Pericentriolar Material Research Articles

The Drosophila Pericentrin-like-protein (PLP) cooperates with Cnn to maintain the integrity of the outer PCM.

ABSTRACTCentrosomes comprise a pair of centrioles surrounded by a matrix of pericentriolar material (PCM). In vertebrate cells, Pericentrin plays an important part in mitotic PCM assembly, but the Drosophila Pericentrin-like protein (PLP) appears to have a more minor role in mitotic fly cells. Here we investigate the function of PLP during the rapid mitotic cycles of the early Drosophila embryo. Unexpectedly, we find that PLP is specifically enriched in the outer-most regions of the PCM, where it largely co-localizes with the PCM scaffold protein Cnn. In the absence of PLP the outer PCM appears to be structurally weakened, and it rapidly disperses along the centrosomal microtubules (MTs). As a result, centrosomal MTs are subtly disorganized in embryos lacking PLP, although mitosis is largely unperturbed and these embryos develop and hatch at near-normal rates. Y2H analysis reveals that PLP can potentially form multiple interactions with itself and with the PCM recruiting proteins Asl, Spd-2 and Cnn. A deletion analysis suggests that PLP participates in a complex network of interactions that ultimately help to strengthen the PCM.

Opposing effects of pericentrin and microcephalin on the pericentriolar material regulate CHK1 activation in the DNA damage response.

Genotoxic stresses lead to centrosome amplification, a frequently-observed feature in cancer that may contribute to genome instability and to tumour cell invasion. Here we have explored how the centrosome controls DNA damage responses. For most of the cell cycle, centrosomes consist of two centrioles embedded in the proteinaceous pericentriolar material (PCM). Recent data indicate that the PCM is not an amorphous assembly of proteins, but actually a highly organised scaffold around the centrioles. The large coiled-coil protein, pericentrin, participates in PCM assembly and has been implicated in the control of DNA damage responses (DDRs) through its interactions with checkpoint kinase 1 (CHK1) and microcephalin (MCPH1). CHK1 is required for DNA damage-induced centrosome amplification, whereas MCPH1 deficiency greatly increases the amplification seen after DNA damage. We found that the PCM showed a marked expansion in volume and a noticeable change in higher-order organisation after ionising radiation treatment. PCM expansion was dependent on CHK1 kinase activity and was potentiated by MCPH1 deficiency. Furthermore, pericentrin deficiency or mutation of a separase cleavage site blocked DNA damage-induced PCM expansion. The extent of nuclear CHK1 activation after DNA damage reflected the level of PCM expansion, with a reduction in pericentrin-deficient or separase cleavage site mutant-expressing cells, and an increase in MCPH1-deficient cells that was suppressed by the loss of pericentrin. Deletion of the nuclear export signal of CHK1 led to its hyperphosphorylation after irradiation and reduced centrosome amplification. Deletion of the nuclear localisation signal led to low CHK1 activation and low centrosome amplification. From these data, we propose a feedback loop from the PCM to the nuclear DDR in which CHK1 regulates pericentrin-dependent PCM expansion to control its own activation.

Pericentrin interacts with KASH domain-containing protein Syne-2

Objective Pericentrin, a highly conserved protein of the pericentriolar material, serves as a multifunctional scaffold for numerous proteins and plays an important role in microtubule organization. Mutations in the human PCNT gene are associated with a range of diseases including primordial dwarfism and ciliopathies. In the mouse retina Pericentrin colocalizes with several proteins responsible for transport processes at the connecting cilium between the two photoreceptor compartments. In order to get more insights on the function of Pericentrin in the retina we try to identify new ciliary as well as centrosomal interaction partners.

NSUN6 is a human RNA methyltransferase that catalyzes formation of m5C72 in specific tRNAs.

