Lipid rafts are nanoscale entities in the membranes of eukaryotic cells which provide a mechanism for the functional membrane segregation vital for several cellular processes. This lateral segregation of specific lipid and protein components provides the facilitative platforms for a variety of signaling and trafficking events at the plasma membrane and in the Golgi. Rafts are distinguished from the surrounding membranes by their physical properties and composition - they are relatively tightly packed and enriched in saturated lipids, sterols, and lipid-anchored proteins. Although the existence of rafts has been conclusively confirmed by several independent techniques, questions concerning various aspects of membrane heterogeneity are still to be addressed. Typical experiments investigating raft composition have been designed to evaluate the affinity of a given component for raft domains. In such experiments, the results are usually interpreted in a Boolean fashion, i.e., the component is either a raft molecule, or not. However, this binary point of view overlooks potential complexity that may underlie the nature of membrane heterogeneity. In this work, we systematically investigated the nature of functional cellular membrane heterogeneity. We started by characterizing the model membranes and fluorescent lipid analogs widely used in research into membrane domains. After extensively evaluating the potentials/limits of these approaches and the artifacts that must be avoided or alternatively could be exploited, we applied these tools to understand whether the cell membrane has multiple kinds of raft domains with distinct compositions and physical properties, rather than only one. We found that cell membranes have the potential to form various kinds of functional domains having different physicochemical properties, compositions, and functional outputs. Therefore, we propose continuously variable lipid packing as the principle of the functional membrane lateral heterogeneity. According to this principle, the membrane is not composed of a single variety of raft domain with strictly defined properties coexisting alongside a specific and uniform non-raft environment; rather it is composed of entities having continuously variable lipid packing. Finally, we show that this spectrum of membrane packing modulates the orientation of membrane lipid receptors, which ultimately influences their specific bioactivity. Our results showing continuously variable lipid packing and its ability to fine-tune the activity of membrane molecules comprise a novel model for the structure and function of eukaryotic membranes.

Eulenfalter sind in Deutschland die artenreichste Gruppe der Großschmetterlinge. Die Broschüre informiert über die Gefährdungssituation von 392 Arten in Sachsen. Die meisten Arten sind dämmerungs- und nachtaktiv, einige aber auch tagaktiv. Viele Bewohner trockener Offenlandbiotope, der Moore und des Feuchtgrünlandes sind bereits ausgestorben. Noch immer sind viele Arten dieser Lebensräume vom Aussterben bedroht oder hochgradig gefährdet. Hinzu kommen hochgradig gefährdete Eulenfalterarten verschiedener Feuchtlebensräume an Gewässern. Wesentliche Gefährdungsfaktoren sind die allgemeine Eutrophierung der Standorte, die Nutzungsaufgabe oder der Verlust extensiv genutzter Standorte sowie Veränderungen im Wasserhaushalt von Feuchtlebensräumen. Auch der Einsatz von Insektiziden kann sich direkt auf Raupen und Falter auswirken. Rote Listen werden regelmäßig aktualisiert. Eine Rote Liste Eulenfalter Sachsens erschien zuletzt 1995. Redaktionsschluss: 31.12.2017

Multicellular organisms need to control their size throughout development and adult life in the face of challenges such as rapid growth. Unraveling the mechanisms that regulate tissue growth in epithelial tissues, in order to generate organs of correct size and proportion, remains a crucial goal of developmental biology. A suitable epithelial tissue for studying tissue growth in Drosophila, is the proliferative monolayer epithelial sheet of imaginal wing discs, which gives rise to the adult wing. The Hippo signaling pathway regulates tissue growth in wing development. There are several observations that link tissue growth/Hippo signaling with cell polarity and the actin cytoskeletal organization. The aim of this thesis was the study of the interplay between cell polarity, cytoskeletal organization and tissue growth. To gain further insight into how apical polarity proteins regulate tissue growth, an enhancer/suppressor screen that was previously conducted in our lab by Linda Nemetschke, was used. The screen was based on the modification of a dominant smaller wing phenotype induced upon overexpression of CrbextraTM-GFP. One of the enhancers identified in this screen is a gene called big bang (bbg). The absence of bbg results in smaller wings with a slower cell cycle and increased apoptosis in wing discs. bbg encodes a protein expressed in the apical cortex in wing disc cells and is required for the proper localization of apical proteins, like Crb, in wing disc epithelia. Bbg is also in the same complex with Spaghetti Squash (Sqh) in the apical cortex of the wing disc epithelia. sqh encodes an actin-binding protein that has actin cross-linking and contractile properties. Bbg stabilizes Sqh in the apical compartment of the cell. It is reported that both Crb and Sqh regulate tissue growth through the Hippo signaling pathway. In conclusion, Bbg regulates wing tissue growth, acting as a scaffolding molecule, through the proper localization of apical components of the cells like Crb and the cytoskeletal component Sqh.

