Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 85-103).

Thesis (Ph. D.)--University of Virginia, 1997. / Spine title: Control of anaphase onset checkpoint. Includes bibliographical references (172-192). Also available online through Digital Dissertations.

ABSTRACT The molecular mechanisms controlling longevity have been subject to intense scrutiny in recent years. It is clear that genomic stability, stress response and nutrient signaling all play critical roles in lifespan determination, but the precise molecular mechanisms and their often subtle influence on cellular function remain largely unknown. The Anaphase Promoting Complex (APC) is an evolutionarily conserved ubiquitin-protein ligase composed of 13 subunits in yeast, required for M and G1 cell cycle progression, and is associated with cancer and premature aging in many model systems when defective. The APC targets substrates for proteasome-dependent degradation, yet the full range of APC substrates and their role in mediating genomic stability, stress response and longevity are largely unknown. In this study, we use the model organism Saccharomyces cerevisiae to investigate the results of two screens designed to identify novel APC targets, regulators and/or modifiers, in an effort to better understand the function of the APC. Both of these screens made use of the Apc5 subunit. This subunit is likely an important structural component of the APC and may be targeted by many APC regulatory enzymes. This subunit is essential, but a temperature sensitive (ts) allele of Apc5 was available for these studies. First, a Yeast 2-Hybrid (Y2H) screen utilizing Apc5 as bait recovered the lifespan determinant Fob1 as a potential APC substrate. We hypothesized that the APC targets Fob1 for proteasome- and ubiquitin-dependent degradation. Authenticating Fob1 as a novel APC substrate makes up the first part of this thesis. We have found that Fob1 is unstable specifically in G1, and cycles throughout the cell cycle in a manner similar to Clb2, an APC target. Consistent with the APC mediating Fob1 degradation, Fob1 is stabilized in APC and proteasome mutants. Disruption of FOB1 in WT cells increased replicative lifespan, a measure of how many daughter cells a single mother will produce prior to senescence; moreover, FOB1 disruption improved APC mutant replicative lifespan defects. Increased FOB1 expression decreased replicative lifespan in WT cells, while increased expression in APC mutant cells did not reduce replicative lifespan further, suggesting an epistatic interaction. FOB1 deletion also suppressed cell cycle progression, and rDNA recombination defects observed in apc5CA cells. Mutation to a putative Destruction Box-like motif (Fob1E420V) disrupted Fob1 modification, stabilized the protein and increased rDNA recombination. These results support our hypothesis that Fob1 is a novel APC target and that Fob1 dosage may be regulated by the APC in response to cell cycle and environmental cues to regulate APC-dependent genomic stability and longevity. Second, an aptamer (small peptide) based screen identified peptides capable of suppressing the ts defect of the apc5CA mutant. One aptamer of interest is Y65, which has homology to the ubiquitin ligase Elc1. A Y2H found that this peptide Y65 binds the unstable stress response transcription factor Cin5. We hypothesized that this peptide may stabilize Cin5 by masking ubiquitin-dependent degradation. Stabilized Cin5 may in turn alleviate some apc5CA mutant defects. Characterizing Cin5 and confirming that Cin5 is subject to proteasome and ubiquitin-dependent degradation makes up the second portion of this thesis. During our investigation of Cin5 we identify a phospho-inhibited degradation motif within Cin5 that prevents ubiquitination and subsequent degradation when phosphorylated. We also provide evidence suggesting Cin5 may be targeted by a previously unidentified ubiquitin ligase subcomplex including Elc1 and Grr1. These data have helped elucidate the ubiquitin dependent regulation of Cin5. In summary, this research demonstrates the feasibility of using the Y2H and aptamer screens to identify and characterize molecular networks that interplay with the APC. Additionally, identifying and characterizing proteins where APC activity or function can be modified by aptamer binding has the potential to classify drug targets for therapeutic use in higher eukaryotes. Further understanding of the role the APC plays in cell cycle progression, chromatin assembly, genomic stability, stress response and longevity will be valuable to fundamental biological science, and may also have applications in health science and medicine.

