170,772 research outputs found
On the regulation of centriole duplication in human cells : exploring the interactions of polo-like kinase 4 with the centrosomal proteins Cep192 and STIL
No full textCentrioles duplicate once in each cell cycle to give rise to two centrosomes that form the spindle poles during mitosis. Aberrant centriole duplication can result in the formation of supernumerary centrosomes, leading to incorrect spindle assembly and chromosome segregation errors, thereby possibly contributing to carcinogenesis (Ganem et al., 2009; Nigg, 2002; Zyss and Gergely, 2008). Thus, to ensure genome stability, centriole duplication has to be precisely regulated. Polo-like kinase 4 (PLK4) is a key regulator of centriole duplication (Bettencourt-Dias et al., 2005; Habedanck et al., 2005). PLK4 is characterized by an N-terminal Ser/Thr kinase domain and three C-terminal Polo-boxes (PB1-PB3) (Slevin et al., 2012). The PB1-PB2 domain is required for PLK4's centrosomal localization and binding to Cep152 (Cizmecioglu et al., 2010; Hatch et al., 2010; Slevin et al., 2012). In contrast to PB1-PB2, no binding partners have been described for PB3.
Here, we identify Cep192 and STIL as novel interaction partners of PLK4-PB1-PB2 and PLK4-PB3, respectively. In the first part of this study, we reveal that Cep192 directly binds PB1-PB2 via a short region within its N-terminus, which contains conserved patches of acidic residues. We show that also in the case of Cep152 a short N-terminal acidic region is critical for the binding to PB1-PB2. These acidic regions of Cep192 and Cep152 enable electrostatic interactions with positively charged residues of the PB1-PB2 domain in order to promote PLK4 centriolar recruitment (Sonnen et al., 2013). In the second part of this study, we identify STIL as the first known binding partner of PLK4-PB3. We show that the coiled-coil motif of STIL (STIL-CC) is necessary and sufficient for this interaction and thus important for centriole duplication. Based on a collaboration for crystallographic and NMR analyses, we furthermore demonstrate that PB3 adopts a canonical PB fold, and that the PLK4-PB3/STIL-CC binding mimics coiled-coil formation. Analysis of structure-guided STIL mutants suggests a dual binding mode of STIL-CC to PB3 and L1 of PLK4 (linker between the catalytic domain and the PB domains). Taken together, we propose a speculative model for the initial steps of procentriole assembly according to which PLK4 is recruited to centrioles by electrostatic interactions between PB1-PB2 and Cep192/Cep152, and thereafter is stabilized and activated via STIL-CC binding to PB3 and L1
Control of centriole numbers by Plk4 autophosphorylation and βTrCP-mediated degradation
No full textProper centrosome numbers are imperative for faithful cell division, as aberrant centrosome numbers can lead to chromosomal instability, a hallmark of cancer development (Nigg 2002; Ganem et al., 2009). Hence, initiation of centriole duplication has to be tightly regulated. Recently, we and others demonstrated that Polo-like kinase 4 (Plk4) fulfills a pivotal role in regulating this process (Bettencourt-Dias et al., 2005; Habedanck et al., 2005). Plk4 protein levels and its activity directly correlate with centriole numbers: depletion of Plk4 leads to sequential loss of centrioles in successive cell divisions (Bettencourt-Dias et al., 2005; Habedanck et al., 2005) and its overexpression promotes bona fide overduplication of centrioles (Habedanck et al., 2005; Kleylein-Sohn et al., 2007), while both lead to progressive increase in abnormal spindle formation (reviewed in Nigg 2007). Even though Plk4 is a key regulator of centriole biogenesis and is crucial for maintaining constant centriole number, the mechanisms regulating its activity and expression are only beginning to emerge.