Die weltweit ungleiche Verteilung von Entwicklungschancen birgt unerschöpfliches Konfliktpotenzial. Dies lässt sich an der unterschiedlichen Verfügbarkeit von Nahrungsenergie ebenso festmachen wie an Ungleichgewichten im Bereich technisch erzeugter Energie. Die Umwandlung von Nahrungsmitteln in Treibstoffe kann diese globalen Disparitäten noch verschärfen. Der Hunger der Industrienationen nach Automobilität war nur mit Treibstoffen zu stillen, die auch auf synthetischem Weg gewonnen wurden. Der Beitrag nähert sich der Geschichte energetischer Ungleichheiten in kritischer Auseinandersetzung mit Mobilitäts- und Konsumparadigmen. Dabei werden unterschiedliche Dimensionen technologisch induzierter Ungleichheit sichtbar, die sich von der globalen Ungleichverteilung von Macht, über die soziale Bedingtheit differierender Teilhabechancen, bis hin zu geschlechtsspezifischen Technikaneignungen erstrecken. / The global context of unequal development chances is reflected both by the differing availability of food energy and by imbalances in technically generated energy. The transformation of food into fuels could well aggravate these global disparities still further. It has to date only been possible to satisfy the hunger of the industrial nations for automobility with fuels won by synthetic methods. The paper approaches the history of energetic inequalities in a critical discussion with food and mobility paradigms. A focus is placed on different dimensions of technologically induced inequalities: These include the unequal distribution of power, the social limitations on participation chances and gender-specific technology appropriations.

Wnt proteins have key roles in patterning of multicellular animals, acting at a distance from their sites of production. However, it is not well understood how these molecules propagate. This question has become even more puzzling by the discovery that Wnts harbour post-translational lipid-modifications, which enhance association with membranes and may therefore limit propagation by simple diffusion in an aqueous environment. The cellular mechanisms involved in Wnt propagation are largely unknown for vertebrate organisms. Here, I discuss my findings on the cellular localization of zebrafish Wnt8, as an example of a vertebrate Wnt. Wnt8 is a key signal for positioning the midbrain-hindbrain brain boundary (MHB) organizer along the anterior-posterior axis of the developing brain in vertebrates. However, it is not clear how this protein propagates from its source, the blastoderm margin, to the target cells, in the prospective neural plate. For this purpose, I have analysed a biologically active, fluorescently tagged Wnt8 in live zebrafish embryos. Wnt8 was present in live tissue in membrane associated punctate structures. In Wnt8 expressing cells these puncta localise to filopodial cellular processes, from which the protein is released to neighbouring cells. This filopodial release requires posttranslational palmitoylation. Although palmitoylation-defective Wnt8 retains auto- and juxtacrine signaling activity, it fails to signal over a long-range. Additionally, this Wnt8 palmitoylation is necessary for regulation of its neural plate target genes. These results suggest that vertebrate Wnt proteins use cell-to-cell contact through filopodia as a shortrange propagation mechanism while released palmitoylated Wnt is required for longrange signaling activity. Furthermore, I show that a Wnt8 receptor, Frizzled9 can negatively influence Wnt8 propagation and signaling range. Finally, I was able to determine the presence of an endogenous Wnt8 gradient in the neurectoderm. I discuss these findings in the context of Wnt8 signaling function in mediating anterior-posterior patterning during early brain development.

