Structural centrosome aberrations sensitize polarized epithelia to basal cell extrusion.

Centrosome aberrations disrupt tissue architecture and may confer invasive properties to cancer cells. Here we show that structural centrosome aberrations, induced by overexpression of either Ninein-like protein (NLP) or CEP131/AZI1, sensitize polarized mammalian epithelia to basal cell extrusion. While unperturbed epithelia typically dispose of damaged cells through apical dissemination into luminal cavities, certain oncogenic mutations cause a switch in directionality towards basal cell extrusion, raising the potential for metastatic cell dissemination. Here we report that NLP-induced centrosome aberrations trigger the preferential extrusion of damaged cells towards the basal surface of epithelial monolayers. This switch in directionality from apical to basal dissemination coincides with a profound reorganization of the microtubule cytoskeleton, which in turn prevents the contractile ring repositioning that is required to support extrusion towards the apical surface. While the basal extrusion of cells harbouring NLP-induced centrosome aberrations requires exogenously induced cell damage, structural centrosome aberrations induced by excess CEP131 trigger the spontaneous dissemination of dying cells towards the basal surface from MDCK cysts. Thus, similar to oncogenic mutations, structural centrosome aberrations can favour basal extrusion of damaged cells from polarized epithelia. Assuming that additional mutations may promote cell survival, this process could sensitize epithelia to disseminate potentially metastatic cells.

Quantitative proteomic and phosphoproteomic comparison of human colon cancer DLD-1 cells differing in ploidy and chromosome stability.

Although aneuploidy is poorly tolerated during embryogenesis, aneuploidy and whole chromosomal instability (CIN) are common hallmarks of cancer, raising the question of how cancer cells can thrive in spite of chromosome aberrations. Here we present a comprehensive and quantitative proteomics analysis of isogenic DLD-1 colorectal adenocarcinoma cells lines, aimed at identifying cellular responses to changes in ploidy and/or CIN. Specifically, we compared diploid (2N) and tetraploid (4N) cells with posttetraploid aneuploid (PTA) clones and engineered trisomic clones. Our study provides a comparative data set on the proteomes and phosphoproteomes of the above cell lines, comprising several thousand proteins and phosphopeptides. In comparison to the parental 2N line, we observed changes in proteins associated with stress responses and with interferon signaling. Although we did not detect a conspicuous protein signature associated with CIN, we observed many changes in phosphopeptides that relate to fundamental cellular processes, including mitotic progression and spindle function. Most importantly, we found that most changes detectable in PTA cells were already present in the 4N progenitor line. This suggests that activation of mitotic pathways through hyper-phosphorylation likely constitutes an important response to chromosomal burden. In line with this conclusion, cells with extensive chromosome gains showed differential sensitivity toward a number of inhibitors targeting cell cycle kinases, suggesting that the efficacy of anti-mitotic drugs may depend on the karyotype of cancer cells.

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.

The SKP1-Cullin-F-box E3 ligase ßTrCP and CDK2 cooperate to control STIL abundance and centriole number.

Deregulation of centriole duplication has been implicated in cancer and primary microcephaly. Accordingly, it is important to understand how key centriole duplication factors are regulated. E3 ubiquitin ligases have been implicated in controlling the levels of several duplication factors, including PLK4, STIL and SAS-6, but the precise mechanisms ensuring centriole homeostasis remain to be fully understood. Here, we have combined proteomics approaches with the use of MLN4924, a generic inhibitor of SCF E3 ubiquitin ligases, to monitor changes in the cellular abundance of centriole duplication factors. We identified human STIL as a novel substrate of SCF-ßTrCP. The binding of ßTrCP depends on a DSG motif within STIL, and serine 395 within this motif is phosphorylated in vivo SCF-ßTrCP-mediated degradation of STIL occurs throughout interphase and mutations in the DSG motif causes massive centrosome amplification, attesting to the physiological importance of the pathway. We also uncover a connection between this new pathway and CDK2, whose role in centriole biogenesis remains poorly understood. We show that CDK2 activity protects STIL against SCF-ßTrCP-mediated degradation, indicating that CDK2 and SCF-ßTrCP cooperate via STIL to control centriole biogenesis.

