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      CLASP2 binding to curved microtubule tips promotes flux and stabilizes kinetochore attachments

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          Abstract

          Girão et al. use structure-guided functional mutants of CLASP2 to show that recognition of growing microtubule plus-ends through EB–protein interaction and the ability to associate with curved microtubule protofilaments through TOG2 and TOG3 domains promote growth and stabilization of kinetochore–microtubules required for poleward flux.

          Abstract

          CLASPs are conserved microtubule plus-end–tracking proteins that suppress microtubule catastrophes and independently localize to kinetochores during mitosis. Thus, CLASPs are ideally positioned to regulate kinetochore–microtubule dynamics required for chromosome segregation fidelity, but the underlying mechanism remains unknown. Here, we found that human CLASP2 exists predominantly as a monomer in solution, but it can self-associate through its C-terminal kinetochore-binding domain. Kinetochore localization was independent of self-association, and driving monomeric CLASP2 to kinetochores fully rescued normal kinetochore–microtubule dynamics, while partially sustaining mitosis. CLASP2 kinetochore localization, recognition of growing microtubule plus-ends through EB–protein interaction, and the ability to associate with curved microtubule protofilaments through TOG2 and TOG3 domains independently sustained normal spindle length, timely spindle assembly checkpoint satisfaction, chromosome congression, and faithful segregation. Measurements of kinetochore–microtubule half-life and poleward flux revealed that CLASP2 regulates kinetochore–microtubule dynamics by integrating distinctive microtubule-binding properties at the kinetochore–microtubule interface. We propose that kinetochore CLASP2 suppresses microtubule depolymerization and detachment by binding to curved protofilaments at microtubule plus-ends.

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          Fluorogenic probes for live-cell imaging of the cytoskeleton.

          We introduce far-red, fluorogenic probes that combine minimal cytotoxicity with excellent brightness and photostability for fluorescence imaging of actin and tubulin in living cells. Applied in stimulated emission depletion (STED) microscopy, they reveal the ninefold symmetry of the centrosome and the spatial organization of actin in the axon of cultured rat neurons with a resolution unprecedented for imaging cytoskeletal structures in living cells.
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            Tracking the ends: a dynamic protein network controls the fate of microtubule tips.

            Microtubule plus-end tracking proteins (+TIPs) are a diverse group of evolutionarily conserved cellular factors that accumulate at the ends of growing microtubules. They form dynamic networks through the interaction of a limited set of protein modules, repeat sequences and linear motifs that bind to each other with moderate affinities. +TIPs regulate different aspects of cell architecture by controlling microtubule dynamics, microtubule interactions with cellular structures and signalling factors, and the forces that are exerted on microtubule networks.
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              An EB1-binding motif acts as a microtubule tip localization signal.

              Microtubules are filamentous polymers essential for cell viability. Microtubule plus-end tracking proteins (+TIPs) associate with growing microtubule plus ends and control microtubule dynamics and interactions with different cellular structures during cell division, migration, and morphogenesis. EB1 and its homologs are highly conserved proteins that play an important role in the targeting of +TIPs to microtubule ends, but the underlying molecular mechanism remains elusive. By using live cell experiments and in vitro reconstitution assays, we demonstrate that a short polypeptide motif, Ser-x-Ile-Pro (SxIP), is used by numerous +TIPs, including the tumor suppressor APC, the transmembrane protein STIM1, and the kinesin MCAK, for localization to microtubule tips in an EB1-dependent manner. Structural and biochemical data reveal the molecular basis of the EB1-SxIP interaction and explain its negative regulation by phosphorylation. Our findings establish a general "microtubule tip localization signal" (MtLS) and delineate a unifying mechanism for this subcellular protein targeting process.
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                Author and article information

                Journal
                J Cell Biol
                J. Cell Biol
                jcb
                jcb
                The Journal of Cell Biology
                Rockefeller University Press
                0021-9525
                1540-8140
                3 February 2020
                22 November 2019
                22 November 2019
                : 219
                : 2
                : e201905080
                Affiliations
                [1 ]Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal
                [2 ]Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Porto, Portugal
                [3 ]Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto, Porto, Portugal
                [4 ]International Graduate School of Arts and Sciences, Yokohama City University, Yokohama, Japan
                [5 ]Cell Division Group, Experimental Biology Unit, Department of Biomedicine, Faculdade de Medicina, Universidade do Porto, Porto, Portugal
                Author notes
                Correspondence to Helder Maiato: maiato@ 123456i3s.up.pt
                Author information
                https://orcid.org/0000-0002-1549-9233
                https://orcid.org/0000-0003-2311-8258
                https://orcid.org/0000-0003-4784-8933
                https://orcid.org/0000-0001-7714-5302
                https://orcid.org/0000-0002-7698-1170
                https://orcid.org/0000-0002-6200-9997
                Article
                201905080
                10.1083/jcb.201905080
                7041679
                31757788
                e5fc7883-a572-4524-8199-550f8e77ba18
                © 2019 Girão et al.

                This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

                History
                : 10 May 2019
                : 17 October 2019
                : 04 November 2019
                Page count
                Pages: 20
                Funding
                Funded by: Fundação para a Ciência e a Tecnologia, DOI https://doi.org/10.13039/501100001871;
                Award ID: SFRH/BD/141066/2018
                Funded by: Grant-in-Aid for Scientific Research
                Award ID: 22570190
                Funded by: European Research Council, DOI https://doi.org/10.13039/501100000781;
                Award ID: 681443
                Funded by: B2Tech i3S Scientific Platform
                Categories
                Research Articles
                Article
                35
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                Cell biology
                Cell biology

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