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«Titel der Dissertation „Roles of SUN-1 phosphorylations during C. elegans meiosis“ Verfasser Mag.rer.nat. Alexander Woglar angestrebter ...»

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Titel der Dissertation

„Roles of SUN-1 phosphorylations during C. elegans



Mag.rer.nat. Alexander Woglar

angestrebter akademischer Grad

Doktor der Naturwissenschaften (Dr.rer.nat.)

Wien, 2013

Studienkennzahl lt. Studienblatt: A 091 441

Dissertationsgebiet lt. Studienblatt: Genetik-Mikrobiologie

Betreuerin: Univ.-Prof. Mag. Dr. Verena Jantsch-Plunger Contents Acknowledgement




Proposed aims of this thesis

Chapter 1.0 Contribution to the initial description of SUN-1 phosphorylation, aggregate formation and mobility

Chapter 1.1 SUN-1 forms mobile aggregates at chromosome ends and is phosphorylated at the onset of leptotene

Chapter 1.2 Mobile SUN-1 aggregates mediate homologous chromosome pairing by shuffling chromosome ends

Chapter 2.1 The SUN-1 phosphorylation patterns correlate with hallmarks of meiotic progression

Chapter 2.2 Persistent SUN-1 phosphorylation correlates with synaptic and recombinational errors in the wild type

Chapter 2.3 Synaptic and recombination failure independently prolong phosphorylation of SUN-1

Chapter 2.4 SUN-1 cannot be rephosphorylated in pachytene

Chapter 2.5 A recombination intermediate is required for dephosphorylation of SUN-1 in late pachytene

Chapter 2.6 Analysis of SUN-1 phosphosite mutants in meiosis

Chapter 2.7 SUN-1 phosphorylation is required for wild-type kinetics of synaptonemal complex formation and genomic integrity under challenged conditions

Chapter 2.8 SUN-1 phosphorylation is required for aggregate persistence and chromosome mobility beyond the TZ

Chapter 2.9 SUN-1 phosphorylation and aggregate formation is dependent on Polo kinases, PLk-2 (and PLK-1)

Chapter 2.10 SUN-1 phosphorylation is required for stable PLK-2 localization under challenged conditions


Material and Methods



Curriculum vitae


Acknowledgement Ich möchte mich bei meiner Familie, vor allem meinen Eltern, Robert und Helga, und meinen Freunden bedanken die mich über die Jahre meines Doktorats unermüdlich unterstützt haben.

I want thank my supervisor, Verena Jantsch, for an interesting time with a lot of exiting questions raised and some of them even answered.

I want to thank Alexandra Penkner, Antoine Baudrimont and Thomas Machacek for teaching me how to deal with worms. Also I want to thank the rest of the Jantsch and Loidl group for the amazing atmosphere, fruitful discussions and the countless occasions they helped me out over the past years.

I would not be able to assemble a complete list of people (in collaborations, in the Department of Chromosome Biology, the Campus and in the community) who contributed one or the other way to my work during the time of my PhD. Nevertheless I want to cordially thank all of them.

Thank you!

Abstract Sexual reproducing organisms have to halve their genomes prior to gamete formation to preserve genome size over generations. During meiosis, a diploid cell undergoes two rounds of succeeding chromosome segregation to give rise to four haploid products that can develop into sperms and oocytes. Therefore, prior to the first meiotic division, homologous chromosomes have to be paired and connected to each other by a crossover to be subsequently separated in meiotic anaphase I.

Besides pairing of chromosomes, crossover formation requires the introduction of DNA double strand breaks (DSBs) and repair of them by use of the homologous chromosome as a repair template. The therefore required proximity between the homologous chromosomes is established by the synaptonemal complex (SC), a proteinacious structure established between them. The C. elegans inner nuclear membrane protein Matefin/SUN-1 transmits cytoplasm-generated kinetic forces onto chromosomes at the onset of leptotene/zygotene. This leads to individualized movement of chromosomes and eventual paring of homologous chromosomes with subsequent formation of the SC between them. Meiotic progression and sequential achievement of these meiotic tasks (pairing, SC formation, DSB repair and recombination) have to be tightly coordinated.

We have found that meiotic chromosome movement is accompanied by formation of SUN-1 aggregates at the putative chromosomal attachment sites at the nuclear envelope and phosphorylation of SUN-1 at multiple residues. The phosphorylations depend on CHK-2 and PLK-2. PLK-2 colocalizes with the SUN-1 aggregates during the time of homology search and interacts physically with SUN-1. Failures in recombination and synapsis lead to the persistence of mobile and phosphorylated SUN-1 aggregates and to persisting recruitment of PLK-2 to chromosome ends.

