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      PLK4 is a microtubule-associated protein that self-assembles promoting de novo MTOC formation


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          The centrosome is an important microtubule-organising centre (MTOC) in animal cells. It consists of two barrel-shaped structures, the centrioles, surrounded by the pericentriolar material (PCM), which nucleates microtubules. Centrosomes can form close to an existing structure (canonical duplication) or de novo. How centrosomes form de novo is not known. The master driver of centrosome biogenesis, PLK4, is critical for the recruitment of several centriole components. Here, we investigate the beginning of centrosome biogenesis, taking advantage of Xenopus egg extracts, where PLK4 can induce de novo MTOC formation ( Eckerdt et al., 2011; Zitouni et al., 2016). Surprisingly, we observe that in vitro, PLK4 can self-assemble into condensates that recruit α- and β-tubulins. In Xenopus extracts, PLK4 assemblies additionally recruit STIL, a substrate of PLK4, and the microtubule nucleator γ-tubulin, forming acentriolar MTOCs de novo. The assembly of these robust microtubule asters is independent of dynein, similar to what is found for centrosomes. We suggest a new mechanism of action for PLK4, where it forms a self-organising catalytic scaffold that recruits centriole components, PCM factors and α- and β-tubulins, leading to MTOC formation.

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          Summary: PLK4 binds to microtubules and self-assembles into condensates that recruit tubulin and trigger de novo microtubule-organising centre formation in vitro.

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          A chemical switch for inhibitor-sensitive alleles of any protein kinase.

          Protein kinases have proved to be largely resistant to the design of highly specific inhibitors, even with the aid of combinatorial chemistry. The lack of these reagents has complicated efforts to assign specific signalling roles to individual kinases. Here we describe a chemical genetic strategy for sensitizing protein kinases to cell-permeable molecules that do not inhibit wild-type kinases. From two inhibitor scaffolds, we have identified potent and selective inhibitors for sensitized kinases from five distinct subfamilies. Tyrosine and serine/threonine kinases are equally amenable to this approach. We have analysed a budding yeast strain carrying an inhibitor-sensitive form of the cyclin-dependent kinase Cdc28 (CDK1) in place of the wild-type protein. Specific inhibition of Cdc28 in vivo caused a pre-mitotic cell-cycle arrest that is distinct from the G1 arrest typically observed in temperature-sensitive cdc28 mutants. The mutation that confers inhibitor-sensitivity is easily identifiable from primary sequence alignments. Thus, this approach can be used to systematically generate conditional alleles of protein kinases, allowing for rapid functional characterization of members of this important gene family.
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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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              SAK/PLK4 is required for centriole duplication and flagella development.

              SAK/PLK4 is a distinct member of the polo-like kinase family. SAK-/- mice die during embryogenesis, whereas SAK+/- mice develop liver and lung tumors and SAK+/- MEFs show mitotic abnormalities. However, the mechanism underlying these phenotypes is still not known. Here, we show that downregulation of SAK in Drosophila cells, by mutation or RNAi, leads to loss of centrioles, the core structures of centrosomes. Such cells are able to undergo repeated rounds of cell division, but display broad disorganized mitotic spindle poles. We also show that SAK mutants lose their centrioles during the mitotic divisions preceding male meiosis but still produce cysts of 16 primary spermatocytes as in the wild-type. Mathematical modeling of the stereotyped cell divisions of spermatogenesis can account for such loss by defective centriole duplication. The majority of spermatids in SAK mutants lack centrioles and so are unable to make sperm axonemes. Finally, we show that depletion of SAK in human cells also prevents centriole duplication and gives rise to mitotic abnormalities. SAK/PLK4 is necessary for centriole duplication both in Drosophila and human cells. Drosophila cells tolerate the lack of centrioles and undertake mitosis but cannot form basal bodies and hence flagella. Human cells depleted of SAK show error-prone mitosis, likely to underlie its tumor-suppressor role.

                Author and article information

                J Cell Sci
                J. Cell. Sci
                Journal of Cell Science
                The Company of Biologists Ltd
                15 February 2019
                9 November 2018
                9 November 2018
                : 132
                : 4 , SPECIAL ISSUE: Reconstituting Cell Biology
                : jcs219501
                [1 ]Cell Cycle Regulation Laboratory, Instituto Gulbenkian de Ciência , Rua da Quinta Grande 6, Oeiras, 2780–156, Portugal
                [2 ]Laboratory of Protein Dynamics and Signalling, National Institutes of Health/National Cancer Institute/Center for Cancer Research , Frederick, MD 21702, USA
                [3 ]Max Planck Institute of Molecular Biology and Genetics , Pfotenhauerstrasse 108, 01307 Dresden, Germany
                Author notes

                These authors contributed equally to this work

                Author information
                © 2018. Published by The Company of Biologists Ltd

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

                : 9 May 2018
                : 10 September 2018
                Funded by: European Molecular Biology Organization, http://dx.doi.org/10.13039/100004410;
                Award ID: ALTF 1088-2009
                Funded by: Marie-Curie Actions;
                Award ID: 253373
                Funded by: Fundação para a Ciência e a Tecnologia, http://dx.doi.org/10.13039/501100001871;
                Funded by: The Company of Biologists, http://dx.doi.org/10.13039/501100000522;
                Funded by: European Research Council, http://dx.doi.org/10.13039/100010663;
                Award ID: ERC-COG-683258
                Funded by: National Institutes of Health, http://dx.doi.org/10.13039/100000002;
                Funded by: National Cancer Institute, http://dx.doi.org/10.13039/100000054;
                Research Article

                Cell biology
                plk4,mtocs,in vitro reconstitution,microtubule nucleation,pcm,centrosome,de novo assembly,supramolecular assembly


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