Kim, Doyeon and Park, Pojeong and Li, Xiuyuan and Wong-Campos, J David and Tian, He and Moult, Eric M and Grimm, Jonathan B and Lavis, Luke D and Cohen, Adam E} } @article {70169, title = {Optimizing multifunctional fluorescent ligands for intracellular labeling.}, journal = {Proc Natl Acad Sci U S A}, volume = {122}, year = {2025}, month = {2025 Nov 04}, pages = {e2510046122}, abstract = {

Enzyme-based self-labeling tags enable the covalent attachment of synthetic molecules to proteins inside living cells. A frontier of this field is designing cell-permeable multifunctional ligands that contain fluorophores in combination with affinity tags or pharmacological agents. This is challenging since attachment of additional chemical moieties onto fluorescent ligands can adversely affect membrane permeability. To address this problem, we examined the chemical properties of rhodamine-based self-labeling tag ligands through the lens of medicinal chemistry. We found that the lactone-zwitterion equilibrium constant () of rhodamines inversely correlates with their distribution coefficients (log), suggesting that ligands based on dyes exhibiting low and high log values, such as Si-rhodamines, would efficiently enter cells. We designed cell-permeable multifunctional HaloTag ligands with a biotin moiety to purify mitochondria or a JQ1 appendage to translocate BRD4 within the nucleus. We found that translocation of BRD4 to constitutive heterochromatin in cells leads to apparent increases in transcriptional activity. These fluorescent reagents enable affinity capture and translocation of intracellular proteins in living cells, and our general design concepts will facilitate the design of multifunctional chemical tools for biology.

Preprint:\ https://doi.org/10.1101/2022.07.02.498544
Preprint:\ https://doi.org/10.32388/0xcyuc

}, keywords = {Bromodomain Containing Proteins, Cell Cycle Proteins, Fluorescent Dyes, Humans, Ligands, Mitochondria, Nuclear Proteins, Rhodamines, Staining and Labeling, Transcription Factors}, issn = {1091-6490}, doi = {10.1073/pnas.2510046122}, url = {https://doi.org/10.1073/pnas.2510046122}, author = {Kumar, Pratik and Vevea, Jason D and Tkachuk, Ariana N and Campbell, Kirby R and Watson, Emma T and Ayala, Anthony X and Grimm, Jonathan B and Chapman, Edwin R and Solecki, David J and Lavis, Luke D} } @article {69622, title = {A modular chemigenetic calcium indicator for multiplexed in vivo functional imaging.}, journal = {Nat Methods}, year = {2024}, month = {2024 Sep 20}, abstract = {

Genetically encoded fluorescent calcium indicators allow cellular-resolution recording of physiology. However, bright, genetically targetable indicators that can be multiplexed with existing tools in vivo are needed for simultaneous imaging of multiple signals. Here we describe WHaloCaMP, a modular chemigenetic calcium indicator built from bright dye-ligands and protein sensor domains. Fluorescence change in WHaloCaMP results from reversible quenching of the bound dye via a strategically placed tryptophan. WHaloCaMP is compatible with rhodamine dye-ligands that fluoresce from green to near-infrared, including several that efficiently label the brain in animals. When bound to a near-infrared dye-ligand, WHaloCaMP shows a 7{\texttimes} increase in fluorescence intensity and a 2.1-ns increase in fluorescence lifetime upon calcium binding. We use WHaloCaMP1a to image Ca responses in vivo in flies and mice, to perform three-color multiplexed functional imaging of hundreds of neurons and astrocytes in zebrafish larvae and to quantify Ca concentration using fluorescence lifetime imaging microscopy (FLIM).

}, issn = {1548-7105}, doi = {10.1038/s41592-024-02411-6}, url = {https://doi.org/10.1038/s41592-024-02411-6}, author = {Farrants, Helen and Shuai, Yichun and Lemon, William C and Monroy Hernandez, Christian and Zhang, Deng and Yang, Shang and Patel, Ronak and Qiao, Guanda and Frei, Michelle S and Plutkis, Sarah E and Grimm, Jonathan B and Hanson, Timothy L and Tomaska, Filip and Turner, Glenn C and Stringer, Carsen and Keller, Philipp J and Beyene, Abraham G and Chen, Yao and Liang, Yajie and Lavis, Luke D and Schreiter, Eric R} } @article {68561, title = {Time-tagged ticker tapes for intracellular recordings.}, journal = {Nature Biotechnology}, volume = {41}, year = {2023}, month = {2023 May 01}, pages = {631-9}, abstract = {

Recording transcriptional histories of a cell would enable deeper understanding of cellular developmental trajectories and responses to external perturbations. Here we describe an engineered protein fiber that incorporates diverse fluorescent marks during its growth to store a ticker tape-like history. An embedded HaloTag reporter incorporates user-supplied dyes, leading to colored stripes that map the growth of each individual fiber to wall clock time. A co-expressed eGFP tag driven by a promoter of interest records a history of transcriptional activation. High-resolution multi-spectral imaging on fixed samples reads the cellular histories, and interpolation of eGFP marks relative to HaloTag timestamps provides accurate absolute timing. We demonstrate recordings of doxycycline-induced transcription in HEK cells and cFos promoter activation in cultured neurons, with a single-cell absolute accuracy of 30-40 minutes over a 12-hour recording. The protein-based ticker tape design we present here could be generalized to achieve massively parallel single-cell recordings of diverse physiological modalities.

}, issn = {1546-1696}, doi = {10.1038/s41587-022-01524-7}, author = {Lin, Dingchang and Li, Xiuyuan and Moult, Eric and Park, Pojeong and Tang, Benjamin and Shen, Hao and Grimm, Jonathan B and Falco, Natalie and Jia, Bill Z and Baker, David and Lavis, Luke D and Cohen, Adam E} } @article {67846, title = {Caveat fluorophore: an insiders{\textquoteright} guide to small-molecule fluorescent labels.}, journal = {Nature Methods}, volume = {19}, year = {2022}, month = {2022 Feb 01}, pages = {149-58}, abstract = {

The last three decades have brought a revolution in fluorescence microscopy. The development of new microscopes, fluorescent labels and analysis techniques has pushed the frontiers of biological imaging forward, moving from fixed to live cells, from diffraction-limited to super-resolution imaging and from simple cell culture systems to experiments in vivo. The large and ever-evolving collection of tools can be daunting for biologists, who must invest substantial time and effort in adopting new technologies to answer their specific questions. This is particularly relevant when working with small-molecule fluorescent labels, where users must navigate the jargon, idiosyncrasies and caveats of chemistry. Here, we present an overview of chemical dyes used in biology and provide frank advice from a chemist{\textquoteright}s perspective.

}, issn = {1548-7105}, doi = {10.1038/s41592-021-01338-6}, author = {Grimm, Jonathan B and Lavis, Luke D} } @article {67752, title = {Biosensors based on peptide exposure show single molecule conformations in live cells.}, journal = {Cell}, volume = {184}, year = {2021}, month = {2021 Oct 28}, pages = {5670-5685}, abstract = {

We describe an approach to study the conformation of individual proteins during single particle tracking (SPT) in living cells. "Binder/tag" is based on incorporation of a 7-mer peptide (the tag) into a protein where its solvent exposure is controlled by protein conformation. Only upon exposure can the peptide specifically interact with a reporter protein (the binder). Thus, simple fluorescence localization reflects protein conformation. Through direct excitation of bright dyes, the trajectory and conformation of individual proteins can be followed. Simple protein engineering provides highly specific biosensors suitable for SPT and FRET. We describe tagSrc, tagFyn, tagSyk, tagFAK, and an orthogonal binder/tag pair. SPT showed slowly diffusing islands of activated Src within Src clusters and dynamics of activation in adhesions. Quantitative analysis and stochastic modeling revealed in\ vivo Src kinetics. The simplicity of binder/tag can provide access to diverse proteins.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2021.09.026}, author = {Liu, Bei and Stone, Orrin J and Pablo, Michael and Herron, J Cody and Nogueira, Ana T and Dagliyan, Onur and Grimm, Jonathan B and Lavis, Luke D and Elston, Timothy C and Hahn, Klaus M} } @article {67633, title = {Enabling In Vivo Photocatalytic Activation of Rapid Bioorthogonal Chemistry by Repurposing Silicon-Rhodamine Fluorophores as Cytocompatible Far-Red Photocatalysts.}, journal = {Journal of the American Chemical Society}, volume = {143}, year = {2021}, month = {2021 Jul 21}, pages = {10793-10803}, abstract = {

Chromophores that absorb in the tissue-penetrant far-red/near-infrared window have long served as photocatalysts to generate singlet oxygen for photodynamic therapy. However, the cytotoxicity and side reactions associated with singlet oxygen sensitization have posed a problem for using long-wavelength photocatalysis to initiate other types of chemical reactions in biological environments. Herein, silicon-Rhodamine compounds (SiRs) are described as photocatalysts for inducing rapid bioorthogonal chemistry using 660 nm light through the oxidation of a dihydrotetrazine to a tetrazine in the presence of cyclooctene dienophiles. SiRs have been commonly used as fluorophores for bioimaging but have not been applied to catalyze chemical reactions. A series of SiR derivatives were evaluated, and the Janelia Fluor-SiR dyes were found to be especially effective in catalyzing photooxidation (typically 3\%). A dihydrotetrazine/tetrazine pair is described that displays high stability in both oxidation states. A protein that was site-selectively modified by cyclooctene was quantitatively conjugated upon exposure to 660 nm light and a dihydrotetrazine. By contrast, a previously described methylene blue catalyst was found to rapidly degrade the protein. SiR-red light photocatalysis was used to cross-link hyaluronic acid derivatives functionalized by dihydrotetrazine and cyclooctenes, enabling 3D culture of human prostate cancer cells. Photoinducible hydrogel formation could also be carried out in live mice through subcutaneous injection of a Cy7-labeled hydrogel precursor solution, followed by brief irradiation to produce a stable hydrogel. This cytocompatible method for using red light photocatalysis to activate bioorthogonal chemistry is anticipated to find broad applications where spatiotemporal control is needed in biological environments.