Thesis (Ph. D.)--Case Western Reserve University, 2005. / [School of Medicine] Department of Genetics. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Meiosis is a specialized cell cycle, which generates haploid gametes from diploid parental cells. During meiosis one round of cohesion establishment during premeiotic DNA replication mediates two rounds of chromosome segregation. During meiosis I homologous chromosomes separate, whereas sister chromatids segregate during the second meiotic division without an intervening round of DNA replication. Both rounds of chromosome segregation are triggered by an ubiquitin ligase called the Anaphase-Promoting Complex or Cyclosome (APC/C). APC/C-dependent destruction of securin/Pds1 is required to activate separase, a thiol protease that mediates chromosome segregation by cleavage of the cohesin complex. The first meiotic division is preceded by an extended prophase I, during which maternal and paternal chromatids undergo recombination. The persistence of cohesion during premeiotic S- and prophase I is essential for recombination and both meiotic nuclear divisions. In order to prevent premature loss of cohesion, the APC/C has to be inactivated during early meiosis. How the APC/C is kept inactive during premeiotic S- and prophase I was unknown. This question has been addressed by studying the APC/C subunit Mnd2 from the budding yeast Saccharomyces cerevisiae. This work demonstrates that Mnd2 is required for the persistence of cohesion during premeiotic S- and prophase I. Mnd2 prevents premature activation of the APC/C by the meiosis-specific substrate recognition factor Ama1. In cells lacking Mnd2, the APC/C-Ama1 enzyme triggers premature ubiquitin-dependent degradation of Pds1, which leads to premature separation of sister chromatids due to an unrestrained activity of separase. Thus, chromosome segregation during meiosis depends on both inhibition of a meiosis-specific APC/C and timely activation of APC/C- dependent proteolysis. / Die Meiose ist ein spezialisierter Zellzyklus, der zum Ziel hat haploide Gameten aus diploiden Vorläuferzellen zu produzieren. Dafür erfolgen nach der prä-meiotischen DNA Replikation zwei aufeinanderfolgende Kernteilungen. In der ersten meiotischen Teilung erfolgt die Trennung der homologen Chromosomen. In einer zweiten meiotischen Teilung werden dann die Schwesterchromatiden getrennt. Die Trennung der Chromosomen wird durch den Anaphase-Promoting Complex oder Cyclosome (APC/C), einer Ubiquitin Ligase, reguliert. Der APC/C initiiert den Abbau von Securin/Pds1, einem Inhibitor der Thiol-Protease Separase, welche für die Trennung der Chromosomen zum Beginn der Anaphase verantwortlich ist. In einer im Vergleich zur Mitose extrem langen meiotischen Prophase I findet Rekombination zwischen maternalen und paternalen Chromosomen statt. Für diesen Vorgang, sowie für die beiden folgenden meiotischen Teilungen, wird Kohäsion zwischen den Schwesterchromatiden benötigt. Ein frühzeitiger Verlust der Kohäsion führt zur frühzeitigen Trennnung der Schwesterchromatiden, wodurch aneuploide Gameten produziert werden können. Daher muss die Aktivität des APC/C während der meiotischen Prophase I inhibiert werden. Wie der APC/C während der Prophase I inaktiviert wird, war bisher unbekannt. Einsicht in dieses Problem ergab sich aus der Untersuchung der APC/C Untereinheit Mnd2 aus der Bäckerhefe Saccharomyces cerevisiae. Es wird gezeigt, dass Mnd2 für den Verbleib der Kohäsion zwischen den Schwesterchromatiden während der meiotischen S- und Prophase I benötigt wird. Während dieser Phase verhindert Mnd2 die frühzeitige Aktivierung der Meiose-spezifischen Form des APC/C-Ama1. In meiotischen Zellen, die kein Mnd2 besitzen, löst das APC/C-Ama1 Enzym die Ubiquitin-abhängige Zerstörung von Pds1 aus. Dies führt zu einer frühzeitigen Aktivierung von Separase, welches die Trennung der Schwesterchromatiden schon während der meiotischen S- und Prophase I zur Folge hat. Die korrekte Verteilung der Chromosomen hängt daher sowohl von der Inhibierung als auch der Aktivierung des APC/C ab.