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.

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.

Cellular Prion Protein PrPC and Ecto-5'-Nucleotidase Are Markers of the Cellular Stress Response to Aneuploidy.

Aneuploidy is a hallmark of most human tumors, but the molecular physiology of aneuploid cells is not well characterized. In this study, we screened cell surface biomarkers of approximately 300 proteins by multiparameter flow cytometry using multiple aneuploid model systems such as cell lines, patient samples, and mouse models. Several new biomarkers were identified with altered expression in aneuploid cells, including overexpression of the cellular prion protein CD230/PrPC and the immunosuppressive cell surface enzyme ecto-5'-nucleotidase CD73. Functional analyses associated these alterations with increased cellular stress. An increased number of CD73+ cells was observed in confluent cultures in aneuploid cells relative to their diploid counterparts. An elevated expression in CD230/PrPC was observed in serum-deprived cells in association with increased generation of reactive oxygen species. Overall, our work identified biomarkers of aneuploid karyotypes, which suggest insights into the underlying molecular physiology of aneuploid cells. Cancer Res; 77(11); 2914-26. ©2017 AACR.

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.

Impact of Centrosome Aberrations on Chromosome Segregation and Tissue Architecture in Cancer.

Centrosomes determine the disposition of microtubule networks and thereby contribute to regulate cell shape, polarity, and motility, as well as chromosome segregation during cell division. Additionally, centrioles, the core components of centrosomes, are required for the formation of cilia and flagella. Mutations in genes coding for centrosomal and centriolar proteins are responsible for several human diseases, foremost ciliopathies and developmental disorders resulting in small brains (primary microcephaly) or small body size (dwarfism). Moreover, a long-standing postulate implicates numerical and/or structural centrosome aberrations in the etiology of cancer. In this review, we will discuss recent work on the role of centrosome aberrations in the promotion of genome instability and the disruption of tissue architecture, two hallmarks of human cancers. We will emphasize recent studies on the impact of centrosome aberrations on the polarity of epithelial cells cultured in three-dimensional spheroid models. Collectively, the results from these in vitro systems suggest that different types of centrosome aberrations can promote invasive behavior through different pathways. Particularly exciting is recent evidence indicating that centrosome aberrations may trigger the dissemination of potentially metastatic cells through a non-cell-autonomous mechanism.

The PLK4-STIL-SAS-6 module at the core of centriole duplication.

Centrioles are microtubule-based core components of centrosomes and cilia. They are duplicated exactly once during S-phase progression. Central to formation of each new (daughter) centriole is the formation of a nine-fold symmetrical cartwheel structure onto which microtubule triplets are deposited. In recent years, a module comprising the protein kinase polo-like kinase 4 (PLK4) and the two proteins STIL and SAS-6 have been shown to stay at the core of centriole duplication. Depletion of any one of these three proteins blocks centriole duplication and, conversely, overexpression causes centriole amplification. In this short review article, we summarize recent insights into how PLK4, STIL and SAS-6 co-operate in space and time to form a new centriole. These advances begin to shed light on the very first steps of centriole biogenesis.

The Ska complex promotes Aurora B activity to ensure chromosome biorientation.

