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      Rho-Kinase Planar Polarization at Tissue Boundaries Depends on Phospho-regulation of Membrane Residence Time

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          Summary

          The myosin II activator Rho-kinase (Rok) is planar polarized at the tissue boundary of the Drosophila embryonic salivary gland placode through a negative regulation by the apical polarity protein Crumbs that is anisotropically localized at the boundary. However, in inner cells of the placode, both Crumbs and Rok are isotropically enriched at junctions. We propose that modulation of Rok membrane residence time by Crumbs’ downstream effectors can reconcile both behaviors. Using FRAP combined with in silico simulations, we find that the lower membrane dissociation rate (k off) of Rok at the tissue boundary with low Crumbs explains this boundary-specific effect. The S/T kinase Pak1, recruited by Crumbs and Cdc42, negatively affects Rok membrane association in vivo and in vitro can phosphorylate Rok near the pleckstrin homology (PH) domain that mediates membrane association. These data reveal an important mechanism of the modulation of Rok membrane residence time via affecting the k off that may be widely employed during tissue morphogenesis.

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          Highlights

          • Rho-kinase is planar polarized at tissue boundaries, complementary to Crumbs

          • Crumbs and downstream Pak1 modulate Rok residence time by affecting k off

          • Pak1 can phosphorylate Rok near the PH and Rho-binding domains

          • Rok phosphorylation affects residence time and allows polarization at boundaries

          Abstract

          Sidor et al. show that a modulation of Rok’s k off through phosphorylation of its membrane association regions by Pak1 downstream of Crumbs can drive planar polarization of Rok at tissue boundaries.

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          Most cited references56

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          Despite the existence of fluorescent proteins spanning the entire visual spectrum, the bulk of modern imaging experiments continue to rely on variants of the green fluorescent protein derived from Aequorea victoria. Meanwhile, a great deal of recent effort has been devoted to engineering and improving red fluorescent proteins, and relatively little attention has been given to green and yellow variants. Here we report a novel monomeric yellow-green fluorescent protein, mNeonGreen, which is derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. This fluorescent protein is the brightest monomeric green or yellow fluorescent protein yet described, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging, and is an excellent FRET acceptor for the newest generation of cyan fluorescent proteins.
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            Identification of functional elements and regulatory circuits by Drosophila modENCODE.

            To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.
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              Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.

              The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged recently as a powerful method to manipulate the genomes of various organisms. Here, we report a toolbox for high-efficiency genome engineering of Drosophila melanogaster consisting of transgenic Cas9 lines and versatile guide RNA (gRNA) expression plasmids. Systematic evaluation reveals Cas9 lines with ubiquitous or germ-line-restricted patterns of activity. We also demonstrate differential activity of the same gRNA expressed from different U6 snRNA promoters, with the previously untested U6:3 promoter giving the most potent effect. An appropriate combination of Cas9 and gRNA allows targeting of essential and nonessential genes with transmission rates ranging from 25-100%. We also demonstrate that our optimized CRISPR/Cas tools can be used for offset nicking-based mutagenesis. Furthermore, in combination with oligonucleotide or long double-stranded donor templates, our reagents allow precise genome editing by homology-directed repair with rates that make selection markers unnecessary. Last, we demonstrate a novel application of CRISPR/Cas-mediated technology in revealing loss-of-function phenotypes in somatic cells following efficient biallelic targeting by Cas9 expressed in a ubiquitous or tissue-restricted manner. Our CRISPR/Cas tools will facilitate the rapid evaluation of mutant phenotypes of specific genes and the precise modification of the genome with single-nucleotide precision. Our results also pave the way for high-throughput genetic screening with CRISPR/Cas.
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                Author and article information

                Contributors
                Journal
                Dev Cell
                Dev. Cell
                Developmental Cell
                Cell Press
                1534-5807
                1878-1551
                10 February 2020
                10 February 2020
                : 52
                : 3
                : 364-378.e7
                Affiliations
                [1 ]MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, UK
                Author notes
                []Corresponding author clara.sidor@ 123456univ-amu.fr
                [∗∗ ]Corresponding author kroeper@ 123456mrc-lmb.cam.ac.uk
                [2]

                Present address: IBDM-Institut de Biologie du Développement de Marseille (UMR 7288), CNRS & Aix-Marseille Université, Case 907, Parc Scientifique de Luminy, 13288 Marseille Cedex 9, France

                [3]

                Lead Contact

                Article
                S1534-5807(19)31024-X
                10.1016/j.devcel.2019.12.003
                7008249
                31902655
                5acdafad-580c-4da9-8712-6e6e90375f8e
                © 2019 MRC Laboratory of Molecular Biology

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

                History
                : 20 April 2019
                : 24 October 2019
                : 9 December 2019
                Categories
                Article

                Developmental biology
                morphogenesis,polarity,cytoskeleton,tubulogenesis,rho-kinase,anisotropy,actomyosin
                Developmental biology
                morphogenesis, polarity, cytoskeleton, tubulogenesis, rho-kinase, anisotropy, actomyosin

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