Controlling cytokinesis in the fission yeast Schizosaccharomyces pombe : the role of dma1; and ubc8

ABSTRACT The fission yeast Schizosaccharomyces pombe is a single celled eukaryote that has proved to be an excellent model system for the study of cell cycle control. S. pombe cells are rod shaped and grow mainly by elongation at their tips. They divide by formation of medially-placed cell wall, or septum, which cleaves the cell in two. Once the cell commits itself to mitosis the site of division is determined by formation of an acto-myosin based contractile ring at the cell cortex. The ring is assembled in stages throughout mitosis and contracts at the end of anaphase, coincident with spindle disassembly. The contraction, but not the assembly, of the ring requires the signal transduction network called the septation initiation network or SIN. The core components of the SIN are three protein kinases (cdc7p, sidl p and sid2p) and their regulatory subunits (spg1 p, cdcl4p and moblp, respectively). Signalling is dependent upon the nucleotide status of the GTPase spgl p, which is regulated by a two-component GAP protein, cdc16p-byr4p. Signalling is thought to emanate from the spindle pole body, where core SIN components are anchored to a scaffold comprised of sid4p and cdc11p. Activation of the SIN requires the protein kinase plolp, which also has additional roles in mitosis. SIN signalling is tightly regulated to assure the proper co-ordination of mitosis and cytokinesis. Ectopic activation of the SIN in interphase can uncouple septum formation from mitosis, while deregulated SIN signalling leads to formation of cells with multiple septa that do not cleave. Regulators of SIN activity are therefore of considerable interest. This study has concentrated upon two of these, dma1 and ubc8. I have demonstrated that dmal becomes essential when SIN signalling is activated. This leads me to propose a tripartite model for regulation of the SIN during the mitotic cell cycle. Increased expression of dma1 inhibits SIN signalling and prevents cell division. To identify potential targets and mediators of this, multicopy suppressors of dma1 toxicity were identified. One of these, ubc8, is the subject of this thesis. Genetic and molecular analyses are consistent with the view that ubc8p acts as an inhibitor of the SIN Localisation of ubc8p indicates that it is a nuclear protein. The ubc8 gene is not essential, but in its absence cells are unable to prevent septum formation if progression through mitosis is impaired. These data suggest that it may be an effector of the spindle assembly checkpoint. Together, these data shed new light upon the mechanisms by which cytokinesis is regulated in S. pombe. RESUME La levure Schizosaccharomyces pombe est un eucaryote unicellulaire qui est un bon système d'étude du cycle cellulaire. Les cellules de S. pombe sont en forme de bâtonnets et poussent par allongement aux deux bouts. Elles se divisent en formant une paroi au milieu de la cellule, qui s'appelle un septum et qui sépare la cellule en deux. Une fois que la cellule est engagée dans la mitose, le site de clivage est déterminé par la formation d'un anneau contractile d'acto-myosine au niveau du cortex cellulaire. Cet anneau est séquentiellement assemblé au cours de la mitose et se contacte à la fin de l'anaphase, au moment où le fuseau mitotique et désassemblé. La contraction, mais non pas l'assemblage, de l'anneau dépend d'un réseau de signalisation appelé septation initiation netvvork' ou SIN. Les composants centraux du SIN sont trois kinases (cdc7, sidi et sid2) ainsi que leurs sous-unités régulatrices (spgl, cdc14 et mob1, respectivement). La signalisation dépend du nucléotide rattaché à la GTPase spgl qui est régulée par une GAP comprenant deux sous-unités cdc16 et byr4. La signalisation est présumée provenir du pôle du fuseau où les composants centraux du SIN sont ancrés grâce à un échafaudage comprenant sid4 et cdcl 1. La signalisation est étroitement régulée pour assurer une bonne coordination entre mitose et cytokinèse. Une activation ectopique du SIN en interphase peut découpler la formation du septum de la mitose, engendrant des cellules à multiples septa qui ne sont pas clivés. C'est pourquoi les régulateurs du SIN sont d'un intérêt considérable. Cette étude se concentre autour de deux ces régulateurs, dma1 et ubc8. J'ai montré que dma1 devient essentiel quand la signalisation du SIN est activée. Ceci m'amène à proposer un modèle en trois parties pour la régulation du SIN durant la mitose. Une expression élevée de dma1 inhibe la signalisation du SIN et empêche la division cellulaire. Afin d'identifier des substrats ou médiateurs potentiels de la toxicité de dma1, des supresseurs en copies multiples ont été identifiés. Un de ces supresseurs, ubc8, constitue le deuxième sujet de cette thèse. Les études génétiques et moléculaires suggèrent un rôle inhibiteur du SIN par ubc8. Ubc8p est une protéine nucléaire, non essentielle, mais en son absence les cellules ne peuvent pas restreindre la fomation du septum, lorsque la progression de la mitose est perturbée. Les données suggèrent que ubc8 pourrait être un effecteur de point de contrôle de l'assemblage du fuseau mitotique. Prises dans leur ensemble, ces données apportent un nouvel éclairage sur les mécanismes de régulation de la cytokinèse dans S. pombe.

