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113 Publications

Showing 61-70 of 113 results
04/07/17 | Teaching old dyes new tricks: biological probes built from fluoresceins and rhodamines.
Lavis LD
Annual Review of Biochemistry. 2017 Apr 07;86:825-43. doi: 10.1146/annurev-biochem-061516-044839

Small-molecule fluorophores, such as fluorescein and rhodamine derivatives, are critical tools in modern biochemical and biological research. The field of chemical dyes is old; colored molecules were first discovered in the 1800s, and the fluorescein and rhodamine scaffolds have been known for over a century. Nevertheless, there has been a renaissance in using these dyes to create tools for biochemistry and biology. The application of modern chemistry, biochemistry, molecular genetics, and optical physics to these old structures enables and drives the development of novel, sophisticated fluorescent dyes. This critical review focuses on an important example of chemical biology-the melding of old and new chemical knowledge-leading to useful molecules for advanced biochemical and biological experiments. Expected final online publication date for the Annual Review of Biochemistry Volume 86 is June 20, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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03/15/17 | Quantifying transcription factor binding dynamics at the single-molecule level in live cells.
Presman DM, Ball DA, Paakinaho V, Grimm JB, Lavis LD, Karpova TS, Hager GL
Methods (San Diego, Calif.). 2017 Mar 15:. doi: 10.1016/j.ymeth.2017.03.014

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.

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03/13/17 | Stochastic protein labeling enables long-term single molecule observation in vivo.
Liu H, Dong P, Ioannou MS, Li L, Shea J, Pasolli HA, Grimm JB, Rivlin PK, Lavis LD, Koyama M, Liu Z
bioRxiv. 2017 Mar 13:. doi: 10.1101/116186

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.

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10/24/16 | Bright photoactivatable fluorophores for single-molecule imaging.
Lavis LD, Grimm JB, English BP, Choi H, Muthusamy AK, Mehl BP, Dong P, Brown TA, Lippincott-Schwartz J, Liu Z, Lionnet T
Nature Methods. 2016 Oct 24;13(12):985-8. doi: 10.1038/nmeth.4034

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.

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10/10/16 | Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin.
Zhen CY, Tatavosian R, Huynh TN, Duc HN, Das R, Kokotovic M, Grimm JB, Lavis LD, Lee J, Mejia FJ, Li Y, Yao T, Ren X
eLife. 2016 Oct 10;5:. doi: 10.7554/eLife.17667

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.

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09/15/16 | Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis.
Zhang Z, English BP, Grimm JB, Kazane SA, Hu W, Tsai A, Inouye C, You C, Piehler J, Schultz PG, Lavis LD, Revyakin A, Tjian R
Genes and Development. 2016 Sep 15;30:2106-18. doi: 10.1101/gad.285395.116

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 “step-wise” 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–promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II–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.

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Lavis LabSinger Lab
09/13/16 | Glutamate-induced RNA localization and translation in neurons.
Yoon YJ, Wu B, Buxbaum AR, Das S, Tsai A, English BP, Grimm JB, Lavis LD, Singer RH
Proceedings of the National Academy of Sciences of the United States of America. 2016 Sep 13:. doi: 10.1073/pnas.1614267113

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.

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08/03/16 | Real-time imaging of Huntingtin aggregates diverting target search and gene transcription.
Li L, Liu H, Dong P, Li D, Legant WR, Grimm JB, Lavis LD, Betzig E, Tjian R, Liu Z
eLife. 2016 Aug 03;5:. doi: 10.7554/eLife.17056

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 'sticky' 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.

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07/11/16 | Evaluation of the Ser-His dipeptide, a putative catalyst of amide and ester hydrolysis.
MacDonald MJ, Lavis LD, Hilvert D, Gellman SH
Organic Letters. 2016 Jul 11:. doi: 10.1021/acs.orglett.6b01279

Efficient hydrolysis of amide bonds has long been a reaction of interest for organic chemists. The rate constants of proteases are unmatched by those of any synthetic catalyst. It has been proposed that a dipeptide containing serine and histidine is an effective catalyst of amide hydrolysis, based on an apparent ability to degrade a protein. The capacity of the Ser-His dipeptide to catalyze the hydrolysis of several discrete ester and amide substrates is investigated using previously described conditions. This dipeptide does not catalyze the hydrolysis of amide or unactivated ester groups in any of the substrates under the conditions evaluated.

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05/24/16 | Design and synthesis of a calcium-sensitive photocage.
Heckman LM, Grimm JB, Schreiter ER, Kim C, Verdecia MA, Shields BC, Lavis LD
Angewandte Chemie (International ed. in English). 2016 May 24:. doi: 10.1002/anie.201602941

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.

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