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77 Publications

Showing 1-10 of 77 results
10/03/18 | A toolbox for multiplexed super-resolution imaging of the E. coli nucleoid and membrane using novel PAINT labels.
Spahn CK, Glaesmann M, Grimm JB, Ayala AX, Lavis LD, Heilemann M
Scientific Reports. 2018 Oct 03;8(1):14768. doi: 10.1038/s41598-018-33052-3

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.

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08/29/18 | Cell-specific chemical delivery using a selective Nitroreductase-Nitroaryl pair.
Gruber TD, Krishnamurthy C, Grimm JB, Tadross MR, Wysocki LM, Gartner ZJ, Lavis LD
ACS Chemical Biology. 2018 Aug 29:. doi: 10.1021/acschembio.8b00524

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.

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08/15/18 | Optimization of fluorophores for chemical tagging and immunohistochemistry of Drosophila neurons.
Meissner GW, Grimm JB, Johnston RM, Sutcliffe B, Ng J, Jefferis GS, Cachero S, Lavis LD, Malkesman O
PLoS One. 2018 Aug 15;13(8):e0200759. doi: 10.1371/journal.pone.0200759

The use of genetically encoded 'self-labeling tags' 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.

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07/13/18 | Fluorogenic structure activity library pinpoints molecular variations in substrate specificity of structurally homologous esterases.
White A, Koelper A, Russell A, Larsen EM, Kim C, Lavis LD, Hoops GC, Johnson RJ
The Journal of Biological Chemistry. 2018 Jul 13;293(36):13851-62. doi: 10.1074/jbc.RA118.003972

Cellular esterases catalyze many essential biological functions by performing hydrolysis reactions on diverse substrates. The promiscuity of esterases complicates assignment of their substrate preferences and biological functions. To identify universal factors controlling esterase substrate recognition, we designed a 32-member structure-activity relationship (SAR) library of fluorogenic ester substrates and used this library to systematically interrogate esterase preference for chain length, branching patterns, and polarity to differentiate common classes of esterase substrates. Two structurally homologous bacterial esterases were screened against this library, refining their previously broad overlapping substrate specificity. esterase ybfF displayed a preference for γ-position thioethers and ethers, whereas Rv0045c from displayed a preference for branched substrates with and without thioethers. We determined that this substrate differentiation was partially controlled by individual substrate selectivity residues Tyr119 in ybfF and His187 in Rv0045c; reciprocal substitution of these residues shifted each esterase's substrate preference. This work demonstrates that the selectivity of esterases is tuned based on transition state stabilization, identifies thioethers as an underutilized functional group for esterase substrates, and provides a rapid method for differentiating structural isozymes. This SAR library could have multi-faceted future applications including in vivo imaging, biocatalyst screening, molecular fingerprinting, and inhibitor design.

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06/21/18 | Imaging dynamic and selective low-complexity domain interactions that control gene transcription.
Chong S, Dugast-Darzacq C, Liu Z, Dong P, Dailey GM, Cattoglio C, Heckert A, Banala S, Lavis L, Darzacq X, Tjian R
Science (New York, N.Y.). 2018 Jun 21;361(6400):eaar2555. doi: 10.1126/science.aar2555

Many eukaryotic transcription factors (TFs) contain intrinsically disordered low-complexity domains (LCDs), but how they drive transactivation remains unclear. Here, live-cell single-molecule imaging reveals that TF-LCDs form local high-concentration interaction hubs at synthetic and endogenous genomic loci. TF-LCD hubs stabilize DNA binding, recruit RNA polymerase II (Pol II), and activate transcription. LCD-LCD interactions within hubs are highly dynamic, display selectivity with binding partners, and are differentially sensitive to disruption by hexanediols. Under physiological conditions, rapid and reversible LCD-LCD interactions occur between TFs and the Pol II machinery without detectable phase separation. Our findings reveal fundamental mechanisms underpinning transcriptional control and suggest a framework for developing single-molecule imaging screens for novel drugs targeting gene regulatory interactions implicated in disease.

