The FAcilitates Chromatin Transcription (FACT) complex is a dimeric histone chaperone that operates on chromatin during transcription and replication. FACT also interacts with a specialized centromeric nucleosome containing the histone H3 variant centromere protein A (CENP-A) and with CENP-TW, two subunits of the constitutive centromere-associated network (CCAN), a 16-protein complex associated with CENP-A. The significance of these interactions remains elusive. Here, we show that FACT has multiple additional binding sites on CCAN. The interaction with CCAN is strongly stimulated by casein kinase II phosphorylation of FACT. Mitotic localization of FACT to kinetochores is strictly dependent on specific CCAN subcomplexes. Conversely, CENP-TW requires FACT for stable localization. Unexpectedly, we also find that DNA readily displaces FACT from CCAN, supporting the speculation that FACT becomes recruited through a pool of CCAN that is not stably integrated into chromatin. Collectively, our results point to a potential role of FACT in chaperoning CCAN during transcription or in the stabilization of CCAN at the centromere during the cell cycle.

MYCBP2 (PAM) is a large signaling hub that plays a key role in various processes, including neuronal connectivity and growth, cell division, and protein ubiquitination. Together with the substrate specificity factor FBXO45, MYCBP2 forms an E3 ligase complex that is involved in mitotic cell fate decision. During extended mitotic arrest caused by anti-microtubule drugs, cells may either experience cell death or escape mitosis through mitotic slippage. E3 ligase mediated ubiquitination is antagonized by deubiquitinating enzymes (DUBs). In this study, we show that despite their opposing activities, DUB-E3 ligase complexes can form and cooperate. We identify an E3 ligase complex consisting of MYCBP2 and a new substrate specificity factor, SPRYD3. Interestingly, SPRYD3-MYCBP2 promotes bipolar spindle formation by facilitating non-canonical ubiquitination on the DUB USP11 cysteine 318. We find that this process promotes bipolar spindle formation and mitotic slippage in presence of microtubule targeting drugs.

Centriole duplication is regulated by polo-like kinase 4 (PLK4) and several conserved initiator proteins. The precise timing and regulation of PLK4 activation are critical for ensuring that centriole duplication occurs only once per cell cycle. Although significant progress has been made in understanding how PLK4 is activated, many aspects remain unclear. Here, we show how CEP152 contributes to the activation of PLK4. We utilize human cell lines that have been genetically engineered to rapidly degrade CEP152. Upon degradation of CEP152, localization of PLK4 at the proximal end of the centriole is disrupted. We show that binding of CEP152 N-terminal part to PLK4 increases phosphorylation and kinase activation. CEP152 controls the localization and levels of phosphorylated PLK4 at the proximal end of the centriole. CEP152 binding to PLK4 leads to phosphorylation and activation of PLK4 that might stabilize PLK4 dimer formation, thus allowing autophosphorylation. We propose that CEP152 activates PLK4 to ensure proper centriole duplication at the onset of S-phase.

Centrioles play important roles in the assembly of centrosomes and cilia. Centriole duplication occurs once per cell cycle and is dependent on polo-like kinase 4 (PLK4). To prevent centriole amplification, which is a hallmark of cancer, PLK4 protein levels need to be tightly regulated. Here, we show that the Cullin4A/B-DDB1-DCAF1, CRL4DCAF1, E3 ligase targets PLK4 for degradation in human cells. DCAF1 binds and ubiquitylates PLK4 in the G2 phase to prevent premature centriole duplication in mitosis. In contrast to the regulation of PLK4 by SCFβ-TrCP, the interaction between PLK4 and DCAF1 is independent of PLK4 kinase activity and mediated by polo-boxes 1 and 2 of PLK4, suggesting that DCAF1 promotes PLK4 ubiquitylation independently of β-TrCP. Thus, the SCFSlimb/β-TrCPpathway, targeting PLK4 for ubiquitylation based on its phosphorylation state and CRL4DCAF1, which ubiquitylates PLK4 by binding to the conserved PB1-PB2 domain, appear to be complementary ways to control PLK4 abundance to prevent centriole overduplication.