Many cellular RNAs require modification of specific residues for their biogenesis, structure, and function. 5-methylcytosine (m5C) is a common chemical modification in DNA and RNA but in contrast to the DNA modifying enzymes, only little is known about the methyltransferases that establish m5C modifications in RNA. The putative RNA methyltransferase NSUN6 belongs to the family of Nol1/Nop2/SUN domain (NSUN) proteins, but so far its cellular function has remained unknown. To reveal the target spectrum of human NSUN6, we applied UV crosslinking and analysis of cDNA (CRAC) as well as chemical crosslinking with 5-azacytidine. We found that human NSUN6 is associated with tRNAs and acts as a tRNA methyltransferase. Furthermore, we uncovered tRNACys and tRNAThr as RNA substrates of NSUN6 and identified the cytosine C72 at the 3′ end of the tRNA acceptor stem as the target nucleoside. Interestingly, target recognition in vitro depends on the presence of the 3′-CCA tail. Together with the finding that NSUN6 localizes to the cytoplasm and largely colocalizes with marker proteins for the Golgi apparatus and pericentriolar matrix, our data suggest that NSUN6 modifies tRNAs in a late step in their biogenesis.

Interphase centrosome organization by the PLP-Cnn scaffold is required for centrosome function.

Pericentriolar material (PCM) mediates the microtubule (MT) nucleation and anchoring activity of centrosomes. A scaffold organized by Centrosomin (Cnn) serves to ensure proper PCM architecture and functional changes in centrosome activity with each cell cycle. Here, we investigate the mechanisms that spatially restrict and temporally coordinate centrosome scaffold formation. Focusing on the mitotic-to-interphase transition in Drosophila melanogaster embryos, we show that the elaboration of the interphase Cnn scaffold defines a major structural rearrangement of the centrosome. We identify an unprecedented role for Pericentrin-like protein (PLP), which localizes to the tips of extended Cnn flares, to maintain robust interphase centrosome activity and promote the formation of interphase MT asters required for normal nuclear spacing, centrosome segregation, and compartmentalization of the syncytial embryo. Our data reveal that Cnn and PLP directly interact at two defined sites to coordinate the cell cycle-dependent rearrangement and scaffolding activity of the centrosome to permit normal centrosome organization, cell division, and embryonic viability.

ATF5 Connects the Pericentriolar Materials to the Proximal End of the Mother Centriole

Although it is known that the centrioles play instructive roles in pericentriolar material (PCM) assembly and that the PCM is essential for proper centriole formation, the mechanism that governs centriole-PCM interaction is poorly understood. Here, we show that ATF5 forms a characteristic 9-fold symmetrical ring structure in the inner layer of the PCM outfitting the proximal end of the mother centriole. ATF5 controls the centriole-PCM interaction in a cell-cycle- and centriole-age-dependent manner. Interaction of ATF5 with polyglutamylated tubulin (PGT) on the mother centriole and with PCNT in the PCM renders ATF5 as a required molecule in mother centriole-directed PCM accumulation and in PCM-dependent centriole formation. ATF5 depletion blocks PCM accumulation at the centrosome and causes fragmentation of centrioles, leading to the formation of multi-polar mitotic spindles and genomic instability. These data show that ATF5 is an essential structural protein that is required for the interaction between the mother centriole and the PCM.

SPD-2/CEP192 and CDK Are Limiting for Microtubule-Organizing Center Function at the Centrosome

The centrosome acts as the microtubule-organizing center (MTOC) during mitosis in animal cells. Microtubules are nucleated and anchored by γ-tubulin ring complexes (γ-TuRCs) embedded within the centrosome's pericentriolar material (PCM). The PCM is required for the localization of γ-TuRCs, and both are steadily recruited to the centrosome, culminating in a peak in MTOC function in metaphase. In differentiated cells, the centrosome is often attenuated as an MTOC and MTOC function is reassigned to non-centrosomal sites such as the apical membrane in epithelial cells, the nuclear envelope in skeletal muscle, and down the lengths of axons and dendrites in neurons. Hyperactive MTOC function at the centrosome is associated with epithelial cancers and with invasive behavior in tumor cells. Little is known about the mechanisms that limit MTOC activation at the centrosome. Here, we find that MTOC function at the centrosome is completely inactivated during cell differentiation in C. elegans embryonic intestinal cells and MTOC function is reassigned to the apical membrane. In cells that divide after differentiation, the cellular MTOC state switches between the membrane and the centrosome. Using cell fusion experiments in live embryos, we find that the centrosome MTOC state is dominant and that the inactive MTOC state of the centrosome is malleable; fusion of a mitotic cell to a differentiated or interphase cell results in rapid reactivation of the centrosome MTOC. We show that conversion of MTOC state involves the conserved centrosome protein SPD-2/CEP192 and CDK activity from the mitotic cell.