Here, we show that human Plk4 is subject to beta-TrCP-dependent proteasomal degradation, indicating that this pathway is conserved from Drosophila to human (Cunha-Ferreira et al., 2009; Rogers et al., 2009). Unexpectedly, we found that stable overexpression of kinase-dead Plk4 leads to centriole overduplication. Our data indicate that this phenotype depends on the presence of endogenous wild-type Plk4 and that centriole overduplication results from disruption of Plk4 trans-autophosphorylation by kinase-dead Plk4, which then shields endogenous Plk4 from recognition by beta-TrCP. We conclude that active Plk4 promotes its own degradation by catalyzing beta-TrCP binding through trans-autophosphorylation within homodimers which has been independently confirmed by others (Holland et al., 2010). Additionally, we propose that Plk4 autophosphorylation is not sufficient for its degradation and that instead an additional kinase is required for this process
Expression of chicken lamin B2 in Escherichia coli : characterization of its structure, assembly, and molecular interactions
No full textChicken lamin B2, a nuclear member of the intermediate-type filament (IF) protein family, was expressed as a full-length protein in Escherichia coli. After purification, its structure and assembly properties were explored by EM, using both glycerol spraying/low-angle rotary metal shadowing and negative staining for preparation, as well as by analytical ultracentrifugation. At its first level of structural organization, lamin B2 formed "myosin-like" 3.1S dimers consisting of a 52-nm-long tail flanked at one end by two globular heads. These myosin-like molecules are interpreted to represent two lamin polypeptides interacting via their 45-kD central rod domains to form a segmented, parallel and unstaggered 52-nm-long two-stranded alpha-helical coiled-coil, and their COOH-terminal end domains folding into globular heads. At the second level of organization, lamin B2 dimers associated longitudinally to form polar head-to-tail polymers. This longitudinal mode of association of laminin dimers is in striking contrast to the lateral mode of association observed previously for cytoplasmic IF dimers. At the third level of organization, these polar head-to-tail polymers further associated laterally, in an approximately half-staggered fashion, to form filamentous and eventually paracrystal-like structures revealing a pronounced 24.5-nm axial repeat. Finally, following up on recent studies implicating the mitotic cdc2 kinase in the control of lamin polymerization (Peter, M., J. Nakagawa, M. Dorée, J. C. Labbé, and E. A. Nigg. 1990. Cell. 61:591-602), we have examined the effect of phosphorylation by purified cdc2 kinase on the assembly properties and molecular interactions of the bacterially expressed lamin B2. Phosphorylation of chicken lamin B2 by cdc2 kinase interferes with the head-to-tail polymerization of the lamin dimers. This finding supports the notion that cdc2 kinase plays a major, direct role in triggering mitotic disassembly of the nuclear lamina
Investigation of human centrosomes - with a special focus on the function of Cep152 and Cep192 in centriole duplication
No full textCentrioles are symmetrical, barrel-shaped, microtubule-based structures, that serve as building platforms for the formation of centrosomes and cilia. In dividing cells, they recruit a matrix of proteins called pericentriolar material (PCM) to form the centrosome, the major microtubule-organizing centre in animal cells. In differentiating cells, centrioles also function as basal bodies for the formation of flagella and cilia. To ensure the proper segregation of centrioles during cell division, centriole duplication is tightly controlled and coordinated with the cell cycle. To date, several factors have been identified in human cells that are recruited in a consecutive fashion to nascent centrioles to template the outgrowth of one procentriole orthogonally to the pre-existing one during each cell cycle. In addition to structural components, these duplication factors include the kinase Plk4 (polo-like kinase 4), which is pivotal for the initiation of centriole duplication.
Here, we have taken two approaches to investigate centrosomes in more detail: first, the human centrosomal proteins Cep192 and Cep152 were functionally characterized; second, the localization of key centrosomal and centriolar components was analyzed using super-resolution three-dimensional structured illumination microscopy (3D-SIM).
1. Previously, Cep192 and Cep152 had been identified as novel centrosomal proteins in a proteomic screen. Homologues of Cep152 in flies and zebrafish had been implicated in PCM recruitment and centrosome duplication, but Cep152 had not been investigated in humans. Likewise, the worm homologue of Cep192 also functions in PCM recruitment and centrosome duplication. However, in humans its role in centriole duplication remained controversial.
Here, we have established that Cep152 is dispensable, whereas Cep192 is essential for PCM recruitment in mitotic cells. This functional difference is further illustrated by their differential subcellular localizations during interphase and mitosis. The stable centrosomal integration of Cep152 depended on Cep192, whereas Cep192 localized independently of Cep152.