During prolonged mitotic arrest induced by anti-microtubule drugs, cell fate decision is determined by two alternative pathways, one leading to cell death, the other inducing premature escape from mitosis by mitotic slippage. FBWX7, a member of the F-box family of proteins and substrate-targeting subunit of the SCF (SKP1-CUL1-F-Box) E3 ubiquitin ligase complex promotes mitotic cell death and prevents mitotic slippage. In this study, we report that WDR5, a component of the mixed lineage leukemia (MLL) complex of Histone 3 Lysine 4 (H3K4) methyltransferases is a substrate of FBXW7. WDR5 binds to FBXW7 in vivo and in vitro and its ubiquitin-mediated proteasomal degradation is mediated by FBXW7. Furthermore, we find that WDR5 depletion counteracts FBXW7 loss-of-function by reducing mitotic slippage and polyploidization. Our data elucidate a new mechanism in mitotic cell fate regulation which might contribute to prevent chemotherapy resistance in patients after anti-microtubule drug treatment.

Centrioles play important roles in the assembly of centrosomes and cilia. Centriole duplication occurs once per cell cycle and is dependent on polo-like kinase 4 (PLK4). To prevent centriole amplification, which is a hallmark of cancer, PLK4 protein levels need to be tightly regulated. Here, we show that the Cullin(CUL)4A/B-DDB1-DCAF1, CRL4 DCAF1 , E3 ligase targets PLK4 for degradation in human cells. DCAF1 binds and ubiquitylates PLK4 in G2 phase to prevent premature centriole duplication in mitosis. In contrast to the regulation of PLK4 by SCF β-TrCP , the interaction between PLK4 and DCAF1 is independent of PLK4 kinase activity and mediated by polo-boxes 1 and 2 of PLK4, suggesting that DCAF1 promotes PLK4 ubiquitylation independently of β-TrCP. Thus, the SCF Slimb/β-TrCP pathway, targeting PLK4 for ubiquitylation based on its phosphorylation state and CRL4 DCAF1 , which ubiquitylates PLK4 by binding to the conserved PB1-PB2 domain, appear to be complementary ways to control PLK4 abundance to prevent centriole overduplication.

Centromeres are specialized chromosome loci that seed the kinetochore, a large protein complex that effects chromosome segregation. The organization of the interface between the kinetochore and the specialized centromeric chromatin, marked by the histone H3 variant CENP-A, remains incompletely understood. A 16-subunit complex, the constitutive centromere associated network (CCAN), bridges CENP-A to the spindle-binding moiety of the kinetochore. Here, we report a cryo-electron microscopy structure of human CCAN. We highlight unique features such as the pseudo GTPase CENP-M and report how a crucial CENP-C motif binds the CENP-LN complex. The CCAN structure has also important implications for the mechanism of specific recognition of the CENP-A nucleosome. A supported model depicts the interaction as fuzzy and identifies the disordered CCAN subunit CENP-C as only determinant of specificity. A more speculative model identifies both CENP-C and CENP-N as specificity determinants, but implies CENP-A may be in a hemisome rather than in a classical octamer.

During prolonged mitotic arrest induced by antimicrotubule drugs, cell fate decision is determined by two alternative pathways, one leading to cell death and the other inducing premature escape from mitosis by mitotic slippage. FBWX7, a member of the F-box family of proteins and substrate-targeting subunit of the SKP1-CUL1-F-Box E3 ubiquitin ligase complex, promotes mitotic cell death and prevents mitotic slippage, but molecular details underlying these roles for FBWX7 are unclear. In this study, we report that WDR5 (WD-repeat containing protein 5), a component of the mixed lineage leukemia complex of histone 3 lysine 4 methyltransferases, is a substrate of FBXW7. We determined by coimmunoprecipitation experiments and in vitro binding assays that WDR5 interacts with FBXW7 in vivo and in vitro. SKP1-CUL1-F-Box-FBXW7 mediates ubiquitination of WDR5 and targets it for proteasomal degradation. Furthermore, we find that WDR5 depletion counteracts FBXW7 loss of function by reducing mitotic slippage and polyploidization. In conclusion, our data elucidate a new mechanism in mitotic cell fate regulation, which might contribute to prevent chemotherapy resistance in patients after antimicrotubule drug treatment.