The centrosomal linker and microtubules provide dual levels of spatial coordination of centrosomes.

The centrosome is the principal microtubule organizing center in most animal cells. It consists of a pair of centrioles surrounded by pericentriolar material. The centrosome, like DNA, duplicates exactly once per cell cycle. During interphase duplicated centrosomes remain closely linked by a proteinaceous linker. This centrosomal linker is composed of rootletin filaments that are anchored to the centrioles via the protein C-Nap1. At the onset of mitosis the linker is dissolved by Nek2A kinase to support the formation of the bipolar mitotic spindle. The importance of the centrosomal linker for cell function during interphase awaits characterization. Here we assessed the phenotype of human RPE1 C-Nap1 knockout (KO) cells. The absence of the linker led to a modest increase in the average centrosome separation from 1 to 2.5 μm. This small impact on the degree of separation is indicative of a second level of spatial organization of centrosomes. Microtubule depolymerisation or stabilization in C-Nap1 KO cells dramatically increased the inter-centrosomal separation (> 8 μm). Thus, microtubules position centrosomes relatively close to one another in the absence of linker function. C-Nap1 KO cells had a Golgi organization defect with a two-fold expansion of the area occupied by the Golgi. When the centrosomes of C-Nap1 KO cells showed considerable separation, two spatially distinct Golgi stacks could be observed. Furthermore, migration of C-Nap1 KO cells was slower than their wild type RPE1 counterparts. These data show that the spatial organization of centrosomes is modulated by a combination of centrosomal cohesion and microtubule forces. Furthermore a modest increase in centrosome separation has major impact on Golgi organization and cell migration.

Centrosomes. Regulated assembly of a supramolecular centrosome scaffold in vitro.

The centrosome organizes microtubule arrays within animal cells and comprises two centrioles surrounded by an amorphous protein mass called the pericentriolar material (PCM). Despite the importance of centrosomes as microtubule-organizing centers, the mechanism and regulation of PCM assembly are not well understood. In Caenorhabditis elegans, PCM assembly requires the coiled-coil protein SPD-5. We found that recombinant SPD-5 could polymerize to form micrometer-sized porous networks in vitro. Network assembly was accelerated by two conserved regulators that control PCM assembly in vivo, Polo-like kinase-1 and SPD-2/Cep192. Only the assembled SPD-5 networks, and not unassembled SPD-5 protein, functioned as a scaffold for other PCM proteins. Thus, PCM size and binding capacity emerge from the regulated polymerization of one coiled-coil protein to form a porous network.

Growth factor dependent regulation of centrosome function and genomic instability by HuR.

The mRNA binding protein HuR is over expressed in cancer cells and contributes to disease progression through post-transcriptional regulation of mRNA. The regulation of HuR and how this relates to glioma is the focus of this report. SRC and c-Abl kinases regulate HuR sub-cellular trafficking and influence accumulation in the pericentriolar matrix (PCM) via a growth factor dependent signaling mechanism. Growth factor stimulation of glioma cell lines results in the associate of HuR with the PCM and amplification of centrosome number. This process is regulated by tyrosine phosphorylation of HuR and is abolished by mutating tyrosine residues. HuR is overexpressed in tumor samples from patients with glioblastoma and associated with a reduced survival. These findings suggest HuR plays a significant role in centrosome amplification and genomic instability, which contributes to a worse disease outcome.

SEPT12/SPAG4/LAMINB1 complexes are required for maintaining the integrity of the nuclear envelope in postmeiotic male germ cells.

