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113 Publications

Showing 71-80 of 113 results
05/05/16 | Real-time quantification of single RNA translation dynamics in living cells.
Morisaki T, Lyon K, DeLuca KF, DeLuca JG, English BP, Zhang Z, Lavis LD, Grimm JB, Viswanathan S, Looger LL
Science. 2016 May 05;352(6292):1425-9. doi: 10.1126/science.aaf0899

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.

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05/03/16 | RNA Polymerase II cluster dynamics predict mRNA output in living cells.
Cho W, Jayanth N, English BP, Inoue T, Andrews JO, Conway W, Grimm JB, Spille J, Lavis LD, Lionnet T, Cisse II
eLife. 2016 May 03;5:. doi: 10.7554/eLife.13617

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.

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04/06/16 | Steroid receptors reprogram FoxA1 occupancy through dynamic chromatin transitions.
Swinstead EE, Miranda TB, Paakinaho V, Baek S, Goldstein I, Hawkins M, Karpova TS, Ball D, Mazza D, Lavis LD, Grimm JB, Morisaki T, Grøntved L, Presman DM, Hager GL
Cell. 2016 Apr 6:. doi: 10.1016/j.cell.2016.02.067

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.

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03/07/16 | High-density three-dimensional localization microscopy across large volumes.
Legant WR, Shao L, Grimm JB, Brown TA, Milkie DE, Avants BB, Lavis LD, Betzig E
Nature Methods. 2016 Mar 7:. doi: 10.1038/nmeth.3797

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.

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02/17/16 | Virginia Orange: A versatile, red-shifted fluorescein scaffold for single- and dual-input fluorogenic probes.
Grimm JB, Gruber TD, Ortiz G, Brown TA, Lavis LD
Bioconjugate Chemistry. 2016 Feb 17;27(2):474-80. doi: 10.1021/acs.bioconjchem.5b00566

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.

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01/20/16 | A platform for brain-wide imaging and reconstruction of individual neurons.
Economo MN, Clack NG, Lavis LD, Gerfen CR, Svoboda K, Myers EW, Chandrashekar J
eLife. 2016 Jan 20;5:. doi: 10.7554/eLife.10566

The structure of axonal arbors controls how signals from individual neurons are routed within the mammalian brain. However, the arbors of very few long-range projection neurons have been reconstructed in their entirety, as axons with diameters as small as 100 nm arborize in target regions dispersed over many millimeters of tissue. We introduce a platform for high-resolution, three-dimensional fluorescence imaging of complete tissue volumes that enables the visualization and reconstruction of long-range axonal arbors. This platform relies on a high-speed two-photon microscope integrated with a tissue vibratome and a suite of computational tools for large-scale image data. We demonstrate the power of this approach by reconstructing the axonal arbors of multiple neurons in the motor cortex across a single mouse brain.

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12/11/15 | Synthesis of a far-red photoactivatable silicon-containing rhodamine for super-resolution microscopy.
Grimm JB, Klein T, Kopek BG, Shtengel G, Hess HF, Sauer M, Lavis LD
Angewandte Chemie (International ed. in English). 2015 Dec 11;55(5):1723-7. doi: 10.1002/anie.201509649

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.

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07/17/15 | Ketamine Inside Neurons?
Lester HA, Lavis LD, Dougherty DA
American Journal of Psychiatry. 2015 Jul 17;172(11):1064-6. doi: 10.1176/appi.ajp.2015.14121537
05/21/15 | Imaging live-cell dynamics and structure at the single-molecule level.
Liu Z, Lavis LD, Betzig E
Molecular Cell. 2015 May 21;58(4):644-59. doi: 10.1016/j.molcel.2015.02.033

Observation of molecular processes inside living cells is fundamental to a quantitative understanding of how biological systems function. Specifically, decoding the complex behavior of single molecules enables us to measure kinetics, transport, and self-assembly at this fundamental level that is often veiled in ensemble experiments. In the past decade, rapid developments in fluorescence microscopy, fluorescence correlation spectroscopy, and fluorescent labeling techniques have enabled new experiments to investigate the robustness and stochasticity of diverse molecular mechanisms with high spatiotemporal resolution. This review discusses the concepts and strategies of structural and functional imaging in living cells at the single-molecule level with minimal perturbations to the specimen.

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02/10/15 | A sensitive and robust enzyme kinetic experiment using microplates and fluorogenic ester substrates
Johnson RJ, Hoops GC, Savas CJ, Kartje Z, Lavis LD
Journal of Chemical Education. 2015 Feb;92(2):385-8. doi: 10.1021/ed500452f

Enzyme kinetics measurements are a standard component of undergraduate biochemistry laboratories. The combination of serine hydrolases and fluorogenic enzyme substrates provides a rapid, sensitive, and general method for measuring enzyme kinetics in an undergraduate biochemistry laboratory. In this method, the kinetic activity of multiple protein variants is determined in parallel using a microplate reader, multichannel pipets, serial dilutions, and fluorogenic ester substrates. The utility of this methodology is illustrated by the measurement of differential enzyme activity in microplate volumes in triplicate with small protein samples and low activity enzyme variants. Enzyme kinetic measurements using fluorogenic substrates are, thus, adaptable for use with student-purified enzyme variants and for comparative enzyme kinetics studies. The rapid setup and analysis of these kinetic experiments not only provides advanced undergraduates with experience in a fundamental biochemical technique, but also provides the adaptability for use in inquiry-based laboratories.

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