Centrosomes nucleate and anchor microtubules and therefore play major roles in spindle formation and chromosome segregation during mitosis. Duplication of the centrosome occurs, similar to DNA, only once during the cell cycle. Aberration of the centrosome number is common in human tumors. At the core of centriole duplication is the conserved polo-like kinase 4, Plk4, and two structural proteins, STIL and Sas-6. In this review, I summarize and discuss developments in our understanding of the first steps of centriole duplication and their regulation.

The Epstein-Barr virus (EBV) transforms resting B cells and is involved in the development of B cell lymphomas. We report here that the viral noncoding RNA EBER2 accelerates B cell growth by potentiating expression of the UCHL1 deubiquitinase that itself increased expression of the Aurora kinases and of cyclin B1. Importantly, this effect was also visible in Burkitt's lymphoma cells that express none of the virus's known oncogenes. Mechanistically, EBER2 bound the UCHL1 messenger RNA (mRNA), thereby bringing a protein complex that includes PU.1, a UCHL1 transactivator, to the vicinity of its promoter. Although the EBV oncogene LMP1 has been suggested to induce UCHL1, we show here that EBER2 plays a much more important role to reach significant levels of the deubiquitinase in infected cells. However, some viruses that carried a polymorphic LMP1 had an increased ability to achieve full UCHL1 expression. This work identifies a direct cellular target of a viral noncoding RNA that is likely to be central to EBV's oncogenic properties.

The centrosome is present in most animal cells and functions as the major microtubule-organizing center to ensure faithful chromosome segregation during cell division. As cells transition from interphase to mitosis, the duplicated centrosomes separate and move to opposite sides of the cell where the spindle assembles. Centrosomes not only nucleate but also organize microtubules of the mitotic spindle. The mitotic spindle is anchored to the cell cortex by the astral microtubules emanating from the centrosomes. Proper orientation of the mitotic spindle is essential for correct cell division. Centrosome-localized polo-like kinase Plk1 has been linked to regulation of proper spindle orientation. A number of proteins including MISP and NuMA have been implicated in the Plk1-mediated spindle orientation pathway.

Cell fate decision upon prolonged mitotic arrest induced by microtubule-targeting agents depends on the activity of the tumor suppressor and F-box protein FBXW7. FBXW7 promotes mitotic cell death and prevents premature escape from mitosis through mitotic slippage. Mitotic slippage is a process that can cause chemoresistance and tumor relapse. Therefore, understanding the mechanisms that regulate the balance between mitotic cell death and mitotic slippage is an important task. Here we report that FBXW7 protein levels markedly decline during extended mitotic arrest. FBXO45 binds to a conserved acidic N-terminal motif of FBXW7 specifically under a prolonged delay in mitosis, leading to ubiquitylation and subsequent proteasomal degradation of FBXW7 by the FBXO45-MYCBP2 E3 ubiquitin ligase. Moreover, we find that FBXO45-MYCBP2 counteracts FBXW7 in that it promotes mitotic slippage and prevents cell death in mitosis. Targeting this interaction represents a promising strategy to prevent chemotherapy resistance.