Male infertility affects approximately 50% of all infertile couples. The male-related causes of intracytoplasmic sperm injection failure include the absence of sperm, immotile or immature sperm, and sperm with structural defects such as those caused by premature chromosomal condensation and DNA damage. Our previous studies based on a knockout mice model indicated that SEPT12 proteins are critical for the terminal morphological formation of sperm. SEPT12 mutations in men result in teratozospermia and oligozospermia. In addition, the spermatozoa exhibit morphological defects of the head and tail, premature chromosomal condensation, and nuclear damage. However, the molecular functions of SEPT12 during spermatogenesis remain unclear. To determine the molecular functions of SEPT12, we applied a yeast 2-hybrid system to identify SEPT12 interactors. Seven proteins that interact with SEPT12 were identified: SEPT family proteins (SEPT4 and SEPT6), nuclear or nuclear membrane proteins (protamine 2, sperm-associated antigen 4, and NDC1 transmembrane nucleoproine), and sperm-related structural proteins (pericentriolar material 1 and obscurin-like 1). Sperm-associated antigen 4 (SPAG4; also known as SUN4) belongs to the SUN family of proteins and acts as a linker protein between nucleoskeleton and cytoskeleton proteins and localizes in the nuclear membrane. We determined that SEPT12 interacts with SPAG4 in a male germ cell line through coimmunoprecipitation. During human spermiogenesis, SEPT12 is colocalized with SPAG4 near the nuclear periphery in round spermatids and in the centrosome region in elongating spermatids. Furthermore, we observed that SEPT12/SPAG4/LAMINB1 formed complexes and were coexpressed in the nuclear periphery of round spermatids. In addition, mutated SEPT12, which was screened from an infertile man, affected the integration of these nuclear envelope complexes through coimmunoprecipitation. This was the first study that suggested that SEPT proteins link to the SUN/LAMIN complexes during the formation of nuclear envelopes and are involved in the development of postmeiotic germ cells.

Neuroanatomical and behavioral deficits in mice haploinsufficient for Pericentriolar material 1 (Pcm1)

The pericentriolar material (PCM) is composed of proteins responsible for microtubule nucleation/anchoring at the centrosome, some of which have been associated with genetic susceptibility to schizophrenia. Here, we show that mice haploinsufficient for Pericentriolar material 1 (Pcm1+/−), which encodes a component of the PCM found to bear rare loss of function mutations in patients with psychiatric illness, manifest neuroanatomical phenotypes and behavioral abnormalities. Using ex vivo magnetic resonance imaging of the Pcm1+/− brain, we detect reduced whole brain volume. Pcm1 mutant mice show impairment in social interaction, specifically in the social novelty phase, but not in the sociability phase of the three-chamber social interaction test. In contrast, Pcm1+/− mice show normal preference for a novel object, suggesting specific impairment in response to novel social stimulus. In addition, Pcm1+/− mice display significantly reduced rearing activity in the open field. Pcm1+/− mice behave normally in the elevated plus maze, rotarod, prepulse inhibition, and progressive ratio tests. Together, our results suggest that haploinsufficiency at the Pcm1 locus can induce a range of neuroanatomical and behavioral phenotypes that support the candidacy of this locus in neuropsychiatric disorders.

Centrosomes and mitotic spindle poles: a recent liaison?

Centrosomes comprise two cylindrical centrioles embedded in the pericentriolar material (PCM). The PCM is an ordered assembly of large scaffolding molecules, providing an interaction platform for proteins involved in signalling, trafficking and most importantly microtubule nucleation and organization. In mitotic cells, centrosomes are located at the spindle poles, sites where spindle microtubules converge. However, certain cell typesand organisms lack centrosomes, yet contain focused spindle poles, highlighting that despite their juxtaposition in cells, centrosomes and mitotic spindle poles are distinct physical entities. In the present paper, we discuss the origin of centrosomes and summarize their contribution to mitotic spindle assembly and cell division. We then describe the key molecular players that mediate centrosome attachment to mitotic spindle poles and explore why co-segregation of centrosomes and spindle poles into daughter cells is of potential benefit to organisms.

Method: In vitro analysis of pericentriolar material assembly.