Neuerliche Fortschritte im Verständnis biomolekularer Motoren rücken ihre Anwendung als Nanomaschinen in den Bereich des Möglichen. So könnten sie zum Beispiel als Nanoroboter arbeiten, um in molekularen Fabriken kleine – aber dennoch komplizierte – Strukturen auf winzigen Förderbändern herzustellen, um Netzwerke molekularer Nanodrähte und Transistoren für elektronische Anwendungen zu assemblieren oder sie könnten in adaptiven Materialien patrouillieren und diese, wenn nötig, reparieren. In diesem Sinne besitzen biomolekulare Motoren das Potenzial, die Basis für die Konstruktion, Strukturierung und Wartung nanoskaliger Materialien zu bilden. / Recent advances in understanding how biomolecular motors work have raised the possibility that they might find applications as nanomachines. For example, they could be used as molecule- sized robots that work in molecular factories where small, but intricate structures are made on tiny assembly lines, that construct networks of molecular conductors and transistors for use as electrical circuits, or that continually patrol inside “adaptive” materials and repair them when necessary. Thus biomolecular motors could form the basis of bottom-up approaches for constructing, active structuring and maintenance at the nanometer scale.

The aim of this dissertation was to improve our understanding of the mechanisms underlying biodiversity ecosystem functioning (BEF) relationships and the provisioning of ecosystem functions in temperate forest soils. I studied the role of tree diversity on soil microbial properties, standard litter decomposition, abiotic soil properties, and soil surface temperature. All studies took place in the Kreinitz tree diversity experiment in Central Germany which was established in 2005. It spans a tree diversity gradient from 1 to 6 different commonly cultivated tree species. My experiments included measurements within the whole diversity gradient in November 2017 and a high-resolution time series on monocultures and five-species mixtures in 2017 and 2018. In addition, I utilized tree inventory data about tree mortality, height, diameter and biomass. Chapter I aimed to assess general BEF relationships in soil. Chapter II followed a spatio-temporal framework explaining BEF relationships in forest soils via the spatial and temporal stability of biotic and abiotic properties, based on concepts like species asynchrony and complementarity. Chapter III utilized the perturbation of the extreme summer drought of 2018. It focused on biodiversity-resistance relationships and assessed how tree species richness and identity affected tree mortality rates in the experiment. All three studies showed that tree species identity and community composition are essential in shaping BEF relationships in temperate forest soils and are pivotal for the stable provisioning of ecosystem functions. The influence of tree species identity and community composition could be related to changes in abiotic soil properties and microclimatic conditions (i.e. soil surface temperature). I found evidence that spatio-temporal dynamics are indeed crucial determinants in BEF relationships in forest ecosystems. Overall, my thesis indicated how climate change and other global change factors will likely influence the provisioning and stability of soil ecosystem functions in forest via their intense pressure on tree community composition and the perturbation of spatiotemporal patterns. Overall, this dissertation advanced our mechanistic understanding of BEF relationships in temperate forest soils. While it underlined the dangers of global change for the provisioning of ecosystem functions, it also offered vantage points to prepare our forests for a changing future.