Study of Vaccinia and Cowpox viruses' replication in Rac1-N17 dominant-negative cells

Interfering with cellular signal transduction pathways is a common strategy used by many viruses to create a propitious intracellular environment for an efficient replication. Our group has been studying cellular signalling pathways activated by the orthopoxviruses Vaccinia (VACV) and Cowpox (CPXV) and their significance to viral replication. In the present study our aim was to investigate whether the GTPase Rac1 was an upstream signal that led to the activation of MEK/ERK1/2, JNK1/2 or Akt pathways upon VACV or CPXV' infections. Therefore, we generated stable murine fibroblasts exhibiting negative dominance to Rac1-N17 to evaluate viral growth and the phosphorylation status of ERK1/2, JNK1/2 and Akt. Our results demonstrated that VACV replication, but not CPXV, was affected in dominant-negative (DN) Rac1-N17 cell lines in which viral yield was reduced in about 10-fold. Viral late gene expression, but not early, was also reduced. Furthermore, our data showed that Akt phosphorylation was diminished upon VACV infection in DN Rac1-N17 cells, suggesting that Rac1 participates in the phosphoinositide-3 kinase pathway leading to the activation of Akt. In conclusion, our results indicate that while Rac1 indeed plays a role in VACV biology, perhaps another GTPase may be involved in CPXV replication.

Survival-independent function of NF-kappaB/Rel during late stages of thymocyte differentiation.

Transcription factors of the NF-kappaB/Rel family are important mediators of extracellular signals. Their implication in positive selection of thymocytes is suggested by a defective thymic development in transgenic mice that over-express IkappaB in thymocytes. These mice exhibit an accumulation of an unusually prominent population of TCRhigh/CD4/CD8 double positive cells in the thymus and a dramatic reduction of CD4+ and CD8+ cells in the periphery. The present study addresses the role of NF-kappaB in survival and differentiation processes of maturing thymocytes using IkappaB/bcl-2 and IkappaB/HY double-transgenic mice. Neither the introduction of the anti-apoptosis gene bcl-2 nor the positively selecting background in female HY transgenic mice resulted in a rescue of the maturational defects observed in the thymus of IkappaB transgenic mice. Thus, rather than promoting survival the main role of NF-kappaB/Rel proteins during positive selection of thymocytes appears to be the mediation of differentiation signals.

Peroxisome proliferator-activated receptor-beta signaling contributes to enhanced proliferation of hepatic stellate cells.