SUN-1 phosphorylations are required for the continuous localization of PLK-2 to chromosome ends and persistent chromosome mobility, characteristic for a zygotene arrest. Furthermore SUN-1 phosphorylation enables the formation of the SC with wild-type kinetics.

In our data we present evidence for a checkpoint that monitors the presence of the obligate crossover mediated by SUN-1 phosphorylation. We propose that signals emanating from failures to successfully finish meiotic tasks are integrated at the nuclear periphery to regulate chromosome end-led movement and meiotic progression.


Vor der Gamtenfusion müssen sexuell reproduzierende Organismen ihre Genome halbieren, um die Chromosomenanzahl über Generationen konstant zu halten. Dafür durchläuft eine diploide Gametenvorläuferzelle zwei unmittelbar aufeinanderfolgend Chromosomenteilungen während der Meiose. Dies führt zu vier haploiden Tochterzellkernen die sich prinzipiell in Spermien oder Eizellen entwickeln können.

Damit homologe Chromosomen während der ersten meiotischen Teilung voneinander seggregieren können müssen sie gepaart und durch ein Crossover verbunden werden. Für die Etablierung eines meiotischen Crossovers werden, abgesehen von Chromosomenpaarung, auch DNS Doppelstrangbrüche (DSB) und Reperatur der Selbigen unter Verwendung des homologen Chromosomes als Reperturvorlage für homologe Rekombination, benötigt. Die hierfür erforderliche Nähe von homologen Chromosomen wird durch den Synaptonemalen Komplex (SC), einer Proteinstruktur, die zwischen den homologen Partnern aufgebaut wird, hergestellt. Am Beginn der meiotischen Prophase überträgt SUN-1, ein Transmembranprotein in der inneren nukleären Membrane, kinetische Kräfte aus dem Zytoplasma in den Kern. Dies führt zur individuellen Bewegung von Chromosomen, die zu dieser Zeit mit einem Ende an der Kernmembran verankert sind. Die Bewegung der einzellnen Chromosomen wird benötigt um die homologen Chromosomen zu paaren und um ungewollter, nicht homologer SC Formierung entgegenzuwirken. Der koordiniert Ablauf dieser einzellnen Funktionen (homologe Paarung, Aufbau des SC, DSB Generierung und Reperatur) während der Meiose bedarf einem hohen Ausmass and Regulation.

Wir haben herausgefunden, dass SUN-1 Aggregate an diesen Chromosomenbindungsstellen an der Kernmembran formt um die Chromosomenbewegung zu gewährleisten. Gleichzeitig mit dem Beginn seiner Aggregation wird SUN-1 an mehreren Aminosäuren an seinem nukleären N-terminus phosphoryliert. Diese Phosphorylierungen sind von CHK-2 und PLK-2 abhängig.

PLK-2 lokalisiert an den SUN-1 Aggregaten und co-prezipitiert mit SUN-1. SUN-1 Aggregate und Phosphorylierung persistieren wenn Synapsis oder Rekombination inhibiert sind und Chromosomenmobiltät wird unter diesen Umstände aufrechterhalten. Für diesen Arrest in der frühen meiotischen Prophase wird die Phosphorylierung von SUN-1 benötigt, um PLK-2 stabil and den mobilen Chromosomenenden zu halten um die damit einhergehende persistierende Chromosomenmobilität zu gewährleisten. Weiters ist die Phosphorylierung von SUNvon Nöten, um den SC mit wildtypischer Kinetik aufzubauen.

Unsere Beobachtungen lassen den Schluss zu, dass SUN-1 Phosphorylierung einen Zellzyklusprogressions Checkpunkt mediert, der das ‚obligate Crossover’ gewährleistet.

Introduction Meiosis – an overview To maintain the genome size over generations sexually reproducing organism have to halve their number of chromosomes before gamete formation. For this purpose most organisms make use of a specialized form of cell division: meiosis. Fusion of the resulting haploid cells (e.g.: fertilization of an egg by a sperm) yields a diploid zygote with the same number of chromosomes as the parental generation. Over the course of meiosis genetic diversity is generated by random segregation of homologous chromosomes and by reciprocal exchange of genetic information among them.

Mechanistically the halving of the chromosomal number is achieved by one cycle of pre-meiotic DNA replication followed by two successive rounds of cell or nuclear divisions resulting in four haploid nuclei. During the first, or reductional meiotic division, the homologous chromosomes, or more correct, centromeres, are moved to opposite poles of the cell (nucleus). In most sexually reproducing organisms, like yeast, human, mouse or C. elegans, a physical link between the homologous chromosomes has to be established to ensure their correct segregation during the first meiotic division by the cellular spindle apparatus. This linkage is formed by crossovers, which are manifestation of physical recombination events between homologous chromosomes during the extended meiotic prophase I. Requirements for crossover formation are sister chromatid cohesion, homolog juxtaposition, formation of DNA double strand breaks (DSBs) and their repair via the homologous chromosome.

