SAS-4 is recruited to a dynamic structure in newly forming centrioles that is stabilized by the gamma-tubulin-mediated addition of centriolar microtubules.

Centrioles are surrounded by pericentriolar material (PCM), which is proposed to promote new centriole assembly by concentrating gamma-tubulin. Here, we quantitatively monitor new centriole assembly in living Caenorhabditis elegans embryos, focusing on the conserved components SAS-4 and SAS-6. We show that SAS-4 and SAS-6 are coordinately recruited to the site of new centriole assembly and reach their maximum levels during S phase. Centriolar SAS-6 is subsequently reduced by a mechanism intrinsic to the early assembly pathway that does not require progression into mitosis. Centriolar SAS-4 remains in dynamic equilibrium with the cytoplasmic pool until late prophase, when it is stably incorporated in a step that requires gamma-tubulin and microtubule assembly. These results indicate that gamma-tubulin in the PCM stabilizes the nascent daughter centriole by promoting microtubule addition to its outer wall. Such a mechanism may help restrict new centriole assembly to the vicinity of preexisting parent centrioles that recruit PCM

Basal body stability and ciliogenesis requires the conserved component Poc1

Centrioles are the foundation for centrosome and cilia formation. The biogenesis of centrioles is initiated by an assembly mechanism that first synthesizes the ninefold symmetrical cartwheel and subsequently leads to a stable cylindrical microtubule scaffold that is capable of withstanding microtubule-based forces generated by centrosomes and cilia. We report that the conserved WD40 repeat domain–containing cartwheel protein Poc1 is required for the structural maintenance of centrioles in Tetrahymena thermophila. Furthermore, human Poc1B is required for primary ciliogenesis, and in zebrafish, DrPoc1B knockdown causes ciliary defects and morphological phenotypes consistent with human ciliopathies. T. thermophila Poc1 exhibits a protein incorporation profile commonly associated with structural centriole components in which the majority of Poc1 is stably incorporated during new centriole assembly. A second dynamic population assembles throughout the cell cycle. Our experiments identify novel roles for Poc1 in centriole stability and ciliogenesis

Assembly of centrosomal proteins and microtubule organization depends on PCM-1

The protein PCM-1 localizes to cytoplasmic granules known as “centriolar satellites” that are partly enriched around the centrosome. We inhibited PCM-1 function using a variety of approaches: microinjection of antibodies into cultured cells, overexpression of a PCM-1 deletion mutant, and specific depletion of PCM-1 by siRNA. All approaches led to reduced targeting of centrin, pericentrin, and ninein to the centrosome. Similar effects were seen upon inhibition of dynactin by dynamitin, and after prolonged treatment of cells with the microtubule inhibitor nocodazole. Inhibition or depletion of PCM-1 function further disrupted the radial organization of microtubules without affecting microtubule nucleation. Loss of microtubule organization was also observed after centrin or ninein depletion. Our data suggest that PCM-1–containing centriolar satellites are involved in the microtubule- and dynactin-dependent recruitment of proteins to the centrosome, of which centrin and ninein are required for interphase microtubule organization

Inhibition of centrosome protein assembly leads to p53-dependent exit from the cell cycle

Previous evidence has indicated that an intact centrosome is essential for cell cycle progress and that elimination of the centrosome or depletion of individual centrosome proteins prevents the entry into S phase. To investigate the molecular mechanisms of centrosome-dependent cell cycle progress, we performed RNA silencing experiments of two centrosome-associated proteins, pericentriolar material 1 (PCM-1) and pericentrin, in primary human fibroblasts. We found that cells depleted of PCM-1 or pericentrin show lower levels of markers for S phase and cell proliferation, including cyclin A, Ki-67, proliferating cell nuclear antigen, minichromosome maintenance deficient 3, and phosphorylated retinoblastoma protein. Also, the percentage of cells undergoing DNA replication was reduced by >50%. At the same time, levels of p53 and p21 increased in these cells, and cells were predisposed to undergo senescence. Conversely, depletion of centrosome proteins in cells lacking p53 did not cause any cell cycle arrest. Inhibition of p38 mitogen-activated protein kinase rescued cell cycle activity after centrosome protein depletion, indicating that p53 is activated by the p38 stress pathway

The bornavirus-derived human protein EBLN1 promotes efficient cell cycle transit, microtubule organisation and genome stability.

