Once and only once: mechanisms of centriole duplication and their deregulation in disease.

Centrioles are conserved microtubule-based organelles that form the core of the centrosome and act as templates for the formation of cilia and flagella. Centrioles have important roles in most microtubule-related processes, including motility, cell division and cell signalling. To coordinate these diverse cellular processes, centriole number must be tightly controlled. In cycling cells, one new centriole is formed next to each pre-existing centriole in every cell cycle. Advances in imaging, proteomics, structural biology and genome editing have revealed new insights into centriole biogenesis, how centriole numbers are controlled and how alterations in these processes contribute to diseases such as cancer and neurodevelopmental disorders. Moreover, recent work has uncovered the existence of surveillance pathways that limit the proliferation of cells with numerical centriole aberrations. Owing to this progress, we now have a better understanding of the molecular mechanisms governing centriole biogenesis, opening up new possibilities for targeting these pathways in the context of human disease.

The PIDDosome activates p53 in response to supernumerary centrosomes.

Centrosomes, the main microtubule-organizing centers in animal cells, are replicated exactly once during the cell division cycle to form the poles of the mitotic spindle. Supernumerary centrosomes can lead to aberrant cell division and have been causally linked to chromosomal instability and cancer. Here, we report that an increase in the number of mature centrosomes, generated by disrupting cytokinesis or forcing centrosome overduplication, triggers the activation of the PIDDosome multiprotein complex, leading to Caspase-2-mediated MDM2 cleavage, p53 stabilization, and p21-dependent cell cycle arrest. This pathway also restrains the extent of developmentally scheduled polyploidization by regulating p53 levels in hepatocytes during liver organogenesis. Taken together, the PIDDosome acts as a first barrier, engaging p53 to halt the proliferation of cells carrying more than one mature centrosome to maintain genome integrity.

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.

Structural centrosome aberrations promote non-cell-autonomous invasiveness.

Centrosomes are the main microtubule-organizing centers of animal cells. Although centrosome aberrations are common in tumors, their consequences remain subject to debate. Here, we studied the impact of structural centrosome aberrations, induced by deregulated expression of ninein-like protein (NLP), on epithelial spheres grown in Matrigel matrices. We demonstrate that NLP-induced structural centrosome aberrations trigger the escape ("budding") of living cells from epithelia. Remarkably, all cells disseminating into the matrix were undergoing mitosis. This invasive behavior reflects a novel mechanism that depends on the acquisition of two distinct properties. First, NLP-induced centrosome aberrations trigger a re-organization of the cytoskeleton, which stabilizes microtubules and weakens E-cadherin junctions during mitosis. Second, atomic force microscopy reveals that cells harboring these centrosome aberrations display increased stiffness. As a consequence, mitotic cells are pushed out of mosaic epithelia, particularly if they lack centrosome aberrations. We conclude that centrosome aberrations can trigger cell dissemination through a novel, non-cell-autonomous mechanism, raising the prospect that centrosome aberrations contribute to the dissemination of metastatic cells harboring normal centrosomes.

PICH: a DNA translocase specially adapted for processing anaphase bridge DNA.

The Plk1-interacting checkpoint helicase (PICH) protein localizes to ultrafine anaphase bridges (UFBs) in mitosis alongside a complex of DNA repair proteins, including the Bloom's syndrome protein (BLM). However, very little is known about the function of PICH or how it is recruited to UFBs. Using a combination of microfluidics, fluorescence microscopy, and optical tweezers, we have defined the properties of PICH in an in vitro model of an anaphase bridge. We show that PICH binds with a remarkably high affinity to duplex DNA, resulting in ATP-dependent protein translocation and extension of the DNA. Most strikingly, the affinity of PICH for binding DNA increases with tension-induced DNA stretching, which mimics the effect of the mitotic spindle on a UFB. PICH binding also appears to diminish force-induced DNA melting. We propose a model in which PICH recognizes and stabilizes DNA under tension during anaphase, thereby facilitating the resolution of entangled sister chromatids.

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging.