Chromosome biorientation and accurate segregation rely on the plasticity of kinetochore-microtubule (KT-MT) attachments. Aurora B facilitates KT-MT dynamics by phosphorylating kinetochore proteins that are critical for KT-MT interactions. Among the substrates whose microtubule and kinetochore binding is curtailed by Aurora B is the spindle and kinetochore-associated (Ska) complex, a key factor for KT-MT stability. Here, we show that Ska is not only a substrate of Aurora B, but is also required for Aurora B activity. Ska-deficient cells fail to biorient and display chromosome segregation errors underlying suppressed KT-MT turnover. These defects coincide with KNL1-Mis12-Ndc80 network hypophosphorylation, reduced mitotic centromere-associated kinesin localization, and Aurora B T-loop phosphorylation at kinetochores. We further show that Ska requires its microtubule-binding capability to promote Aurora B activity in cells and stimulates Aurora B catalytic activity in vitro. Finally, we show that protein phosphatase 1 counteracts Aurora B activity to enable Ska kinetochore accumulation once biorientation is achieved. We propose that Ska promotes Aurora B activity to limit its own microtubule and kinetochore association and to ensure that KT-MT dynamics and stability fall within an optimal balance for biorientation.

Ska3 Ensures Timely Mitotic Progression by Interacting Directly With Microtubules and Ska1 Microtubule Binding Domain.

The establishment of physical attachment between the kinetochore and dynamic spindle microtubules, which undergo cycles of polymerization and depolymerization generating straight and curved microtubule structures, is essential for accurate chromosome segregation. The Ndc80 and Ska complexes are the major microtubule-binding factors of the kinetochore responsible for maintaining chromosome-microtubule coupling during chromosome segregation. We previously showed that the Ska1 subunit of the Ska complex binds dynamic microtubules using multiple contact sites in a mode that allows conformation-independent binding. Here, we show that the Ska3 subunit is required to modulate the microtubule binding capability of the Ska complex (i) by directly interacting with tubulin monomers and (ii) indirectly by interacting with tubulin contacting regions of Ska1 suggesting an allosteric regulation. Perturbing either the Ska3-microtubule interaction or the Ska3-Ska1 interactions negatively influences microtubule binding by the Ska complex in vitro and affects the timely onset of anaphase in cells. Thus, Ska3 employs additional modulatory elements within the Ska complex to ensure robust kinetochore-microtubule attachments and timely progression of mitosis.

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.

Evaluation and Improvement of Quantification Accuracy in Isobaric Mass Tag-Based Protein Quantification Experiments.

The multiplexing capabilities of isobaric mass tag-based protein quantification, such as Tandem Mass Tags or Isobaric Tag for Relative and Absolute Quantitation have dramatically increased the scope of mass spectrometry-based proteomics studies. Not only does the technology allow for the simultaneous quantification of multiple samples in a single MS injection, but its seamless compatibility with extensive sample prefractionation methods allows for comprehensive studies of complex proteomes. However, reporter ion-based quantification has often been criticized for limited quantification accuracy due to interference from coeluting peptides and peptide fragments. In this study, we investigate the extent of this problem and propose an effective and easy-to-implement remedy that relies on spiking a 6-protein calibration mixture to the samples. We evaluated our ratio adjustment approach using two large scale TMT 10-plex data sets derived from a human cancer and noncancer cell line as well as E. coli cells grown at two different conditions. Furthermore, we analyzed a complex 2-proteome artificial sample mixture and investigated the precision of TMT and precursor ion intensity-based label free quantification. Studying the protein set identified by both methods, we found that differentially abundant proteins were assigned dramatically higher statistical significance when quantified using TMT. Data are available via ProteomeXchange with identifier PXD003346.

Probing the catalytic functions of Bub1 kinase using the small molecule inhibitors BAY-320 and BAY-524.

The kinase Bub1 functions in the spindle assembly checkpoint (SAC) and in chromosome congression, but the role of its catalytic activity remains controversial. Here, we use two novel Bub1 inhibitors, BAY-320 and BAY-524, to demonstrate potent Bub1 kinase inhibition both in vitro and in intact cells. Then, we compared the cellular phenotypes of Bub1 kinase inhibition in HeLa and RPE1 cells with those of protein depletion, indicative of catalytic or scaffolding functions, respectively. Bub1 inhibition affected chromosome association of Shugoshin and the chromosomal passenger complex (CPC), without abolishing global Aurora B function. Consequently, inhibition of Bub1 kinase impaired chromosome arm resolution but exerted only minor effects on mitotic progression or SAC function. Importantly, BAY-320 and BAY-524 treatment sensitized cells to low doses of Paclitaxel, impairing both chromosome segregation and cell proliferation. These findings are relevant to our understanding of Bub1 kinase function and the prospects of targeting Bub1 for therapeutic applications.