Centrosomes are major microtubule-organizing centers in eukaryotic cells and play a critical role in embryonic development and asymmetric cell division. Centrosomes comprise a pair of centrioles surrounded by an amorphous proteinaceous meshwork called the pericentriolar material (PCM). Robust deposition of PCM around the centrioles is essential for a centrosome to achieve full microtubule nucleating potential. Despite the wealth of information on PCM composition and function, the mechanism and regulation of PCM assembly have been difficult to ascertain, due in part to the lack of an in vitro system. Here, we describe methods to establish an in vitro system to study PCM assembly in Caenorhabditis elegans. Specifically, we describe (1) how to express and purify the C. elegans PCM proteins SPD-5, SPD-2, and PLK-1 from baculovirus-infected insect cells, (2) how to assemble these proteins into PCM-like structures in vitro, and (3) how to quantify this assembly process in a semiautomated fashion.

Purification of centrosomes from mammalian cell lines.

Centrosomes act as the main microtubule-organizing centre of animal cells and play critical roles in the cell, such as mitotic spindle organization, cell polarity, and motility. They are composed of two barrel-shaped structures, the centrioles, surrounded by the pericentriolar matrix. In mammalian cells, the two centrioles differ structurally due to generational difference, the oldest one bearing appendages which allow the transient docking of the centriole at the plasma membrane in order to grow a primary cilium. Centrosome components are highly conserved throughout evolution and several pathologies have been associated with centrosomal defects. The understanding of such a complex organelle has therefore been a challenge for many researchers and has led to the development of centrosomal purification procedures to assess molecular composition, biological function, and structural organization of centrosomes. In this paper, we detail a step-by-step procedure to generate high yield of purified centrosome obtained from various mammalian cell lines.

Degradation of Cep68 and PCNT cleavage mediate Cep215 removal from the PCM to allow centriole separation, disengagement and licensing.

An intercentrosomal linker keeps a cell's two centrosomes joined together until it is dissolved at the onset of mitosis. A second connection keeps daughter centrioles engaged to their mothers until they lose their orthogonal arrangement at the end of mitosis. Centriole disengagement is required to license centrioles for duplication. We show that the intercentrosomal linker protein Cep68 is degraded in prometaphase through the SCF(βTrCP) (Skp1-Cul1-F-box protein) ubiquitin ligase complex. Cep68 degradation is initiated by PLK1 phosphorylation of Cep68 on Ser332, allowing recognition by βTrCP. We also found that Cep68 forms a complex with Cep215 (also known as Cdk5Rap2) and PCNT (also known as pericentrin), two PCM (pericentriolar material) proteins involved in centriole engagement. Cep68 and PCNT bind to different pools of Cep215.We propose that Cep68 degradation allows Cep215 removal from the peripheral PCM preventing centriole separation following disengagement, whereas PCNT cleavage mediates Cep215 removal from the core of the PCM to inhibit centriole disengagement and duplication.

A unique insertion in STARD9's motor domain regulates its stability.

STARD9 is a largely uncharacterized mitotic kinesin and putative cancer target that is critical for regulating pericentriolar material cohesion during bipolar spindle assembly. To begin to understand the mechanisms regulating STARD9 function and their importance to cell division, we took a multidisciplinary approach to define the cis and trans factors that regulate the stability of the STARD9 motor domain. We show that, unlike the other ∼50 mammalian kinesins, STARD9 contains an insertion in loop 12 of its motor domain (MD). Working with the STARD9-MD, we show that it is phosphorylated in mitosis by mitotic kinases that include Plk1. These phosphorylation events are important for targeting a pool of STARD9-MD for ubiquitination by the SCFβ-TrCP ubiquitin ligase and proteasome-dependent degradation. Of interest, overexpression of nonphosphorylatable/nondegradable STARD9-MD mutants leads to spindle assembly defects. Our results with STARD9-MD imply that in vivo the protein levels of full-length STARD9 could be regulated by Plk1 and SCFβ-TrCP to promote proper mitotic spindle assembly.

RBM14 prevents assembly of centriolar protein complexes and maintains mitotic spindle integrity.

