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Die mitotische Spindel ist ideal für die Erforschung der Selbstorganisation und Plastizität molekularer Kollektive im Zytoplasma. Die Geometrie der Spindel ist entscheidend für die korrekte Chromosomentrennung, muss sich aber an die Zellgröße anpassen. Es ist unbekannt, ob und wie Zellen während ihrer Differenzierung die Spindelarchitektur anpassen, was insbesondere während der Gehirnentwicklung relevant ist. Wir untersuchten dies mit Maus-Embryonalstammzellen, die in frühe neuronale Vorläuferzellen differenziert wurden. Wir entwickelten ein automatisiertes Mikroskopieprotokoll um einen umfassenden Datensatz von mitotischen Zellen zu generieren. Außerdem entwickelten wir Spindle3D, ein Werkzeug zur dreidimensionalen Analyse von Spindeln. Überraschenderweise waren die Spindelvolumina in differenzierenden Zellen bis zu 24% kleiner als in pluripotenten Zellen. Während die Wachstumsgeschwindigkeit der Mikrotubuli unverändert blieb, verschob sich in sich differenzierenden Zellen die Nukleation von Mikrotubuli zugunsten der astralen Population. Diese Veränderung der Spindelarchitektur basierte auf der differenzierungsbedingten Verdünnung des Zytoplasmas. Dies aktivierte CPAP, ein Protein, das die Zentrosomenreifung reguliert, was zur Superskalierung des perizentriolären Materials und verstärkte Rekrutierung von gamma-Tubulin an den Zentrosomen und somit zur Umlagerung von Mikrotubuli innerhalb der Spindel führte. Diese Veränderungen der mitotischen Architektur konnten durch externe osmotische Einwirkung in undifferenzierten Zellen nachgestellt werden. Insgesamt verbinden unsere Ergebnisse zelltypspezifische zytoplasmatische Materialeigenschaften mit der Spindelarchitektur., The mitotic spindle provides an excellent system in which to study the plasticity of molecular collectives. To segregate chromosomes accurately, the spindle’s geometry must be adaptive to changes in cell size. It is unknown whether and how differentiating cells adjust spindle architecture, specifically during brain development when spindle defects have severe pathological consequences. Using murine embryonic stem cells, we recapitulated the transition from pluripotency to early neural cell identities in vitro. Aiming at a systematic exploration of spindle and cell morphology throughout this process, we developed an automated microscopy protocol and Spindle3D, a morphometric tool for the analysis of spindles in confocal images. Intriguingly, in cells with comparable cell volume, spindle volumes were up to 24% smaller in cells undergoing differentiation. While microtubule growth speed remained equal, we measured increased nucleation of astral microtubules at the expense of the spindle bulk in differentiating cells. The shift in spindle architecture was explained by the differentiation-driven cytoplasmic dilution. This activated the centrosomal regulator CPAP, causing the superscaling of the pericentriolar material and the concomitant increased recruitment of gamma-tubulin to the centrosomes, redistributing microtubule numbers within the spindle. Mimicking the dilution effect by osmotic challenge reproduced the same mitotic architecture in undifferentiated cells. Collectively, our results link cell state-specific cytoplasmic material properties to spindle architecture.

In the intricate realm of biology, meiosis stands as the remarkable process responsible for generating genetically diverse haploid gametes from diploid cells. In 2000, Pezzi et al., identified STAG3 as a novel meiotic-specific synaptonemal-complex associated protein belonging to the highly conserved family of stromalin nuclear proteins. Later, over the years, research groups characterised the depletion phenotype of STAG3 in mice, where deficiency of STAG3 causes severe chromosomal defects and early meiotic arrest. These studies together collectively highlighted STAG3 as the most important meiotic cohesin. Traditionally, the role of the cohesin complex was understood as maintaining cohesion between chromatids during cell division. However, over the years, this perception has evolved significantly, expanding to include the regulation of dynamic chromosomal configurations during meiosis. With the realisation of STAG3's importance in meiotic progression, the next pressing question becomes: how does STAG3 coordinate this intricate process? This study sought to address that question by examining the STAG3 interactome in male germ cells, aiming to uncover novel pathways through which STAG3 contributes to maintaining meiotic progression. Through successful purification of the STAG3-REC8 complex, its ability to form functional complexes in-vitro was demonstrated. During my PhD thesis, I discovered links between STAG3 and DNA repair mechanisms beyond the well-known homologous recombination /non-homologous end joining pathway in meiotic recombination. By looking at the meiotic-specific protein interactome bound to the STAG3-REC8 complex through Mass Spectroscopic analysis, we identified STAG3 involvement in PARP-1-mediated repair of DNA double-strand breaks occurring outside of the programmed DSB repair during the zygotene stage of prophase I. PARP-1 is an ADP-ribose polymerase which acts as a first responder that detects DNA damage and facilitates the activation of the DNA re

