Centrosome Architecture Research Articles

Conformational Plasticity of Centrin 1 from Toxoplasma gondii in Binding to the Centrosomal Protein SFI1.

Centrins are calcium (Ca2+)-binding proteins that are involved in many cellular functions including centrosome regulation. A known cellular target of centrins is SFI1, a large centrosomal protein containing multiple repeats that represent centrin-binding motifs. Recently, a protein homologous to yeast and mammalian SFI1, denominated TgSFI1, which shares SFI1-repeat organization, was shown to colocalize at centrosomes with centrin 1 from Toxoplasma gondii (TgCEN1). However, the molecular details of the interaction between TgCEN1 and TgSFI1 remain largely unknown. Herein, combining different biophysical methods, including isothermal titration calorimetry, nuclear magnetic resonance, circular dichroism, and fluorescence spectroscopy, we determined the binding properties of TgCEN1 and its individual N- and C-terminal domains to synthetic peptides derived from distinct repeats of TgSFI1. Overall, our data indicate that the repeats in TgSFI1 constitute binding sites for TgCEN1, but the binding modes of TgCEN1 to the repeats differ appreciably in terms of binding affinity, Ca2+ sensitivity, and lobe-specific interaction. These results suggest that TgCEN1 displays remarkable conformational plasticity, allowing for the distinct repeats in TgSFI1 to possess precise modes of TgCEN1 binding and regulation during Ca2+ sensing, which appears to be crucial for the dynamic association of TgCEN1 with TgSFI1 in the centrosome architecture.

Zika virus alters centrosome organization to suppress the innate immune response.

Zika virus (ZIKV) is a flavivirus transmitted via mosquitoes and sex to cause congenital neurodevelopmental defects, including microcephaly. Inherited forms of microcephaly (MCPH) are associated with disrupted centrosome organization. Similarly, we found that ZIKV infection disrupted centrosome organization. ZIKV infection disrupted the organization of centrosomal proteins including CEP63, a MCPH-associated protein. The ZIKV nonstructural protein NS3 bound CEP63, and expression of NS3 was sufficient to alter centrosome architecture and CEP63 localization. Loss of CEP63 suppressed ZIKV-induced centrosome disorganization, indicating that ZIKV requires CEP63 to disrupt centrosome organization. ZIKV infection or CEP63 loss decreased the centrosomal localization and stability of TANK-binding kinase 1 (TBK1), a regulator of the innate immune response. ZIKV infection also increased the centrosomal accumulation of the CEP63 interactor DTX4, a ubiquitin ligase that degrades TBK1. Therefore, we propose that ZIKV disrupts CEP63 function to increase centrosomal DTX4 localization and destabilization of TBK1, thereby tempering the innate immune response.

A modified TurboID approach identifies tissue-specific centriolar components in C. elegans.

Proximity-dependent labeling approaches such as BioID have been a great boon to studies of protein-protein interactions in the context of cytoskeletal structures such as centrosomes which are poorly amenable to traditional biochemical approaches like immunoprecipitation and tandem affinity purification. Yet, these methods have so far not been applied extensively to invertebrate experimental models such as C. elegans given the long labeling times required for the original promiscuous biotin ligase variant BirA*. Here, we show that the recently developed variant TurboID successfully probes the interactomes of both stably associated (SPD-5) and dynamically localized (PLK-1) centrosomal components. We further develop an indirect proximity labeling method employing a GFP nanobody-TurboID fusion, which allows the identification of protein interactors in a tissue-specific manner in the context of the whole animal. Critically, this approach utilizes available endogenous GFP fusions, avoiding the need to generate multiple additional strains for each target protein and the potential complications associated with overexpressing the protein from transgenes. Using this method, we identify homologs of two highly conserved centriolar components, Cep97 and BLD10/Cep135, which are present in various somatic tissues of the worm. Surprisingly, neither protein is expressed in early embryos, likely explaining why these proteins have escaped attention until now. Our work expands the experimental repertoire for C. elegans and opens the door for further studies of tissue-specific variation in centrosome architecture.

3D-Structured Illumination Microscopy of Centrosomes in Human Cell Lines.