Furthermore, both Cep152 and Cep192 were required for proper centriole duplication, thereby clarifying the controversy about the implication of Cep192 in this process. We also show that Cep192 and Cep152 co-operate in the centriolar recruitment of the kinase Plk4. Concomitantly, centriole duplication was only inhibited to a similar extent as in Plk4- or Sas-6-depleted cells, if both Cep152 and Cep192 were depleted. In agreement, we have identified and characterized interactions of Plk4 with the N termini of both Cep152 and Cep192. Finally, not only the recruitment of Plk4, but also of other duplication factors such as CPAP and Sas-6 was impaired in Cep152- and/ or Cep192-depleted cells.
We have also addressed the regulation of Cep152 and Cep192. Centrosomal levels of both proteins increased towards mitosis. Similarly, cytoplasmic Cep152 levels peaked when cells approached mitosis, whereas Cep192 levels were stable.
Hence, we show that both Cep152 and Cep192 function as centriole duplication factors. Both proteins exert a partly redundant function and their co-operation orchestrates recruitment of Plk4 as well as other centriole duplication factors and thus canonical centriole duplication.
2. Using 3D-SIM we have analysed the spatial relationship of 18 centriole and PCM components of human centrosomes at different cell cycle stages.
During mitosis, PCM proteins formed extended networks with interspersed gamma-Tubulin. In interphase, most proteins were arranged at specific and defined distances from the walls of centrioles, resulting in ring-like staining. Additionally, orientation of Cep152 with its C-terminus close to centriole walls and its N-terminus facing outwards was visualised using site-specific antibodies against either terminus of the protein. At the distal end of centrioles, appendage proteins formed rings with several density masses, usually with a multiplicity below that expected from the 9-fold symmetry of centrioles. Although Cep164 remained centriolar throughout the cell cycle, the number of discernible density masses was clearly reduced during mitosis. At the proximal end, Sas-6 formed a dot at the site of daughter centriole formation, consistent with its role in cartwheel formation. Plk4 and STIL co-localized with Sas-6, but the bulk of the cartwheel protein Cep135 was associated with mother centrioles. Remarkably, Plk4 formed a dot on the surface of the mother centriole even before Sas-6 staining became detectable, indicating that Plk4 constitutes an early marker for the site of nascent centriole formation
Are language production problems apparent in adults who no longer meet diagnostic criteria for attention-deficit/hyperactivity disorder?
No full textIn this study, we examined sentence production in a sample of adults (N = 21) who had had attention-deficit/hyperactivity disorder (ADHD) as children, but as adults no longer met DSM-IV diagnostic criteria (APA, 2000). This “remitted” group was assessed on a sentence production task. On each trial, participants saw two objects and a verb. Their task was to construct a sentence using the objects as arguments of the verb. Results showed more ungrammatical and disfluent utterances with one particular type of verb (i.e., participle). In a second set of analyses, we compared the remitted group to both control participants and a “persistent” group, who had ADHD as children and as adults. Results showed that remitters were more likely to produce ungrammatical utterances and to make repair disfluencies compared to controls, and they patterned more similarly to ADHD participants. Conclusions focus on language output in remitted ADHD, and the role of executive functions in language production
Characterization of mitotic checkpoint complexes
No full textIn eukaryotes, chromosome segregation critically depends on the establishment of productive contacts between kinetochores (KTs), specialized chromosomal structures, and the spindle microtubules (MTs). In mitosis, the spindle assembly checkpoint (SAC) is the major surveillance mechanism that restrains anaphase onset until all KTs become bi-oriented by spindle MTs. Several SAC proteins act in concert to relay the presence of unattached KTs to the cell cycle machinery in the cytoplasm. The SAC protein Mad2 plays a pivotal role in this signal transduction cascade, contributing both to the KT sensor and to the SAC cytoplasmic effector. Mad2 can fold into two distinct conformers, Open (O) and Closed (C), and can asymmetrically dimerize. Biophysical and structural work had demonstrated that the conformational dynamics of Mad2 is crucial for its activation in vitro, but models arising from this work could not be exhaustively tested in cells. Here, we describe a monoclonal antibody that specifically recognizes the dimerization interface of C-Mad2. This antibody revealed several conformation specific features of Mad2 in human cells. Notably, we show that Mad2 requires association with its KT-receptor Mad1 to adopt the Closed conformation. Furthermore, C-Mad2 antibody microinjection interfered with Mad2 asymmetric dimerization and abrogated the SAC, accelerating mitotic progression. Remarkably, microinjection of a Mad1-neutralizing antibody triggered a comparable mitotic acceleration. Finally, we show that the activity of the Mad1:C-Mad2 complex undergoes regulation by p31comet-dependent ‘capping’. We also suggest that p31comet capping is negatively regulated by the SAC kinase Mps1 and the SAC regulator Tpr. Collectively, this work provides direct in vivo evidence for the model that a KT complex of Mad1:C-Mad2 acts as a template to sustain the SAC and it challenges the distinction between SAC and mitotic timer
