Control of elongation of replication-activated histone genes

Am Beginn meiner Arbeit stand die Hypothese, dass die Transkription von replikationsabhängigen Histongenen nicht nur durch Faktoren wie dem negativen Elongationsfaktor NELF oder Cap-Bindekomplex CBC reguliert wird, sondern dass ein Zellzyklus abhängiger Checkpoint auf Ebene der transkriptionellen Elongation bestimmt, ob Polymerase II über das 3’ Prozessierungssignal dieser Gene hinaus lies oder nicht. Anders als andere proteinkodierende mRNAs enden jene von replikationsabhängigen Histongenen in einer 3’ Stamm-Schleifen-Struktur anstatt am 3’ Ende polyadenyliert zu sein. Meine Chromatin Immunopräzipitation und reverse-Transkriptase-PCR Ergebnisse bestätigen unsere vorangegangene Annahme nicht, allerdings bedürfte es sensitiverer Methoden um eine sichere Aussage treffen zu können. Leider war die generelle Transkriptionsrate der replikationsabhängigen Histongene relativ gering verglichen zu jener von Referenzgenen wie dem U2 snRNA Gen und ACTB, folglich war es schwer Beweise für einen signifikanten Unterschied in den Pol II Profilen abhängig vom Zellzyklus zu finden. Zusätzlich wurden weitere Transkriptionsfaktoren und post-translationelle Histonmodifikationen wie H3K36me3 (siehe Appendix) oder H2B- Monoubiquitinierung untersucht, allerdings bedarf es weiterer Analysen um eine genaue Aussage machen zu können. Andere Gene könnten in diesem Bereich liegen und auf Grund derer bestimmte Histonprofile und Modifikationen auftreten. Die Synchronisation der Zellen durch doppelten Thymidin Block selbst funktionierte ausgezeichnet, brachte neue Daten betreffend der Dauer der einzelnen Zellzyklus Phasen der verwendeten HeLa-Zellen und erwies sich als sehr gut Methode zur Untersuchung von Zellzyklus abhängigen Faktoren. Das zweite Projekt war eine genomweite ChIP-Sequenzierung von mono- ubiquitiniertem H2B. Dies ist eine Histonmodifizierung die mit transkriptioneller Aktivität einhergeht. Die Ausführung funktionierte sehr gut nachdem Parameter der Immunopräzipitation optimiert wurden. Das Resultat ist sehr viel versprechend da es klar erkennbar eine bimodale Verteilung mit einem Minimum an der Startstelle der Transkription aufweist. Die komplette Analyse der Daten wird den Rahmen dieser Diplomarbeit in Bezug auf Zeit und Umfang allerdings leider sprengen.At the beginning of my work was the assumption that the transcription of replication-activated histone genes is not only regulated by various factor such as negative elongation factor NELF or cap binding complex CBC, but that there is a cell cycle dependent check-point at the level of transcription elongation that regulates whether polymerase II goes beyond the 3’ processing signal of these genes. Contrary to other protein coding genes, replication-activated histone genes end in a 3’ stem-loop instead of being polyadenylated. My ChIP/ RT-PCR results suggest that our hypothesis is not the case. However, more sensitive methods would be required to be conclusive. Unfortunately, the total transcription rate of the replication-activated histone genes was quite low compared to reference genes like the U2 snRNA gene or the ACTB gene, and it was hard to get evidence for significant differences of Pol II profiles on these genes depending on the cell cycle. In addition, other transcription factors and histone modification marks as H3K36me3 (see appendix) or H2Bub were investigated and gave interesting and novel results. Other genes might lie within that region studied and the histone occupancy and modifications marks may relate to these. The synchronisation of the cells by double thymidine blocking worked very well, providing novel data concerning the duration of the single cell cycle phases of the HeLa cells used and turned out to be a powerful tool for investigating cell cycle dependent factors. The second project was a genome-wide ChIP-sequencing of H2Bub, which is known to be positively linked with transcriptional activity. It worked very well after parameters for the ChIP were optimized. The result is promising as it shows a bi-modal distribution with its minimum at the transcription stat side. However, further analysis is beyond the scope of this thesis concerning data and time

Advantages of acute brain slices prepared at physiological temperature in the characterization of synaptic functions

