Centrosomal Activity Research Articles

Kinesin-1 promotes centrosome clustering and nuclear migration in the Drosophila oocyte.

Microtubules and their associated motors are important players in nucleus positioning. Although nuclear migration in Drosophila oocytes is controlled by microtubules, a precise role for microtubule-associated molecular motors in nuclear migration has yet to be reported. We characterize novel landmarks that allow a precise description of the pre-migratory stages. Using these newly defined stages, we report that, prior to migration, the nucleus moves from the oocyte anterior side toward the center and concomitantly the centrosomes cluster at the posterior of the nucleus. In absence of Kinesin-1, centrosome clustering is impaired and the nucleus fails to position and migrate properly. The maintenance of a high level of Polo-kinase at centrosomes prevents centrosome clustering and impairs nuclear positioning. In absence of Kinesin-1, SPD-2 an essential component of the pericentriolar material is increased at the centrosomes, suggesting that Kinesin-1 associated defects result from a failure to reduce centrosome activity. Consistently, depleting centrosomes rescues the nuclear migration defects induced by Kinesin-1 inactivation. Our results suggest that Kinesin-1 controls nuclear migration in the oocyte by modulating centrosome activity.

Organization of cortical microtubules in differentiated cells.

The microtubule cytoskeleton plays a critical role in a variety of cellular activities, and its structures and functions have been extensively studied. However, little is known about cell differentiation-related microtubule remodeling, its regulatory mechanisms, and its physiological functions. Recent studies have shown that microtubule-binding proteins as well as cell junctions, such as desmosomes and adherens junctions, are involved in the remodeling of microtubules in response to cell differentiation. In addition, the microtubule-organizing activity and structural integrity of centrosomes undergo dramatic changes during cell differentiation to promote microtubule remodeling. Here we summarize recent advances revealing the dynamic changes in microtubule organization and functions during cell differentiation. We also highlight the molecular mechanisms underlying microtubule modeling in differentiated cells, focusing on the key roles played by microtubule-binding proteins, cell junctions, and centrosomes. This article is protected by copyright. All rights reserved.

Pericentrin interacts with Kinesin-1 to drive centriole motility.

Centrosome positioning is essential for their function. Typically, centrosomes are transported to various cellular locations through the interaction of centrosomal microtubules (MTs) with motor proteins anchored at the cortex or the nuclear surface. However, it remains unknown how centrioles migrate in cellular contexts in which they do not nucleate MTs. Here, we demonstrate that during interphase, inactive centrioles move directly along the interphase MT network as Kinesin-1 cargo. We identify Pericentrin-Like-Protein (PLP) as a novel Kinesin-1 interacting molecule essential for centriole motility. In vitro assays show that PLP directly interacts with the cargo binding domain of Kinesin-1, allowing PLP to migrate on MTs. Binding assays using purified proteins revealed that relief of Kinesin-1 autoinhibition is critical for its interaction with PLP. Finally, our studies of neural stem cell asymmetric divisions in the Drosophila brain show that the PLP-Kinesin-1 interaction is essential for the timely separation of centrioles, the asymmetry of centrosome activity, and the age-dependent centrosome inheritance.

Multifaceted Microcephaly-Related Gene MCPH1.

MCPH1, or BRIT1, is often mutated in human primary microcephaly type 1, a neurodevelopmental disorder characterized by a smaller brain size at birth, due to its dysfunction in regulating the proliferation and self-renewal of neuroprogenitor cells. In the last 20 years or so, genetic and cellular studies have identified MCPH1 as a multifaceted protein in various cellular functions, including DNA damage signaling and repair, the regulation of chromosome condensation, cell-cycle progression, centrosome activity and the metabolism. Yet, genetic and animal model studies have revealed an unpredicted essential function of MPCH1 in gonad development and tumorigenesis, although the underlying mechanism remains elusive. These studies have begun to shed light on the role of MPCH1 in controlling various pathobiological processes of the disorder. Here, we summarize the biological functions of MCPH1, and lessons learnt from cellular and mouse models of MCPH1.