The centrosome is the main microtubule-organizing center of animal cells, and is composed of two barrel-shaped microtubule-based centrioles embedded in protein dense pericentriolar material. Compositional and architectural re-organization of the centrosome drives its duplication, and enables its microtubule-organizing activity and capability to form the primary cilium, which extends from the mature (mother) centriole, as the cell exits the cell cycle. Centrosomes and primary cilia are essential to human health, signified by the causal role of centrosome- and cilia-aberrations in numerous congenic disorders, as well as in the etiology and progression of cancer. The list of disease-associated centrosomal proteins and their proximitomes is steadily expanding, emphasizing the need for high resolution mapping of such proteins to specific substructures of the organelle. Here, we provide a detailed 3D-structured illumination microscopy (3D-SIM) protocol for comparative localization analysis of fluorescently labeled proteins at the centrosome in fixed human cell lines, at approximately 120 nm lateral and 300 nm axial resolution. The procedure was optimized to work with primary antibodies previously known to depend on more disruptive fixation reagents, yet largely preserves centriole and centrosome architecture, as shown by transposing acquired images of landmark proteins on previously published transmission electron microscopy (TEM) images of centrosomes. Even more advantageously, it is compatible with fluorescent protein tags. Finally, we introduce an internal reference to ensure correct 3D channel alignment. This protocol hence enables flexible, swift, and information-rich localization and interdependence analyses of centrosomal proteins, as well as their disorder-associated mutations.

Human NINEIN polymorphism at codon 1111 is associated with the risk of colorectal cancer.

NINEIN serves an essential role in centrosome function as a microtubule organizing center, and in the reformation of the interphase centrosome architecture following mitosis. In the present study, the association between NINEIN Pro1111Ala (rs2236316), a missense single nucleotide polymorphism, and the risk of colorectal cancer (CRC), related to smoking and alcohol consumption habits in 200 patients with CRC and 1,141 cancer-free control participants were assessed in a case-control study performed in Japan. The results showed that the NINEIN Ala/Ala genotype compared with the Pro/Pro genotype was significantly more associated with an increased risk of CRC, and the males with the Ala/Ala genotype exhibited a significantly increased risk of CRC compared with those with Pro/Pro and Pro/Ala genotypes. Stratified analyses of the Ala/Ala genotype with CRC risk further showed an increased association in never/light drinkers (<23 g of ethanol/day), in male never/light drinkers and in male patients with rectal cancer. These findings suggest that the genetic variant of the NINEIN Pro1111Ala polymorphism has a significant effect on CRC susceptibility in the Japanese population.

C14orf166 Is a Biomarker for Predicting Hepatocellular Carcinoma Recurrence

Aim: Chromosome 14 open reading frame 166 (C14orf166) acts as a transcriptional repressor and is correlated with centrosome architecture manipulation. Nevertheless, the function of C14orf166 in hepatocellular carcinoma (HCC) progression remains unclear. We aimed to investigate the role C14orf166 plays in HCC and further compared the prognostic value of C14orf166 with that of clinicopathological features. Methods: C14orf166 expression was evaluated in a human liver cell line, HCC cell lines, HCC tissues and adjacent noncancerous liver tissues with qRT-PCR, western blot and immunohistochemistry. Patients were divided into two different groups according to C14orf166 level. The relationship between C14orf166 expression and clinicopathological features was assessed by Pearson chi-squared test and receiver operating characteristic curves. Cumulative disease-free survival (DFS) and overall survival (OS) curves were evaluated using the Kaplan–Meier method. Results: C14orf166 mRNA and protein expression is upregulated in HCC cell lines and tissues. The level of C14orf166 was correlated with serum alpha-fetoprotein level, lymph node metastasis, tumor size and recurrence, with high C14orf166 expression correlating with high HCC recurrence risk. The poor OS and DFS of HCC patients are partly due to the persistently high HCC recurrence risk. When combined with serum alpha-fetoprotein level, the predictive accuracy of C14orf166 for HCC recurrence was enhanced (AUC = 0.712, 95% CI 0.603–0.821; p = 0.001). Conclusions: This study demonstrated that C14orf166 is a high-risk biomarker and predictive factor for HCC recurrence, providing information for the selection of appropriate treatment strategies.

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.

C14ORF166 overexpression is associated with pelvic lymph node metastasis and poor prognosis in uterine cervical cancer.

C14ORF166 (chromosome 14 open reading frame 166) is a transcriptional repressor related to the regulation of centrosome architecture. However, the role of C14ORF166 in the development and progression of cancer remains largely unknown. The aim of this study was to investigate the expression and clinicopathological significance of C14ORF166 in cervical cancer. C14ORF166 expression was analyzed using quantitative real-time PCR (RT-PCR) and Western blotting in cervical cancer cell lines and eight paired cervical cancer samples and the adjacent normal tissues. Immunohistochemistry was used to analyze C14ORF166 protein expression in 148 clinicopathologically characterized cervical cancer specimens. Statistical analyses were performed to evaluate the relationship between the expression of C14ORF166 and clinicopathologic features and prognosis. C14ORF166 mRNA and protein expression were significantly upregulated in cervical cancer cell lines and tissue samples (P < 0.05). Immunohistochemical analysis revealed a high expression of C14ORF166 was observed in 39.9 % (59/148) of the cervical cancer specimens; the remaining samples expressed low levels or did not express any detectable C14ORF166. The chi-square test indicated that high-level expression of C14ORF166 was significantly associated with International Federation of Gynecology and Obstetrics (FIGO) stage (P < 0.001), vital status (P = 0.026), tumor size (P = 0.034), serum squamous cell carcinoma antigen level (SCC-Ag; P = 0.035), and pelvic lymph node metastasis (P < 0.001). Patients with highly expressed C14ORF166 showed a tendency to receive postoperative chemotherapy (P = 0.005) and postoperative radiation (P = 0.008). Furthermore, high C14ORF166 expression was associated with poorer overall survival compared to low C14ORF166 expression, and C14ORF166 was a significant prognostic factor in univariate and multivariate analysis (P < 0.05). High C14ORF166 expression had prognostic value for poor outcome in cervical cancer. C14ORF166 may represent a biomarker of pelvic lymph node metastasis and enable the identification of high-risk patients along with selection of appropriate treatment strategies.