}, issn = {1520-5126}, doi = {10.1021/jacs.1c05547}, author = {Wang, Chuanqi and Zhang, He and Zhang, Tao and Zou, Xiaoyu and Wang, Hui and Rosenberger, Julia E and Vannam, Raghu and Trout, William S and Grimm, Jonathan B and Lavis, Luke D and Thorpe, Colin and Jia, Xinqiao and Li, Zibo and Fox, Joseph M} } @article {65667, title = {A general method to optimize and functionalize red-shifted rhodamine dyes.}, journal = {Nature Methods}, year = {2020}, month = {2020 Jul 27}, abstract = {

Expanding the palette of fluorescent dyes is vital to push the frontier of biological imaging. Although rhodamine dyes remain the premier type of small-molecule fluorophore owing to their bioavailability and brightness, variants excited with far-red or near-infrared light suffer from poor performance due to their propensity to adopt a lipophilic, nonfluorescent form. We report a framework for rationalizing rhodamine behavior in biological environments and a general chemical modification for rhodamines that optimizes long-wavelength variants and enables facile functionalization with different chemical groups. This strategy yields red-shifted {\textquoteright}Janelia Fluor{\textquoteright} (JF) dyes useful for biological imaging experiments in cells and in vivo.

}, issn = {1548-7105}, doi = {10.1038/s41592-020-0909-6}, author = {Grimm, Jonathan B and Tkachuk, Ariana N and Xie, Liangqi and Choi, Heejun and Mohar, Boaz and Falco, Natalie and Schaefer, Kathy and Patel, Ronak and Zheng, Qinsi and Liu, Zhe and Lippincott-Schwartz, Jennifer and Brown, Timothy A and Lavis, Luke D} } @article {65262, title = {Nanoscale subcellular architecture revealed by multicolor three-dimensional salvaged fluorescence imaging.}, journal = {Nature Methods}, volume = {17}, year = {2020}, month = {2020 Jan 06}, pages = {225-231}, abstract = {

Combining the molecular specificity of fluorescent probes with three-dimensional imaging at nanoscale resolution is critical for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a 4Pi single-molecule switching super-resolution microscope that enables ratiometric multicolor imaging of mammalian cells at 5-10-nm localization precision in three dimensions using {\textquoteright}salvaged fluorescence{\textquoteright}. Imaging two or three fluorophores simultaneously, we show fluorescence images that resolve the highly convoluted Golgi apparatus and the close contacts between the endoplasmic reticulum and the plasma membrane, structures that have traditionally been the imaging realm of electron microscopy. The salvaged fluorescence approach is equally applicable in most single-objective microscopes.

}, issn = {1548-7105}, doi = {10.1038/s41592-019-0676-4}, author = {Zhang, Yongdeng and Schroeder, Lena K and Lessard, Mark D and Kidd, Phylicia and Chung, Jeeyun and Song, Yuanbin and Benedetti, Lorena and Li, Yiming and Ries, Jonas and Grimm, Jonathan B and Lavis, Luke D and De Camilli, Pietro and Rothman, James E and Baddeley, David and Bewersdorf, Joerg} } @article {65724, title = {Novel fluorescent ligands enable single-molecule localization microscopy of the dopamine transporter.}, journal = {ACS Chemical Neuroscience}, year = {2020}, month = {2020 Sep 14}, abstract = {

The dopamine transporter (DAT) is critical for spatiotemporal control of dopaminergic neurotransmission and the target for therapeutic agents, including ADHD medications, and abused substances, such as cocaine. Here, we develop new fluorescently labeled ligands that bind DAT with high affinity and enable single-molecule detection of the transporter. The cocaine analogue MFZ2-12 (1) was conjugated to novel rhodamine-based Janelia Fluorophores (JF549 and JF646). High affinity binding of the resulting ligands to DAT was demonstrated by potent inhibition of [3H]dopamine uptake in DAT transfected CAD cells and by competition radioligand binding experiments on rat striatal membranes. Visualization of binding was substantiated by confocal or TIRF microscopy revealing selective binding of the analogues to DAT transfected CAD cells. Single particle tracking experiments were performed with JF549-conjugated DG3-80 (3) and JF646-conjugated DG4-91 (4) on DAT transfected CAD cells enabling quantification and categorization of the dynamic behavior of DAT into four distinct motion classes (immobile, confined, Brownian, and directed). Finally, we show that the ligands can be used in direct stochastic optical reconstruction microscopy (dSTORM) experiments permitting further analyses of DAT distribution on the nanoscale. In summary, these novel fluorescent ligands are promising new tools for studying DAT localization and regulation with single-molecule resolution.

}, issn = {1948-7193}, doi = {10.1021/acschemneuro.0c00397}, author = {Guthrie, Daryl and Klein Herenbrink, Carmen and Lycas, Matthew and Ku, Therese and Bonifazi, Alessandro and DeVree, Brian and Mathiasen, Signe and Javitch, Jonathan and Grimm, Jonathan B and Lavis, Luke D and Gether, Ulrik and Newman, Amy Hauck} } @article {65668, title = {Rational design of bioavailable photosensitizers for manipulation and imaging of biological systems.}, journal = {Cell Chemical Biology}, volume = {27}, year = {2020}, month = {2020 Aug 20}, pages = {1063-72}, abstract = {

Light-mediated chemical reactions are powerful methods for manipulating and interrogating biological systems. Photosensitizers, compounds that generate reactive oxygen species upon excitation with light, can be utilized for numerous biological experiments, but the repertoire of bioavailable photosensitizers is limited. Here, we describe the synthesis, characterization, and utility of two photosensitizers based upon the widely used rhodamine scaffold and demonstrate their efficacy for chromophore-assisted light inactivation, cell ablation in culture and in\ vivo, and photopolymerization of diaminobenzidine for electron microscopy. These chemical tools will facilitate a broad range of applications spanning from targeted destruction of proteins to high-resolution imaging.

}, issn = {2451-9448}, doi = {10.1016/j.chembiol.2020.07.001}, author = {Binns, Thomas C and Ayala, Anthony X and Grimm, Jonathan B and Tkachuk, Ariana N and Castillon, Guillaume A and Phan, Sebastien and Zhang, Lixia and Brown, Timothy A and Liu, Zhe and Adams, Stephen R and Ellisman, Mark H and Koyama, Minoru and Lavis, Luke D} } @article {65081, title = {Bright and photostable chemigenetic indicators for extended in vivo voltage imaging.}, journal = {Science}, volume = {365}, year = {2019}, month = {2019 Aug 13}, pages = {699-704}, abstract = {

Imaging changes in membrane potential using genetically encoded fluorescent voltage indicators (GEVIs) has great potential for monitoring neuronal activity with high spatial and temporal resolution. Brightness and photostability of fluorescent proteins and rhodopsins have limited the utility of existing GEVIs. We engineered a novel GEVI, "Voltron", that utilizes bright and photostable synthetic dyes instead of protein-based fluorophores, extending the combined duration of imaging and number of neurons imaged simultaneously by more than tenfold relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously, over 15 min of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.

