As cells enter mitosis, centrosomes dramatically increase in size and ability to nucleate microtubules. This process, termed centrosome maturation, is driven by the accumulation and activation of gamma-tubulin and other proteins that form the pericentriolar material on centrosomes during G2/prophase. Here, we show that the human centrosomal protein, Cep192 (centrosomal protein of 192 kDa), is an essential component of the maturation machinery. Specifically, we have found that siRNA depletion of Cep192 results in a complete loss of functional centrosomes in mitotic but not interphase cells. In mitotic cells lacking Cep192, microtubules become organized around chromosomes but rarely acquire stable bipolar configurations. These cells contain normal numbers of centrioles but cannot assemble gamma-tubulin, pericentrin, or other pericentriolar proteins into an organized PCM. Alternatively, overexpression of Cep192 results in the formation of multiple, extracentriolar foci of gamma-tubulin and pericentrin. Together, our findings support the hypothesis that Cep192 stimulates the formation of the scaffolding upon which gamma-tubulin ring complexes and other proteins involved in microtubule nucleation and spindle assembly become functional during mitosis.

The actions of thyroid hormones are mediated by binding to nuclear receptors, TR␣ 4 and TR␤, that act as ligand-dependent transcription factors by association, generally as heterodimers with retinoid X receptors, with thyroid hormone response elements located in regulatory regions of target genes (1). TRs can also modulate expression of genes that do not contain a hormone response element by modulating the activity of other transcription factors and signaling pathways (2), including activator protein-1 (AP-1)-, cyclic AMP-response element-, and nuclear factor-B (NF-B)-dependent pathways (3-7).Studies with knock-out (KO) mice for TRs obtained by homologous recombination have indicated that KO mice for ␣1 and ␤ TR isoforms have different phenotypes (8 -10) and that the double KO mice, surprisingly, survive (11), indicating that these receptors are not required for viability. Although these animals show extreme resistance to thyroid hormones, they exhibit a milder phenotype than hypothyroid mice, indicating divergent consequences for hormone versus receptor deficiency for some actions.Although the skin is a target tissue for thyroid hormone action, no studies on the skin phenotype in TR KO mice have been reported. TR␣ and TR␤ mRNAs are present in the skin (12-15), and these receptors can regulate either positively or negatively the expression of selected keratins in cultured cells (16 -19). Clinical evidence (19 -27) as well as studies in hypothyroid mice and rats (28 -30) also suggest that thyroid hormones could be involved in epidermal proliferation and differentiation, hair growth, and wound healing besides affecting the function of dermal fibroblasts. A question emerging from these studies is how to distinguish between effects due to altered thyroid hormone levels and effects due to expression of specific TR isoforms.The TR KO mice represent an excellent model for the analysis of the role of these receptors in the skin and its response to hyperproliferative stimuli. Topical application of 12-O-tetradecanolyphorbol-13-acetate (TPA) to mouse skin is a well known model for induction of skin hyperproliferation and also promotes a strong inflammatory reaction that activates multiple immunostimulatory pathways. The skin response to TPA is associated with activation of several intracellular pathways (e.g. mitogen-activated protein kinases (MAPKs), AKT, NF-B, STAT3, and AP-1) as well as an increase in the content of chemical mediators, such as cytokines, chemokines, vasoactive peptides, prostaglandins, leukotrienes, and nitric oxide among others (31)(32)(33)(34).In this work, we have investigated skin proliferation and inflammation, before and after TPA application, in mice lacking TR␣1, TR␤, or both genes, comparing these responses with those of hypothyroid animals to distinguish the specific contributions of receptor expression and activation. We found that TRs and thyroid hormones are required for skin homeostasis after TPA treatment and that both receptor genes contribute to

IB kinase 2 (IKK2 or IKK␤) is a component of the IKK complex that coordinates the cellular response to a diverse set of extracellular stimuli, including cytokines, microbial infection, and stress. In response to an external stimulus, the complex is activated, resulting in the phosphorylation and subsequent proteasome-mediated degradation of IB proteins. This event triggers the nuclear import of the NF-B transcription factor, which activates the transcription of genes that regulate a variety of fundamental biological processes, including immune response, cell survival, and development. Here, we define an essential role for IKK2 in normal mitotic progression and the maintenance of spindle bipolarity. Chemical and genetic perturbation of IKK2 promotes the formation of multipolar spindles and chromosome missegregation. Depletion of IKK2 results in the deregulation of Aurora A protein stability and coincident hyperactivation of a putative Aurora A substrate, the mitotic motor KIF11. These data support a function for IKK2 as an antagonist of Aurora A signaling during mitosis. Additionally, our results indicate a direct role for IKK2 in the maintenance of genome stability and underscore the potential for oncogenic consequences in targeting this kinase for therapeutic intervention.Aurora A ͉ mitosis ͉ NF-B ͉ spindle polarity

A loss-of-function screen for siRNAs that arrest human cells in metaphase reveals genes involved in mitotic spindle integrity.