BACKGROUND & AIMS: The peroxisome proliferator-activated nuclear receptors (PPAR-alpha, PPAR-beta, and PPAR-gamma), which modulate the expression of genes involved in energy homeostasis, cell cycle, and immune function, may play a role in hepatic stellate cell activation. Previous studies focused on the decreased expression of PPAR-gamma in hepatic stellate cell activation but did not investigate the expression and role of the PPAR-alpha and -beta isotypes. The aim of this study was to evaluate the expression of the different PPARs during hepatic stellate cell activation in vitro and in situ and to analyze possible factors that might contribute to their expression. In a second part of the study, the effect of a PPAR-beta agonist on acute liver injury was evaluated. METHODS: The effects of PPAR isotype-specific ligands on hepatic stellate cell transition were evaluated by bromodeoxyuridine incorporation, gel shifts, immunoprecipitation, and use of antisense PPAR-beta RNA-expressing adenoviruses. Tumor necrosis factor alpha-induced PPAR-beta phosphorylation and expression was evaluated by metabolic labeling and by using specific P38 inhibitors. RESULTS: Hepatic stellate cells constitutively express high levels of PPAR-beta, which become further induced during culture activation and in vivo fibrogenesis. No significant expression of PPAR-alpha or -gamma was found. Stimulation of the P38 mitogen-activated protein kinase pathway modulated the expression of PPAR-beta. Transcriptional activation of PPAR-beta by L165041 enhanced hepatic stellate cell proliferation. Treatment of rats with a single bolus of CCl(4) in combination with L165041 further enhanced the expression of fibrotic markers. CONCLUSIONS: PPAR-beta is an important signal-transducing factor contributing to hepatic stellate cell proliferation during acute and chronic liver inflammation.

Negative control of keratinocyte differentiation by Rho/CRIK signaling coupled with up-regulation of KyoT1/2 (FHL1) expression.

Rho GTPases integrate control of cell structure and adhesion with downstream signaling events. In keratinocytes, RhoA is activated at early times of differentiation and plays an essential function in establishment of cell-cell adhesion. We report here that, surprisingly, Rho signaling suppresses downstream gene expression events associated with differentiation. Similar inhibitory effects are exerted by a specific Rho effector, CRIK (Citron kinase), which is selectively down-modulated with differentiation, thereby allowing the normal process to occur. The suppressing function of Rho/CRIK on differentiation is associated with induction of KyoT1/2, a LIM domain protein gene implicated in integrin-mediated processes and/or Notch signaling. Like activated Rho and CRIK, elevated KyoT1/2 expression suppresses differentiation. Thus, Rho signaling exerts an unexpectedly complex role in keratinocyte differentiation, which is coupled with induction of KyoT1/2, a LIM domain protein gene with a potentially important role in control of cell self renewal.

Function of TRADD in tumor necrosis factor receptor 1 signaling and in TRIF-dependent inflammatory responses.

Tumor necrosis factor receptor 1 (TNFR1) and Toll-like receptors (TLRs) regulate immune and inflammatory responses. Here we show that the TNFR1-associated death domain protein (TRADD) is critical in TNFR1, TLR3 and TLR4 signaling. TRADD deficiency abrogated TNF-induced apoptosis, prevented recruitment of the ubiquitin ligase TRAF2 and ubiquitination of the adaptor RIP1 in the TNFR1 signaling complex, and considerably inhibited but did not completely abolish activation of the transcription factor NF-kappaB and mitogen-activated protein kinases 'downstream' of TNFR1. TRIF-dependent cytokine production induced by the synthetic double-stranded RNA poly(I:C) and lipopolysaccharide was lower in TRADD-deficient mice than in wild-type mice. Moreover, TRADD deficiency inhibited poly(I:C)-mediated RIP1 ubiquitination and activation of NF-kappaB and mitogen-activated protein kinase signaling in fibroblasts but not in bone marrow macrophages. Thus, TRADD is an essential component of TNFR1 signaling and has a critical but apparently cell type-specific function in TRIF-dependent TLR responses.

Intrinsically photosensitive retinal ganglion cells.

The recent discovery of melanopsin-expressing retinal ganglion cells that mediate the pupil light reflex has provided new insights into how the pupil responds to different properties of light. These ganglion cells are unique in their ability to transduce light into electrical energy. There are parallels between the electrophysiologic behavior of these cells in primates and the clinical pupil response to chromatic stimuli. Under photopic conditions, a red light stimulus produces a pupil constriction mediated predominantly by cone input via trans-synaptic activation of melanopsin-expressing retinal ganglion cells, whereas a blue light stimulus at high intensity produces a steady-state pupil constriction mediated primarily by direct intrinsic photoactivation of the melanopsin-expressing ganglion cells. Preliminary data in humans suggest that under photopic conditions, cones primarily drive the transient phase of the pupil light reflex, whereas intrinsic activation of the melanopsin-expressing ganglion cells contributes heavily to sustained pupil constriction. The use of chromatic light stimuli to elicit transient and sustained pupil light reflexes may become a clinical pupil test that allows differentiation between disorders affecting photoreceptors and those affecting retinal ganglion cells.

