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Kodani et al. identify a role for the mammalian homologue of yeast SFI1 as a regulator of centriole duplication. They show that SFI1 localizes USP9X to the centrosome to stabilize the microcephaly protein STIL to promote centriole duplication., In mammals, centrioles participate in brain development, and human mutations affecting centriole duplication cause microcephaly. Here, we identify a role for the mammalian homologue of yeast SFI1, involved in the duplication of the yeast spindle pole body, as a critical regulator of centriole duplication in mammalian cells. Mammalian SFI1 interacts with USP9X, a deubiquitylase associated with human syndromic mental retardation. SFI1 localizes USP9X to the centrosome during S phase to deubiquitylate STIL, a critical regulator of centriole duplication. USP9X-mediated deubiquitylation protects STIL from degradation. Consistent with a role for USP9X in stabilizing STIL, cells from patients with USP9X loss-of-function mutations have reduced STIL levels. Together, these results demonstrate that SFI1 is a centrosomal protein that localizes USP9X to the centrosome to stabilize STIL and promote centriole duplication. We propose that the USP9X protection of STIL to facilitate centriole duplication underlies roles of both proteins in human neurodevelopment.

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., eLife digest 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.

The ability to deplete a protein of interest is critical for dissecting cellular processes. Traditional methods of protein depletion are often slow acting, which can be problematic when characterizing a cellular process that occurs within a short period of time, such as mitosis. Furthermore, these methods are usually not reversible. Recent advances to achieve protein depletion function by inducibly trafficking proteins of interest to an endogenous E3 ubiquitin ligase complex to promote ubiquitination and subsequent degradation by the proteasome. One of these systems, the auxin-inducible degron (AID) system, has been shown to permit rapid and inducible degradation of AID-tagged target proteins in mammalian cells. The AID system can control the abundance of a diverse set of cellular proteins, including those contained within protein complexes, and is active in all phases of the cell cycle. Here we discuss considerations for the successful implementation of the AID system and describe a protocol using CRISPR/Cas9 to achieve biallelic insertion of an AID in human cells. This method can also be adapted to insert other tags, such as fluorescent proteins, at defined genomic locations.

The ability to deplete a protein of interest is critical for dissecting cellular processes. Traditional methods of protein depletion are often slow acting, which can be problematic when characterizing a cellular process that occurs within a short period of time, such as mitosis. Furthermore, these methods are usually not reversible. Recent advances to achieve protein depletion function by inducibly trafficking proteins of interest to an endogenous E3 ubiquitin ligase complex to promote ubiquitination and subsequent degradation by the proteasome. One of these systems, the auxin-inducible degron (AID) system, has been shown to permit rapid and inducible degradation of AID-tagged target proteins in mammalian cells. The AID system can control the abundance of a diverse set of cellular proteins, including those contained within protein complexes, and is active in all phases of the cell cycle. Here we discuss considerations for the successful implementation of the AID system and describe a protocol using CRISPR/Cas9 to achieve biallelic insertion of an AID in human cells. This method can also be adapted to insert other tags, such as fluorescent proteins, at defined genomic locations.

Binding of STIL activates Plk4, and the subsequent phosphorylation of STIL by Plk4 primes the binding of STIL to SAS6 to promote centriole assembly., Centriole duplication occurs once per cell cycle in order to maintain control of centrosome number and ensure genome integrity. Polo-like kinase 4 (Plk4) is a master regulator of centriole biogenesis, but how its activity is regulated to control centriole assembly is unclear. Here we used gene editing in human cells to create a chemical genetic system in which endogenous Plk4 can be specifically inhibited using a cell-permeable ATP analogue. Using this system, we demonstrate that STIL localization to the centriole requires continued Plk4 activity. Most importantly, we show that direct binding of STIL activates Plk4 by promoting self-phosphorylation of the activation loop of the kinase. Plk4 subsequently phosphorylates STIL to promote centriole assembly in two steps. First, Plk4 activity promotes the recruitment of STIL to the centriole. Second, Plk4 primes the direct binding of STIL to the C terminus of SAS6. Our findings uncover a molecular basis for the timing of Plk4 activation through the cell cycle–regulated accumulation of STIL.

The deposited article is a post-print version (author's manuscript from PMC and available in PMC 2017 May 9). This publication hasn't any creative commons license associated. This deposit is composed by the main article and the supplementary materials are present in the publisher's page in the following link: https://www.sciencedirect.com/science/article/pii/S0960982216303001?via%3Dihub#sec4 Centrioles are essential for the assembly of both centrosomes and cilia. Centriole biogenesis occurs once and only once per cell cycle and is temporally coordinated with cell-cycle progression, ensuring the formation of the right number of centrioles at the right time. The formation of new daughter centrioles is guided by a pre-existing, mother centriole. The proximity between mother and daughter centrioles was proposed to restrict new centriole formation until they separate beyond a critical distance. Paradoxically, mother and daughter centrioles overcome this distance in early mitosis, at a time when triggers for centriole biogenesis Polo-like kinase 4 (PLK4) and its substrate STIL are abundant. Here we show that in mitosis, the mitotic kinase CDK1-CyclinB binds STIL and prevents formation of the PLK4-STIL complex and STIL phosphorylation by PLK4, thus inhibiting untimely onset of centriole biogenesis. After CDK1-CyclinB inactivation upon mitotic exit, PLK4 can bind and phosphorylate STIL in G1, allowing pro-centriole assembly in the subsequent S phase. Our work shows that complementary mechanisms, such as mother-daughter centriole proximity and CDK1-CyclinB interaction with centriolar components, ensure that centriole biogenesis occurs once and only once per cell cycle, raising parallels to the cell-cycle regulation of DNA replication and centromere formation. ERC grant: (ERC-2010-StG-261344); FCT grants: (FCT Investigator, EXPL/BIM-ONC/0830/2013, PTDC/SAU-BD/105616/2008); EMBO installation grant. info:eu-repo/semantics/publishedVersion