Dissertation

Bacterial viruses, also known as phages, are intrinsic components of the Earth’s Critical Zone (CZ). Together with diverse communities of bacteria and fungi, they occupy habitats of the CZ extending from the vegetation canopy, through the soils and into the aquifers. In this thesis, I aimed to study the transport of phages in the upper CZ and their interactions with non-host soil bacteria and fungi to reveal their role in regulating the CZ microbial life. To reflect transport processes of CZ-inhabiting phages in soil tracers are highly useful. Thus, in the first study, I evaluated the transport efficiency and particle intactness of marine tracer phages passing through soil. Marine phages were selected as tracer, because they are non-pathogenic, non-toxic, naturally absent and thus non-multiplying in the terrestrial subsurface. I found that the marine phages PSA-HM1 and PSA-HS2 retained high phage particle intactness in contrast to commonly used Escherichia virus T4. This suggests their potential as particle tracers to mimic the transport of (bio-) colloids of similar traits in soil. Soil in the CZ is often unsaturated and restricts mobility of microorganisms. Fungi bridge unsaturated zones in soil and hence provide network for microbial transport. In the second study, I developed a hyphosphere model system mimicking unsaturated soil environment, and reported on the ability of hyphal-riding bacteria to co-transport lytic phages and to utilize phages as “weapons” for improved colonization of water-unsaturated habitats. As the findings emphasize the importance of hyphal transport of bacteria and associated phages, in the third study, I developed a hyphae-assisted approach and isolated five soil bacteria able to co-transport phages. In analogy to invasion frameworks in macroecology, the hyphosphere model system with bacteria and co-transported phages can be useful models to simulate processes of biological invasion at micro-scale. In the fourth study, I investigated dormant phages (i.e. prophages) that are widespread in the CZ and can be induced under environmental stress. I found that volatile fungal metabolites can act as triggers for prophage induction and may exert long-distance manipulation of prophage activity thereby affecting microbial community and nutrient cycling in soil. Altogether, the findings may help to elucidate transport processes of phages in the CZ and to reveal the role of phages in the CZ microbial ecosystem. Approaches (e.g. phage as tracers) and findings (e.g. phage-bacterial co-transport) may also serve as useful tools for testing hypothesis in other disciplines, such as hydrogeology, invasion ecology and chemical ecology.

Cells use energy to maintain order, as living systems are inherently non-equilibrium. Or- der in the cytoplasm is achieved by compartmentalization. One type of compartment that gained interest in recent years is membraneless organelles (MLOs). Observations of the liquid-like properties of MLOs led to their interpretation in analogy to Liquid-Liquid Phase Separation (LLPS). However, LLPS alone implies a passive closed system that tends towards equilibrium, which is incompatible with the physical nature of the cell. It is unclear then what non-equilibrium interactions give rise to the dynamics of MLOs in the cell. We sought to decipher the regulatory interactions that give rise to active condensation in the actomyosin cortex of C. elegans. The components of the actomyosin cortex, F- actin and its branching nucleation module Arp2/3 and N-WASP (WSP-1 in C. elegans) have been described as a phase separated system in previous reports. In vitro, phase separated N-WASP compartments do not have the non-equilibrium growth and disas- sembly dynamics observed in the multicomponent clusters in vivo. Therefore, our goal is to examine WSP-1, Arp2/3 and F-actin interactions in the endogenous context. We chose the stage in which the quiescent oocyte cortex becomes actively contractile. During the transition out of quiescence, we observed transient WSP-1 Arp2/3 F-actin puncta