Meiosis is a specialized cell cycle, which generates haploid gametes from diploid parental cells. During meiosis one round of cohesion establishment during premeiotic DNA replication mediates two rounds of chromosome segregation. During meiosis I homologous chromosomes separate, whereas sister chromatids segregate during the second meiotic division without an intervening round of DNA replication. Both rounds of chromosome segregation are triggered by an ubiquitin ligase called the Anaphase-Promoting Complex or Cyclosome (APC/C). APC/C-dependent destruction of securin/Pds1 is required to activate separase, a thiol protease that mediates chromosome segregation by cleavage of the cohesin complex. The first meiotic division is preceded by an extended prophase I, during which maternal and paternal chromatids undergo recombination. The persistence of cohesion during premeiotic S- and prophase I is essential for recombination and both meiotic nuclear divisions. In order to prevent premature loss of cohesion, the APC/C has to be inactivated during early meiosis. How the APC/C is kept inactive during premeiotic S- and prophase I was unknown. This question has been addressed by studying the APC/C subunit Mnd2 from the budding yeast Saccharomyces cerevisiae. This work demonstrates that Mnd2 is required for the persistence of cohesion during premeiotic S- and prophase I. Mnd2 prevents premature activation of the APC/C by the meiosis-specific substrate recognition factor Ama1. In cells lacking Mnd2, the APC/C-Ama1 enzyme triggers premature ubiquitin-dependent degradation of Pds1, which leads to premature separation of sister chromatids due to an unrestrained activity of separase. Thus, chromosome segregation during meiosis depends on both inhibition of a meiosis-specific APC/C and timely activation of APC/C- dependent proteolysis. / Die Meiose ist ein spezialisierter Zellzyklus, der zum Ziel hat haploide Gameten aus diploiden Vorläuferzellen zu produzieren. Dafür erfolgen nach der prä-meiotischen DNA Replikation zwei aufeinanderfolgende Kernteilungen. In der ersten meiotischen Teilung erfolgt die Trennung der homologen Chromosomen. In einer zweiten meiotischen Teilung werden dann die Schwesterchromatiden getrennt. Die Trennung der Chromosomen wird durch den Anaphase-Promoting Complex oder Cyclosome (APC/C), einer Ubiquitin Ligase, reguliert. Der APC/C initiiert den Abbau von Securin/Pds1, einem Inhibitor der Thiol-Protease Separase, welche für die Trennung der Chromosomen zum Beginn der Anaphase verantwortlich ist. In einer im Vergleich zur Mitose extrem langen meiotischen Prophase I findet Rekombination zwischen maternalen und paternalen Chromosomen statt. Für diesen Vorgang, sowie für die beiden folgenden meiotischen Teilungen, wird Kohäsion zwischen den Schwesterchromatiden benötigt. Ein frühzeitiger Verlust der Kohäsion führt zur frühzeitigen Trennnung der Schwesterchromatiden, wodurch aneuploide Gameten produziert werden können. Daher muss die Aktivität des APC/C während der meiotischen Prophase I inhibiert werden. Wie der APC/C während der Prophase I inaktiviert wird, war bisher unbekannt. Einsicht in dieses Problem ergab sich aus der Untersuchung der APC/C Untereinheit Mnd2 aus der Bäckerhefe Saccharomyces cerevisiae. Es wird gezeigt, dass Mnd2 für den Verbleib der Kohäsion zwischen den Schwesterchromatiden während der meiotischen S- und Prophase I benötigt wird. Während dieser Phase verhindert Mnd2 die frühzeitige Aktivierung der Meiose-spezifischen Form des APC/C-Ama1. In meiotischen Zellen, die kein Mnd2 besitzen, löst das APC/C-Ama1 Enzym die Ubiquitin-abhängige Zerstörung von Pds1 aus. Dies führt zu einer frühzeitigen Aktivierung von Separase, welches die Trennung der Schwesterchromatiden schon während der meiotischen S- und Prophase I zur Folge hat. Die korrekte Verteilung der Chromosomen hängt daher sowohl von der Inhibierung als auch der Aktivierung des APC/C ab.

Asymmetric positioning of the mitotic spindle in C. elegans embryos is mediated by force-generating complexes that are anchored at the plasma membrane and that pull on microtubules growing out from the spindle poles. Although asymmetric distribution of the force generators is thought to underlie asymmetric positioning of the spindle, the number and location of the force generators has not been well defined. In particular, it has not been possible to visualize individual force generating events at the cortex. We discovered that perturbation of the acto-myosin cortex leads to the formation of long membrane invaginations that are pulled from the plasma membrane toward the spindle poles. Several lines of evidence show that the invaginations, which also occur in unperturbed embryos though at lower frequency, are pulled by the same force generators responsible for spindle positioning. Thus, the invaginations serve as a tool to localize the sites of force generation at the cortex and allow us to estimate a lower limit on the number of cortical force generators within the cell.