}, issn = {1095-9203}, doi = {10.1126/science.aav6416}, author = {Abdelfattah, Ahmed S and Kawashima, Takashi and Singh, Amrita and Novak, Ondrej and Liu, Hui and Shuai, Yichun and Huang, Yi-Chieh and Campagnola, Luke and Seeman, Stephanie C and Yu, Jianing and Zheng, Jihong and Grimm, Jonathan B and Patel, Ronak and Friedrich, Johannes and Mensh, Brett D and Paninski, Liam and Macklin, John J and Murphy, Gabe J and Podgorski, Kaspar and Lin, Bei-Jung and Chen, Tsai-Wen and Turner, Glenn C and Liu, Zhe and Koyama, Minoru and Svoboda, Karel and Ahrens, Misha B and Lavis, Luke D and Schreiter, Eric R} } @article {65176, title = {Rational design of fluorogenic and spontaneously blinking labels for super-resolution imaging.}, journal = {ACS Central Science}, volume = {5}, year = {2019}, month = {2019 Sep 25}, pages = {1602-1613}, abstract = {

Rhodamine dyes exist in equilibrium between a fluorescent zwitterion and a nonfluorescent lactone. Tuning this equilibrium toward the nonfluorescent lactone form can improve cell-permeability and allow creation of "fluorogenic" compounds-ligands that shift to the fluorescent zwitterion upon binding a biomolecular target. An archetype fluorogenic dye is the far-red tetramethyl-Si-rhodamine (SiR), which has been used to create exceptionally useful labels for advanced microscopy. Here, we develop a quantitative framework for the development of new fluorogenic dyes, determining that the lactone-zwitterion equilibrium constant () is sufficient to predict fluorogenicity. This rubric emerged from our analysis of known fluorophores and yielded new fluorescent and fluorogenic labels with improved performance in cellular imaging experiments. We then designed a novel fluorophore-Janelia Fluor 526 (JF)-with SiR-like properties but shorter fluorescence excitation and emission wavelengths. JF is a versatile scaffold for fluorogenic probes including ligands for self-labeling tags, stains for endogenous structures, and spontaneously blinking labels for super-resolution immunofluorescence. JF constitutes a new label for advanced microscopy experiments, and our quantitative framework will enable the rational design of other fluorogenic probes for bioimaging.

}, issn = {2374-7943}, doi = {10.1021/acscentsci.9b00676}, author = {Zheng, Qinsi and Ayala, Anthony X and Chung, Inhee and Weigel, Aubrey V and Ranjan, Anand and Falco, Natalie and Grimm, Jonathan B and Tkachuk, Ariana N and Wu, Carl and Lippincott-Schwartz, Jennifer and Singer, Robert H and Lavis, Luke D} } @article {49406, title = {Cell-specific chemical delivery using a selective Nitroreductase-Nitroaryl pair.}, journal = {ACS Chemical Biology}, volume = {13}, year = {2018}, month = {2018 Aug 29}, pages = {1888-96}, abstract = {

The utility of small molecules to probe or perturb biological systems is limited by the lack of cell-specificity. "Masking" the activity of small molecules using a general chemical modification and "unmasking" it only within target cells overcomes this limitation. To this end, we have developed a selective enzyme-substrate pair consisting of engineered variants of E. coli nitroreductase (NTR) and a 2-nitro- N-methylimidazolyl (NM) masking group. To discover and optimize this NTR-NM system, we synthesized a series of fluorogenic substrates containing different nitroaromatic masking groups, confirmed their stability in cells, and identified the best substrate for NTR. We then engineered the enzyme for improved activity in mammalian cells, ultimately yielding an enzyme variant (enhanced NTR, or eNTR) that possesses up to 100-fold increased activity over wild-type NTR. These improved NTR enzymes combined with the optimal NM masking group enable rapid, selective unmasking of dyes, indicators, and drugs to genetically defined populations of cells.

}, issn = {1554-8937}, doi = {10.1021/acschembio.8b00524}, author = {Gruber, Todd D and Krishnamurthy, Chithra and Grimm, Jonathan B and Tadross, Michael R and Wysocki, Laura M and Gartner, Zev J and Lavis, Luke D} } @article {49366, title = {Optimization of fluorophores for chemical tagging and immunohistochemistry of Drosophila neurons.}, journal = {PLoS One}, volume = {13}, year = {2018}, month = {2018 Aug 15}, pages = {e0200759}, abstract = {

The use of genetically encoded {\textquoteright}self-labeling tags{\textquoteright} with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved Drosophila melanogaster transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.

}, issn = {1932-6203}, doi = {10.1371/journal.pone.0200759}, author = {Meissner, Geoffrey W and Grimm, Jonathan B and Johnston, Rebecca M and Sutcliffe, Ben and Ng, Julian and Jefferis, Gregory S X E and Cachero, Sebastian and Lavis, Luke D and Malkesman, Oz} } @article {48997, title = {Robust model-based analysis of single-particle tracking experiments with Spot-On.}, journal = {eLife}, volume = {7}, year = {2018}, month = {2018 Jan 04}, abstract = {

Single-particle tracking (SPT) has become an important method to bridge biochemistry and cell biology since it allows direct observation of protein binding and diffusion dynamics in live cells. However, accurately inferring information from SPT studies is challenging due to biases in both data analysis and experimental design. To address analysis bias, we introduce {\textquoteright}Spot-On{\textquoteright}, an intuitive web-interface. Spot-On implements a kinetic modeling framework that accounts for known biases, including molecules moving out-of-focus, and robustly infers diffusion constants and subpopulations from pooled single-molecule trajectories. To minimize inherent experimental biases, we implement and validate stroboscopic photo-activation SPT (spaSPT), which minimizes motion-blur bias and tracking errors. We validate Spot-On using experimentally realistic simulations and show that Spot-On outperforms other methods. We then apply Spot-On to spaSPT data from live mammalian cells spanning a wide range of nuclear dynamics and demonstrate that Spot-On consistently and robustly infers subpopulation fractions and diffusion constants.

}, issn = {2050-084X}, doi = {10.7554/eLife.33125}, author = {Hansen, Anders S and Woringer, Maxime and Grimm, Jonathan B and Lavis, Luke D and Tjian, Robert and Darzacq, Xavier} } @article {49456, title = {A toolbox for multiplexed super-resolution imaging of the E. coli nucleoid and membrane using novel PAINT labels.}, journal = {Scientific Reports}, volume = {8}, year = {2018}, month = {2018 Oct 03}, pages = {14768}, abstract = {

Maintenance of the bacterial homeostasis initially emanates from interactions between proteins and the bacterial nucleoid. Investigating their spatial correlation requires high spatial resolution, especially in tiny, highly confined and crowded bacterial cells. Here, we present super-resolution microscopy using a palette of fluorescent labels that bind transiently to either the membrane or the nucleoid of fixed E. coli cells. The presented labels are easily applicable, versatile and allow long-term single-molecule super-resolution imaging independent of photobleaching. The different spectral properties allow for multiplexed imaging in combination with other localisation-based super-resolution imaging techniques. As examples for applications, we demonstrate correlated super-resolution imaging of the bacterial nucleoid with the position of genetic loci, of nascent DNA in correlation to the entire nucleoid, and of the nucleoid of metabolically arrested cells. We furthermore show that DNA- and membrane-targeting labels can be combined with photoactivatable fluorescent proteins and visualise the nano-scale distribution of RNA polymerase relative to the nucleoid in drug-treated E. coli cells.

}, issn = {2045-2322}, doi = {10.1038/s41598-018-33052-3}, author = {Spahn, Christoph K and Glaesmann, Mathilda and Grimm, Jonathan B and Ayala, Anthony X and Lavis, Luke D and Heilemann, Mike} } @article {49576, title = {Whole-cell, 3D and multi-color STED imaging with exchangeable fluorophores.}, journal = {Nano Letters}, volume = {19}, year = {2018}, month = {2018 Dec 10}, pages = {500-5}, abstract = {

We demonstrate STED microscopy of whole bacterial and eukaryotic cells using fluorogenic labels that reversibly bind to their target structure. A constant exchange of labels guarantees the removal of photobleached fluorophores and their replacement by intact fluorophores, thereby circumventing bleaching-related limitations of STED super-resolution imaging. We achieve a constant labeling density and demonstrate a fluorescence signal for long and theoretically unlimited acquisition times. Using this concept, we demonstrate whole-cell, 3D, multi-color and live cell STED microscopy.