that assemble and disassemble. To capture growth dynamics for all puncta, we devel- oped a novel phase portrait analysis tool. The phase portrait allows us to simultaneously study puncta growth and disassembly rates as a function of internal composition. The growth rate dependence on internal composition reflects the non-trivial changes to nu- cleation profiles that accompany condensation in active, open, multi-component systems. We observed superlinear WSP-1 growth rates consistent with condensation. Further, we identified the in vivo equivalent of a nucleation barrier for WSP-1 condensation. The in vivo nucleation barrier increases with branching F-actin reaction, which tunes con- densation. Correspondingly, the reactive components WSP-1 and Arp2/3 are important for condensate dynamics. Combining condensation and the branching reaction, we for- mulated a coarse-grained model which captures non-equilibrium condensate dynamics. Altogether, our results showed that WSP-1 grows like condensation, and its growth is steered away from equilibrium by Arp2/3 mediated branching reaction. In summary, combining high-resolution imaging, quantitative analysis and theory, we identified the interactions that could explain non-equilibrium condensation in the acto- myosin cortex. The living dynamics that arise from the interplay between condensation and reaction. The interplay between physical processes (like condensation) and biological regulation (such as reactions) may be a common organizing principle behind MLO for- mation, as well as other non-equilibrium processes in the cell. The methods and concepts developed in this work hold the promise to deepen our understanding of how living cells regulate their dynamic organization, in order to maintain themselves in a non-equilibrium ordered state.:1 Introduction 1 1.1 Evolving concepts of cellular organization 1 1.2 Condensation of biomolecules 3 1.2.1 Terminology for biomolecular condensates 5 1.2.2 Technical considerations for identifying liquid-like properties and LLPS 7 1.2.3 Thermodynamics of condensation 10 1.2.4 The problem of an equilibrium description of living systems 13 1.2.5 Towards active condensation 14 1.3 Actomyosin cortex self-organization 16 1.3.1 F-actin treadmilling and nucleation 17 1.3.2 N-WASP and Arp2/3 regulation 18 1.3.3 Multivalent interactions in condensation of transmembrane receptors and actin regulators 22 1.3.4 Cortex activation in C. elegans 23 2 Aims 25 3 Results 26 3.1 C. elegans cortical activation begins at fertilization 26 3.1.1 C. elegans oocytes as an ex utero model for cortex self-organization 27 3.2 WSP-1, Arp2/3 and F-actin form dynamic multicomponent phases 32 3.2.1 Capping proteins outcompete Formin in WSP-1 Arp2/3 puncta preventing F-actin elongation 32 3.2.2 WSP-1 and Arp2/3 are required for punctate F-actin formation and dynamics 34 3.2.3 Summary of the characterization of cortical activation 34 3.3 Establishment of systematic phase portrait analysis for multicomponent clusters 36 3.3.1 Non-equilibrium features of the multicomponent puncta 36 3.3.2 Recording intensity traces of multicomponent cluster over time 37 3.3.3 Probability flux of composition in the phase portrait show a closed cycle 38 3.3.4 WSP-1 F-actin puncta have a preferred joint concentration 38 3.3.5 The phase portrait is robust to cell-to-cell noise 41 3.3.6 Choosing the appropriate bin size 41 3.4 Existence of a tuned critical size and signatures of active condensation 45 3.4.1 Growth rate dependence on internal composition 45 3.4.2 Stoichiometric growth laws of WSP-1 F-actin clusters 47 3.4.3 Estimation of WSP-1 cluster critical size in vivo 47 3.4.4 Theoretical description of WSP-1 and F-actin interactions in regulating puncta dynamics 48 3.4.5 Summary of 2D phase portrait findings 52 3.5 Towards three dimensional phase portrait analysis of the reaction network 54 3.6 Initial assessment of the compartment’s external environment 54 3.7 Identification of modulators of puncta dynamics 56 3.7.1 CDC-42 controls cortical levels of WSP-1 56 3.7.2 RHO-1 and Formin CYK-1 are not involved in WSP-1 F-actin condensate dynamics 58 3.7.3 WSP-1 and Arp2/3 dynamics are independent of NCK-1 and VAB-1 58 3.7.4 Arp2/3 regulates condensate dynamics 60 3.8 Summary of perturbations 63 4 Conclusions and outlook 64 4.1 Concluding remarks 64 4.2 Discussion 66 4.3 Future directions 67 4.3.1 Realizing the full potential of the phase portraits in identifying biochemical interactions 67 4.3.2 Resolving the ultrastructure of condensates . 