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      Drosophila germ granules are structured and contain homotypic mRNA clusters

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          Abstract

          Germ granules, specialized ribonucleoprotein particles, are a hallmark of all germ cells. In Drosophila, an estimated 200 mRNAs are enriched in the germ plasm, and some of these have important, often conserved roles in germ cell formation, specification, survival and migration. How mRNAs are spatially distributed within a germ granule and whether their position defines functional properties is unclear. Here we show, using single-molecule FISH and structured illumination microscopy, a super-resolution approach, that mRNAs are spatially organized within the granule whereas core germ plasm proteins are distributed evenly throughout the granule. Multiple copies of single mRNAs organize into ‘homotypic clusters' that occupy defined positions within the center or periphery of the granule. This organization, which is maintained during embryogenesis and independent of the translational or degradation activity of mRNAs, reveals new regulatory mechanisms for germ plasm mRNAs that may be applicable to other mRNA granules.

          Abstract

          What regulates mRNAs transcript localization in the germ granules in Drosophila is unclear. Here Trcek et al. identify that germ plasm proteins are homogeneously distributed in germ granules but once localized, individual mRNAs form homotypic clusters, contributing structure to the germ granules.

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          Most cited references35

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          Instant super-resolution imaging in live cells and embryos via analog image processing

          Existing super-resolution fluorescence microscopes compromise acquisition speed to provide subdiffractive sample information. We report an analog implementation of structured illumination microscopy that enables 3D super-resolution imaging with 145 nm lateral and 350 nm axial resolution, at acquisition speeds up to 100 Hz. By performing image processing operations optically instead of digitally, we removed the need to capture, store, and combine multiple camera exposures, increasing data acquisition rates 10–100x over other super-resolution microscopes and acquiring and displaying super-resolution images in real-time. Low excitation intensities allow imaging over hundreds of 2D sections, and combined physical and computational sectioning allow similar depth penetration to confocal microscopy. We demonstrate the capability of our system by imaging fine, rapidly moving structures including motor-driven organelles in human lung fibroblasts and the cytoskeleton of flowing blood cells within developing zebrafish embryos.
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            A transgenic mouse for in vivo detection of endogenous labeled mRNA

            Live-cell single mRNA imaging is a powerful tool, but has been restricted in higher eukaryotes to artificial cell lines and reporter genes. We describe an approach that enables live-cell imaging of single endogenous labeled mRNA molecules transcribed in primary mammalian cells and tissue. We generated a knock-in mouse line in which an MS2 binding site (MBS) cassette was targeted to the 3′UTR of the essential β-actin gene. As β-actin-MBS was ubiquitously expressed, we were able to uniquely address endogenous mRNA regulation in any tissue or cell type. We simultaneously followed transcription from the β-actin alleles in real-time and observed transcriptional bursting in response to serum stimulation with precise temporal resolution. We performed tracking of single endogenous labeled mRNA particles being transported in primary hippocampal neurons. The MBS also provided a means for high sensitivity Fluorescence In Situ Hybridization (FISH), allowing detection and localization of single β-actin mRNA molecules in various mouse tissues.
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              RNA granules in germ cells.

              "Germ granules" are cytoplasmic, nonmembrane-bound organelles unique to germline. Germ granules share components with the P bodies and stress granules of somatic cells, but also contain proteins and RNAs uniquely required for germ cell development. In this review, we focus on recent advances in our understanding of germ granule assembly, dynamics, and function. One hypothesis is that germ granules operate as hubs for the posttranscriptional control of gene expression, a function at the core of the germ cell differentiation program.
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                Author and article information

                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Publishing Group
                2041-1723
                05 August 2015
                2015
                : 6
                : 7962
                Affiliations
                [1 ]Department of Cell Biology, HHMI, Skirball Institute of Biomolecular Medicine, NYU School of Medicine , 540 First Avenue, New York, New York 10016, USA
                [2 ]Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, NIH , BG 13 RM G800, 13 South Dr, Bethesda, Maryland 20814, USA
                [3 ]Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute , 19700 Helix Drive, Ashburn, Virginia 20147, USA
                Author notes
                Article
                ncomms8962
                10.1038/ncomms8962
                4918342
                26242323
                367245e4-8d2c-40c6-a001-cbb1cf9e3ee6
                Copyright © 2015, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 22 April 2015
                : 26 June 2015
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