Acute brain slice preparation is a powerful experimental model for investigating the characteristics of synaptic function in the brain. Although brain tissue is usually cut at ice-cold temperature (CT) to facilitate slicing and avoid neuronal damage, exposure to CT causes molecular and architectural changes of synapses. To address these issues, we investigated ultrastructural and electrophysiological features of synapses in mouse acute cerebellar slices prepared at ice-cold and physiological temperature (PT). In the slices prepared at CT, we found significant spine loss and reconstruction, synaptic vesicle rearrangement and decrease in synaptic proteins, all of which were not detected in slices prepared at PT. Consistent with these structural findings, slices prepared at PT showed higher release probability. Furthermore, preparation at PT allows electrophysiological recording immediately after slicing resulting in higher detectability of long-term depression (LTD) after motor learning compared with that at CT. These results indicate substantial advantages of the slice preparation at PT for investigating synaptic functions in different physiological conditions

Dense 4D nanoscale reconstruction of living brain tissue

Three-dimensional (3D) reconstruction of living brain tissue down to an individual synapse level would create opportunities for decoding the dynamics and structure–function relationships of the brain’s complex and dense information processing network; however, this has been hindered by insufficient 3D resolution, inadequate signal-to-noise ratio and prohibitive light burden in optical imaging, whereas electron microscopy is inherently static. Here we solved these challenges by developing an integrated optical/machine-learning technology, LIONESS (live information-optimized nanoscopy enabling saturated segmentation). This leverages optical modifications to stimulated emission depletion microscopy in comprehensively, extracellularly labeled tissue and previous information on sample structure via machine learning to simultaneously achieve isotropic super-resolution, high signal-to-noise ratio and compatibility with living tissue. This allows dense deep-learning-based instance segmentation and 3D reconstruction at a synapse level, incorporating molecular, activity and morphodynamic information. LIONESS opens up avenues for studying the dynamic functional (nano-)architecture of living brain tissue

Control of human endometrial stromal cell motility by PDGF-BB, HB-EGF and trophoblast-secreted factors

Human implantation involves extensive tissue remodeling at the fetal-maternal interface. It is becoming increasingly evident that not only trophoblast, but also decidualizing endometrial stromal cells are inherently motile and invasive, and likely contribute to the highly dynamic processes at the implantation site. The present study was undertaken to further characterize the mechanisms involved in the regulation of endometrial stromal cell motility and to identify trophoblast-derived factors that modulate migration. Among local growth factors known to be present at the time of implantation, heparin-binding epidermal growth factor-like growth factor (HB-EGF) triggered chemotaxis (directed locomotion), whereas platelet-derived growth factor (PDGF)-BB elicited both chemotaxis and chemokinesis (non-directed locomotion) of endometrial stromal cells. Supernatants of the trophoblast cell line AC-1M88 and of first trimester villous explant cultures stimulated chemotaxis but not chemokinesis. Proteome profiling for cytokines and angiogenesis factors revealed neither PDGF-BB nor HB-EGF in conditioned media from trophoblast cells or villous explants, while placental growth factor, vascular endothelial growth factor and PDGF-AA were identified as prominent secretory products. Among these, only PDGF-AA triggered endometrial stromal cell chemotaxis. Neutralization of PDGF-AA in trophoblast conditioned media, however, did not diminish chemoattractant activity, suggesting the presence of additional trophoblast-derived chemotactic factors. Pathway inhibitor studies revealed ERK1/2, PI3 kinase/Akt and p38 signaling as relevant for chemotactic motility, whereas chemokinesis depended primarily on PI3 kinase/Akt activation. Both chemotaxis and chemokinesis were stimulated upon inhibition of Rho-associated, coiled-coil containing protein kinase. The chemotactic response to trophoblast secretions was not blunted by inhibition of isolated signaling cascades, indicating activation of overlapping pathways in trophoblast-endometrial communication. In conclusion, trophoblast signals attract endometrial stromal cells, while PDGF-BB and HB-EGF, although not identified as trophoblast-derived, are local growth factors that may serve to fine-tune directed and non-directed migration at the implantation site