The second or equational meiotic division resembles a mitotic division by separating the sister chromatids giving raise to for haploid gametes or gamete-precursors (reviewed in: (Petronczki, Siomos et al. 2003; Gerton and Hawley 2005))

Why using C. elegans as a meiotic model system?

The roundworm represents an easily tractable genetic system for both forward and reverse genetic approaches to identify and analysis meiotic mutants. C. elegans offers the possibility to isolate mutants that are defective in homologous pairing, synapsis and/or crossover formation by following increased spontaneous occurrence of males (5A; XO) among the self-progeny of hermaphroditic worms (5A; XX). Males arise due to X chromosome non-disjunction in meiosis I; only 0,02% of the progeny of a self-fertilized wild-type are males; mutations that result in more males are referred to as him (high incidence of males) (Hodgkin, Horvitz et al. 1979).

RNA-mediated interference (RNAi) was not only discovered in worms (Fire, Xu et al.

1998), but is easy to apply by soaking (Tabara, Grishok et al. 1998), feeding (Timmons and Fire 1998) or injecting (Fire, Xu et al. 1998) dsRNA. Recently techniques for targeted genomic deletions (Frokjaer-Jensen, Davis et al. 2010;

Wood, Lo et al. 2011) and single copy insertion transgenes (Frokjaer-Jensen, Davis et al. 2008) were developed and made available.

The body wall and also the gonad of C. elegans are transparent. This fact makes this model highly suitable for immunofluorescence on whole fixed tissues and also for live imaging of meiocytes progressing through the gonad. Further advantages of C.

elegans as a meiotic model are the short generation time (2.5 days on 25°C), the appearance as self-fertile hermaphrodites or males respectively and the ability to be frozen for permanent storage.

Meiosis I in the C. elegans gonad – A cytological overview

In the two gonad tubes of C. elegans hermaphrodites (one in the males) nuclei undergo the complete meiotic prophase I in a timely and spatially organized manner.

In the adult worm meiocytes progress through the meiotic cell cycle as they move down the gonad tube from the distal tip to the spermatheca. By that, at a given position of the syncytial gonad the nuclei have spent the same time in meiosis (Hirsh, Oppenheim et al. 1976; Crittenden, Troemel et al. 1994). Therefore the gonad represents a highly synchronous meiotic time course. The germline comprises half of the total number of nuclei in an adult hermaphrodite worm (Hirsh, Oppenheim et al.


The distal end of the gonad contains germ cell nuclei undergoing mitotic divisions and pre-meiotic S-phase (Crittenden, Leonhard et al. 2006). Further done the gonad, about 20 cell rows from the distal tip cell, a meiotic stem cell niche is induced by the distal tip cell causing rows of meiotic cells to form and move down to the proximal end, while they undergo the differed stages of prophase (Kimble and White 1981;

Watt and Hogan 2000).

Meiosis is entered in leptotene/zygotene. In contrast to other organisms studied these first two stages of meiotic prophase I cannot be readily distinguished cytologically in C. elegans. Leptotene/zygotene in worms corresponds to the transition zone in the gonad, in which chromatin adapts a half moon shaped configuration (Hirsh, Oppenheim et al. 1976). In the transition zone homologous chromosomes pair and synapse (Dernburg, McDonald et al. 1998; MacQueen, Colaiacovo et al. 2002; Colaiacovo, MacQueen et al. 2003). Also meiotic DSBs start to be induced in this stage (Alpi, Pasierbek et al. 2003; Colaiacovo, MacQueen et al.

2003; Mets and Meyer 2009).

Meiocytes move down the gonad with an approximate speed of one cell row per hour (Crittenden, Leonhard et al. 2006). After 7 to 12 cell rows synapsis between homologous chromosomes is completed and cells enter pachytene. With beginning of pachytene chromosomes start to redistribute throughout the nuclear volume. In this meiotic stage DSB repair and recombination take place along fully synapsed chromosomes (MacQueen, Colaiacovo et al. 2002; Alpi, Pasierbek et al. 2003;

Colaiacovo, MacQueen et al. 2003).

Diplotene starts with the disassembly of the SC (Martinez-Perez and Villeneuve 2005). Indicative for diplotene chiasmata, the cytological manifestations of crossovers, become apparent.

In the stage of diakinesis meiocytes leave the syncytium and cellularize. Due to further chromosome condensation, six DAPI positive structures become visible, which correspond to the six bivalents. These pairs of homologs are held together only by one chiasma per chromosome (Villeneuve 1994).

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