Centrioles are essential for the formation of centrosomes and cilia. While numerical and/or structural centrosomes aberrations are implicated in cancer, mutations in centriolar and centrosomal proteins are genetically linked to ciliopathies, microcephaly, and dwarfism. The evolutionarily conserved mechanisms underlying centrosome biogenesis are centered on a set of key proteins, including Plk4, Sas-6, and STIL, whose exact levels are critical to ensure accurate reproduction of centrioles during cell cycle progression. However, neither the intracellular levels of centrosomal proteins nor their stoichiometry within centrosomes is presently known. Here, we have used two complementary approaches, targeted proteomics and EGFP-tagging of centrosomal proteins at endogenous loci, to measure protein abundance in cultured human cells and purified centrosomes. Our results provide a first assessment of the absolute and relative amounts of major components of the human centrosome. Specifically, they predict that human centriolar cartwheels comprise up to 16 stacked hubs and 1 molecule of STIL for every dimer of Sas-6. This type of quantitative information will help guide future studies of the molecular basis of centrosome assembly and function.

Structural and functional organization of the Ska complex, a key component of the kinetochore-microtubule interface.

The Ska complex is an essential mitotic component required for accurate cell division in human cells. It is composed of three subunits that function together to establish stable kinetochore-microtubule interactions in concert with the Ndc80 network. We show that the structure of the Ska core complex is a W-shaped dimer of coiled coils, formed by intertwined interactions between Ska1, Ska2, and Ska3. The C-terminal domains of Ska1 and Ska3 protrude at each end of the homodimer, bind microtubules in vitro when connected to the central core, and are essential in vivo. Mutations disrupting the central coiled coil or the dimerization interface result in chromosome congression failure followed by cell death. The Ska complex is thus endowed with bipartite and cooperative tubulin-binding properties at the ends of a 350 Å-long molecule. We discuss how this symmetric architecture might complement and stabilize the Ndc80-microtubule attachments with analogies to the yeast Dam1/DASH complex.

Direct regulation of tRNA and 5S rRNA gene transcription by Polo-like kinase 1.

Polo-like kinase Plk1 controls numerous aspects of cell-cycle progression. We show that it associates with tRNA and 5S rRNA genes and regulates their transcription by RNA polymerase III (pol III) through direct binding and phosphorylation of transcription factor Brf1. During interphase, Plk1 promotes tRNA and 5S rRNA expression by phosphorylating Brf1 directly on serine 450. However, this stimulatory modification is overridden at mitosis, when elevated Plk1 activity causes Brf1 phosphorylation on threonine 270 (T270), which prevents pol III recruitment. Thus, although Plk1 enhances net tRNA and 5S rRNA production, consistent with its proliferation-stimulating function, it also suppresses untimely transcription when cells divide. Genomic instability is apparent in cells with Brf1 T270 mutated to alanine to resist Plk1-directed inactivation, suggesting that chromosome segregation is vulnerable to inappropriate pol III activity.

The autoregulated instability of Polo-like kinase 4 limits centrosome duplication to once per cell cycle.

Centrioles organize the centrosome, and accurate control of their number is critical for the maintenance of genomic integrity. Centriole duplication occurs once per cell cycle and is controlled by Polo-like kinase 4 (Plk4). We showed previously that Plk4 phosphorylates itself to promote its degradation by the proteasome. Here we demonstrate that this autoregulated instability controls the abundance of endogenous Plk4. Preventing Plk4 autoregulation causes centrosome amplification, stabilization of p53, and loss of cell proliferation; moreover, suppression of p53 allows growth of cells carrying amplified centrosomes. Plk4 autoregulation thus guards against genome instability by limiting centrosome duplication to once per cell cycle.

A novel TPR-BEN domain interaction mediates PICH-BEND3 association.

PICH is a DNA translocase required for the maintenance of chromosome stability in human cells. Recent data indicate that PICH co-operates with topoisomerase IIα to suppress pathological chromosome missegregation through promoting the resolution of ultra-fine anaphase bridges (UFBs). Here, we identify the BEN domain-containing protein 3 (BEND3) as an interaction partner of PICH in human cells in mitosis. We have purified full length PICH and BEND3 and shown that they exhibit a functional biochemical interaction in vitro. We demonstrate that the PICH-BEND3 interaction occurs via a novel interface between a TPR domain in PICH and a BEN domain in BEND3, and have determined the crystal structure of this TPR-BEN complex at 2.2 Å resolution. Based on the structure, we identified amino acids important for the TPR-BEN domain interaction, and for the functional interaction of the full-length proteins. Our data reveal a proposed new function for BEND3 in association with PICH, and the first example of a specific protein-protein interaction mediated by a BEN domain.