PICH promotes sister chromatid disjunction and co-operates with topoisomerase II in mitosis.

PICH is a SNF2 family DNA translocase that binds to ultra-fine DNA bridges (UFBs) in mitosis. Numerous roles for PICH have been proposed from protein depletion experiments, but a consensus has failed to emerge. Here, we report that deletion of PICH in avian cells causes chromosome structural abnormalities, and hypersensitivity to an inhibitor of Topoisomerase II (Topo II), ICRF-193. ICRF-193-treated PICH(-/-) cells undergo sister chromatid non-disjunction in anaphase, and frequently abort cytokinesis. PICH co-localizes with Topo IIα on UFBs and at the ribosomal DNA locus, and the timely resolution of both structures depends on the ATPase activity of PICH. Purified PICH protein strongly stimulates the catalytic activity of Topo II in vitro. Consistent with this, a human PICH(-/-) cell line exhibits chromosome instability and chromosome condensation and decatenation defects similar to those of ICRF-193-treated cells. We propose that PICH and Topo II cooperate to prevent chromosome missegregation events in mitosis.

A bacterial type III secretion-based protein delivery tool for broad applications in cell biology.

Methods enabling the delivery of proteins into eukaryotic cells are essential to address protein functions. Here we propose broad applications to cell biology for a protein delivery tool based on bacterial type III secretion (T3S). We show that bacterial, viral, and human proteins, fused to the N-terminal fragment of the Yersinia enterocolitica T3S substrate YopE, are effectively delivered into target cells in a fast and controllable manner via the injectisome of extracellular bacteria. This method enables functional interaction studies by the simultaneous injection of multiple proteins and allows the targeting of proteins to different subcellular locations by use of nanobody-fusion proteins. After delivery, proteins can be freed from the YopE fragment by a T3S-translocated viral protease or fusion to ubiquitin and cleavage by endogenous ubiquitin proteases. Finally, we show that this delivery tool is suitable to inject proteins in living animals and combine it with phosphoproteomics to characterize the systems-level impact of proapoptotic human truncated BID on the cellular network.

Assessment of current mass spectrometric workflows for the quantification of low abundant proteins and phosphorylation sites.

The data described here provide a systematic performance evaluation of popular data-dependent (DDA) and independent (DIA) mass spectrometric (MS) workflows currently used in quantitative proteomics. We assessed the limits of identification, quantification and detection for each method by analyzing a dilution series of 20 unmodified and 10 phosphorylated synthetic heavy labeled reference peptides, respectively, covering six orders of magnitude in peptide concentration with and without a complex human cell digest background. We found that all methods performed very similarly in the absence of background proteins, however, when analyzing whole cell lysates, targeted methods were at least 5-10 times more sensitive than directed or DDA methods. In particular, higher stage fragmentation (MS3) of the neutral loss peak using a linear ion trap increased dynamic quantification range of some phosphopeptides up to 100-fold. We illustrate the power of this targeted MS3 approach for phosphopeptide monitoring by successfully quantifying 9 phosphorylation sites of the kinetochore and spindle assembly checkpoint component Mad1 over different cell cycle states from non-enriched pull-down samples. The data are associated to the research article 'Evaluation of data-dependent and data-independent mass spectrometric workflows for sensitive quantification of proteins and phosphorylation sites׳ (Bauer et al., 2014) [1]. The mass spectrometry and the analysis dataset have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000964.