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5 Publications

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    02/01/22 | Organization of translating secretome mRNAS on endoplasmic reticulum
    Choi H, Liao Y, Yoon YJ, Grimm J, Lavis LD, Singer RH, Lippincott-Schwartz J
    Biophysical Journal. 2022 Feb 01;121(3):33a. doi: 10.1016/j.bpj.2021.11.2550

    The endoplasmic reticulum (ER) has a complex morphology comprised of stacked sheets, tubules, and three-way junctions, which together function as a platform for protein synthesis of membrane and secretory proteins. Specific ER subdomains are thought to be spatially organized to enable protein synthesis activity, but precisely where these domains are localized is unclear, especially relative to the plethora of organelle interactions taking place on the ER. Here, we use single-molecule tracking of ribosomes and mRNA in combination with simultaneous imaging of ER to assess the sites of membrane protein synthesis on the ER. We found that ribosomes were widely distributed throughout different ER morphologies, but the synthesis of membrane proteins (including Type I, II, and multi-spanning) and an ER luminal protein (Calreticulin) occurred primarily at three-way junctions. Lunapark played a key role in stabilizing transmembrane protein mRNA at three-way junctions. We additionally found that translating mRNAs coding for transmembrane proteins are in the vicinity of lysosomes and translate through a cap-independent but eIF2-dependent mechanism. These results support the idea that discrete ER subdomains co-exist with lysosomes to support specific types of protein synthesis activities, with ER-lysosome interactions playing an important role in the translation of secretome mRNAs.

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    05/24/21 | A general method to improve fluorophores using deuterated auxochromes.
    Grimm JB, Xie L, Casler JC, Patel R, Tkachuk AN, Falco N, Choi H, Lippincott-Schwartz J, Brown TA, Glick BS, Liu Z, Lavis LD
    JACS Au. 2021 May 24;1(5):690-6. doi: 10.1021/jacsau.1c00006

    Fluorescence microscopy relies on dyes that absorb and then emit photons. In addition to fluorescence, fluorophores can undergo photochemical processes that decrease quantum yield or result in spectral shifts and irreversible photobleaching. Chemical strategies that suppress these undesirable pathways—thereby increasing the brightness and photostability of fluorophores—are crucial for advancing the frontier of bioimaging. Here, we describe a general method to improve small-molecule fluorophores by incorporating deuterium into the alkylamino auxochromes of rhodamines and other dyes. This strategy increases fluorescence quantum yield, inhibits photochemically induced spectral shifts, and slows irreparable photobleaching, yielding next-generation labels with improved performance in cellular imaging experiments.

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    07/27/20 | A general method to optimize and functionalize red-shifted rhodamine dyes.
    Grimm JB, Tkachuk AN, Xie L, Choi H, Mohar B, Falco N, Schaefer K, Patel R, Zheng Q, Liu Z, Lippincott-Schwartz J, Brown TA, Lavis LD
    Nature Methods. 2020 Jul 27:. doi: 10.1038/s41592-020-0909-6

    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 'Janelia Fluor' (JF) dyes useful for biological imaging experiments in cells and in vivo.

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    09/25/19 | Rational design of fluorogenic and spontaneously blinking labels for super-resolution imaging.
    Zheng Q, Ayala AX, Chung I, Weigel AV, Ranjan A, Falco N, Grimm JB, Tkachuk AN, Wu C, Lippincott-Schwartz J, Singer RH, Lavis LD
    ACS Central Science. 2019 Sep 25;5(9):1602-1613. doi: 10.1021/acscentsci.9b00676

    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.

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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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