The role of Notch-mediator RBPJkappa in first trimester human trophoblasts

Die Plazenta ist jenes Organ das während der Schwangerschaft die Verbindung zwischen der Mutter und ihrem Embryo herstellt. Hauptaufgaben umfassen den Nähr- und Sauerstofftransport von mütterlichem zu embryonalem Blut und Abtransport von Abbauprodukten des embryonalen Stoffwechsels. Um das zu bewerkstelligen verbindet sich die Plazenta mit der Schleimhaut des Uterus, der Dezidua, und plazentale Trophoblasten wandern in das mütterliche Gewebe ein. Hier haben Trophoblasten sowohl eine endokrine Funktion als auch die Aufgabe die zur Plazenta führenden Spiralarterien umzubauen um die Blutzufuhr den Bedürfnissen des Embryos anzupassen. Fehler in diesem Prozess stehen in engem Zusammenhang mit der Entwicklung von schweren Schwangerschaftskomplikationen wie Präeklampsie oder Fehlgeburten. Umso mehr überrascht es, dass sowohl die Entwicklung als auch die Details zur Funktionenweise der menschlichen Plazenta bisweilen eher dürftig erforscht worden sind. Bis jetzt wurden mehrere zelluläre Signaltransduktionswege in der Differenzierung von Trophoblasten nachgewiesen, doch die Rolle des kanonischen Notch-Signalweges über seinen zentralen Transkriptionsfaktor RBPJkappa und dessen Ko-Aktivatoren MAML1-3 im Menschen ist bislang unbekannt. In dieser Studie zeige ich mittels Immunfluoreszenzmikroskopie und Western Blot erstmalig, dass sowohl RBPJkappa als auch die MAML Proteine in villösen wie extravillösen Trophoblasten vorhanden sind. Des Weiteren konnte eine Abnahme des Signals eines RBPJkappa-abhängigen Luziferase-Reporters während der in vitro Differenzierung von primären Trophoblasten nachgewiesen werden, was auch mit einer geringer werdenden Expression des Notch-Zielgenes HES1 während dieser Entwicklung einhergeht. Der Knock-Down von RBPJkappa mittels siRNA führte zu einer erhöhten Proliferation von sowohl primären kultivierten Trophoblasten als auch in Zellsäulen von villösen Explants. Interessanterweise führte dies auch zu einer vermehrten Differenzierung, was durch die erhöhte Expression von Differenzierungsmarkern wie ADAM12, ITGA1 und T-cell factor 4 gezeigt werden konnte. Nicht beeinträchtigte waren hingegen Apoptose und Motilität der Zellen durch den Knock-Down. Zusammengefasst weisen die Daten auf eine durch den kanonischen Notch Signalweg kontrollierte Proliferation und Differenzierung von Vorläuferzellen der Zellsäulentrophoblasten hin. Dies könnte ein Mechanismus sein um übermäßiges Wachstum und vorzeitige Differenzierung der Zellen zu verhindern und so einen kontrollierten Ablauf zu bewahren.The placenta is the connecting organ between an embryo and its mother and it fulfils a multitude of tasks that later in life are accomplished by a number of individual organs. Despite its pivotal function during pregnancy and its postulated involvement in the pathogenesis of major gestational disorders such as pre-eclampsia, miscarriage or intrauterine growth restriction it is a rather neglected organ. Placental functions involve nutrient and oxygen supply of the embryo as well as waste disposal. To realise that, formation of an accurate foetal-maternal interface and proper trophoblast invasion into uterine tissue with subsequent vascular remodelling is essential. Nevertheless, molecular signalling pathways controlling this tightly regulated process are still poorly elucidated. In this study I provide evidence that the canonical Notch signalling pathway, acting through its central transcription factor RBPJkappa, plays a major role in controlling proliferation and differentiation of first trimester primary trophoblasts and villous explant cultures. RBPJkappa and its co-activators MAML1-3 were detected in human villous cytotrophoblasts (CTBs) and extravillous trophoblasts (EVTs) of tissue sections using immunofluorescence. Basic Notch activity was measured in isolated CTBs after transfection with an RBPJκ-dependent luciferase reporter. However, RBPJkappa expression, transcript levels of its target gene HES1 and reporter activity declined during in vitro formation of EVTs. SiRNA-mediated gene knock-down of RBPJkappa extinguished Notch reporter expression and enhanced proliferation of both, isolated CTBs and cell column trophoblasts in villous explant cultures, promoting an increased outgrowth of the latter. Accordingly, markers of the differentiated EVT, i.e. ADAM12, ITGA1 and T-cell factor 4 were upregulated upon gene silencing. Notably, motility and apoptosis were not affected. These data suggest a Notch-dependent control of cell column trophoblast progenitors that could represents a mechanism to prevent exaggerated placental growth by balancing rates of differentiation and proliferation.Arbeit an der Bibliothek noch nicht eingelangt - Daten nicht geprüftAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität Wien, Dissertation, 2016OeBB(VLID)171515

LIONESS enables 4D nanoscale reconstruction of living brain tissue

We developed LIONESS, a technology that leverages improvements to optical super-resolution microscopy and prior information on sample structure via machine learning to overcome the limitations (in 3D-resolution, signal-to-noise ratio and light exposure) of optical microscopy of living biological specimens. LIONESS enables dense reconstruction of living brain tissue and morphodynamics visualization at the nanoscale

