We study the influence of finite shear deformations on the microstructure and rheology of solutions of entangled semiflexible polymers theoretically and by numerical simulations and experiments with filamentous actin. Based on the tube model of semiflexible polymers, we predict that large finite shear deformations strongly affect the average tube width and curvature, thereby exciting considerable restoring stresses. In contrast, the associated shear alignment is moderate, with little impact on the average tube parameters, and thus expected to be long-lived and detectable after cessation of shear. Similarly, topologically preserved hairpin configurations are predicted to leave a long-lived fingerprint in the shape of the distributions of tube widths and curvatures. Our numerical and experimental data support the theory.

Formins are proteins involved in regulation and construction of actin filaments of eucaryotic organism. They parcipitate in regulating cytokinesis, polar tip growth, and thus participate in development of whole organisms. There are 2 classes of formins in Arabidopsis thaliana. Both classes include FH1 and FH2 domains (formin homology 1 a 2). Class I formins have N-terminal transmembrane domain, unlike class II formins. Some formins of class II have a N-terminal PTEN domain (Phosphatase and Tensin Homolog). Sequence analyses predicted membrane binding via phosphatase or C2 subdomain of PTEN. This thesis was focused on the formin AtFH14, specifically its PTEN domain. Based on predicted sequence, a DNA fragment encoding the PTEN domain was amplified, sequenced and cloned to destination vectors for YFP and EOS phusions. Marked protein was visualized by transient expression in Nicotiana benthamiana. Stably transformed Arabidopsis lines were prepared for stably expression of protein. The tagged protein was localized in cortical cytoplasm, cytoplasmatical strands, probably in nuclear membrane or perinuclear cytoplasm, as well as in peculiar "folicle-like" structures that might be due to binding of PTEN at the periphery of some membrane organelles. Also were seen filament structures, maybe caused by PTEN binding...

ARPC2 protein localization in a plant cell Kateřina Šlajcherová 1 Abstract Actin cytoskeleton is an ubiquitous structure which plays numerous irreplacable roles. Actin nucleation is, beside formins, performed by ARP2/3 complex (actin-related protein), comprising of seven subunits (ARP2, 3, C1-C5) and activated by protein SCAR/WAVE complex. ARP2/3 complex is attached to the membrane and branches existing microfilaments, apart from nucleating them de novo. ARP2/3 mutants in most organisms show severe defects. However, plant mutants exhibit only mild phenotype, for example, Arabidopsis thaliana ARPC2 mutant (dis2-1) has deformed trichomes and leaf epidermal cells, but its viability is not impaired. The aim of the thesis is to map ARPC2 localization within the cell and broaden our understanding of ARP2/3 complex role in plant cell morphogenesis. Tobacco ARPC2 (NtArpC2) subunit was visualized in Arabidopsis plants, using the GFP fusion protein as well as imunofluorescence and anti-ARPC2 antibody. Experiments were undertaken to collocalize the subunit with actin and microtubular cytoskeleton, with mitochondrions, endosomes and other membrane organelles. The specimens were observed in confocal and TIRF microscope. The GFP-NtARPC2 protein shows as motile dots; their movement, but not their existence, is dependent...

Actin filaments and microtubules are involved in cell development and morphogenesis. Plant Class II formins regulate both cytoskeletal polymers. However their function has not yet been fully described. This study examines effects of LOF mutations in Arabidopsis thaliana FH13 (AT5G58160) and FH14 (AT1G31810) genes on early root system development using a pharmacological approach. Since measuring root length of numerous mutant lines in multiple conditions is laborious and time consuming, this thesis also involves optimization of this process with the aim to establish a reliable method of fast visualisation and measurement of Arabidopsis seedlings in a time series in the laboratory. Furthermore, statistical analysis for a large amount of data gathered in multiple conditions had to be optimized. While no significant phenotype in terms of root length was found in fh13, fh14 and double fh13 fh14 LOF mutants under standard conditions, treatment with cytoskeletal drugs revealed possible changes in lateral root branching in an fh14 mutant. Nevertheless, specific function of FH13 and FH14 remains a question.