A la fin de la vie d’une cellule, différentes forces mécaniques permettent la séparation des chromosomes. Nos données préliminaires suggèrent l’existence d’un autre mécanisme provenant du centre du fuseau mitotique, non décrit dans l’embryon une cellule de C. elegans qui permettrait la séparation des chromosomes. Dans cette cellule, les microtubules kinétochoriens n’appliquent aucune force mécaniques sur les chromosomes durant l’anaphase. Il a été décrit que les chromosomes sont séparés grâce au déplacement des centrosomes via les forces de traction corticales. A l’aide de la microchirurgie laser dans les embryons une cellule de C. elegans, j’ai montré qu’en détruisant physiquement un ou deux centrosomes, les chromosomes continuent de se séparer, révélant l’existence d’une force de propulsion interne au fuseau mitotique (Nahaboo et al., 2015). En combinant la destruction de centrosomes et l’inactivation génétique, nous avons caractérisé les rôles de gènes favorisant ou freinant cette force de propulsion. J’ai observé que la kinésine-5, BMK-1, et le crosslinker MAP-65/SPD-1 freinent cette force de propulsion. Alors que dans d’autres espèces ces protéines favorisent la séparation des chromosomes. Nous avons remarqué que les protéines RanGTP et CLASP, favorisant de la nucléation et la polymérisation des microtubules, aident cette force de propulsion. Ces propriétés suggèrent que la polymérisation des microtubules au centre du fuseau est requise pour permettre la séparation des chromosomes durant la mitose.Par manque d’outils adéquats afin d’altérer la dynamique des microtubules, nous avons collaboré avec l’équipe de biochimistes du Dr. D. Trauner à Munich en Allemagne. Ils ont synthétisé la molécule photoactivable, Photostatin (PST), permettant la dépolymérisation des microtubules en quelques secondes (Borowiak et al., 2015). Entre 390 - 430 nm, PST est activé, dépolymérisant les microtubules, alors qu’entre 500 – 530 nm, PST est inactivé, permettant la polymérisation normale des microtubules. J’ai mesuré que la croissance des microtubules avec PST actif est absente dans des cellules Hela. J’ai montré que le cycle cellulaire dans l’embryon de C. elegans est arrêté localement en présence de PST actif. Nous avons alors montré que PST contrôle optiquement la dynamique des microtubules, in vitro, in cellulo et in vivo, de manière non invasive, rapide, locale et réversible. En résume, j’ai identifié une nouvelle force permettant la séparation des chromosomes à l’aide des approches moléculaires et biophysiques, et j’ai aidé à la caractérisation PST, un antimicrotubule photoactivable de manière locale et réversible. / In mitosis, different mechanical forces are involved in chromosome segregation. In C. elegans one-cell embryos, preliminary data suggest that an unknown mechanism, coming from inside the mitotic spindle, could influence chromosome separation. In those cells, it has been showed that kinetochore microtubule activity is absent. Thanks to external pulling forces, centrosome separation drives chromosome segregation. By using microsurgery inside the one-cell C. elegans embryos, we have shown that destroying one or two centrosomes did not prevent chromosome separation, revealing the existence of an outward pushing force (Nahaboo et al., 2015). By combining gene inactivation and centrosome destruction, we showed that the kinesin-5 and the crosslinker SPD-1 act as a brake on this pushing force, whereas they enhance chromosome segregation in other species. Moreover, we identified a novel role for the two microtubule-growth and nucleation agents, RanGTP and CLASP, in the establishment of the centrosome-independent force during anaphase. Their involvement raises the interesting possibility that microtubule polymerization of midzone microtubules is required to sustain chromosome segregation during mitosis. Then, we aim to reversibility affect microtubule dynamics in the central spindle. Because of the lack of adequate tools, we have collaborated with biochemists from Dr. D. Trauner lab, in Munich, Germany, who are specialized in photoactivable drugs. They have synthetized a photoswitable drug, Photostatin (PST), which can depolymerize microtubules in few seconds in an on/off manner (Borowiak et al., 2015). Under blue light (390 - 430 nm), PST is activated leading to microtubule depolymerization, whereas under green light (500 - 530 nm), PST is activated which does not affect microtubule dynamics. I measured that microtubule growing is absent in presence of activated PST in Hela cells. I also showed that cell cycle can be stopped thank to activated PST in multiple cell C. elegans embryos. We have shown that PST can control microtubule dynamics thanks to visible light in vitro, in cellulo and in vivo, as an on/off switch, in a non-invasive, local and reversible manner.

Asymmetric positioning of the mitotic spindle in C. elegans embryos is mediated by force-generating complexes that are anchored at the plasma membrane and that pull on microtubules growing out from the spindle poles. Although asymmetric distribution of the force generators is thought to underlie asymmetric positioning of the spindle, the number and location of the force generators has not been well defined. In particular, it has not been possible to visualize individual force generating events at the cortex. We discovered that perturbation of the acto-myosin cortex leads to the formation of long membrane invaginations that are pulled from the plasma membrane toward the spindle poles. Several lines of evidence show that the invaginations, which also occur in unperturbed embryos though at lower frequency, are pulled by the same force generators responsible for spindle positioning. Thus, the invaginations serve as a tool to localize the sites of force generation at the cortex and allow us to estimate a lower limit on the number of cortical force generators within the cell.