The nematode Auanema rhodensis has recently been established as a new model organism. A. rhodensis is characterized by the simultaneous existence of three phenotypical sexes (males, females and hermaphrodites; called trioecy), skewed sex ratios which do not follow Mendel's laws, and variant segregation patterns according to sex and type of gametogenesis. Recently, A. rhodensis has been used to study the possible mechanisms for sex determination in three-sexed species and the variability of basic processes during sexual reproduction including meiotic divisions. During male meiosis, a diploid primary spermatocyte undergoes two consecutive divisions to form four haploid spermatids. Surprisingly, male meiosis in A. rhodensis results in two functional and two nonfunctional spermatids depending on the presence of an X-chromatid. A. rhodensis males exhibit a set of paired autosomes and one single X-chromosome, like males of the wellestablished nematode model organism Caenorhabditis elegans. In contrast to C. elegans, however, the X-chromosome in A. rhodensis divides precociously into its sister chromatids during the first meiotic division followed by a lagging X-chromatid and its uneven distribution during meiosis II. Additionally, the second meiotic divi-sion within this species is characterized by an asymmetric organelle distribution and a spindle structure reminiscent of a monopolar spindle. In this study, serial section electron tomography was used to analyse the ultrastructure of the microtubule skeleton in spermatocytes of A. rhodensis. The analysis of spermiogenesis using electron tomography posed some key advantages compared to standard transmission electron microscopy. First, the microtubule (MT) network could be studied in detail including spindle formation, organization of spindle poles, rearrangement of MTs, and inter-action between MTs and chromosomes. Second, the number and shape of chromosomes could be visualized. And third, the morphology of organelles coul, Der Fadenwurm Auanema rhodensis hat sich in den letzten Jahren mehr und mehr zu einem neuen Modellorganismus entwickelt. Typisch für A. rhodensis ist das zeitgleiche Vor-kommen dreier phänotypischer Geschlechter (Männchen, Weibchen und Hermaphroditen; die sogenannte Triözie) und deren zahlenmäßig ungleiches Verhältnis zueinander, welches sich nicht durch Mendelsche Regeln erklären lässt. Ebenfalls auffällig sind nach Geschlecht und Gametogenese (Oogenese oder Spermatogenese) abweichende chromosomale Segregationsmuster. Unlängst hat A. rhodensis zu Erkenntnissen über die Geschlechtsdeterminierung in dreigeschlechtlichen Arten und die Varianz grundlegender Prozesse in der Meiose beigetragen. Während der männlichen Meiose (Spermatogenese) teilt sich eine diploide primäre Vorläuferzelle (primäre Spermatozyte) in zwei aufeinanderfolgenden Teilungen in insgesamt vier haploide Spermatiden. Bei A. rhodensis führt die Spermatogenese ungewöhnlicher-weise zu zwei funktionalen und zwei nicht-funktionalen Spermatiden, wobei die Funktionsfähigkeit vom Auftreten eines X-Chromatids abhängt. Männchen von A. rhodensis besitzen, ähnlich wie im Modellorganismus Caenorhabditis elegans, eine Reihe gepaarter Autosomen sowie ein einzelnes X-Chromosom. Im Gegensatz zu C. elegans teilt sich das ungepaarte X-Chromosom in A. rhodensis vorzeitig schon während der ersten meiotischen Teilung in seine Chromatiden, wodurch es zu einer verzögerten und ungleichen Verteilung des X-Chromatids während der zweiten meiotischen Teilung kommt. Diese zweite meiotische Teilung bei A. rhodensis ist außerdem durch eine asymmetrische Verteilung der Organellen und Mikrotubuli gekennzeichnet, letztere ähneln einer monopolaren Spindel. In dieser Arbeit wurde die Methode der seriellen Elektronentomographie genutzt, um die Ultrastruktur der Mikrotubuli in meiotischen Spindeln in Spermatozyten von A. rhodensis zu untersuchen. Zum einen wurden mittels Elektronentomographie das Netzwerk der Mikrotubuli und die Spindelor

Eukaryotic cells have to divide constantly in order to promote the growth of certain organs, to replace dying or damaged cells, or to set up an entire organism. These essential processes are called mitosis in the case of somatic cell division. Mitotic cell division is the process during which chromosomes, centrosomes, and microtubules (MTs) are involved to set up a bipolar structure called the “mitotic spindle”. This bipolar spindle is formed by MTs, which are presumably mainly organized from the centrosomes. However, more data are being published that suggest MTs nucleation can occur from other MTs or even a chromosome surface. These biopolymers are built from α/β-tubulin heterodimers and can dynamically grow and shrink to exert forces necessary for chromosome segregation. Previous studies of spindles during mitosis have allowed the identification of different MT classes