70 4.3.3 Further investigation of the biological function 71 4.3.4 Applying full-dynamic data acquisition to other membraneless organelles 71 5 Materials and Methods 72 5.1 C.elegans maintenance and strains 72 5.2 Sample preparation 72 5.2.1 In utero imaging 72 5.2.2 Oocyte imaging 73 5.2.3 C.elegans HaloTag staining 73 5.2.4 Oocyte chemical inhibitor treatments 73 5.3 RNAi Feeding 73 5.4 Microscopy 73 5.4.1 Spinning disk microscopy 73 5.4.2 SIM-TIRF microscopy 74 5.5 TIRF microscopy 74 5.6 Phase portrait analysis pipeline 74 5.7 Kymographs 76 / Zellen verbrauchen Energie, um Ordnung aufrechtzuerhalten, da lebende Systeme von Natur aus ungleichgewichtig sind. Ordnung im Zytoplasma wird durch Kompartimen- tierung erreicht. Eine Art von Kompartiment, das in den letzten Jahren an Interesse gewonnen hat, sind membranlose Organellen (engl.: membraneless organelles, MLOs). Beobachtungen der flu ̈ssigkeits ̈ahnlichen Eigenschaften dieser MLOs fu ̈hrten zu ihrer In- terpretation in Analogie zur Flu ̈ssig-Flu ̈ssig-Phasentrennung (engl.: liquid-liquid phase separation, LLPS). Die LLPS allein impliziert jedoch ein passives geschlossenes System, das zum Gleichgewicht neigt und mit der physikalischen Natur der Zelle nicht kompatibel ist. Es war bisher nicht bekannt, welche Ungleichgewichtswechselwirkungen die Dynamik von MLOs in der Zelle hervorrufen. Wir wollten die regulatorischen Wechselwirkungen entschlu ̈sseln, die zu aktiver Konden- sation im Aktomyosin-Kortex von C. elegans fu ̈hren. Die Komponenten des Aktomyosin- Kortex, F-Aktin und seines verzweigten Nukleationsmoduls Arp2/3 und N-WASP (WSP- 1 in C. elegans) wurden in fru ̈heren Studien als phasengetrenntes System beschrieben. In vitro weisen phasengetrennte N-WASP-Kompartimente allerdings nicht dieselben un- gleichgewichtigen Wachstums- und Zerlegungsdynamiken auf, die in kultivierten Zellen beobachtet werden. Daher wollten wir die Wechselwirkungen zwischen WSP-1, Arp2/3 und F-Aktin im Kontext des Fadenwurms C. elegans untersuchen. Wir haben das C.elegans Lebenstadium gew ̈ahlt, in dem die ruhende Eizellenrinde aktiv kontraktil wird. Wa ̈hrend des U ̈bergangs aus der ruhigen in die aktive Periode konnten wir voru ̈bergehende WSP- 1 Arp2/3 F-Aktin-Puncta beobachten, die sich zusammensetzen und zerlegen. Um die Wachstumsdynamik fu ̈r alle Puncta zu erfassen, haben wir ein neuartiges Tool zur Anal- yse von Phasenportr ̈ats entwickelt. Das Phasenportr ̈at ermo ̈glicht es uns, gleichzeitig die Wachstums- und die Zerlegungsraten von Puncta in Abha ̈ngigkeit der inneren Zusam- mensetzung zu messen. Die Abha ̈ngigkeit der Wachstumsrate von der inneren Zusam- mensetzung spiegelt die nicht trivialen A ̈nderungen der Nukleationsprofile wider, die mit der Kondensation in aktiven, offenen Mehrkomponentensystemen einhergehen. Wir kon- nten superlineare WSP-1-Wachstumsraten beobachten, die mit der Kondensation u ̈bere- instimmen. Ferner konnten wir das In-vivo-A ̈quivalent einer Nukleationsbarriere fu ̈r die WSP-1-Kondensation identifizieren. Die In-vivo-Nukleationsbarriere nimmt mit der verzweigten F-Actin-Reaktion zu, die die Kondensation reguliert. Dementsprechend sind die reaktiven Komponenten WSP-1 und Arp2/3 wichtig fu ̈r die Dynamik des Konden- sats. Wir haben die Kondensations- und Verzweigungsreaktionen kombiniert, um damit ein grobko ̈rniges Modell zu formulieren, das die Ungleichgewichtskondensationsdynamik erfasst. Insgesamt haben unsere Ergebnisse gezeigt, dass WSP-1 kondensiert und diese Kondensation durch Arp2/3-vermittelte Verzweigungsreaktionen aus dem Gleichgewicht gebracht wird. Zusammenfassend konnten wir durch Kombination