Introduction: Cryptosporidium parvum is a protozoan parasite that causes diarrhoea in many host species worldwide. CpCDPK1 appears to be essential for invasion and a promising drug target. Aim of the study: The aims of this study were to expand the knowledge of CpCDPK1. To achieve that, attempts were made to inhibit this gene by BKI-1294 in vitro and generate CpCDPK1 KO C. parvum. To maintain the genetically modified parasites, I studied the suitability of infection and propagation in a new animal model RAGgc × IFN-gamma mouse and in vitro model COLO - 680 N cell line. Animals, materials and methods: 4×106 freshly excysted C. parvum sporozoites were seeded into transfected GFP-MDBK cultures at the confluency of 70 – 80 % and simultaneously exposed to 500 nM of BKI-1294. IFA was applied to observe the invasion and host cell actin accumulation. Guide RNA (gRNA) for CRISPR-mediated transfection was designed and the Nluc-neoR repair cassette was flanked with 50 bp long 5’- and 3’UTR of CpCDPK1 by PCR. Transfection was performed by octaarginine transportation and compared to electroporation. COLO - 680 N cells with the confluency of 70 – 80% were infected with 4×106 non-transfected and transfected sporozoites of C. parvum. To establish a laboratory animal model for propagation of C. parvum and drug screening RAGgc × IFN-gamma mice were infected with 500 (G2), 1000 (G3) and 5000 (G4) of oocysts. BALB/c WT mice were inoculated with 5000 (G1) oocysts as control. Faeces were sampled for C. parvum DNA extraction. Real time PCR was applied to calculate the oocyst yield. Results: In the presence of 500 nM BKI-1294, parasite-induced host cell actin accumulation was not observed at 24 and 48 h after inoculation in vitro pointing at altered infectivity of CDPK inhibited sporozoites. Extracellular noninvasive sporozoites were found at 24 h p.i., only one meront was observed in a host cell at 72 h p.i. CRISPR-mediated gene editing was applied to C. parvum to knock out CDPK1. Transfected C. parvum were found in COLO-680 N cells through 6 passages. However, no newly generated oocysts were harvested. RAGgc × IFN-gamma mice were tested suitable as an animal model for C. parvum infection studies and oocyst propagation. These crossbred mice are very sensitive to infection at doses as low as 500 oocysts. They displayed emaciation, rough fur and trembling. The survival percentage was 71.4 % (G2), 85.7 % (G3), 57.1 % (G4) and 100 % (G1) at the end of study. Oocyst yield of 108 OPG was calculated in the crossbred mice whereas only 104 OPG were counted in Balb/C mice. Yields did not differ significantly (P > 0.05) in crossbred mice infected with different oocysts doses. Conclusions: 1.The function of CpCDPK1 is obviously important to the invasion process including attachment and utilization of host cell actin to form PV. This assumption was confirmed by CDPK inhibition and genetic KO. However, methods that increase the transfection efficiency are needed to enhance the generation of KO C. parvum. 2. The transfection method mediated by octargninine is superior to electroporation in consideration of DNA consumption and requirement of device. 3. Due to the low required infection dose and clinical manifestation RAGgc × IFN-gamma mice appear very well suited to serve as an in vivo laboratory model of C. parvum infection and for propagation of particularly transgenic C. parvum strains. 4. COLO – 680 N cells appear suited to be an in vitro model for C. parvum infection and transfection study, however, not qualified for propagation.:Contents 1. Introduction 1 2. Literature Review 2 2.1 Biology 2 2.1.1 Systematics 2 2.1.2 Life cycle 2 2.1.3 Tenacity of oocysts 4 2.1.4 Excystation of oocysts and invasion of host cells 4 2.1.5 Formation of the PV 6 2.1.6 Nutrient supply by the host 7 2.2 Epidemiology 8 2.2.1 Human Cryptosporidiosis 8 2.2.2 Animal Cryptosporidiosis 9 2.3 Detection and Diagnosis 11 2.4 Treatment options 12 2.5 Hygiene 14 2.6 Vaccine 16 2.7 In vitro and vivo Models 16 2.8 Structure and function of Calcium-dependent protein kinases 18 3. Animals, materials and methods 21 3.1 Animals and materials 21 3.1.3 Mice 21 3.1.4 Cells 21 3.1.5 C. parvum oocysts 21 3.1.6 Reagents 21 3.1.7 Plasmids and oligonucleotides 23 3.1.7.1 Plasmids 23 3.1.7.2 Primers and probes 24 3.1.8 Kits 25 3.1.9 Instruments and software 25 3.2 Methods 26 3.2.1 Preparation of reagents 26 3.2.2 C. parvum oocysts maintaince 27 3.2.3 PCR 27 3.2.3.1 Amplification of NdeI and AatII flanked 5’CDPK1 27 3.2.3.2 Annealing of gRNA 27 3.2.3.3 Amplification of repair cassette via Touchdown PCR (TD-PCR) 28 3.2.3.4 Colony PCR 29 3.2.3.5 Real-time PCR for C. parvum hsp70 30 3.2.4 Restriction enzyme digestion 31 3.2.4.1 Restriction enzyme digestion of pA - pD 31 3.2.4.2 Enzyme digestion and dephosphorylation of p185 31 3.2.5 Agarose gel electrophoresis 32 3.2.6 Gel purification 32 3.2.7 Ligation 33 3.2.7.1 Ligation of CDPK1 KO plasmids 33 3.2.7.2 Ligation of gRNA and p185 33 3.2.8 Transformation 34 3.2.9 Plasmid extraction 34 3.2.10 C. parvum oocysts excystation 35 3.2.11 C. parvum infection 35 3.2.11.1 In vitro infection 35 3.2.11.2 C. parvum infection in mice 36 3.2.12 Transfection 36 3.2.12.1 Electroporation for MDBK transfection 36 3.2.12.2 Electroporation for C. parvum transfection 37 3.2.12.3 CpCDPK1 knock out through Cell penetrating peptide (CPP) - octaarginine mediated transfection 38 3.2.13 Geneticin screening for GFP-MDBK cells 39 3.2.14 Indirect immunofluorescent assay (IFA) 39 3.2.15 Animal feeding and body conditioning score (BCS) monitoring 40 3.2.16 Faecal sample collection 43 3.2.17 DNA extraction and oocysts per gram (OPG) determination of fecal samples 43 3.2.18 Statistical analysis 44 4. Results 45 4.1 CDPK1 knockout by REMI 45 4.1.1 Construction of Knockout plasmid 45 4.1.2 Electroporation protocol and in vitro analysis 49 4.2 CDPK1 knockout by CRISPR/Cas 9-mediated gene editing 50 4.2.1 Constructing CRISPR/Cas9_CpCDPK1_7 plasmid 51 4.2.2 Amplification of CDPK1 flanked repair cassette 52 4.2.3 Knockout CDPK1 via CRISPR/cas 9 53 4.2.3.1 Electroporation and in vitro analysis 53 4.2.3.2 CPP transfection and in vitro analysis 55 4.2.3.3 Genetic assay of transfection 57 4.3 In vitro and in vivo model for infection and propagation 58 4.3.1 In vitro model - C. parvum cultivation in COLO - 680 N cells 58 4.3.2 In vivo model Infection pattern of C. parvum in RAGgc x IFN-g KO mice 60 4.3.2.1 Clinical symptoms 60 4.3.2.2 Oocysts excretion 63 4.4 In vitro inhibition of CDPK1 66 4.4.1 Generating bAct-GFP-MDBK cells 67 4.4.2 Influence of CDPK1 inhibition on infection 70 5. Discussion 73 5.1 Sub-cloning 73 5.2 Inhibition of CpCDPK1 delays the host cell actin accumulation in vitro 73 5.3 RAGgc x IFN-gamma KO mice for C. parvum propagation 76 5.4 CpCDPK1 knockout by CRISPR/cas 9 79 5.5 COLO-680 N cells are not suited to propagate C. parvum in vitro 82 6. Summary 85 7. Zusammenfassung 87 8. References 89