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05/22/18 | Nicotinic cholinergic receptors in VTA glutamate neurons modulate excitatory transmission.
Yan Y, Peng C, Arvin MC, Jin X, Kim VJ, Ramsey MD, Wang Y, Banala S, Wokosin DL, McIntosh JM, Lavis LD, Drenan RM
Cell Reports. 2018 May 22;23(8):2236-2244. doi: 10.1016/j.celrep.2018.04.062

Ventral tegmental area (VTA) glutamate neurons are important components of reward circuitry, but whether they are subject to cholinergic modulation is unknown. To study this, we used molecular, physiological, and photostimulation techniques to examine nicotinic acetylcholine receptors (nAChRs) in VTA glutamate neurons. Cells in the medial VTA, where glutamate neurons are enriched, are responsive to acetylcholine (ACh) released from cholinergic axons. VTA VGLUT2 neurons express mRNA and protein subunits known to comprise heteromeric nAChRs. Electrophysiology, coupled with two-photon microscopy and laser flash photolysis of photoactivatable nicotine, was used to demonstrate nAChR functional activity in the somatodendritic subcellular compartment of VTA VGLUT2 neurons. Finally, optogenetic isolation of intrinsic VTA glutamatergic microcircuits along with gene-editing techniques demonstrated that nicotine potently modulates excitatory transmission within the VTA via heteromeric nAChRs. These results indicate that VTA glutamate neurons are modulated by cholinergic mechanisms and participate in the cascade of physiological responses to nicotine exposure.

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04/16/18 | Measuring the global substrate specificity of mycobacterial serine hydrolases using a library of fluorogenic ester substrates.
Bassett B, Waibel B, White A, Hansen H, Stephens D, Koelper A, Larsen EM, Kim C, Glanzer A, Lavis LD, Hoops GC, Johnson RJ
ACS Infectious Diseases. 2018 Apr 16:. doi: 10.1021/acsinfecdis.7b00263

Among the proteins required for lipid metabolism in Mycobacterium tuberculosis are a significant number of uncharacterized serine hydrolases, especially lipases and esterases. Using a streamlined synthetic method, a library of immolative fluorogenic ester substrates was expanded to better represent the natural lipidomic diversity of Mycobacterium. This expanded fluorogenic library was then used to rapidly characterize the global structure activity relationship (SAR) of mycobacterial serine hydrolases in M. smegmatis under different growth conditions. Confirmation of fluorogenic substrate activation by mycobacterial serine hydrolases was performed using nonspecific serine hydrolase inhibitors and reinforced the biological significance of the SAR. The hydrolases responsible for the global SAR were then assigned using gel-resolved activity measurements, and these assignments were used to rapidly identify the relative substrate specificity of previously uncharacterized mycobacterial hydrolases. These measurements provide a global SAR of mycobacterial hydrolase activity, a picture of cycling hydrolase activity, and a detailed substrate specificity profile for previously uncharacterized hydrolases.

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03/26/18 | Photoactivatable drugs for nicotinic optopharmacology.
Banala S, Arvin MC, Bannon NM, Jin X, Macklin JJ, Wang Y, Peng C, Zhao G, Marshall JJ, Gee KR, Wokosin DL, Kim VJ, McIntosh JM, Contractor A, Lester HA, Kozorovitskiy Y, Drenan RM, Lavis LD
Nature Methods. 2018 Mar 26;15(5):347-50. doi: 10.1038/nmeth.4637

Photoactivatable pharmacological agents have revolutionized neuroscience, but the palette of available compounds is limited. We describe a general method for caging tertiary amines by using a stable quaternary ammonium linkage that elicits a red shift in the activation wavelength. We prepared a photoactivatable nicotine (PA-Nic), uncageable via one- or two-photon excitation, that is useful to study nicotinic acetylcholine receptors (nAChRs) in different experimental preparations and spatiotemporal scales.

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02/14/18 | Synthetic and genetically encoded fluorescent neural activity indicators.
Deo C, Lavis LD
Current Opinion in Neurobiology. 2018 Feb 14;50:101-108. doi: 10.1016/j.conb.2018.01.003

The ultimate goal of neuroscience is to relate the complex activity of cells and cell-networks to behavior and cognition. This requires tools and techniques to visualize neuronal activity. Fluorescence microscopy is an ideal tool to measure activity of cells in the brain due to the high sensitivity of the technique and the growing portfolio of optical hardware and fluorescent sensors. Here, we give a chemist's perspective on the recent progress of fluorescent activity indicators that enable the measurement of cellular events in the living brain. We discuss advances in both chemical and genetically encoded sensors and look forward to hybrid indicators, which incorporate synthetic organic dyes into genetically encoded protein constructs.

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01/04/18 | Robust model-based analysis of single-particle tracking experiments with Spot-On.
Hansen AS, Woringer M, Grimm JB, Lavis LD, Tjian R, Darzacq X
eLife. 2018 Jan 04;7:. doi: 10.7554/eLife.33125

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 'Spot-On', 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.

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