}, issn = {1530-6992}, doi = {10.1021/acs.nanolett.8b04385}, author = {Spahn, Christoph and Grimm, Jonathan B and Lavis, Luke D and Lampe, Marko and Heilemann, Mike} } @article {48809, title = {Cohesin can remain associated with chromosomes during DNA replication.}, journal = {Cell Reports}, volume = {20}, year = {2017}, month = {2017 Sep 19}, pages = {2749-55}, abstract = {

To ensure disjunction to opposite poles during anaphase, sister chromatids must be held together following DNA replication. This is mediated by cohesin, which is thought to entrap sister DNAs inside a tripartite ring composed of its Smc and kleisin (Scc1) subunits. How such structures are created during S phase is poorly understood, in particular whether they are derived from complexes that had entrapped DNAs prior to replication. To address this, we used selective photobleaching to determine whether cohesin associated with chromatin in G1 persists in\ situ after replication. We developed a non-fluorescent HaloTag ligand to discriminate the fluorescence recovery signal from labeling of newly synthesized Halo-tagged Scc1 protein (pulse-chase or pcFRAP). In cells where cohesin turnover is inactivated by deletion of WAPL, Scc1 can remain associated with chromatin throughout S phase. These findings suggest that cohesion might be generated by cohesin that is already bound to un-replicated DNA.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2017.08.092}, author = {Rhodes, James D P and Haarhuis, Judith H I and Grimm, Jonathan B and Rowland, Benjamin D and Lavis, Luke D and Nasmyth, Kim A} } @article {48648, title = {Desensitized D2 autoreceptors are resistant to trafficking.}, journal = {Scientific Reports}, volume = {7}, year = {2017}, month = {2017 Jun 29}, pages = {4379}, abstract = {

Dendritic release of dopamine activates dopamine D2 autoreceptors, which are inhibitory G protein-coupled receptors (GPCRs), to decrease the excitability of dopamine neurons. This study used tagged D2 receptors to identify the localization and distribution of these receptors in living midbrain dopamine neurons. GFP-tagged D2 receptors were found to be unevenly clustered on the soma and dendrites of dopamine neurons within the substantia nigra pars compacta (SNc). Physiological signaling and desensitization of the tagged receptors were not different from wild type receptors. Unexpectedly, upon desensitization the tagged D2 receptors were not internalized. When tagged D2 receptors were expressed in locus coeruleus neurons, a desensitizing protocol induced significant internalization. Likewise, when tagged {\textmu}-opioid receptors were expressed in dopamine neurons they too were internalized. The distribution and lack of agonist-induced internalization of D2 receptors on dopamine neurons indicate a purposefully regulated localization of these receptors.

}, issn = {2045-2322}, doi = {10.1038/s41598-017-04728-z}, author = {Robinson, Brooks G and Bunzow, James R and Grimm, Jonathan B and Lavis, Luke D and Dudman, Joshua T and Brown, Jennifer and Neve, Kim A and Williams, John T} } @article {48891, title = {A general method to fine-tune fluorophores for live-cell and in vivo imaging.}, journal = {Nature Methods}, volume = {14}, year = {2017}, month = {2017 Oct}, pages = {987-994}, abstract = {

Pushing the frontier of fluorescence microscopy requires the design of enhanced fluorophores with finely tuned properties. We recently discovered that incorporation of four-membered azetidine rings into classic fluorophore structures elicits substantial increases in brightness and photostability, resulting in the Janelia Fluor (JF) series of dyes. We refined and extended this strategy, finding that incorporation of 3-substituted azetidine groups allows rational tuning of the spectral and chemical properties of rhodamine dyes with unprecedented precision. This strategy allowed us to establish principles for fine-tuning the properties of fluorophores and to develop a palette of new fluorescent and fluorogenic labels with excitation ranging from blue to the far-red. Our results demonstrate the versatility of these new dyes in cells, tissues and animals.

}, keywords = {Animals, Brain, Cell Line, Coloring Agents, Drosophila, Image Processing, Computer-Assisted, Larva, Mice, Microscopy, Fluorescence, Photochemical Processes, Staining and Labeling}, issn = {1548-7105}, doi = {10.1038/nmeth.4403}, author = {Grimm, Jonathan B and Muthusamy, Anand K and Liang, Yajie and Brown, Timothy A and Lemon, William C and Patel, Ronak and Lu, Rongwen and Macklin, John J and Keller, Philipp J and Ji, Na and Lavis, Luke D} } @article {48733, title = {General synthetic method for Si-Fluoresceins and Si-Rhodamines.}, journal = {ACS Central Science}, volume = {3}, year = {2017}, month = {2017 Aug 09}, pages = {975-85}, abstract = {

The century-old fluoresceins and rhodamines persist as flexible scaffolds for fluorescent and fluorogenic compounds. Extensive exploration of these xanthene dyes has yielded general structure{\textendash}activity relationships where the development of new probes is limited only by imagination and organic chemistry. In particular, replacement of the xanthene oxygen with silicon has resulted in new red-shifted Si-fluoresceins and Si-rhodamines, whose high brightness and photostability enable advanced imaging experiments. Nevertheless, efforts to tune the chemical and spectral properties of these dyes have been hindered by difficult synthetic routes. Here, we report a general strategy for the efficient preparation of Si-fluoresceins and Si-rhodamines from readily synthesized bis(2-bromophenyl)silane intermediates. These dibromides undergo metal/bromide exchange to give bis-aryllithium or bis(aryl Grignard) intermediates, which can then add to anhydride or ester electrophiles to afford a variety of Si-xanthenes. This strategy enabled efficient (3{\textendash}5 step) syntheses of known and novel Si-fluoresceins, Si-rhodamines, and related dye structures. In particular, we discovered that previously inaccessible tetrafluorination of the bottom aryl ring of the Si-rhodamines resulted in dyes with improved visible absorbance in solution, and a convenient derivatization through fluoride-thiol substitution. This modular, divergent synthetic method will expand the palette of accessible xanthenoid dyes across the visible spectrum, thereby pushing further the frontiers of biological imaging.

}, doi = {10.1021/acscentsci.7b00247}, author = {Grimm, Jonathan B and Brown, Timothy A and Tkachuk, Ariana N and Lavis, Luke D} } @article {48343, title = {Quantifying transcription factor binding dynamics at the single-molecule level in live cells.}, journal = {Methods (San Diego, Calif.)}, year = {2017}, month = {2017 Mar 15}, abstract = {

Progressive, technological achievements in the quantitative fluorescence microscopy field are allowing researches from many different areas to start unraveling the dynamic intricacies of biological processes inside living cells. From super-resolution microscopy techniques to tracking of individual proteins, fluorescence microscopy is changing our perspective on how the cell works. Fortunately, a growing number of research groups are exploring single-molecule studies in living cells. However, no clear consensus exists on several key aspects of the technique such as image acquisition conditions, or analysis of the obtained data. Here, we describe a detailed approach to perform single-molecule tracking (SMT) of transcription factors in living cells to obtain key binding characteristics, namely their residence time and bound fractions. We discuss different types of fluorophores, labeling density, microscope, cameras, data acquisition, and data analysis. Using the glucocorticoid receptor as a model transcription factor, we compared alternate tags (GFP, mEOS, HaloTag, SNAP-tag, CLIP-tag) for potential multicolor applications. We also examine different methods to extract the dissociation rates and compare them with simulated data. Finally, we discuss several challenges that this exciting technique still faces.

}, issn = {1095-9130}, doi = {10.1016/j.ymeth.2017.03.014}, author = {Presman, Diego M and Ball, David A and Paakinaho, Ville and Grimm, Jonathan B and Lavis, Luke D and Karpova, Tatiana S and Hager, Gordon L} } @article {48938, title = {Semisynthetic fluorescent pH sensors for imaging exocytosis and endocytosis.}, journal = {Nature Communications}, volume = {8}, year = {2017}, month = {2017 Nov 10}, pages = {1412}, abstract = {

The GFP-based superecliptic pHluorin (SEP) enables detection of exocytosis and endocytosis, but its performance has not been duplicated in red fluorescent protein scaffolds. Here we describe "semisynthetic" pH-sensitive protein conjugates with organic fluorophores, carbofluorescein, and Virginia Orange that match the properties of SEP. Conjugation to genetically encoded self-labeling tags or antibodies allows visualization of both exocytosis and endocytosis, constituting new bright sensors for these key steps of synaptic transmission.

}, issn = {2041-1723}, doi = {10.1038/s41467-017-01752-5}, author = {Martineau, Magalie and Somasundaram, Agila and Grimm, Jonathan B and Gruber, Todd D and Choquet, Daniel and Taraska, Justin W and Lavis, Luke D and Perrais, David} } @article {48346, title = {Stochastic protein labeling enables long-term single molecule observation in vivo.}, journal = {bioRxiv}, year = {2017}, month = {2017 Mar 13}, abstract = {Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control protein copy number in a cell. This system has a dynamic titration range of more than 10,000 fold, enabling sparse labeling of proteins expressed at widely different levels. Combined with fluorescence signal amplification tags, this system extends the duration of automated single-molecule tracking by 2 orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in live zebrafish. We found that axon initial segment utilizes a waterfall mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor Sox2 samples clustered binding sites in spatially-restricted sub-nuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements for a quantitative understanding of complex control of molecular dynamics in vivo.}, doi = {10.1101/116186}, author = {Liu, Hui and Dong, Peng and Ioannou, Maria S and Li, Li and Shea, Jamien and Pasolli, H Amalia and Grimm, Jonathan B and Rivlin, Patricia K and Lavis, Luke D and Koyama, Minoru and Liu, Zhe} } @article {48804, title = {Synthesis of Janelia Fluor HaloTag and SNAP-Tag Ligands and Their Use in Cellular Imaging Experiments.}, journal = {Methods in Molecular Biology (Clifton, N.J.)}, volume = {1663}, year = {2017}, month = {2017}, pages = {179-188}, abstract = {

The development of genetically encoded self-labeling protein tags such as the HaloTag and SNAP-tag has expanded the utility of chemical dyes in microscopy. Intracellular labeling using these systems requires small, cell-permeable dyes with high brightness and photostability. We recently discovered a general method to improve the properties of classic fluorophores by replacing N,N-dimethylamino groups with four-membered azetidine rings to create the "Janelia Fluor" dyes. Here, we describe the synthesis of the HaloTag and SNAP-tag ligands of Janelia Fluor 549 and Janelia Fluor 646 as well as standard labeling protocols for use in ensemble and single-molecule cellular imaging.