Cdk1 phosphorylation negatively regulates the activity of Net1 towards RhoA during mitosis

The Neuroepithelial transforming gene 1 (Net1) is a RhoA subfamily guanine nucleotide exchange factor that is overexpressed in a number of cancers and contributes to cancer cell motility and proliferation. Net1 also plays a Rho GTPase independent role in mitotic progression, where it promotes centrosomal activation of Aurora A and Pak2, and aids in chromosome alignment during prometaphase. To understand regulatory mechanisms controlling the mitotic function of Net1, we examined whether it was phosphorylated by the mitotic kinase Cdk1. We observed that Cdk1 phosphorylated Net1 on multiple sites in its N-terminal regulatory domain and C-terminus in vitro. By raising phospho-specific antibodies to two of these sites, we also demonstrated that both endogenous and transfected Net1 were phosphorylated by Cdk1 in cells. Substitution of the major Cdk1 phosphorylation sites with aliphatic or acidic residues inhibited the interaction of Net1 with RhoA, and treatment of metaphase cells with a Cdk1 inhibitor increased Net1 activity. Cdk1 inhibition also increased Net1 localization to the plasma membrane and stimulated cortical F-actin accumulation. Moreover, Net1 overexpression caused spindle polarity defects that were reduced in frequency by acidic substitution of the major Cdk1 phosphorylation sites. These data indicate that Cdk1 phosphorylates Net1 during mitosis and suggest that this negatively regulates its ability to signal to RhoA and alter actin cytoskeletal organization.

Centrocortin RNA localization to centrosomes is regulated by FMRP and facilitates error-free mitosis.

Centrosomes are microtubule-organizing centers required for error-free mitosis and embryonic development. The microtubule-nucleating activity of centrosomes is conferred by the pericentriolar material (PCM), a composite of numerous proteins subject to cell cycle-dependent oscillations in levels and organization. In diverse cell types, mRNAs localize to centrosomes and may contribute to changes in PCM abundance. Here, we investigate the regulation of mRNA localization to centrosomes in the rapidly cycling Drosophila melanogaster embryo. We find that RNA localization to centrosomes is regulated during the cell cycle and developmentally. We identify a novel role for the fragile-X mental retardation protein in the posttranscriptional regulation of a model centrosomal mRNA, centrocortin (cen). Further, mistargeting cen mRNA is sufficient to alter cognate protein localization to centrosomes and impair spindle morphogenesis and genome stability.

Alpha-synuclein aggresomes inhibit ciliogenesis and multiple functions of the centrosome.

ABSTRACTProtein aggregates are the pathogenic hallmarks of many different neurodegenerative diseases and include the accumulation of α-synuclein, the main component of Lewy bodies found in Parkinson's disease. Aggresomes are closely-related, cellular accumulations of misfolded proteins. They develop in a juxtanuclear position, adjacent to the centrosome, the microtubule organizing centre of the cell, and share some protein components. Despite the long-standing observation that aggresomes/Lewy bodies and the centrosome sit side-by-side in the cell, no studies have been done to see whether these protein accumulations impede organelle function. We investigated whether the formation of aggresomes affected key centrosome functions: its ability to organise the microtubule network and to promote cilia formation. We find that when aggresomes are present, neuronal cells are unable to organise their microtubule network. New microtubules are not nucleated and extended, and the cells fail to respond to polarity cues. Since neurons are polarised, ensuring correct localisation of organelles and the effective intracellular transport of neurotransmitter vesicles, loss of centrosome activity could contribute to functional deficits and neuronal cell death in Parkinson's disease. In addition, we provide evidence that many cell types, including dopaminergic neurons, cannot form cilia when aggresomes are present, which would affect their ability to receive extracellular signals.