Overexpression of chromosome 14 open reading frame 166 correlates with disease progression and poorer prognosis in human NPC.

Chromosome 14 open reading frame 166 (c14orf166) contributes to regulation of centrosome architecture, transcription initiation, and RNA elongation and processing. However, the role of c14orf166 in human cancer remains unclear. This study aimed to investigate the expression and clinicopathological significance of c14orf166 in nasopharyngeal carcinoma (NPC). Real-time PCR and Western blotting analyses were employed to examine c14orf166 expression in immortalized primary nasopharyngeal epithelial cells (NP69), six NPC cell lines, three paired tumor samples and the adjacent noncancerous tissues from the same patients, and three additional tumor samples. Immunohistochemistry was performed to examine c14orf166 protein expression in archived paraffin-embedded tumor samples from 109 patients with clinicopathologically confirmed NPC. The association of c14orf166 expression with clinicopathological features and survival outcomes was assessed. C14orf166 messenger RNA and protein expression were upregulated in NPC cell lines and human tumor tissues compared to NP69 cells and noncancerous tissue samples, respectively. Immunohistochemical analyses revealed 104/109 (95.4 %) of the NPC tissue samples expressed c14orf166. High expression of c14orf166 (cutoff score >6) was significantly associated with gender (P = 0.016), clinical stage (P = 0.010), T classification (P = 0.013), N classification (P = 0.029), distant metastasis (P < 0.001), vital status (P = 0.001), and treatment method (P = 0.028; chi-square test). High c14orf166 expression was associated with significantly shorter overall survival (OS) and disease-free survival (DFS) than low c14orf166 expression. Multivariate analysis revealed c14orf166 was an independent prognostic indicator of OS and DFS in all patients (P = 0.046, P = 0.006) and was associated with poor OS and DFS in the subgroups with advanced clinical stage (P = 0.037, P = 0.001), lymph node metastasis (P = 0.001, P < 0.001), and T3-T4 disease (P = 0.005, P = 0.001), respectively. C14orf166 is overexpressed and may represent a novel prognostic factor in NPC.

Interaction Proteomics Identify NEURL4 and the HECT E3 Ligase HERC2 as Novel Modulators of Centrosome Architecture

Centrosomes are composed of a centriole pair surrounded by an intricate proteinaceous matrix referred to as pericentriolar material. Although the mechanisms underpinning the control of centriole duplication are now well understood, we know relatively little about the control of centrosome size and shape. Here we used interaction proteomics to identify the E3 ligase HERC2 and the neuralized homologue NEURL4 as novel interaction partners of the centrosomal protein CP110. Using high resolution imaging, we find that HERC2 and NEURL4 localize to the centrosome and that interfering with their function alters centrosome morphology through the appearance of aberrant filamentous structures that stain for a subset of pericentriolar material proteins including pericentrin and CEP135. Using an RNA interference-resistant transgene approach in combination with structure-function analyses, we show that the association between CP110 and HERC2 depends on nonoverlapping regions of NEURL4. Whereas CP110 binding to NEURL4 is dispensable for the regulation of pericentriolar material architecture, its association with HERC2 is required to maintain normal centrosome integrity. NEURL4 is a substrate of HERC2, and together these results indicate that the NEURL4-HERC2 complex participates in the ubiquitin-dependent regulation of centrosome architecture.

The Plk1 target Kizuna stabilizes mitotic centrosomes to ensure spindle bipolarity

Formation of a bipolar spindle is essential for faithful chromosome segregation at mitosis. Because centrosomes define spindle poles, defects in centrosome number and structural organization can lead to a loss of bipolarity. In addition, microtubule-mediated pulling and pushing forces acting on centrosomes and chromosomes are also important for bipolar spindle formation. Polo-like kinase 1 (Plk1) is a highly conserved Ser/Thr kinase that has essential roles in the formation of a bipolar spindle with focused poles. However, the mechanism by which Plk1 regulates spindle-pole formation is poorly understood. Here, we identify a novel centrosomal substrate of Plk1, Kizuna (Kiz), depletion of which causes fragmentation and dissociation of the pericentriolar material from centrioles at prometaphase, resulting in multipolar spindles. We demonstrate that Kiz is critical for establishing a robust mitotic centrosome architecture that can endure the forces that converge on the centrosomes during spindle formation, and suggest that Plk1 maintains the integrity of the spindle poles by phosphorylating Kiz.