}, issn = {1940-6029}, doi = {10.1007/978-1-4939-7265-4_15}, author = {Grimm, Jonathan B and Brown, Timothy A and English, Brian P and Lionnet, Timoth{\'e}e and Lavis, Luke D} } @article {48752, title = {Visualizing long-term single-molecule dynamics in vivo by stochastic protein labeling.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {115}, year = {2017}, month = {2017 Jan 09}, pages = {343-8}, abstract = {

Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control copy number of fluorescently labeled molecules in a cell. This system has a dynamic titration range of \>10,000 fold, enabling sparse labeling of proteins expressed at different abundance levels. Combined with photostable labels, this system extends the duration of automated single-molecule tracking by 2 orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in intact\ zebrafish. We found axon initial segment utilizes a "waterfall" mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor hops between clustered binding sites in spatially-restricted sub-nuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements, enabling new experiments to quantitatively understand complex control of molecular dynamics\ in vivo.

}, doi = {10.1073/pnas.1713895115}, author = {Liu, Hui and Dong, Peng and Ioannou, Maria S and Li, Li and Shea, Jamien and Pasolli, H Amalia and Grimm, Jonathan B and Rivlin, Patricia K and Lavis, Luke D and Koyama, Minoru and Liu, Zhe} } @article {47729, title = {Bright photoactivatable fluorophores for single-molecule imaging.}, journal = {Nature Methods}, volume = {13}, year = {2016}, month = {2016 Oct 24}, pages = {985-8}, abstract = {

Small molecule fluorophores are important tools for advanced imaging experiments. The development of self-labeling protein tags such as the HaloTag and SNAP-tag has expanded the utility of chemical dyes in live-cell microscopy. We recently described a general method for improving the brightness and photostability of small, cell-permeable fluorophores, resulting in the novel azetidine-containing "Janelia Fluor" (JF) dyes. Here, we refine and extend the utility of the JF dyes by synthesizing photoactivatable derivatives that are compatible with live cell labeling strategies. These compounds retain the superior brightness of the JF dyes once activated, but their facile photoactivation also enables improved single-particle tracking and localization microscopy experiments.

}, doi = {10.1038/nmeth.4034}, author = {Lavis, Luke D and Grimm, Jonathan B and English, Brian P and Choi, Heejin and Muthusamy, Anand K and Mehl, Brian P and Dong, Peng and Brown, Timothy A and Lippincott-Schwartz, Jennifer and Liu, Zhe and Lionnet, Timoth{\'e}e} } @article {47508, title = {Design and synthesis of a calcium-sensitive photocage.}, journal = {Angewandte Chemie (International ed. in English)}, year = {2016}, month = {2016 May 24}, abstract = {

Photolabile protecting groups (or "photocages") enable precise spatiotemporal control of chemical functionality and facilitate advanced biological experiments. Extant photocages exhibit a simple input-output relationship, however, where application of light elicits a photochemical reaction irrespective of the environment. Herein, we refine and extend the concept of photolabile groups, synthesizing the first Ca(2+) -sensitive photocage. This system functions as a chemical coincidence detector, releasing small molecules only in the presence of both light and elevated [Ca(2+) ]. Caging a fluorophore with this ion-sensitive moiety yields an "ion integrator" that permanently marks cells undergoing high Ca(2+) flux during an illumination-defined time period. Our general design concept demonstrates a new class of light-sensitive material for cellular imaging, sensing, and targeted molecular delivery.

}, issn = {1521-3773}, doi = {10.1002/anie.201602941}, author = {Heckman, Laurel M and Grimm, Jonathan B and Schreiter, Eric R and Kim, Charles and Verdecia, Mark A and Shields, Brenda C and Lavis, Luke D} } @article {47967, title = {Glutamate-induced RNA localization and translation in neurons.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, year = {2016}, month = {2016 Sep 13}, abstract = {

Localization of mRNA is required for protein synthesis to occur within discrete intracellular compartments. Neurons represent an ideal system for studying the precision of mRNA trafficking because of their polarized structure and the need for synapse-specific targeting. To investigate this targeting, we derived a quantitative and analytical approach. Dendritic spines were stimulated by glutamate uncaging at a diffraction-limited spot, and the localization of single β-actin mRNAs was measured in space and time. Localization required NMDA receptor activity, a dynamic actin cytoskeleton, and the transacting RNA-binding protein, Zipcode-binding protein 1 (ZBP1). The ability of the mRNA to direct newly synthesized proteins to the site of localization was evaluated using a Halo-actin reporter so that RNA and protein were detected simultaneously. Newly synthesized Halo-actin was enriched at the site of stimulation, required NMDA receptor activity, and localized preferentially at the periphery of spines. This work demonstrates that synaptic activity can induce mRNA localization and local translation of β-actin where the new actin participates in stabilizing the expanding synapse in dendritic spines.

}, doi = {10.1073/pnas.1614267113}, author = {Yoon, Young J and Wu, Bin and Buxbaum, Adina R and Das, Sulagna and Tsai, Albert and English, Brian P and Grimm, Jonathan B and Lavis, Luke D and Singer, Robert H} } @article {47374, title = {High-density three-dimensional localization microscopy across large volumes.}, journal = {Nature Methods}, year = {2016}, month = {2016 Mar 7}, abstract = {

Extending three-dimensional (3D) single-molecule localization microscopy away from the coverslip and into thicker specimens will greatly broaden its biological utility. However, because of the limitations of both conventional imaging modalities and conventional labeling techniques, it is a challenge to localize molecules in three dimensions with high precision in such samples while simultaneously achieving the labeling densities required for high resolution of densely crowded structures. Here we combined lattice light-sheet microscopy with newly developed, freely diffusing, cell-permeable chemical probes with targeted affinity for DNA, intracellular membranes or the plasma membrane. We used this combination to perform high-localization precision, ultrahigh-labeling density, multicolor localization microscopy in samples up to 20 μm thick, including dividing cells and the neuromast organ of a zebrafish embryo. We also demonstrate super-resolution correlative imaging with protein-specific photoactivable fluorophores, providing a mutually compatible, single-platform alternative to correlative light-electron microscopy over large volumes.

}, issn = {1548-7105}, doi = {10.1038/nmeth.3797}, author = {Legant, Wesley R and Shao, Lin and Grimm, Jonathan B and Brown, Timothy A and Milkie, Daniel E and Avants, Brian B and Lavis, Luke D and Betzig, Eric} } @article {47933, title = {Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin.}, journal = {eLife}, volume = {5}, year = {2016}, month = {2016 Oct 10}, abstract = {

The Polycomb PRC1 plays essential roles in development and disease pathogenesis. Targeting of PRC1 to chromatin is thought to be mediated by the Cbx family proteins (Cbx2/4/6/7/8) binding to histone H3 with a K27me3 modification (H3K27me3). Despite this prevailing view, the molecular mechanisms of targeting remain poorly understood. Here, by combining live-cell single-molecule tracking (SMT) and genetic engineering, we reveal that H3K27me3 contributes significantly to the targeting of Cbx7 and Cbx8 to chromatin, but less to Cbx2, Cbx4, and Cbx6. Genetic disruption of the complex formation of PRC1 facilitates the targeting of Cbx7 to chromatin. Biochemical analyses uncover that the CD and AT-hook-like (ATL) motif of Cbx7 constitute a functional DNA-binding unit. Live-cell SMT of Cbx7 mutants demonstrates that Cbx7 is targeted to chromatin by co-recognizing of H3K27me3 and DNA. Our data suggest a novel hierarchical cooperation mechanism by which histone modifications and DNA coordinate to target chromatin regulatory complexes.

}, issn = {2050-084X}, doi = {10.7554/eLife.17667}, author = {Zhen, Chao Yu and Tatavosian, Roubina and Huynh, Thao Ngoc and Duc, Huy Nguyen and Das, Raibatak and Kokotovic, Marko and Grimm, Jonathan B and Lavis, Luke D and Lee, Jun and Mejia, Frances J and Li, Yang and Yao, Tingting and Ren, Xiaojun} } @article {47938, title = {Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis.}, journal = {Genes and Development}, volume = {30}, year = {2016}, month = {2016 Sep 15}, pages = {2106-18}, abstract = {

Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II). A {\textquotedblleft}step-wise{\textquotedblright} preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently. To gain a clearer dynamic picture of PIC assembly, we established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented it by live-cell imaging. Here we performed real-time measurements of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB. Surprisingly, we found that while promoter binding of TFIID and TFIIA is stable, promoter binding by TFIIB is highly transient and dynamic (with an average residence time of 1.5 sec). Stable TFIIB{\textendash}promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II{\textendash}TFIIF. This transient-to-stable transition of TFIIB-binding dynamics has gone undetected previously and underscores the advantages of single-molecule assays for revealing the dynamic nature of complex biological reactions.