Radial somatic F‐actin organization affects growth cone dynamics during early neuronal development

The centrosome is thought to be the major neuronal microtubule‐organizing center (MTOC) in early neuronal development, producing microtubules with a radial organization. In addition, albeit in vitro, recent work showed that isolated centrosomes could serve as an actin‐organizing center, raising the possibility that neuronal development may, in addition, require a centrosome‐based actin radial organization. Here, we report, using super‐resolution microscopy and live‐cell imaging of cultured rodent neurons, F‐actin organization around the centrosome with dynamic F‐actin aster‐like structures with F‐actin fibers extending and retracting actively. Photoactivation/photoconversion experiments and molecular manipulations of F‐actin stability reveal a robust flux of somatic F‐actin toward the cell periphery. Finally, we show that somatic F‐actin intermingles with centrosomal PCM‐1 (pericentriolar material 1 protein) satellites. Knockdown of PCM‐1 and disruption of centrosomal activity not only affect F‐actin dynamics near the centrosome but also in distal growth cones. Collectively, the data show a radial F‐actin organization during early neuronal development, which might be a cellular mechanism for providing peripheral regions with a fast and continuous source of actin polymers, hence sustaining initial neuronal development.

The dual role of the centrosome in organizing the microtubule network in interphase

Here, we address the regulation of microtubule nucleation during interphase by genetically ablating one, or two, of three major mammalian γ-TuRC-binding factors namely pericentrin, CDK5Rap2, and AKAP450. Unexpectedly, we find that while all of them participate in microtubule nucleation at the Golgi apparatus, they only modestly contribute at the centrosome where CEP192 has a more predominant function. We also show that inhibiting microtubule nucleation at the Golgi does not affect centrosomal activity, whereas manipulating the number of centrosomes with centrinone modifies microtubule nucleation activity of the Golgi apparatus. In centrosome-free cells, inhibition of Golgi-based microtubule nucleation triggers pericentrin-dependent formation of cytoplasmic-nucleating structures. Further depletion of pericentrin under these conditions leads to the generation of individual microtubules in a γ-tubulin-dependent manner. In all cases, a conspicuous MT network forms. Strikingly, centrosome loss increases microtubule number independently of where they were growing from. Our results lead to an unexpected view of the interphase centrosome that would control microtubule network organization not only by nucleating microtubules, but also by modulating the activity of alternative microtubule-organizing centers.

The HCMV Assembly Compartment Is a Dynamic Golgi-Derived MTOC that Controls Nuclear Rotation and Virus Spread

Human cytomegalovirus (HCMV), a leading cause ofcongenital birth defects, forms an unusual cytoplasmic virion maturation site termed the "assembly compartment" (AC). Here, we show that the AC alsoacts as a microtubule-organizing center (MTOC) wherein centrosome activity is suppressedand Golgi-based microtubule (MT) nucleation is enhanced. This involved viral manipulation of discrete functions of MT plus-end-binding (EB) proteins. In particular, EB3, but not EB1 or EB2, was recruited to the AC and was required to nucleate MTs that were rapidly acetylated. EB3-regulated acetylated MTs were necessary for nuclear rotation prior to cell migration, maintenance of AC structure, and optimal virus replication. Independently, a myristoylated peptide that blocked EB3-mediated enrichment of MT regulatory proteins at Golgi regions of theAC also suppressed acetylated MT formation, nuclear rotation, and infection. Thus, HCMV offers new insights into the regulation and functions of Golgi-derived MTs and the therapeutic potential of targeting EB3.

Inhibition of ectopic microtubule assembly by the kinesin-13 KLP-7 prevents chromosome segregation and cytokinesis defects in oocytes

In most species, oocytes lack centrosomes. Accurate meiotic spindle assembly and chromosome segregation - essential to prevent miscarriage or developmental defects - thus occur through atypical mechanisms that are not well characterized. Using quantitative in vitro and in vivo functional assays in the C. elegans oocyte, we provide novel evidence that the kinesin-13 KLP-7 promotes destabilization of the whole cellular microtubule network. By counteracting ectopic microtubule assembly and disorganization of the microtubule network, this function is strictly required for spindle organization, chromosome segregation and cytokinesis in meiotic cells. Strikingly, when centrosome activity was experimentally reduced, the absence of KLP-7 or the mammalian kinesin-13 protein MCAK (KIF2C) also resulted in ectopic microtubule asters during mitosis in C. elegans zygotes or HeLa cells, respectively. Our results highlight the general function of kinesin-13 microtubule depolymerases in preventing ectopic, spontaneous microtubule assembly when centrosome activity is defective or absent, which would otherwise lead to spindle microtubule disorganization and aneuploidy.