Microtubule nucleation was uncovered as a key principle of spindle assembly. However, the mechanistic details about microtubule nucleation and the organization of spindle formation and symmetry are currently being revealed. Here we describe the function of coiled-coil domain containing 61 (Ccdc61), a so far uncharacterized centrosomal protein, in spindle assembly and symmetry. Our data describe that Ccdc61 is required for spindle assembly and precise chromosome alignments in mitosis. Microtubule tip-tracking experiments in the absence of Ccdc61 reveal a clear loss of the intrinsic symmetry of microtubule tracks within the spindle. Furthermore, we show that Ccdc61 controls the centrosomal localization of centrosomal protein of 170 kDa (Cep170), a protein that was shown previously to localize to centrosomes as well as spindle microtubules and promotes microtubule organization and microtubule assembly. Interestingly, selective disruption of Ccdc61 impairs the binding between Cep170 and TANK binding kinase 1, an interaction that is required for microtubule stability. In summary, we have discovered Ccdc61 as a centrosomal protein with an important function in mitotic microtubule organization.

ABSTRACT Correct spindle orientation is achieved through signaling pathways that provide a molecular link between the cell cortex and spindle microtubules in an F-actin-dependent manner. A conserved cortical protein complex, composed of LGN (also known as GPSM2), NuMA (also known as NUMA1) and dynein–dynactin, plays a key role in establishing proper spindle orientation. It has also been shown that the actin-binding protein MISP and the ERM family, which are activated by lymphocyte-oriented kinase (LOK, also known as STK10) and Ste20-like kinase (SLK) (hereafter, SLK/LOK) in mitosis, regulate spindle orientation. Here, we report that MISP functions downstream of the ERM family member ezrin and upstream of NuMA to allow optimal spindle positioning. We show that MISP directly interacts with ezrin and that SLK/LOK-activated ezrin ensures appropriate cortical MISP levels in mitosis by competing with MISP for actin-binding sites at the cell cortex. Furthermore, we found that regulation of the correct cortical MISP levels, by preventing its excessive accumulation, is essential for crescent-like polarized NuMA localization at the cortex and, as a consequence, leads to highly dynamic astral microtubules. Our results uncover how appropriate MISP levels at the cortex are required for proper NuMA polarization and, therefore, an optimal placement of the mitotic spindle within the cell. This article has an associated First Person interview with the first author of the paper.

Precise mitotic spindle orientation is essential for both cell fate and tissue organization while defects in this process are associated with tumorigenesis and other diseases. In most animal cell types, the dynein motor complex is anchored at the cell cortex and exerts pulling forces on astral microtubules to position the spindle. The actin-binding protein MISP controls spindle orientation and mitotic progression in human cells. However, the exact underlying mechanism remains to be elucidated. Here we report that MISP interacts with the multidomain scaffolding protein IQGAP1. We further show that MISP binds to the active form of Cdc42 through IQGAP1. Depletion of MISP promotes increased accumulation of IQGAP1 at the cell cortex and a decrease in its Cdc42-binding capacity leading to reduced active Cdc42 levels. Interestingly, overexpression of IQGAP1 can rescue mitotic defects caused by MISP downregulation including spindle misorientation, loss of astral microtubules and prolonged mitosis and also restores active Cdc42 levels. Importantly, we find that IQGAP1 acts downsteam of MISP in regulating astral microtubule dynamics and the localization of the dynactin subunit p150glued that is crucial for proper spindle positioning. We propose that MISP regulates IQGAP1 and Cdc42 to ensure proper mitotic progression and correct spindle orientation.

Infections with Epstein-Barr virus (EBV) are associated with cancer development, and EBV lytic replication (the process that generates virus progeny) is a strong risk factor for some cancer types. Here we report that EBV infection of B-lymphocytes (in vitro and in a mouse model) leads to an increased rate of centrosome amplification, associated with chromosomal instability. This effect can be reproduced with virus-like particles devoid of EBV DNA, but not with defective virus-like particles that cannot infect host cells. Viral protein BNRF1 induces centrosome amplification, and BNRF1-deficient viruses largely lose this property. These findings identify a new mechanism by which EBV particles can induce chromosomal instability without establishing a chronic infection, thereby conferring a risk for development of tumours that do not necessarily carry the viral genome.