The Nek2 protein kinase: a novel regulator of centrosome structure.

Regulation of the centrosome, the major microtubule organizing centre in an animal cell, is in large part controlled by cell cycle-dependent protein phosphorylation. Along with cyclin dependent kinases, polo kinases and Aurora kinases, NIMA-related kinases are emerging as critical regulators of centrosome structure and function. Nek2 is the most closely related vertebrate protein by sequence to the essential mitotic regulator NIMA of Aspergillus nidulans. Nek2 is highly enriched at the centrosome and functional studies in human and Xenopus systems support a role for Nek2 in both maintenance and modulation of centrosome architecture. In particular, current evidence supports a model in which one function of Nek2 kinase activity is to promote the splitting of duplicated centrosomes at the onset of mitosis through phosphorylation of core centriolar proteins. Recent studies in lower organisms have raised the possibility that kinases related to Nek2 may have conserved functions in MTOC organization, as well as in other aspects of mitotic progression.

CEP110 and ninein are located in a specific domain of the centrosome associated with centrosome maturation.

The mammalian centrosome consists of a pair of centrioles surrounded by pericentriolar material (PCM). The architecture and composition of the centrosome, especially the PCM, changes during the cell cycle. Recently, a subset of PCM proteins have been shown to be arranged in a tubular conformation with an open and a closed end within the centrosome. The presence of such a specific configuration can be used as a landmark for mapping proteins in both a spatial and a temporal fashion. Such mapping studies can provide information about centrosome organization, protein dynamics, protein-protein interactions as well as protein function. In this study, the centrosomal proteins CEP110 and ninein were mapped in relationship to the tubular configuration. Both proteins were found to exhibit a similar distribution pattern. In the mother centrosome, they were found at both ends of the centrosome tube, including the site of centrosome duplication. However, in the daughter centrosome they were present only at the closed end. At the closed end of the mother and daughter centrosome tube, both CEP110 and ninein co-localized with the centriolar protein CEP250/c-Nap1, which confirms ninein's centriole association and places CEP110 in association with this structure. Importantly, the appearance of CEP110 and ninein at the open end of the daughter centrosome occurred during the telophase-G1 transition of the next cell cycle, concomitant with the maturation of the daughter centrosome into a mother centrosome. Microinjection of antibodies against either CEP110 or ninein into metaphase HeLa cells disrupted the reformation of the tubular conformation of proteins within the centrosome following cell division and consequently led to dispersal of centrosomal material throughout the cytosol. Further, microinjection of antibodies to either CEP110 or ninein into metaphase PtK2 cells not only disrupted the tubular configuration within the centrosome but also affected the centrosome's ability to function as a microtubule organizing center (MTOC). This MTOC function was also disrupted when the antibodies were injected into postmitotic cells. Taken together, our results indicate that: (1) a population of CEP110 and ninein is located in a specific domain within the centrosome, which corresponds to the open end of the centrosome tube and is the site of protein addition associated with maturation of a daughter centrosome into a mother centrosome; and (2) the addition of CEP110 and ninein are essential for the reformation of specific aspects of the interphase centrosome architecture following mitosis as well as being required for the centrosome to function as a MTOC.

Regulating centrosomes by protein phosphorylation

Publisher Summary Phosphorylation plays a vital role in regulating centrosome architecture and function during progression of the animal cell cycle. Many of the centrosome-associated protein kinases that catalyze the events are described. This chapter discusses the role of reversible protein phosphorylation in controlling many different aspects of centrosome structure and function, the transitions in centrosome biology that are regulated in a cell-cycle-dependent manner, and the distinct roles of a number of different families of centrosome-associated protein kinases. To ensure equal segregation of chromosomes at mitosis, centrosome duplication must be coordinated with chromosome replication. It is essential to make that all-important connection between centrosomal kinases and substrates to understand how phosphorylation triggers the changes that are observed at the morphological level. Centrosomal kinases themselves are regulated both in respect to cell cycle progression and in response to external signals. The role of the centrosome in determining mitotic spindle orientation and cleavage furrow positioning is investigated in the chapter. Centrosome maturation and separation of the phosphorylation events are used in preparing the centrosome for the establishment of the mitotic spindle and maintaining its bipolarity from one cell division to the next.