Formation of a new centriole adjacent to a pre-existing centriole occurs only once per cell cycle. Despite being crucial for genome integrity, the mechanisms controlling centriole biogenesis remain elusive. Here, we identify RBM14 as a novel suppressor of assembly of centriolar protein complexes. Depletion of RBM14 in human cells induces ectopic formation of centriolar protein complexes through function of the STIL/CPAP complex. Intriguingly, the formation of such structures seems not to require the cartwheel structure that normally acts as a scaffold for centriole formation, whereas they can retain pericentriolar material and microtubule nucleation activity. Moreover, we find that, upon RBM14 depletion, a part of the ectopic centriolar protein complexes in turn assemble into structures more akin to centrioles, presumably by incorporating HsSAS-6, a cartwheel component, and cause multipolar spindle formation. We further demonstrate that such structures assemble in the cytoplasm even in the presence of pre-existing centrioles. This study sheds light on the possibility that ectopic formation of aberrant structures related to centrioles may contribute to genome instability and tumorigenesis.

Centrosome/Cell cycle uncoupling and elimination in the endoreduplicating intestinal cells of C. elegans.

The centrosome cycle is most often coordinated with mitotic cell division through the activity of various essential cell cycle regulators, consequently ensuring that the centriole is duplicated once, and only once, per cell cycle. However, this coupling can be altered in specific developmental contexts; for example, multi-ciliated cells generate hundreds of centrioles without any S-phase requirement for their biogenesis, while Drosophila follicle cells eliminate their centrosomes as they begin to endoreduplicate. In order to better understand how the centrosome cycle and the cell cycle are coordinated in a developmental context we use the endoreduplicating intestinal cell lineage of C. elegans to address how novel variations of the cell cycle impact this important process. In C. elegans, the larval intestinal cells undergo one nuclear division without subsequent cytokinesis, followed by four endocycles that are characterized by successive rounds of S-phase. We monitored the levels of centriolar/centrosomal markers and found that centrosomes lose their pericentriolar material following the nuclear division that occurs during the L1 stage and is thereafter never re-gained. The centrioles then become refractory to S phase regulators that would normally promote duplication during the first endocycle, after which they are eliminated during the L2 stage. Furthermore, we show that SPD-2 plays a central role in the numeral regulation of centrioles as a potential target of CDK activity. On the other hand, the phosphorylation on SPD-2 by Polo-like kinase, the transcriptional regulation of genes that affect centriole biogenesis, and the ubiquitin/proteasome degradation pathway, contribute collectively to the final elimination of the centrioles during the L2 stage.

Abstract 4721: Polymorphisms in cancer-related genes and survival in early stage non-small cell lung cancer

Abstract Background: This study was conducted to identify genetic polymorphisms associated with the prognosis of patients with early stage non-small cell lung cancer (NSCLC). Materials and Methods: We genotyped 1,969 potentially functional single nucleotide polymorphisms (SNPs) of 1,151 genes using the Affymetrix custom-made GeneChip, which were related to the development and progression of cancer, in 166 NSCLC patients who underwent curative surgical resection. A replication study was performed on an independent cohort of 626 patients. Results: Fifty six SNPs which were associated with both overall survival (OS) and disease-free survival (DFS) with log-rank P values lower than 0.05 in a discovery set were selected for validation. Among the 56 SNPs, five SNPs (guanine nucleotide binding protein, beta polypeptide 2-like 1 [GNB2L1] rs1279736C>A and rs3756585T>G, complement component 3 [C3] rs2287845T>C, p300/CBP-associated factor [PCAF] rs17006625A>G, and pericentriolar material 1 [PCM1] rs17691523C>G) were found to be significantly associated with survival outcomes in the same direction as the discovery set. In combined analysis, the rs1279736C>A and s3756585T>G were most significantly associated with OS and DFS in multivariate analysis (P for OS = 4x10-5 and 7x10-5, respectively; and P for DFS = 0.003, both; under a codominant model). Conclusions: We identified five SNPs as markers for prognosis of patients with surgically resected NSCLC. Citation Format: Shin Yup Lee, Yi Young Choi, Hyo-Sung Jeon, Jin Eun Choi, Hyo-Gyoung Kang, Seung Soo Yoo, Eung Bae Lee, Won Kee Lee, Jaehee Lee, Seung Ick Cha, Chang Ho Kim, Ji Woong Son, Jae Yong Park. Polymorphisms in cancer-related genes and survival in early stage non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4721. doi:10.1158/1538-7445.AM2014-4721