In der vorliegenden Arbeit wird die Mechanik von Netzwerken semiflexibler Polymere behandelt. Insbesondere wird der Zusammenhang zwischen der Steifigkeit des Einzelfilaments und der Steifigkeit des Gesamtnetzwerks experimentell untersucht. Der Hintergrund aktueller, einschlägiger theoretische Modelle wird zusammengefasst. Die Möglichkeiten und Limitierungen bisheriger experimenteller Modellsysteme werden diskutiert. Zur Untersuchung des eingangs genannten Zusammenhangs wird ein neuartiges, vielfältiges Modellsystem für semiflexible Polymere auf Basis von DNA Röhren eingeführt und umfassend auf Einzelfilament- und Netzwerkebene charakterisiert. Die Steifigkeit des Netzwerks lässt sich damit und unter Einsatz von Quervernetzern über einen weiten Bereich einstellen. Es zeigt sich, dass bisherige einschlägige Modelle in der korrekten Vorhersage des o.g. Zusammenhangs scheitern. Mögliche Auswege in der Modellierung werden skizziert, sowie konkrete, weitere Anwendungen der DNA Röhren benannt.:Chapter 1 Introduction Chapter 2 Background Section 2.1 Theoretical Models Section 2.2 Rheology Section 2.3 Experimental Model Systems Section 2.4 Existing G_0(l_p) Studies Chapter 3 Materials & Methods Section 3.1 Microscopy Section 3.2 Atomic Force Microscopy Section 3.3 Shear Rheology Section 3.4 Actin Section 3.5 DNA Assembly Section 3.6 Statistical Analysis Tools Chapter 4 Results Section 4.1 Persistence Length of Individual Filaments Section 4.2 Entangled Networks Section 4.3 Reptation Section 4.4 Inextensibility Section 4.5 Cross-Linked DNA n-Helix Tubes Chapter 5 Discussion Section 5.1 Limitations of Established Semiflexible Model Systems Section 5.2 DNA $n$-Helix Tubes as a Tunable Model System Section 5.3 Validation as an Entangled Semiflexible Model System Section 5.4 Impact on Existing Theories Section 5.5 DNA n-Helix Tubes as a Tunable Material Section 5.6 Summary Section 5.7 Outlook Chapter A Further Calculations Section A.1 Detailed Calculations on Worm-Like Chains. Chapter B Protocols Section B.1 Actin Section B.2 DNA n-Helix Tubes Chapter Bibliography Chapter List of Own Publications Chapter Acknowledgments Chapter Zusammenfassung nach §11(4) Promotionsordnung