Thyroid hormone and its receptor act in concert with NRF1 to increase cellular respiration and reactive oxygen species production, leading to DNA damage and premature senescence in susceptible cells.

Cep192 is a centrosomal protein that contributes to the formation and function of the mitotic spindle in mammalian cells. Cep192’s mitotic activities stem largely from its role in the recruitment to the centrosome of numerous additional proteins such as gamma-tubulin and Pericentrin. Here, we examine Cep192’s function in interphase cells. Our data indicate that, as in mitosis, Cep192 stimulates the nucleation of centrosomal microtubules thereby regulating the morphology of interphase microtubule arrays. Interestingly, however, cells lacking Cep192 remain capable of generating normal levels of MTs as the loss of centrosomal microtubules is augmented by MT nucleation from other sites, most notably the Golgi apparatus. The depletion of Cep192 results in a significant decrease in the level of centrosome-associated gamma-tubulin, likely explaining its impact on centrosome microtubule nucleation. However, in stark contrast to mitosis, Cep192 appears to maintain an antagonistic relationship with Pericentrin at interphase centrosomes. Interphase cells depleted of Cep192 display significantly higher levels of centrosome-associated Pericentrin while overexpression of Cep192 reduces the levels of centrosomal Pericentrin. Conversely, depletion of Pericentrin results in elevated levels of centrosomal Cep192 and enhances microtubule nucleation at centrosomes, at least during interphase. Finally, we show that depletion of Cep192 negatively impacts cell motility and alters normal cell polarization. Our current working hypothesis is that the microtubule nucleating capacity of the interphase centrosome is determined by an antagonistic balance of Cep192, which promotes nucleation, and Pericentrin, which inhibits nucleation. This in turn determines the relative abundance of centrosomal and non-centrosomal microtubules that tune cell movement and shape.

Chromosome movements are linked to the active depolymerization of spindle microtubule (MT) ends. Here we identify the kinesin-13 family member, KLP59D, as a novel and uniquely important regulator of spindle MT dynamics and chromosome motility in Drosophila somatic cells. During prometaphase and metaphase, depletion of KLP59D, which targets to centrosomes and outer kinetochores, suppresses the depolymerization of spindle pole-associated MT minus ends, thereby inhibiting poleward tubulin Flux. Subsequently, during anaphase, loss of KLP59D strongly attenuates chromatid-to-pole motion by suppressing the depolymerization of both minus and plus ends of kinetochore-associated MTs. The mechanism of KLP59D's impact on spindle MT plus and minus ends appears to differ. Our data support a model in which KLP59D directly depolymerizes kinetochore-associated plus ends during anaphase, but influences minus ends indirectly by localizing the pole-associated MT depolymerase KLP10A. Finally, electron microscopy indicates that, unlike the other Drosophila kinesin-13s, KLP59D is largely incapable of oligomerizing into MT-associated rings in vitro, suggesting that such structures are not a requisite feature of kinetochore-based MT disassembly and chromosome movements.

CEP192 is a centrosome protein that plays a critical role in centrosome biogenesis and function in mammals, Drosophila and C. elegans. Moreover, CEP192-depleted cells arrest in mitosis with disorganized microtubules, suggesting that CEP192's function in spindle assembly goes beyond its role in centrosome activity and pointing to a potentially more direct role in the regulation of the mitotic microtubule landscape. To better understand CEP192 function in mitosis, we used mass spectrometry to identify CEP192-interacting proteins. We previously reported that CEP192 interacts with NEDD1, a protein that associates with the γ-tubulin ring complex (γ-TuRC) and regulates its phosphorylation status during mitosis. Additionally, within the array of proteins that interact with CEP192, we identified the microtubule binding K63-deubiquitinase CYLD. Further analyses show that co-depletion of CYLD alleviates the bipolar spindle assembly defects observed in CEP192-depleted cells. This functional relationship exposes an intriguing role for CYLD in spindle formation and raises the tantalizing possibility that CEP192 promotes robust mitotic spindle assembly by regulating K63-polyubiquitin-mediated signaling through CYLD.