It was recently discovered that vertebrate genomes contain multiple endogenised nucleotide sequences derived from the non-retroviral RNA bornavirus. Strikingly, some of these elements have been evolutionary maintained as open reading frames in host genomes for over 40 million years, suggesting that some endogenised bornavirus-derived elements (EBL) might encode functional proteins. EBLN1 is one such element established through endogenisation of the bornavirus N gene (BDV N). Here, we functionally characterise human EBLN1 as a novel regulator of genome stability. Cells depleted of human EBLN1 accumulate DNA damage both under non-stressed conditions and following exogenously induced DNA damage. EBLN1-depleted cells also exhibit cell cycle abnormalities and defects in microtubule organisation as well as premature centrosome splitting, which we attribute in part, to improper localisation of the nuclear envelope protein TPR. Our data therefore reveal that human EBLN1 possesses important cellular functions within human cells, and suggest that other EBLs present within vertebrate genomes may also possess important cellular functions

SAS-4 is recruited to a dynamic structure in newly forming centrioles that is stabilized by the γ-tubulin–mediated addition of centriolar microtubules

Centrioles are surrounded by pericentriolar material (PCM), which is proposed to promote new centriole assembly by concentrating γ-tubulin. Here, we quantitatively monitor new centriole assembly in living Caenorhabditis elegans embryos, focusing on the conserved components SAS-4 and SAS-6. We show that SAS-4 and SAS-6 are coordinately recruited to the site of new centriole assembly and reach their maximum levels during S phase. Centriolar SAS-6 is subsequently reduced by a mechanism intrinsic to the early assembly pathway that does not require progression into mitosis. Centriolar SAS-4 remains in dynamic equilibrium with the cytoplasmic pool until late prophase, when it is stably incorporated in a step that requires γ-tubulin and microtubule assembly. These results indicate that γ-tubulin in the PCM stabilizes the nascent daughter centriole by promoting microtubule addition to its outer wall. Such a mechanism may help restrict new centriole assembly to the vicinity of preexisting parent centrioles that recruit PCM

Common themes in centriole and centrosome movements.

addresses: School of Life Sciences, Oxford Brookes University, Gipsy Lane, Oxford, OX3 0BP, UK.Copyright © 2011 Elsevier. NOTICE: this is the author’s version of a work that was accepted for publication in Trends in Cell Biology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Trends in Cell Biology, 2011, Vol. 21, Issue 1, pp. 57 – 66 DOI: 10.1016/j.tcb.2010.09.004Centrioles are found in nearly all eukaryotic cells and are required for growth and maintenance of the radial array of microtubules, the mitotic spindle, and cilia and flagella. Different types of microtubule structures are often required at different places in a given cell; centrioles must move around to nucleate these varied structures. Here, we draw together recent data on diverse centriole movements to decipher common themes in how centrioles move. Par proteins establish and maintain the required cellular asymmetry. The actin cytoskeleton facilitates movement of multiple basal bodies. Microtubule forces acting on the cell cortex, and nuclear-cytoskeletal links, are important for positioning individual centrosomes, and during cell division. Knowledge of these common mechanisms can inform the study of centriole movements across biology

Small molecule antagonists of NAADP-induced Ca2+ release in T-lymphocytes suggest potential therapeutic agents for autoimmune disease

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+-releasing second messenger known to date, but the precise NAADP/Ca2+ signalling mechanisms are still controversial. We report the synthesis of small-molecule inhibitors of NAADP-induced Ca2+ release based upon the nicotinic acid motif. Alkylation of nicotinic acid with a series of bromoacetamides generated a diverse compound library. However, many members were only weakly active or had poor physicochemical properties. Structural optimisation produced the best inhibitors that interact specifically with the NAADP/Ca2+ release mechanism, having no effect on Ca2+ mobilized by the other well-known second messengers D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] or cyclic adenosine 5′-diphospho-ribose (cADPR). Lead compound (2) was an efficient antagonist of NAADP-evoked Ca2+ release in vitro in intact T lymphocytes and ameliorated clinical disease in vivo in a rat experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Compound (3) (also known as BZ194) was synthesized as its bromide salt, confirmed by crystallography, and was more membrane permeant than 2. The corresponding zwitterion (3a), was also prepared and studied by crystallography, but 3 had more desirable physicochemical properties. 3 Is potent in vitro and in vivo and has found widespread use as a tool to modulate NAADP effects in autoimmunity and cardiovascular applications. Taken together, data suggest that the NAADP/Ca2+ signalling mechanism may serve as a potential target for T cell- or cardiomyocyte-related diseases such as multiple sclerosis or arrhythmia. Further modification of these lead compounds may potentially result in drug candidates of clinical use

DISC1 genetics, biology and psychiatric illness

Psychiatric disorders are highly heritable, and in many individuals likely arise from the combined effects of genes and the environment. A substantial body of evidence points towards DISC1 being one of the genes that influence risk of schizophrenia, bipolar disorder and depression, and functional studies of DISC1 consequently have the potential to reveal much about the pathways that lead to major mental illness. Here, we review the evidence that DISC1 influences disease risk through effects upon multiple critical pathways in the developing and adult brain

CDC25B associates with a centrin 2-containing complex and is involved in maintaining centrosome integrity

Background information. CDC25 (cell division cycle 25) phosphatases function as activators of CDK (cyclin-dependent kinase)–cyclin complexes to regulate progression through the CDC. We have recently identified a pool of CDC25B at the centrosome of interphase cells that plays a role in regulating centrosome numbers