Inhibition of ectopic microtubule assembly by the kinesin-13 KLP-7MCAK prevents chromosome segregation and cytokinesis defects in oocytes

In most species, oocytes lack centrosomes. Accurate meiotic spindle assembly and chromosome segregation - essential to prevent miscarriage or developmental defects - thus occur through atypical mechanisms that are not well characterized. Using quantitative in vitro and in vivo functional assays in the C. elegans oocyte, we provide novel evidence that the kinesin-13 KLP-7 promotes destabilization of the whole cellular microtubule network. By counteracting ectopic microtubule assembly and disorganization of the microtubule network, this function is strictly required for spindle organization, chromosome segregation and cytokinesis in meiotic cells. Strikingly, when centrosome activity was experimentally reduced, the absence of KLP-7 or the mammalian kinesin-13 protein MCAK (KIF2C) also resulted in ectopic microtubule asters during mitosis in C. elegans zygotes or HeLa cells, respectively. Our results highlight the general function of kinesin-13 microtubule depolymerases in preventing ectopic, spontaneous microtubule assembly when centrosome activity is defective or absent, which would otherwise lead to spindle microtubule disorganization and aneuploidy.

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.

Abstract 5078: Genome wide association study identifies variants at 16p13 associated with survival in multiple myeloma patients

Abstract Background: Genome wide association studies (GWAS) have recently identified 8 loci identified with multiple myeloma (MM) susceptibility. We hypothesized that germ line genetic variants may also affect MM progression. We performed the first GWAS of MM survival, by conducting a meta-analysis of two existing studies at University of California San Francisco (UCSF) and the Mayo Clinic, and replicating findings in the International Multiple Myeloma rESEarch (IMMEnSE) consortium. Methods: The UCSF study included 353 MM patients genotyped on an Illumina Omni5 and Illumina 660 array. The Mayo clinic study included 239 MM patients genotyped on an Affymetrix 6.0 array. We imputed missing genotypes using the 1000 Genomes dataset and performed a GWAS for survival using proportional hazards models adjusting for age and gender and genetic ancestry using principal components analysis. We conducted a meta-analysis of the results from the two GWAS. We replicated the top SNPs in the IMMEnSE cohort which included 772 patients with survival data from 7 European countries and one North American site. We used the dataset from Grundberg et al (Nat Genetics 2012) to test for an association between SNPs associated with survival and gene expression in immortalized lymphocytes. We used gene expression data from Zhan et al (Blood 2006) to analyze the association between expression and survival. Results: In meta-analysis, we found a genome wide significant association between SNPs on a region at 16p13 and survival (rs72773978; p=2.9x10e-10). Patients with the minor allele were at increased risk for mortality (HR 2.73; 95% CI: 1.99 - 3.73). The top associated SNPs were all in strong linkage disequilibrium and were centered over the gene FOPNL. We replicated the association in the IMMEnSE cohort and found a significant association between the top SNP from the discovery dataset (rs72773978 p=0.037). We used previously published data to determine the association between gene expression and the top associated SNPs from the GWAS. The top SNP (rs72773978) was not in previously published eQTL databases, so we used rs7201759 which was also associated with survival in our data (p=3.8x10e-10) as a proxy. The minor allele for this SNP was associated with increased levels of expression of FOPNL (p=9x10e-5), but not with expression of any other genes within a 1 megabase region. Finally, we also observed an association between increased expression of FOPNL and shorter survival (HR: 2.62 per standard deviation.; 95% CI: 1.08 - 6.32). Conclusion: Germ line variants at 16p13 near the FOPNL gene are associated with survival among MM patients. FOPNL is thought to be involved in centrosomal function, and centrosomal activity has previously been associated with survival in MM patients. Our results support this previous observation. Furthermore, they suggest that germ line variants could also be useful in assessing prognosis for patients with MM. Citation Format: Elad Ziv, Eric Dean, Donglei Hu, Alessandro Martino, Daniel Serie, Daniele Campa, Blake Aftab, Paige Bracci, Gabriele Buda, Jennifer Caswell, Charles Dumontet, Marek Dudziński, Laura Fejerman, Alexandra Greenberg, Scott Hunstman, Artur Jurczyszyn, Krzysztof Jamroziak, Shaji Kumar, Herlander Marques, Thomas Martin, Joaquin Martinez-Lopez, Vincent Rajkumar, Juan Sainz, Annette Juul Vangsted, Marzena Watek, Jeffrey Wolf, Susan Slager, Federico Canzian, Celine Vachon. Genome wide association study identifies variants at 16p13 associated with survival in multiple myeloma patients. [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 5078. doi:10.1158/1538-7445.AM2014-5078