Cell Adhesion and Migration / Function and control of human invasive trophoblast subtypes: Intrinsic vs. maternal control

The establishment of a functional placenta is pivotal for normal fetal development and the maintenance of pregnancy. In the course of early placentation, trophoblast precursors differentiate into highly invasive trophoblast subtypes. These cells, referred to as extravillous trophoblasts (EVTs), penetrate the maternal uterus reaching as far as the inner third of the myometrium. One of the most fundamental functions of EVTs is the transformation of spiral arteries to establish the uteroplacental blood circulation assuring an adequate nutrient and gas supply to the developing fetus. To achieve this, specialized EVT subpopulations interact with maternal immune cells, provoke elastolysis in the arterial wall and replace the endothelial cells lining the spiral arteries to induce intraluminal vascular remodeling. These and other trophoblast-mediated processes are tightly controlled by paracrine signals from the maternal decidua and furthermore underlie an intrinsic cell-type specific program. Various severe pregnancy complications such as preeclampsia or intrauterine growth retardation are associated with abnormal EVT function, shallow invasion, and decreased blood flow to the placenta. Hence a better understanding of human trophoblast invasion seems mandatory to improve therapeutic intervention. This approach, however, requires a profound knowledge of the human placenta, its various trophoblast subtypes and in particular a better understanding of the regulatory network that controls the invasive phenotype of EVTs.P 25187-B13(VLID)310352

Strategies to maximize performance in STimulated Emission Depletion (STED) nanoscopy of biological specimens

Super-resolution fluorescence microscopy has become an important catalyst for discovery in the life sciences. In STimulated Emission Depletion (STED) microscopy, a pattern of light drives fluorophores from a signal-emitting on-state to a non-signalling off-state. Only emitters residing in a sub-diffraction volume around an intensity minimum are allowed to fluoresce, rendering them distinguishable from the nearby, but dark fluorophores. STED routinely achieves resolution in the few tens of nanometers range in biological samples and is suitable for live imaging. Here, we review the working principle of STED and provide general guidelines for successful STED imaging. The strive for ever higher resolution comes at the cost of increased light burden. We discuss techniques to reduce light exposure and mitigate its detrimental effects on the specimen. These include specialized illumination strategies as well as protecting fluorophores from photobleaching mediated by high-intensity STED light. This opens up the prospect of volumetric imaging in living cells and tissues with diffraction-unlimited resolution in all three spatial dimensions

A direct excitatory projection from entorhinal layer 6b neurons to the hippocampus contributes to spatial coding and memory

The mammalian hippocampal formation (HF) plays a key role in several higher brain functions, such as spatial coding, learning and memory. Its simple circuit architecture is often viewed as a trisynaptic loop, processing input originating from the superficial layers of the entorhinal cortex (EC) and sending it back to its deeper layers. Here, we show that excitatory neurons in layer 6b of the mouse EC project to all sub-regions comprising the HF and receive input from the CA1, thalamus and claustrum. Furthermore, their output is characterized by unique slow-decaying excitatory postsynaptic currents capable of driving plateau-like potentials in their postsynaptic targets. Optogenetic inhibition of the EC-6b pathway affects spatial coding in CA1 pyramidal neurons, while cell ablation impairs not only acquisition of new spatial memories, but also degradation of previously acquired ones. Our results provide evidence of a functional role for cortical layer 6b neurons in the adult brain

Chiral and nematic phases of flexible active filaments

The emergence of large-scale order in self-organized systems relies on local interactions between individual components. During bacterial cell division, the tubulin-homolog FtsZ polymerizes into treadmilling filaments that further assemble into a cytoskeletal ring. Although minimal in vitro assays have shown the striking self-organization capacity of FtsZ filaments, such as dynamic chiral assemblies, how these large-scale structures emerge and relate to individual filament properties remains poorly understood. To understand this quantitatively, we combined minimal chiral active matter simulations with biochemical reconstitution experiments. Using STED and TIRF microscopy as well as high-speed AFM, we imaged the behavior of FtsZ filaments on different spatial scales. Simulations and experiments revealed that filament density and flexibility define the local and global order of the system: At intermediate densities, flexible filaments organize into chiral rings and polar bands, while an effectively nematic organization dominates for high filament densities and for mutant filaments with increased rigidity. Our predicted phase diagram captured these features quantitatively, demonstrating how filament flexibility, density and chirality cooperate with activity to give rise to a large repertoire of collective behaviors. These properties are likely important for the dynamic organization of soft chiral matter, including that of treadmilling FtsZ filaments during bacterial cell division