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      PLK4 promotes centriole duplication by phosphorylating STIL to link the procentriole cartwheel to the microtubule wall

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          Abstract

          Centrioles play critical roles in organizing the assembly of the mitotic spindle and templating the formation of primary cilia. Centriole duplication occurs once per cell cycle and is regulated by Polo-like kinase 4 (PLK4). Although significant progress has been made in understanding centriole composition, we have limited knowledge of how PLK4 activity controls specific steps in centriole formation. Here, we show that PLK4 phosphorylates its centriole substrate STIL on a conserved site, S428, to promote STIL binding to CPAP. This phospho-dependent binding interaction is conserved in Drosophila and facilitates the stable incorporation of both STIL and CPAP into the centriole. We propose that procentriole assembly requires PLK4 to phosphorylate STIL in two different regions: phosphorylation of residues in the STAN motif allow STIL to bind SAS6 and initiate cartwheel assembly, while phosphorylation of S428 promotes the binding of STIL to CPAP, linking the cartwheel to microtubules of the centriole wall.

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          A cell’s DNA is the chemical instruction manual for everything it does. Each cell in our bodies contains over two meters of DNA, which is divided into 46 packages of information called chromosomes. When the body needs to make more cells, for example during growth or repair, existing cells divide in two in order to replicate themselves. This means that they also need to copy all of their DNA and then deliver identical sets of chromosomes to each new cell.

          Animal cells use structures called centrioles to help them divide their sets of chromosomes accurately. When cells are about to divide, they make a new set of centrioles by assembling a variety of proteins. This assembly process must be carefully controlled; if too many or too few centrioles are built, cell division errors can occur that lead to the generation of new cells with abnormal numbers of chromosomes.

          The enzyme PLK4 helps to assemble centrioles, but its exact role in the construction process has remained largely unknown. For example, how it might modify different components of the centriole, and why this matters, is poorly understood.

          By performing cell biological and biochemical experiments using human cells, Moyer and Holland show that PLK4 interacts with a protein called STIL that is found in the central part of the centriole. The modification of STIL at a specific location by PLK4 was needed to link it to another protein in the outer wall of the centriole, and was also necessary for the cells to build new centrioles. Cells in which PLK4 was unable to modify STIL had too few centrioles when they were beginning to divide.

          Testing the activity of PLK4 in fruit flies revealed that it plays a similar role as in human cells. This suggests that the modification of STIL by PLK4 is important for normal cell division across different species.

          The results presented by Moyer and Holland help us to understand how dividing cells build the complex machinery that enables them to pass on their genetic material accurately. Future work that builds on these findings could provide insight into human diseases, such as brain development disorders and cancer, where centrioles are either defective or present in the wrong number.

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          Most cited references71

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          Proteomic characterization of the human centrosome by protein correlation profiling.

          The centrosome is the major microtubule-organizing centre of animal cells and through its influence on the cytoskeleton is involved in cell shape, polarity and motility. It also has a crucial function in cell division because it determines the poles of the mitotic spindle that segregates duplicated chromosomes between dividing cells. Despite the importance of this organelle to cell biology and more than 100 years of study, many aspects of its function remain enigmatic and its structure and composition are still largely unknown. We performed a mass-spectrometry-based proteomic analysis of human centrosomes in the interphase of the cell cycle by quantitatively profiling hundreds of proteins across several centrifugation fractions. True centrosomal proteins were revealed by both correlation with already known centrosomal proteins and in vivo localization. We identified and validated 23 novel components and identified 41 likely candidates as well as the vast majority of the known centrosomal proteins in a large background of nonspecific proteins. Protein correlation profiling permits the analysis of any multiprotein complex that can be enriched by fractionation but not purified to homogeneity.
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            Centrioles, centrosomes, and cilia in health and disease.

            Centrioles are barrel-shaped structures that are essential for the formation of centrosomes, cilia, and flagella. Here we review recent advances in our understanding of the function and biogenesis of these organelles, and we emphasize their connection to human disease. Deregulation of centrosome numbers has long been proposed to contribute to genome instability and tumor formation, whereas mutations in centrosomal proteins have recently been genetically linked to microcephaly and dwarfism. Finally, structural or functional centriole aberrations contribute to ciliopathies, a variety of complex diseases that stem from the absence or dysfunction of cilia.
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              Universal and confident phosphorylation site localization using phosphoRS.

              An algorithm for the assignment of phosphorylation sites in peptides is described. The program uses tandem mass spectrometry data in conjunction with the respective peptide sequences to calculate site probabilities for all potential phosphorylation sites. Tandem mass spectra from synthetic phosphopeptides were used for optimization of the scoring parameters employing all commonly used fragmentation techniques. Calculation of probabilities was adapted to the different fragmentation methods and to the maximum mass deviation of the analysis. The software includes a novel approach to peak extraction, required for matching experimental data to the theoretical values of all isoforms, by defining individual peak depths for the different regions of the tandem mass spectrum. Mixtures of synthetic phosphopeptides were used to validate the program by calculation of its false localization rate versus site probability cutoff characteristic. Notably, the empirical obtained precision was higher than indicated by the applied probability cutoff. In addition, the performance of the algorithm was compared to existing approaches to site localization such as Ascore. In order to assess the practical applicability of the algorithm to large data sets, phosphopeptides from a biological sample were analyzed, localizing more than 3000 nonredundant phosphorylation sites. Finally, the results obtained for the different fragmentation methods and localization tools were compared and discussed.
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                Author and article information

                Contributors
                Role: Reviewing Editor
                Role: Senior Editor
                Journal
                eLife
                Elife
                eLife
                eLife
                eLife Sciences Publications, Ltd
                2050-084X
                22 May 2019
                2019
                : 8
                : e46054
                Affiliations
                [1]deptDepartment of Molecular Biology and Genetics Johns Hopkins University School of Medicine BaltimoreUnited States
                University of Oxford United Kingdom
                Utrecht University Netherlands
                University of Oxford United Kingdom
                University of Oxford United Kingdom
                Author information
                https://orcid.org/0000-0003-3728-6367
                Article
                46054
                10.7554/eLife.46054
                6570480
                31115335
                a14a66cb-fbad-4a80-a71f-7252d9dab3e4
                © 2019, Moyer and Holland

                This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

                History
                : 13 February 2019
                : 22 May 2019
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: R01GM114119
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000048, American Cancer Society;
                Award ID: RSG-16-156-01-CCG
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: T32GM007445
                Award Recipient :
                The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Categories
                Research Article
                Cell Biology
                Custom metadata
                Cell biological, biochemical, and genetic analyses reveal that PLK4 controls two different phospho-dependent binding interactions that act together to promote procentriole assembly.

                Life sciences
                centriole,centrosome,mitosis,microtubule,aneuploidy,human
                Life sciences
                centriole, centrosome, mitosis, microtubule, aneuploidy, human

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