TEIF associated centrosome activity is regulated by EGF/PI3K/Akt signaling

Centrosome amplification, which is a characteristic of cancer cells, has been understood as a driving force of genetic instability in the development of cancer. In previous work, we demonstrated that TEIF (transcriptional element-interacting factor) distributes in the centrosomes and regulates centrosome status under both physiologic and pathologic conditions. Here we identify TEIF as a downstream effector in EGF/PI3K/Akt signaling. The addition of EGF or transfection of active Akt stimulates centrosome TEIF distribution, resulting in an increase of centrosome splitting and amplification, while inhibitors of either PI3K or Akt attenuate these changes in TEIF and the associated centrosome status. A consensus motif for Akt phosphorylation (RHRVLT) proved to be involved in centrosomal TEIF localization, and the 469-threonine of this motif may be phosphorylated by Akt both in vitro and in vivo. Elimination of this phosphorylated site on TEIF caused reduced centrosome distribution and centrosome splitting or amplification. Moreover, TEIF closely co-localized with C-NAP1 at the proximal ends of centrioles, and centriolar loading of TEIF stimulated by EGF/Akt could displace C-NAP1, resulting in centrosome splitting. These findings reveal linkage of the EGF/PI3K/Akt signaling pathway to regulation of centrosome status which may act as an oncogenic pathway and induce genetic instability in carcinogenesis.

Centrosomal AKAP350 modulates the G1/S transition

AKAP350 (AKAP450/AKAP9/CG-NAP) is an A-kinase anchoring protein, which recruits multiple signaling proteins to the Golgi apparatus and the centrosomes. Several proteins recruited to the centrosomes by this scaffold participate in the regulation of the cell cycle. Previous studies indicated that AKAP350 participates in centrosome duplication. In the present study we specifically assessed the role of AKAP350 in the progression of the cell cycle. Our results showed that interference with AKAP350 expression inhibits G1/S transition, decreasing the initiation of both DNA synthesis and centrosome duplication. We identified an AKAP350 carboxyl-terminal domain (AKAP350CTD), which contained the centrosomal targeting domain of AKAP350 and induced the initiation of DNA synthesis. Nevertheless, AKAP350CTD expression did not induce centrosomal duplication. AKAP350CTD partially delocalized endogenous AKAP350 from the centrosomes, but increased the centrosomal levels of the cyclin-dependent kinase 2 (Cdk2). Accordingly, the expression of this AKAP350 domain increased the endogenous phosphorylation of nucleophosmin by Cdk2, which occurs at the G1/S transition and is a marker of the centrosomal activity of the cyclin E-Cdk2 complex. Cdk2 recruitment to the centrosomes is a necessary event for the development of the G1/S transition. Altogether, our results indicate that AKAP350 facilitates the initiation of DNA synthesis by scaffolding Cdk2 to the centrosomes, and enabling its specific activity at this organelle. Although this mechanism could also be involved in AKAP350-dependent modulation of centrosomal duplication, it is not sufficient to account for this process.

PLP helps the mother centrosome stay mum

Protein aids centrosome segregation by limiting centrosome activity in neural stem cells.