von hochauflo ̈sender Bildgebung, quan- titativer Analyse und Theorie die Wechselwirkungen identifizieren, die die Ungleichgewicht- skondensation im Aktomyosin-Kortex erkla ̈ren ko ̈nnten. Die Dynamik im lebendem Sys- tem ergibt sich aus dem Zusammenspiel von Kondensation und Reaktion. Die Interaktion zwischen physikalischen Prozessen (wie Kondensation) und biologischen Regulationen (wie Reaktionen) kann ein gemeinsames Organisationsprinzip hinter der MLO-Bildung sowie anderen Ungleichgewichtsprozessen in der Zelle sein. Die in dieser Arbeit entwickel- ten Methoden und Konzepte k ̈onnen daher helfen, unser Versta ̈ndnis daru ̈ber zu vertiefen, wie lebende Zellen ihre r ̈aumlich-zeitlichen Proteinverteilungen dynamisch regulieren, um sich in einem ungleichgewichtigen, geordneten Zustand zu halten.:1 Introduction 1 1.1 Evolving concepts of cellular organization 1 1.2 Condensation of biomolecules 3 1.2.1 Terminology for biomolecular condensates 5 1.2.2 Technical considerations for identifying liquid-like properties and LLPS 7 1.2.3 Thermodynamics of condensation 10 1.2.4 The problem of an equilibrium description of living systems 13 1.2.5 Towards active condensation 14 1.3 Actomyosin cortex self-organization 16 1.3.1 F-actin treadmilling and nucleation 17 1.3.2 N-WASP and Arp2/3 regulation 18 1.3.3 Multivalent interactions in condensation of transmembrane receptors and actin regulators 22 1.3.4 Cortex activation in C. elegans 23 2 Aims 25 3 Results 26 3.1 C. elegans cortical activation begins at fertilization 26 3.1.1 C. elegans oocytes as an ex utero model for cortex self-organization 27 3.2 WSP-1, Arp2/3 and F-actin form dynamic multicomponent phases 32 3.2.1 Capping proteins outcompete Formin in WSP-1 Arp2/3 puncta preventing F-actin elongation 32 3.2.2 WSP-1 and Arp2/3 are required for punctate F-actin formation and dynamics 34 3.2.3 Summary of the characterization of cortical activation 34 3.3 Establishment of systematic phase portrait analysis for multicomponent clusters 36 3.3.1 Non-equilibrium features of the multicomponent puncta 36 3.3.2 Recording intensity traces of multicomponent cluster over time 37 3.3.3 Probability flux of composition in the phase portrait show a closed cycle 38 3.3.4 WSP-1 F-actin puncta have a preferred joint concentration 38 3.3.5 The phase portrait is robust to cell-to-cell noise 41 3.3.6 Choosing the appropriate bin size 41 3.4 Existence of a tuned critical size and signatures of active condensation 45 3.4.1 Growth rate dependence on internal composition 45 3.4.2 Stoichiometric growth laws of WSP-1 F-actin clusters 47 3.4.3 Estimation of WSP-1 cluster critical size in vivo 47 3.4.4 Theoretical description of WSP-1 and F-actin interactions in regulating puncta dynamics 48 3.4.5 Summary of 2D phase portrait findings 52 3.5 Towards three dimensional phase portrait analysis of the reaction network 54 3.6 Initial assessment of the compartment’s external environment 54 3.7 Identification of modulators of puncta dynamics 56 3.7.1 CDC-42 controls cortical levels of WSP-1 56 3.7.2 RHO-1 and Formin CYK-1 are not involved in WSP-1 F-actin condensate dynamics 58 3.7.3 WSP-1 and Arp2/3 dynamics are independent of NCK-1 and VAB-1 58 3.7.4 Arp2/3 regulates condensate dynamics 60 3.8 Summary of perturbations 63 4 Conclusions and outlook 64 4.1 Concluding remarks 64 4.2 Discussion 66 4.3 Future directions 67 4.3.1 Realizing the full potential of the phase portraits in identifying biochemical interactions 67 4.3.2 Resolving the ultrastructure of condensates . 70 4.3.3 Further investigation of the biological function 71 4.3.4 Applying full-dynamic data acquisition to other membraneless organelles 71 5 Materials and Methods 72 5.1 C.elegans maintenance and strains 72 5.2 Sample preparation 72 5.2.1 In utero imaging 72 5.2.2 Oocyte imaging 73 5.2.3 C.elegans HaloTag staining 73 5.2.4 Oocyte chemical inhibitor treatments 73 5.3 RNAi Feeding 73 5.4 Microscopy 73 5.4.1 Spinning disk microscopy 73 5.4.2 SIM-TIRF microscopy 74 5.5 TIRF microscopy 74 5.6 Phase portrait analysis pipeline 74 5.7 Kymographs 76