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Retinoid X Receptors (RXR) were initially identified as nuclear receptors binding with stereo-selectivity the vitamin A derivative 9-cis retinoic acid, although the relevance of this molecule as endogenous activator of RXRs is still elusive. Importantly, within the nuclear receptor superfamily, RXRs occupy a peculiar place, as they are obligatory partners for a number of other nuclear receptors, thus integrating the corresponding signaling pathways. In this chapter, we describe the structural features allowing RXR to form homo- and heterodimers, and the functional consequences of this unique ability. Furthermore, we discuss the importance of studying RXR activity at a genome-wide level in order to comprehensively address the biological implications of their action that is fundamental to understand to what extent RXRs could be exploited as new therapeutic targets.

Peroxisome proliferator-activated receptor beta/delta exerts a strong protection from ischemic acute renal failure.

Ischemic acute renal failure is characterized by damages to the proximal straight tubule in the outer medulla. Lesions include loss of polarity, shedding into the tubule lumen, and eventually necrotic or apoptotic death of epithelial cells. It was recently shown that peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) increases keratinocyte survival after an inflammatory reaction. Therefore, whether PPARbeta/delta could contribute also to the control of tubular epithelium death after renal ischemia/reperfusion was tested. It was found that PPARbeta/delta+/- and PPARbeta/delta-/- mutant mice exhibited much greater kidney dysfunction and injury than wild-type counterparts after a 30-min renal ischemia followed by a 36-h reperfusion. Conversely, wild-type mice that were given the specific PPARbeta/delta ligand L-165041 before renal ischemia were completely protected against renal dysfunction, as indicated by the lack of rise in serum creatinine and fractional excretion of Na+. This protective effect was accompanied by a significant reduction in medullary necrosis, apoptosis, and inflammation. On the basis of in vitro studies, PPARbeta/delta ligands seem to exert their role by activating the antiapoptotic Akt signaling pathway and, unexpectedly, by increasing the spreading of tubular epithelial cells, thus limiting potentially their shedding and anoikis. These results point to PPARbeta/delta as a remarkable new target for preconditioning strategies.

PD-1 is a regulator of NY-ESO-1-specific CD8+ T cell expansion in melanoma patients.

The programmed death 1 (PD-1) receptor is a negative regulator of activated T cells and is up-regulated on exhausted virus-specific CD8(+) T cells in chronically infected mice and humans. Programmed death ligand 1 (PD-L1) is expressed by multiple tumors, and its interaction with PD-1 resulted in tumor escape in experimental models. To investigate the role of PD-1 in impairing spontaneous tumor Ag-specific CD8(+) T cells in melanoma patients, we have examined the effect of PD-1 expression on ex vivo detectable CD8(+) T cells specific to the tumor Ag NY-ESO-1. In contrast to EBV, influenza, or Melan-A/MART-1-specific CD8(+) T cells, NY-ESO-1-specific CD8(+) T cells up-regulated PD-1 expression. PD-1 up-regulation on spontaneous NY-ESO-1-specific CD8(+) T cells occurs along with T cell activation and is not directly associated with an inability to produce cytokines. Importantly, blockade of the PD-1/PD-L1 pathway in combination with prolonged Ag stimulation with PD-L1(+) APCs or melanoma cells augmented the number of cytokine-producing, proliferating, and total NY-ESO-1-specific CD8(+) T cells. Collectively, our findings support the role of PD-1 as a regulator of NY-ESO-1-specific CD8(+) T cell expansion in the context of chronic Ag stimulation. They further support the use of PD-1/PD-L1 pathway blockade in cancer patients to partially restore NY-ESO-1-specific CD8(+) T cell numbers and functions, increasing the likelihood of tumor regression.