C-Nap1, a novel centrosomal coiled-coil protein and candidate substrate of the cell cycle-regulated protein kinase Nek2
No full textNek2 (for NIMA-related kinase 2) is a mammalian cell cycle-regulated kinase structurally related to the mitotic regulator NIMA of Aspergillus nidulans. In human cells, Nek2 associates with centrosomes, and overexpression of active Nek2 has drastic consequences for centrosome structure. Here, we describe the molecular characterization of a novel human centrosomal protein, C-Nap1 (for centrosomal Nek2-associated protein 1), first identified as a Nek2-interacting protein in a yeast two-hybrid screen. Antibodies raised against recombinant C-Nap1 produced strong labeling of centrosomes by immunofluorescence, and immunoelectron microscopy revealed that C-Nap1 is associated specifically with the proximal ends of both mother and daughter centrioles. On Western blots, anti-C-Nap1 antibodies recognized a large protein (<250 kD) that was highly enriched in centrosome preparations. Sequencing of overlapping cDNAs showed that C-Nap1 has a calculated molecular mass of 281 kD and comprises extended domains of predicted coiled-coil structure. Whereas C-Nap1 was concentrated at centrosomes in all interphase cells, immunoreactivity at mitotic spindle poles was strongly diminished. Finally, the COOH-terminal domain of C-Nap1 could readily be phosphorylated by Nek2 in vitro, as well as after coexpression of the two proteins in vivo. Based on these findings, we propose a model implicating both Nek2 and C-Nap1 in the regulation of centriole-centriole cohesion during the cell cycle
Rootletin forms centriole-associated filaments and functions in centrosome cohesion
No full textAfter duplication of the centriole pair during S phase, the centrosome functions as a single microtubule-organizing center until the onset of mitosis, when the duplicated centrosomes separate for bipolar spindle formation. The mechanisms regulating centrosome cohesion and separation during the cell cycle are not well understood. In this study, we analyze the protein rootletin as a candidate centrosome linker component. As shown by immunoelectron microscopy, endogenous rootletin forms striking fibers emanating from the proximal ends of centrioles. Moreover, rootletin interacts with C-Nap1, a protein previously implicated in centrosome cohesion. Similar to C-Nap1, rootletin is phosphorylated by Nek2 kinase and is displaced from centrosomes at the onset of mitosis. Whereas the overexpression of rootletin results in the formation of extensive fibers, small interfering RNA-mediated depletion of either rootletin or C-Nap1 causes centrosome splitting, suggesting that both proteins contribute to maintaining centrosome cohesion. The ability of rootletin to form centriole-associated fibers suggests a dynamic model for centrosome cohesion based on entangling filaments rather than continuous polymeric linkers
The centrosomal protein C-Nap1 is required for cell cycle-regulated centrosome cohesion
No full textDuplicating centrosomes are paired during interphase, but are separated at the onset of mitosis. Although the mechanisms controlling centrosome cohesion and separation are important for centrosome function throughout the cell cycle, they remain poorly understood. Recently, we have proposed that C-Nap1, a novel centrosomal protein, is part of a structure linking parental centrioles in a cell cycle-regulated manner. To test this model, we have performed a detailed structure-function analysis on C-Nap1. We demonstrate that antibody-mediated interference with C-Nap1 function causes centrosome splitting, regardless of the cell cycle phase. Splitting occurs between parental centrioles and is not dependent on the presence of an intact microtubule or microfilament network. Centrosome splitting can also be induced by overexpression of truncated C-Nap1 mutants, but not full-length protein. Antibodies raised against different domains of C-Nap1 prove that this protein dissociates from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Use of the same antibodies in immunoelectron microscopy shows that C-Nap1 is confined to the proximal end domains of centrioles, indicating that a putative linker structure must contain additional proteins. We conclude that C-Nap1 is a key component of a dynamic, cell cycle-regulated structure that mediates centriole-centriole cohesion
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