}, doi = {10.1101/gad.285395.116}, author = {Zhang, Zhengjian and English, Brian P and Grimm, Jonathan B and Kazane, Stephanie A and Hu, Wenxin and Tsai, Albert and Inouye, Carla and You, Changjiang and Piehler, Jacob and Schultz, Peter G and Lavis, Luke D and Revyakin, Andrey and Tjian, Robert} } @article {47741, title = {Real-time imaging of Huntingtin aggregates diverting target search and gene transcription.}, journal = {eLife}, volume = {5}, year = {2016}, month = {2016 Aug 03}, abstract = {

The presumptive altered dynamics of transient molecular interactions in vivo contributing to neurodegenerative diseases have remained elusive. Here, using single-molecule localization microscopy, we show that disease-inducing Huntingtin (mHtt) protein fragments display three distinct dynamic states in living cells - 1) fast diffusion, 2) dynamic clustering and 3) stable aggregation. Large, stable aggregates of mHtt exclude chromatin and form {\textquoteright}sticky{\textquoteright} decoy traps that impede target search processes of key regulators involved in neurological disorders. Functional domain mapping based on super-resolution imaging reveals an unexpected role of aromatic amino acids in promoting protein-mHtt aggregate interactions. Genome-wide expression analysis and numerical simulation experiments suggest mHtt aggregates reduce transcription factor target site sampling frequency and impair critical gene expression programs in striatal neurons. Together, our results provide insights into how mHtt dynamically forms aggregates and disrupts the finely-balanced gene control mechanisms in neuronal cells.

}, issn = {2050-084X}, doi = {10.7554/eLife.17056}, author = {Li, Li and Liu, Hui and Dong, Peng and Li, Dong and Legant, Wesley R and Grimm, Jonathan B and Lavis, Luke D and Betzig, Eric and Tjian, Robert and Liu, Zhe} } @article {47512, title = {Real-time quantification of single RNA translation dynamics in living cells.}, journal = {Science}, volume = {352}, year = {2016}, month = {2016 May 05}, pages = {1425-9}, abstract = {

Although mRNA translation is a fundamental biological process, it has never been imaged in real-time with single molecule precision in vivo. To achieve this, we developed Nascent Chain Tracking (NCT), a technique that uses multi-epitope tags and antibody-based fluorescent probes to quantify single mRNA protein synthesis dynamics. NCT reveals an elongation rate of ~10 amino acids per second, with initiation occurring stochastically every ~30 s. Polysomes contain ~1 ribosome every 200-900 nucleotides and are globular rather than elongated in shape. By developing multi-color probes, we show most polysomes act independently; however, a small fraction (~5\%) form complexes in which two distinct mRNAs can be translated simultaneously. The sensitivity and versatility of NCT make it a powerful new tool for quantifying mRNA translation kinetics.

}, doi = {10.1126/science.aaf0899}, author = {Morisaki, Tatsuya and Lyon, Kenneth and DeLuca, Keith F and DeLuca, Jennifer G and English, Brian P and Zhang, Zhengjian and Lavis, Luke D and Grimm, Jonathan B and Viswanathan, Sarada and Looger, Loren L} } @article {47753, title = {RNA Polymerase II cluster dynamics predict mRNA output in living cells.}, journal = {eLife}, volume = {5}, year = {2016}, month = {2016 May 03}, abstract = {

Protein clustering is a hallmark of genome regulation in mammalian cells. However, the dynamic molecular processes involved make it difficult to correlate clustering with functional consequences in vivo. We developed a live-cell super-resolution approach to uncover the correlation between mRNA synthesis and the dynamics of RNA Polymerase II (Pol II) clusters at a gene locus. For endogenous β-actin genes in mouse embryonic fibroblasts, we observe that short-lived (~8 s) Pol II clusters correlate with basal mRNA output. During serum stimulation, a stereotyped increase in Pol II cluster lifetime correlates with a proportionate increase in the number of mRNAs synthesized. Our findings suggest that transient clustering of Pol II may constitute a pre-transcriptional regulatory event that predictably modulates nascent mRNA output.

}, issn = {2050-084X}, doi = {10.7554/eLife.13617}, author = {Cho, Won-Ki and Jayanth, Namrata and English, Brian P and Inoue, Takuma and Andrews, J Owen and Conway, William and Grimm, Jonathan B and Spille, Jan-Hendrik and Lavis, Luke D and Lionnet, Timoth{\'e}e and Cisse, Ibrahim I} } @article {47420, title = {Steroid receptors reprogram FoxA1 occupancy through dynamic chromatin transitions.}, journal = {Cell}, year = {2016}, month = {2016 Apr 6}, abstract = {

The estrogen receptor (ER), glucocorticoid receptor (GR), and forkhead box protein 1 (FoxA1) are significant factors in breast cancer progression. FoxA1 has been implicated in establishing ER-binding patterns though its unique ability to serve as a pioneer factor. However, the molecular interplay between ER, GR, and FoxA1 requires further investigation. Here we show that ER and GR both have the ability to alter the genomic distribution of the FoxA1 pioneer factor. Single-molecule tracking experiments in live cells reveal a highly dynamic interaction of FoxA1 with chromatin in\ vivo. Furthermore, the FoxA1 factor is not associated with detectable footprints at its binding sites throughout the genome. These findings support a model wherein interactions between transcription factors and pioneer factors are highly dynamic. Moreover, at a subset of genomic sites, the role of pioneer can be reversed, with the steroid receptors serving to enhance binding of FoxA1.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2016.02.067}, author = {Swinstead, Erin E and Miranda, Tina B and Paakinaho, Ville and Baek, Songjoon and Goldstein, Ido and Hawkins, Mary and Karpova, Tatiana S and Ball, David and Mazza, Davide and Lavis, Luke D and Grimm, Jonathan B and Morisaki, Tatsuya and Gr{\o}ntved, Lars and Presman, Diego M and Hager, Gordon L} } @article {47355, title = {Virginia Orange: A versatile, red-shifted fluorescein scaffold for single- and dual-input fluorogenic probes.}, journal = {Bioconjugate Chemistry}, volume = {27}, year = {2016}, month = {2016 Feb 17}, pages = {474-80}, abstract = {

Fluorogenic molecules are important tools for biological and biochemical research. The majority of fluorogenic compounds have a simple input-output relationship, where a single chemical input yields a fluorescent output. Development of new systems where multiple inputs converge to yield an optical signal could refine and extend fluorogenic compounds by allowing greater spatiotemporal control over the fluorescent signal. Here, we introduce a new red-shifted fluorescein derivative, Virginia Orange, as an exceptional scaffold for single- and dual-input fluorogenic molecules. Unlike fluorescein, installation of a single masking group on Virginia Orange is sufficient to fully suppress fluorescence, allowing preparation of fluorogenic enzyme substrates with rapid, single-hit kinetics. Virginia Orange can also be masked with two independent moieties; both of these masking groups must be removed to induce fluorescence. This allows facile construction of multi-input fluorogenic probes for sophisticated sensing regimes and genetic targeting of latent fluorophores to specific cellular populations.

}, issn = {1520-4812}, doi = {10.1021/acs.bioconjchem.5b00566}, author = {Grimm, Jonathan B and Gruber, Todd D and Ortiz, Gloria and Brown, Timothy A and Lavis, Luke D} } @article {46267, title = {A general method to improve fluorophores for live-cell and single-molecule microscopy.}, journal = {Nature Methods}, volume = {12}, year = {2015}, month = {2015 Jan 19}, pages = {244-50}, abstract = {

Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.

}, issn = {1548-7105}, doi = {10.1038/nmeth.3256}, author = {Grimm, Jonathan B and English, Brian P and Chen, Jiji and Slaughter, Joel P and Zhang, Zhengjian and Revyakin, Andrey and Patel, Ronak and Macklin, John J and Normanno, Davide and Singer, Robert H and Lionnet, Timoth{\'e}e and Lavis, Luke D} } @article {47242, title = {Synthesis of a far-red photoactivatable silicon-containing rhodamine for super-resolution microscopy.}, journal = {Angewandte Chemie (International ed. in English)}, volume = {55}, year = {2015}, month = {2015 Dec 11}, pages = {1723-7}, abstract = {

The rhodamine system is a flexible framework for building small-molecule fluorescent probes. Changing N-substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si-containing analogue of Q-rhodamine. This probe is the first example of a "caged" Si-rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red-shifted to allow multicolor imaging. The dye is a useful label for super-resolution imaging and constitutes a new scaffold for far-red fluorogenic molecules.