Pseudomonas aeruginosa Exotoxin Y-Mediated Tau Hyperphosphorylation Impairs Microtubule Assembly in Pulmonary Microvascular Endothelial Cells

Pseudomonas aeruginosa uses a type III secretion system to introduce the adenylyl and guanylyl cyclase exotoxin Y (ExoY) into the cytoplasm of endothelial cells. ExoY induces Tau hyperphosphorylation and insolubility, microtubule breakdown, barrier disruption and edema, although the mechanism(s) responsible for microtubule breakdown remain poorly understood. Here we investigated both microtubule behavior and centrosome activity to test the hypothesis that ExoY disrupts microtubule dynamics. Fluorescence microscopy determined that infected pulmonary microvascular endothelial cells contained fewer microtubules than control cells, and further studies demonstrated that the microtubule-associated protein Tau was hyperphosphorylated following infection and dissociated from microtubules. Disassembly/reassembly studies determined that microtubule assembly was disrupted in infected cells, with no detectable effects on either microtubule disassembly or microtubule nucleation by centrosomes. This effect of ExoY on microtubules was abolished when the cAMP-dependent kinase phosphorylation site (Ser-214) on Tau was mutated to a non-phosphorylatable form. These studies identify Tau in microvascular endothelial cells as the target of ExoY in control of microtubule architecture following pulmonary infection by Pseudomonas aeruginosa and demonstrate that phosphorylation of tau following infection decreases microtubule assembly.

Rho GTPase-independent regulation of mitotic progression by the RhoGEF Net1.

Neuroepithelial transforming gene 1 (Net1) is a RhoA-subfamily-specific guanine nucleotide exchange factor that is overexpressed in multiple human cancers and is required for proliferation. Molecular mechanisms underlying its role in cell proliferation are unknown. Here we show that overexpression or knockdown of Net1 causes mitotic defects. Net1 is required for chromosome congression during metaphase and generation of stable kinetochore microtubule attachments. Accordingly, inhibition of Net1 expression results in spindle assembly checkpoint activation. The ability of Net1 to control mitosis is independent of RhoA or RhoB activation, as knockdown of either GTPase does not phenocopy effects of Net1 knockdown on nuclear morphology, and effects of Net1 knockdown are effectively rescued by expression of catalytically inactive Net1. We also observe that Net1 expression is required for centrosomal activation of p21-activated kinase and its downstream kinase Aurora A, which are critical regulators of centrosome maturation and spindle assembly. These results identify Net1 as a novel regulator of mitosis and indicate that altered expression of Net1, as occurs in human cancers, may adversely affect genomic stability.

The role of Aurora‐A in curcumin‐treated human bladder T24 and breast MCF‐7 cancer cells

Curcumin, a yellow pigment in turmeric, has been proven to induce tumor apoptosis and inhibit tumor proliferation. Aurora‐A, an oncoprotein, plays vital roles in centrosomal activity and chromosome segregation. Although both curcumin and Aurora‐A regulate cell cycle, the effect of curcumin on Aurora‐A has not been concerned. In this study, we observe that curcumin‐induced growth inhibition of human bladder T24 and breast MCF‐7 cancer cells was associated with suppression of Aurora‐A protein and thus kinase activity. Aurora‐A siRNA acted similar to curcumin, resulting in monopolar spindle, G2/M arrest, and cell division reduction. Ectopic Aurora‐A restored the curcumin effects. Of notes, the anticancer effect of curcumin was enhanced by Aurora‐A siRNA, producing additivity and synergism effects in cell division and monopolar phenotype, respectively. The discovery of curcumin‐induced inhibition of oncoprotein Aurora‐A supports the application of curcumin in cancer treatments.(Supported in part by the National Science Council, Taiwan, No. NSC 98‐2313‐B‐003‐002‐MY3; the Ministry of Economic Affairs, Taiwan, No. 99‐EC‐17‐A‐17‐S1‐152; and the National Taiwan Normal University, Taiwan, No. 99‐D and NTNU100‐D‐06).