based on their plus-ends interaction with different cellular target sites. One of the MT classes is the kinetochore microtubules (KMTs), which physically connect chromosomes and centrosomes (i.e. spindle pole) via a specialized protein structure termed the “kinetochore”. This kinetochore-to-spindle pole connection has been studied in many organisms. In budding yeast, this connection is established by only a single KMT. In contrast, multiple KMTs bind to each mammalian kinetochore and form an MT bundle also called “k-fiber”. The ultrastructural architecture of the mammalian k-fiber connection is not well documented. Currently, different models concerning the nature of the kinetochore-to-spindle pole connection via k-fibers are discussed in the literature, i.e. a direct, semi-direct or indirect connection. The widely accepted ‘direct’ model proposes that all k-fibers of the mammalian spindle are formed through tight bundles of up to 20 KMTs, with all MT minus ends associated with the centrosome. However, it is necessary to understand the k-fibers structure in order to interpret its role during chromo

Hintergrund: Schlaf ist ein streng regulierter Zustand körperlicher Ruhe und reduzierten Bewusstseins, der evolutionär im ganzen Tierreich konserviert ist. Schlafmangel ist in der modernen Gesellschaft weit verbreitet und betrifft 10 – 30 % der Erwachsenen. Dies stellt ein ernstes gesundheitliches Problem dar, da Schlafmangel mit vielen Krankheiten assoziiert ist, darunter Depressionen, Krebs und Herz-Kreislauf-Erkrankungen. Umgekehrt beeinflussen auch Krankheiten und das Immunsystem das Schlafverhalten. Trotz der fundamentalen Rolle dieser Wechselbeziehung sind grundlegende molekulare Mechanismen, die Funktionen des Immunsystems und Schlafkontrolle verbinden, bisher kaum verstanden. Da die Schlafregulation in Säugetieren sehr komplex ist, ist es sinnvoll konservierte Mechanismen zuerst in einfacheren Modellorganismen zu untersuchen. Der Rundwurm C. elegans ist ein solcher etablierter, simpler und vielseitiger Modellorganismus für die Schlafforschung. Er schläft sowohl im Rhythmus seiner Larvenentwicklung immer jeweils während des Lethargus kurz vor der Häutung, als auch nach besonderem Stress, wie zum Beispiel Hunger oder Hitze. C. elegans besitzt ein invariantes Nervensystem, in dem eine rapide Depolarisation des einzelnen RIS-Interneurons genügt, um Schlaf zu induzieren. Eine Mutation des AP2 Transkriptionsfaktors APTF-1 verhindert die Expression von FLP-11, dem schlafinduzierenden Neuropeptid von RIS. Dies führt praktisch zu völliger Schlaflosigkeit, die in C. elegans in der Regel nicht tödlich ist, und deshalb ein nützliches Modell für genetisch-chronischen Schlafmangel darstellt. Unser Labor fand heraus, dass eine Gain-of-function-Mutation in der Kollagenase NAS-38 über Signalwege der angeborenen Immunität und RIS-Aktivierung zu vermehrtem Schlaf während des Lethargus führt. Gleichzeitig wird dabei die Expression einer ganzen Familie antimikrobieller Peptide (AMP) hochreguliert. Derselbe Signalweg, einschließlich der AMP, sowie das Schlafverhalten werden auch, Background: Sleep is a tightly regulated state of behavioral quiescence and reduced consciousness, which is conserved throughout the animal kingdom. In modern societies 10 – 30 % of the adult population suffer from insufficient sleep, which poses a serious health problem as sleep deprivation is associated with a variety of diseases including depression, cancer, and cardiovascular diseases. Conversely, sickness and the immune system also influence sleep patterns. Despite the important role of this interrelationship between sleep and immunity, basic molecular mechanisms that link both vital functions are only poorly understood yet. As sleep regulation is complex in mammals and is thus difficult to address experimentally, it is reasonable to investigate its basic conserved mechanisms in simpler models first. The nematode C. elegans is such a well-established, simple, and powerful model organism for sleep research. It displays stress-induced sleep, for example upon starvation or heat shock, but also developmentally-timed sleep during lethargus prior to each larval molt. C. elegans possesses an invariant nervous system in which rapid depolarization of the single RIS interneuron is sufficient to induce sleep. Mutation of the AP2 transcription factor APTF 1 deprives RIS of its sleep-inducing neuropeptide FLP-11 and thus virtually abolishes sleep. This is not per se lethal in C. elegans, thereby presenting a powerful model for genetic sleep deprivation. Our lab found that a gain-of-function mutation in the collagenase NAS-38 strongly increases RIS-dependent sleep during lethargus with a concomitant upregulation of a large family of antimicrobial peptides (AMPs) via immunity pathways. Epidermal wounding also triggers AMP expression via immune signaling and induces sleep in the adult worm. Moreover, genetic sleep deprivation increases mortality upon epidermal injury. Together, this suggests AMPs to act as somnogens from peripheral tissues to the nervous system as part of a pr