}, issn = {1521-3773}, doi = {10.1002/anie.201509649}, author = {Grimm, Jonathan B and Klein, Teresa and Kopek, Benjamin G and Shtengel, Gleb and Hess, Harald F and Sauer, Markus and Lavis, Luke D} } @article {45886, title = {3D imaging of Sox2 enhancer clusters in embryonic stem cells.}, journal = {eLife}, volume = {3}, year = {2014}, month = {2014 Dec 24}, abstract = {

Combinatorial cis-regulatory networks encoded in animal genomes represent the foundational gene expression mechanism for directing cell-fate commitment and maintenance of cell identity by transcription factors (TFs). However, the 3D spatial organization of cis-elements and how such sub-nuclear structures influence TF activity remain poorly understood. Here, we combine lattice light-sheet imaging, single-molecule tracking, numerical simulations, and ChIP-exo mapping to localize and functionally probe Sox2 enhancer-organization in living embryonic stem cells. Sox2 enhancers form 3D-clusters that are segregated from heterochromatin but overlap with a subset of Pol II enriched regions. Sox2 searches for specific binding targets via a 3D-diffusion dominant mode when shuttling long-distances between clusters while chromatin-bound states predominate within individual clusters. Thus, enhancer clustering may reduce global search efficiency but enables rapid local fine-tuning of TF search parameters. Our results suggest an integrated model linking cis-element 3D spatial distribution to local-versus-global target search modalities essential for regulating eukaryotic gene transcription.

}, issn = {2050-084X}, doi = {10.7554/eLife.04236}, author = {Liu, Zhe and Legant, Wesley R and Chen, Bi-Chang and Li, Li and Grimm, Jonathan B and Lavis, Luke D and Betzig, Eric and Tjian, Robert} } @article {6845, title = {Single-molecule tracking of the transcription cycle by sub-second RNA detection.}, journal = {eLife}, volume = {3}, year = {2014}, month = {2014 Jan 28}, pages = {e01775}, abstract = {

Transcription is an inherently stochastic, noisy, and multi-step process, in which fluctuations at every step can cause variations in RNA synthesis, and affect physiology and differentiation decisions in otherwise identical cells. However, it has been an experimental challenge to directly link the stochastic events at the promoter to transcript production. Here we established a fast fluorescence in situ hybridization (fastFISH) method that takes advantage of intrinsically unstructured nucleic acid sequences to achieve exceptionally fast rates of specific hybridization (\~{}10e7 M(-1)s(-1)), and allows deterministic detection of single nascent transcripts. Using a prototypical RNA polymerase, we demonstrated the use of fastFISH to measure the kinetic rates of promoter escape, elongation, and termination in one assay at the single-molecule level, at sub-second temporal resolution. The principles of fastFISH design can be used to study stochasticity in gene regulation, to select targets for gene silencing, and to design nucleic acid nanostructures. DOI: http://dx.doi.org/10.7554/eLife.01775.001.

}, issn = {2050-084X}, doi = {10.7554/eLife.01775}, author = {Zhang, Zhengjian and Revyakin, Andrey and Grimm, Jonathan B and Lavis, Luke D and Tjian, Robert} } @article {6273, title = {Carbofluoresceins and carborhodamines as scaffolds for high-contrast fluorogenic probes.}, journal = {ACS Chemical Biology}, volume = {8}, year = {2013}, month = {2013 Apr 24}, pages = {1303-10}, abstract = {

Fluorogenic molecules are important tools for advanced biochemical and biological experiments. The extant collection of fluorogenic probes is incomplete, however, leaving regions of the electromagnetic spectrum unutilized. Here, we synthesize green-excited fluorescent and fluorogenic analogues of the classic fluorescein and rhodamine 110 fluorophores by replacement of the xanthene oxygen with a quaternary carbon. These anthracenyl "carbofluorescein" and "carborhodamine 110" fluorophores exhibit excellent fluorescent properties and can be masked with enzyme- and photolabile groups to prepare high-contrast fluorogenic molecules useful for live cell imaging experiments and super-resolution microscopy. Our divergent approach to these red-shifted dye scaffolds will enable the preparation of numerous novel fluorogenic probes with high biological utility.

}, issn = {1554-8937}, doi = {10.1021/cb4000822}, author = {Grimm, Jonathan B and Sung, Andrew J and Legant, Wesley R and Hulamm, Phuson and Matlosz, Sylwia M and Betzig, Eric and Lavis, Luke D} } @inbook {6304, title = {The chemistry of small-molecule fluorogenic probes.}, booktitle = {Progress in Molecular Biology and Translational Science}, volume = {113}, year = {2013}, pages = {1-34}, abstract = {

Chemical fluorophores find wide use in biology to detect and visualize different phenomena. A key advantage of small-molecule dyes is the ability to construct compounds where fluorescence is activated by chemical or biochemical processes. Fluorogenic molecules, in which fluorescence is activated by enzymatic activity, light, or environmental changes, enable advanced bioassays and sophisticated imaging experiments. Here, we detail the collection of fluorophores and highlight both general strategies and unique approaches that are employed to control fluorescence using chemistry.

}, issn = {1878-0814}, doi = {10.1016/B978-0-12-386932-6.00001-6}, author = {Grimm, Jonathan B and Heckman, Laurel M and Lavis, Luke D} } @article {6712, title = {Correlative photoactivated localization and scanning electron microscopy.}, journal = {PLoS One}, volume = {8}, year = {2013}, month = {2013 Oct 25}, pages = {e77209}, abstract = {

The ability to localize proteins precisely within subcellular space is crucial to understanding the functioning of biological systems. Recently, we described a protocol that correlates a precise map of fluorescent fusion proteins localized using three-dimensional super-resolution optical microscopy with the fine ultrastructural context of three-dimensional electron micrographs. While it achieved the difficult simultaneous objectives of high photoactivated fluorophore preservation and ultrastructure preservation, it required a super-resolution optical and specialized electron microscope that is not available to many researchers. We present here a faster and more practical protocol with the advantage of a simpler two-dimensional optical (Photoactivated Localization Microscopy (PALM)) and scanning electron microscope (SEM) system that retains the often mutually exclusive attributes of fluorophore preservation and ultrastructure preservation. As before, cryosections were prepared using the Tokuyasu protocol, but the staining protocol was modified to be amenable for use in a standard SEM without the need for focused ion beam ablation. We show the versatility of this technique by labeling different cellular compartments and structures including mitochondrial nucleoids, peroxisomes, and the nuclear lamina. We also demonstrate simultaneous two-color PALM imaging with correlated electron micrographs. Lastly, this technique can be used with small-molecule dyes as demonstrated with actin labeling using phalloidin conjugated to a caged dye. By retaining the dense protein labeling expected for super-resolution microscopy combined with ultrastructural preservation, simplifying the tools required for correlative microscopy, and expanding the number of useful labels we expect this method to be accessible and valuable to a wide variety of researchers.

}, issn = {1932-6203}, doi = {10.1371/journal.pone.0077209}, author = {Kopek, Benjamin G and Shtengel, Gleb and Grimm, Jonathan B and Clayton, David A and Hess, Harald F} } @article {4389, title = {Facile and general synthesis of photoactivatable xanthene dyes.}, journal = {Angewandte Chemie}, volume = {50}, year = {2011}, month = {2011 Nov 18}, pages = {11206-9}, abstract = {

Despite the apparent simplicity of the xanthene fluorophores, the preparation of caged derivatives with free carboxy groups remains a synthetic challenge. A straightforward and flexible strategy for preparing rhodamine and fluorescein derivatives was developed using reduced, {\textquotedblleft}leuco{\textquotedblright} intermediates.

}, issn = {1521-3773}, doi = {10.1002/anie.201104571}, author = {Wysocki, Laura M and Grimm, Jonathan B and Tkachuk, Ariana N and Brown, Timothy A and Betzig, Eric and Lavis, Luke D} } @article {4496, title = {Synthesis of rhodamines from fluoresceins using Pd-catalyzed C-N cross-coupling.}, journal = {Organic Letters}, volume = {13}, year = {2011}, month = {2011 Dec 16}, pages = {6354-7}, abstract = {

A unified, convenient, and efficient strategy for the preparation of rhodamines and N,N{\textquoteright}-diacylated rhodamines has been developed. Fluorescein ditriflates were found to undergo palladium-catalyzed C-N cross-coupling with amines, amides, carbamates, and other nitrogen nucleophiles to provide direct access to known and novel rhodamine derivatives, including fluorescent dyes, quenchers, and latent fluorophores.

