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4 Janelia Publications

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    10/23/14 | Photoswitchable red fluorescent protein with a large stokes shift.
    Piatkevich KD, English BP, Malashkevich VN, Xiao H, Almo SC, Singer RH, Verkhusha VV
    Chemistry & Biology. 2014 Oct 23;21(10):1402-14. doi: 10.1016/j.chembiol.2014.08.010

    A subclass of fluorescent proteins (FPs), large Stokes shift (LSS) FP, are characterized by increased spread between excitation and emission maxima. We report a photoswitchable variant of a red FP with an LSS, PSLSSmKate, which initially exhibits excitation and emission at 445 and 622 nm, but violet irradiation photoswitches PSLSSmKate into a common red form with excitation and emission at 573 and 621 nm. We characterize spectral, photophysical, and biochemical properties of PSLSSmKate in vitro and in mammalian cells and determine its crystal structure in the LSS form. Mass spectrometry, mutagenesis, and spectroscopy of PSLSSmKate allow us to propose molecular mechanisms for the LSS, pH dependence, and light-induced chromophore transformation. We demonstrate the applicability of PSLSSmKate to superresolution photoactivated localization microscopy and protein dynamics in live cells. Given its promising properties, we expect that PSLSSmKate-like phenotype will be further used for photoactivatable imaging and tracking multiple populations of intracellular objects.

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    10/24/14 | Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution.
    Chen B, Legant WR, Wang K, Shao L, Milkie DE, Davidson MW, Janetopoulos C, Wu XS, Hammer JA, Liu Z, English BP, Mimori-Kiyosue Y, Romero DP, Ritter AT, Lippincott-Schwartz J, Fritz-Laylin L, Mullins RD, Mitchell DM, Bembenek JN, Reymann A, Böhme R, Grill SW, Wang JT, Seydoux G, Tulu US, Kiehart DP, Betzig E
    Science. 2014 Oct 24;346(6208):1257998. doi: 10.1126/science.1257998

    Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at subsecond intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and the complexity of living systems.

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    04/20/14 | Efficient Bayesian-based multiview deconvolution.
    Preibisch S, Amat F, Stamataki E, Sarov M, Singer RH, Myers E, Tomancak P
    Nature Methods. 2014 Apr 20;11:645-8. doi: 10.1038/nmeth.2929

    Light-sheet fluorescence microscopy is able to image large specimens with high resolution by capturing the samples from multiple angles. Multiview deconvolution can substantially improve the resolution and contrast of the images, but its application has been limited owing to the large size of the data sets. Here we present a Bayesian-based derivation of multiview deconvolution that drastically improves the convergence time, and we provide a fast implementation using graphics hardware.

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    01/24/14 | Visualization of dynamics of single endogenous mRNA labeled in live mouse.
    Park HY, Lim H, Yoon YJ, Follenzi A, Nwokafor C, Lopez-Jones M, Meng X, Singer RH
    Science. 2014 Jan 24;343(6169):422-4. doi: 10.1126/science.1239200

    The transcription and transport of messenger RNA (mRNA) are critical steps in regulating the spatial and temporal components of gene expression, but it has not been possible to observe the dynamics of endogenous mRNA in primary mammalian tissues. We have developed a transgenic mouse in which all β-actin mRNA is fluorescently labeled. We found that β-actin mRNA in primary fibroblasts localizes predominantly by diffusion and trapping as single mRNAs. In cultured neurons and acute brain slices, we found that multiple β-actin mRNAs can assemble together, travel by active transport, and disassemble upon depolarization by potassium chloride. Imaging of brain slices revealed immediate early induction of β-actin transcription after depolarization. Studying endogenous mRNA in live mouse tissues provides insight into its dynamic regulation within the context of the cellular and tissue microenvironment.

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