Jasmonate signaling pathway.

Jasmonates in plants are cyclic fatty acid-derived regulators structurally similar to prostaglandins in metazoans. These chemicals mediate many of plants' transcriptional responses to wounding and pathogenesis by acting as potent regulators for the expression of numerous frontline immune response genes, including those for defensins and antifungal proteins. Additionally, the pathway is critical for fertility. Ongoing genetic screens and protein-protein interaction assays are identifying components of the canonical jasmonate signaling pathway. A massive molecular machine, based on two multiprotein complexes, SCF(COI1) and the COP9 signalosome (CNS), plays a central role in jasmonate signaling. This machine functions in vivo as a ubiquitin ligase complex, probably targeting regulatory proteins, some of which are expected to be transcriptional repressors. Some defense-related mediators, notably salicylic acid, antagonize jasmonates in controlling the expression of many genes. In Arabidopsis, NONEXPRESSOR OF PR GENES (NPR1) mediates part of this interaction, with another layer of control provided further downstream by the mitogen-activated protein kinase (MAPK) homolog MPK4. Numerous other interpathway connections influence the jasmonate pathway. Insights from Arabidopsis have shown that an allele of the auxin signaling gene AXR1, for example, reduces the sensitivity of plants to jasmonate. APETALA2 (AP2)-domain transcription factors, such as ETHYLENE RESPONSE FACTOR 1 (ERF1), link the jasmonate pathway to the ethylene signaling pathway. As progress in characterizing several new mutants (some of which are hypersensitive to jasmonic acid) augments our understanding of jasmonate signaling, the Connections Map will be updated to include this new information.

Blockade of both alpha1A- and alpha1B-adrenergic receptor subtype signaling is required to inhibit neointimal formation in the mouse femoral artery.

Attenuation of early restenosis after percutaneous coronary intervention (PCI) is important for the successful treatment of coronary artery disease. Some clinical studies have shown that hypertension is a risk factor for early restenosis after PCI. These findings suggest that alpha(1)-adrenergic receptors (alpha(1)-ARs) may facilitate restenosis after PCI because of alpha(1)-AR's remarkable contribution to the onset of hypertension. In this study, we examined the neointimal formation after vascular injury in the femoral artery of alpha(1A)-knockout (alpha(1A)-KO), alpha(1B)-KO, alpha(1D)-KO, alpha(1A)-/alpha(1B)-AR double-KO (alpha(1AB)-KO), and wild-type mice to investigate the functional role of each alpha(1)-AR subtype in neointimal formation, which is known to promote restenosis. Neointimal formation 4 wk after wire injury was significantly (P < 0.05) smaller in alpha(1AB)-KO mice than in any other group of mice, while blood pressures were not altered in any of the groups of mice after wire injury compared with those before it. These results suggest that lack of both alpha(1A)- and alpha(1B)-ARs could be necessary to inhibit neointimal formation in the mouse femoral artery.

A neuron-glia signalling network in the active brain.

Glial cells are active partners of neurons in processing information and synaptic integration. They receive coded signals from synapses and elaborate modulatory responses. The active properties of glia, including long-range signalling and regulated transmitter release, are beginning to be elucidated. Recent insights suggest that the active brain should no longer be regarded as a circuitry of neuronal contacts, but as an integrated network of interactive neurons and glia.

Retinoic acid signaling affects cortical synchrony during sleep.

Delta oscillations, characteristic of the electroencephalogram (EEG) of slow wave sleep, estimate sleep depth and need and are thought to be closely linked to the recovery function of sleep. The cellular mechanisms underlying the generation of delta waves at the cortical and thalamic levels are well documented, but the molecular regulatory mechanisms remain elusive. Here we demonstrate in the mouse that the gene encoding the retinoic acid receptor beta determines the contribution of delta oscillations to the sleep EEG. Thus, retinoic acid signaling, which is involved in the patterning of the brain and dopaminergic pathways, regulates cortical synchrony in the adult.