}, issn = {1523-7052}, doi = {10.1021/ol202618t}, author = {Grimm, Jonathan B and Lavis, Luke D} } @article {1117, title = {A divergent approach to the synthesis of 3-substituted-2-pyrazolines: Suzuki cross-coupling of 3-sulfonyloxy-2-pyrazolines.}, journal = {The Journal of Organic Chemistry}, volume = {74}, year = {2009}, month = {2009 Aug 21}, pages = {6390-3}, abstract = {

The efficient Suzuki cross-coupling of pyrazoline nonaflates with organoboron reagents was achieved to afford diverse 3-substituted-2-pyrazolines in excellent yield. The nonaflates displayed improved reactivity over the corresponding triflates and smoothly coupled to a variety of aryl- and heteroarylboronic acids. This process and its broad scope constitute a rapid, divergent strategy for the synthesis of elaborated 2-pyrazolines that are not readily obtained via conventional methods.

}, keywords = {Kinetics, Pyrazoles, Sulfonic Acids}, issn = {1520-6904}, doi = {10.1021/jo9011859}, author = {Grimm, Jonathan B and Wilson, Kevin J and Witter, David J} } @article {1116, title = {Exploring the pharmacokinetic properties of phosphorus-containing selective HDAC 1 and 2 inhibitors (SHI-1:2).}, journal = {Bioorganic \& Medicinal Chemistry Letters}, volume = {19}, year = {2009}, month = {2009 Apr 1}, pages = {2053-8}, abstract = {

We report the preparation and structure-activity relationships of phosphorus-containing histone deacetylase inhibitors. A strong trend between decreasing phosphorus functional group size and superior mouse pharmacokinetic properties was identified. In addition, optimized candidates showed tumor growth inhibition in xenograft studies.

}, keywords = {Administration, Animals, Antineoplastic Agents, Enzyme Inhibitors, Heterologous, Histone Deacetylase 1, Histone Deacetylase 2, Histone Deacetylase Inhibitors, Histone Deacetylases, Mice, Nude, Oral, Phosphonic Acids, Repressor Proteins, Transplantation}, issn = {1464-3405}, doi = {10.1016/j.bmcl.2009.02.009}, author = {Heidebrecht, Richard W and Chenard, Melissa and Close, Joshua and Dahlberg, William K and Fleming, Judith and Grimm, Jonathan B and Hamill, Julie E and Harsch, Andreas and Haines, Brian B and Hughes, Bethany and Kral, Astrid M and Middleton, Richard E and Mushti, Chandrasekhar and Ozerova, Nicole and Szewczak, Alexander A and Wang, Hongmei and Wilson, Kevin and Witter, David J and Secrist, J Paul and Miller, Thomas A} } @article {1115, title = {Phenylglycine and phenylalanine derivatives as potent and selective HDAC1 inhibitors (SHI-1).}, journal = {Bioorganic \& Medicinal Chemistry Letters}, volume = {18}, year = {2008}, month = {2008 Mar 15}, pages = {1859-63}, abstract = {

An HTS screening campaign identified a series of low molecular weight phenols that showed excellent selectivity (\>100-fold) for HDAC1/HDAC2 over other Class I and Class II HDACs. Evolution and optimization of this HTS hit series provided HDAC1-selective (SHI-1) compounds with excellent anti-proliferative activity and improved physical properties. Dose-dependent efficacy in a mouse HCT116 xenograft model was demonstrated with a phenylglycine SHI-1 analog.

}, keywords = {Acetylation, Amides, Animals, Cell Proliferation, Colonic Neoplasms, Cultured, Dogs, Enzyme Inhibitors, Ether-A-Go-Go Potassium Channels, Glycine, Histone Deacetylase 1, Histone Deacetylase Inhibitors, Humans, Macaca mulatta, Mice, Molecular Structure, Phenylalanine, Structure-Activity Relationship, Tumor Cells, Xenograft Model Antitumor Assays}, issn = {1464-3405}, doi = {10.1016/j.bmcl.2008.02.012}, author = {Wilson, Kevin J and Witter, David J and Grimm, Jonathan B and Siliphaivanh, Phieng and Otte, Karin M and Kral, Astrid M and Fleming, Judith C and Harsch, Andreas and Hamill, Julie E and Cruz, Jonathan C and Chenard, Melissa and Szewczak, Alexander A and Middleton, Richard E and Hughes, Bethany L and Dahlberg, William K and Secrist, J Paul and Miller, Thomas A} } @article {1114, title = {Absence of the Thorpe-Ingold Eet by gem-diphenyl groups in ring-closing enyne metathesis.}, journal = {Tetrahedron Letters}, volume = {48}, year = {2007}, month = {2007 Nov 5}, pages = {7961-4}, abstract = {

In tandem ring-closing metathesis of alkynyl silaketals containing two different tethered olefins, the gem-dimethyl group showed the expected Thorpe-Ingold effect, thereby giving good level of group selectivity. Unexpectedly, however, the corresponding gem-diphenyl group did not show any Thorpe-Ingold effect for the ring closure reaction.

}, issn = {0040-4039}, doi = {10.1016/j.tetlet.2007.09.063}, author = {Kim, Yi Jin and Grimm, Jonathan B and Lee, Daesung} } @article {1112, title = {A new strategy for the synthesis of benzylic sulfonamides: palladium-catalyzed arylation and sulfonamide metathesis.}, journal = {The Journal of Organic Chemistry}, volume = {72}, year = {2007}, month = {2007 Oct 12}, pages = {8135-8}, abstract = {

An efficient two-step strategy has been developed to access diversely functionalized benzylic sulfonamides. Execution of this strategy required the development of two reaction methods: the palladium-catalyzed cross-coupling of aryl halides with CH-acidic methanesulfonamides and a metathesis reaction between the resulting alpha-arylated sulfonamides and diverse amines. The broad scope of the cross-coupling process combined with a versatile sulfonamide metathesis constitutes an efficient strategy for the synthesis of various benzylic sulfonamides.

}, keywords = {Benzyl Compounds, Catalysis, Molecular Structure, Palladium, Sulfonamides}, issn = {0022-3263}, doi = {10.1021/jo701431j}, author = {Grimm, Jonathan B and Katcher, Matthew H and Witter, David J and Northrup, Alan B} } @article {1111, title = {Efficient synthesis of alkynylsilyl ethers and silaketals via base-induced alkynylsilane alcoholysis.}, journal = {The Journal of Organic Chemistry}, volume = {69}, year = {2004}, month = {2004 Dec 10}, pages = {8967-70}, abstract = {

The efficient silylation of alcohols with di- and trialkynylsilanes was achieved under base-catalyzed conditions to afford alkynyl silyl ethers and symmetrical alkynyl silaketals in good yield. A selective alcoholysis of dialkynyl silyl ethers to mixed silaketals was also demonstrated. These products served as substrates for enyne ring-closing metathesis and, consequently, as precursors to stereochemically defined 1,3-dienes.

}, keywords = {Alcohols, Alkalies, Catalysis, Ethers, Molecular Structure, Organometallic Compounds, Silanes}, issn = {0022-3263}, doi = {10.1021/jo048908l}, author = {Grimm, Jonathan B and Lee, Daesung} } @article {1110, title = {Design, synthesis, and development of novel caprolactam anticonvulsants.}, journal = {Bioorganic \& Medicinal Chemistry}, volume = {11}, year = {2003}, month = {2003 Sep 1}, pages = {4133-41}, abstract = {

Epilepsy afflicts 1-2\% of the world{\textquoteright}s population and often goes untreated; nearly 70\% of those with a form of epilepsy fail to receive proper treatment. Therefore, there is great demand for the design of novel, effective anticonvulsants to combat epilepsy in its numerous forms. Previously, alpha-hydroxy-alpha-phenylcaprolactam was found to have rather potent antiepileptic activity [anti-maximal electroshock (MES) ED(50)=63 mg/kg and anti-subcutaneous Metrazol (scMet) ED(50)=74 mg/kg] when administered intraperitoneally in mice. We focused our attention on the development of this compound through traditional medicinal chemistry techniques-including the Topliss approach, isosteric replacement, methylene insertion, and rigid analogue approach-in the hopes of determining the effect of caprolactam alpha-substitution and other structural modifications on anticonvulsant activity. A number of the desired targets were successfully synthesized and submitted to the Anticonvulsant Screening Program of the National Institute of Neurological Disorders and Stroke (NINDS). Phase I results were quite promising for at least three of the compounds: alpha-ethynyl-alpha-hydroxycaprolactam (10), alpha-benzyl-alpha-hydroxycaprolactam (11), and alpha-hydroxy-alpha-(phenylethynyl)caprolactam (13). Phase II results for 11 strongly suggested it as a new structural class for further development, as it exhibited an anti-MES T.I. in excess of 4.0. Further, the potent activity of 13 in all models also pointed to the substituted alkynylcaprolactams as a new anticonvulsant structural class.

}, keywords = {Administration, Animals, Anticonvulsants, Brain, Caprolactam, Drug Design, Epilepsy, Injections, Intraperitoneal, Mice, Oral, Rats, Structure-Activity Relationship}, issn = {0968-0896}, author = {Grimm, Jonathan B and Stables, James P and Brown, Milton L} }