SummaryThe centriole, and the related basal body, is an ancient organelle characterized by a universal 9-fold radial symmetry and is critical for generating cilia, flagella, and centrosomes. The mechanisms directing centriole formation are incompletely understood and represent a fundamental open question in biology. Here, we demonstrate that the centriolar protein SAS-6 forms rod-shaped homodimers that interact through their N-terminal domains to form oligomers. We establish that such oligomerization is essential for centriole formation in C. elegans and human cells. We further generate a structural model of the related protein Bld12p from C. reinhardtii, in which nine homodimers assemble into a ring from which nine coiled-coil rods radiate outward. Moreover, we demonstrate that recombinant Bld12p self-assembles into structures akin to the central hub of the cartwheel, which serves as a scaffold for centriole formation. Overall, our findings establish a structural basis for the universal 9-fold symmetry of centrioles.

Formation of one procentriole next to each pre-existing centriole is essential for centrosome duplication, robust bipolar spindle assembly and maintenance of genome integrity. However, the mechanisms maintaining strict control over centriole copy number are incompletely understood. Here we show that Plk4 and STIL, the key regulators of centriole formation, form a protein complex that provides a scaffold for recruiting HsSAS-6, a major component of the centriolar cartwheel, at the onset of procentriole formation. Furthermore, we demonstrate that phosphorylation of STIL by Plk4 facilitates the STIL/HsSAS-6 interaction and centriolar loading of HsSAS-6. We also provide evidence that negative feedback by centriolar STIL regulates bimodal centriolar distribution of Plk4 and seemingly restricts occurrence of procentriole formation to one site on each parental centriole. Overall, these findings suggest a mechanism whereby coordinated action of three critical factors ensures formation of a single procentriole per parental centriole.

The number of centrioles is tightly controlled to ensure bipolar spindle assembly, which is a prerequisite to maintain genome integrity. However, our understanding of the fundamental principle that governs the formation of a single procentriole per parental centriole is incomplete. Here, we show that the local restriction of Plk4, a master regulator of the procentriole formation, is achieved by a bimodal interaction of STIL with Plk4. We demonstrate that the conserved short coiled-coil region of STIL binds to and protects Plk4 from protein degradation at the site of procentriole formation. On the other hand, the conserved C-terminal region of STIL named truncated in microcephaly (TIM) domain promotes autophosphorylation and degradation of adjacent Plk4 by the direct interaction. Thus, we propose that positive and negative regulation based on the bimodal binding of Plk4 and STIL ensures the formation of a single procentriole per parental centriole.

SummaryPatients with MCPH (autosomal recessive primary microcephaly) exhibit impaired brain development, presumably due to the compromised function of neuronal progenitors. Seven MCPH loci have been identified, including one that encodes centrosome protein 4.1 associated protein (CPAP; also known as centromere protein J, CENPJ). CPAP is a large coiled-coil protein enriched at the centrosome, a structure that comprises two centrioles and surrounding pericentriolar material (PCM). CPAP depletion impairs centriole formation, whereas CPAP overexpression results in overly long centrioles. The mechanisms by which CPAP MCPH patient mutations affect brain development are not clear. Here, we identify CPAP protein domains crucial for its centriolar localization, as well as for the elongation and the formation of centrioles. Furthermore, we demonstrate that conditions that resemble CPAP MCPH patient mutations compromise centriole formation in tissue culture cells. Using adhesive micropatterns, we reveal that such defects correlate with a randomization of spindle position. Moreover, we demonstrate that the MCPH protein SCL/TAL1 interrupting locus (STIL) is also essential for centriole formation and for proper spindle position. Our findings are compatible with the notion that mutations in CPAP and STIL cause MCPH because of aberrant spindle positioning in progenitor cells during brain development.

Stress‐activated protein kinase/c‐Jun N‐terminal kinase (SAPK/JNK) responds to a variety of stress stimuli and controls cell fates such as cell cycle entrance, apoptosis and senescence. Stimuli such as ultraviolet irradiation and chemical reagents that damage genomic DNA induce the activation of the SAPK/JNK signaling pathway. However, it is unclear how the signal arising in the nucleus owing to DNA damage is transmitted to SAPK/JNK in the cytoplasm. Here, we report that the nuclear components Daxx and Ras‐association domain family 1C (RASSF1C) link DNA damage to SAPK/JNK activation in HeLa cells. In response to DNA damage, Daxx localized in promyelocytic leukaemia‐nuclear bodies (PML‐NBs) undergoes ubiquitination and degradation. RASSF1C, a tumor suppressor and newly identified binding partner of Daxx, is constitutively anchored by Daxx in PML‐NBs but is released from the nucleus when Daxx is degraded. This released RASSF1C translocates to cytoplasmic microtubules and participates in the activation of SAPK/JNK. Our data define a novel mechanism by which the Daxx–RASSF1C complex in PML‐NBs couples nuclear DNA damage to the cytoplasmic SAPK/JNK signaling pathway.

Ultraviolet (UV) irradiation stimulates stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/ JNK), which is a member of the mitogen-activated protein kinase (MAPK) superfamily and implicated in stress-induced apoptosis. UV also induces the activation of another MAPK member, extracellular signal-regulated kinase (ERK), which is typically involved in a growth-signaling cascade. However, the UV-induced signaling pathway leading to ERK activation, together with the physiological role, has remained unknown. Here we examined the molecular mechanism and physiological function of UV-induced ERK activation in human epidermoid carcinoma A431 cells that retain a high number of epidermal growth factor (EGF) receptors. UV-induced ERK activation was accompanied with the Tyr phosphorylation of EGF receptors, and both responses were completely abolished in the presence of a selective EGF receptor inhibitor (AG1478) or the Src inhibitor PP2 and by the expression of a kinase-dead Src mutant. On the other hand, SAPK/JNK activation by UV was partially inhibited by these inhibitors. UV stimulated Src activity in a manner similar to the ERK activation, but the Src activation was insensitive to AG1478. UV-induced cell apoptosis measured by DNA fragmentation and caspase 3 activation was enhanced by AG1478 and an ERK kinase inhibitor (U0126) but inhibited by EGF receptor stimulation by the agonist. These results indicate that UV-induced ERK activation, which provides a survival signal against stress-induced apoptosis, is mediated through Src-dependent Tyr phosphorylation of EGF receptors.

A large-scale mutagenesis screen was performed in Medaka to identify genes acting in diverse developmental processes. Mutations were identified in homozygous F3 progeny derived from ENU-treated founder males. In addition to the morphological inspection of live embryos, other approaches were used to detect abnormalities in organogenesis and in specific cellular processes, including germ cell migration, nerve tract formation, sensory organ differentiation and DNA repair. Among 2031 embryonic lethal mutations identified, 312 causing defects in organogenesis were selected for further analyses. From these, 126 mutations were characterized genetically and assigned to 105 genes. The similarity of the development of Medaka and zebrafish facilitated the comparison of mutant phenotypes, which indicated that many mutations in Medaka cause unique phenotypes so far unrecorded in zebrafish. Even when mutations of the two fish species cause a similar phenotype such as one-eyed-pinhead or parachute, more genes were found in Medaka than in zebrafish that produced the same phenotype when mutated. These observations suggest that many Medaka mutants represent new genes and, therefore, are important complements to the collection of zebrafish mutants that have proven so valuable for exploring genomic function in development.

SUMMARY Centrioles are essential for the assembly of both centrosomes and cilia. Centriole biogenesis occurs once and only once per cell cycle and it 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-existent, 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 that distance in early mitosis, at a time where 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 with the cell cycle regulation of DNA replication and centromere formation.