Cardiovascular disease is the leading cause of human death worldwide. Currently, the prevalence of cardiovascular disease and health care costs associated with its onset continue to increase in both developed and developing societies. Concordant with the need to improve preventative measures is the imperative to develop more effective and efficient remedies for incident cardiovascular pathologies. Increased aortic stiffness with aging has recently emerged as an early, independent, and consistent physiological predictor of cardiovascular disease and represents an attractive target for possible therapeutic options. The success of any biomedical strategy in this regard is incumbent upon comprehension of biological processes and mechanical properties attributable to constituent components within the aortic wall. This dissertation tested the hypothesis that aging-induced changes to smooth muscle maintenance of biomechanical homeostasis within the aorta lead to undesirable increases in stiffness, correlative with increased risk of negative cardiovascular outcomes. Conventionally, mechanical studies and models have identified extracellular matrix as the primary determinant of changes in stiffness, but new research presented here shows that this may not be true. In viable ex vivo preparations of aortic tissue, roughly half of the maximal elastic modulus results from alpha-agonist activation of smooth muscle cells. Investigation of the biochemical interactions that characterize this effect revealed a link between aging and decreased expression of Src, a kinase involved in numerous signaling pathways governing cellular growth and survival, as well as defective regulation of focal adhesions between the smooth muscle cells and extracellular matrix. These findings were integrated into a model of aortic contractility and stiffness that establishes an aging-impaired regulatory complex comprising focal adhesions and non-muscle actin cytoskeleton in vascular smooth muscle cells. A better understanding of the mechanisms underlying this model may motivate the design of potential therapeutics, deliverable to previously overlooked target sites within aortic smooth muscle, and ultimately novel treatments for aging-induced cardiovascular disease. / 2017-10-27T00:00:00Z

1. Abstract ARP2/3 complex is well studied in case of animals, it plays key roles in motility of cells and intracellular organels. It's malfunctions result in severe growth disorders and even lethality of affected cells. On the contrary, plant cells do not exhibit such dramatic phenotype of ARP2/3 complex mutations like it is by animals. It is possible that just the different life strategies of plants and animals contribute to differences in a way how animal and plant cells use their cytoskeleton, where ARP2/3 complex is it's part as well. It is highly conserved 7 protein complex from yeast to human. His main functions are creation of new "de novo" actin filaments, actin branched filaments network. Some of the parasite organisms are capable of missusing its nucleator activity to actively move inside of host cell. Because of the plant cells are sourounded by the cell wall, which give them support in creating various shapes and also hinders active movement of the whole cell body, it is likely that ARP 2/3 complex could be possibly involved in novel plant specific functions as well. If we think about the different life strategy of plants and animals we can not ignore all the things these two kingdoms have in common regarding to cytoskeleton processes. That is the need both for vesicular transport and...

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

Capillary Morphogenesis Gene 2 protein (CMG2) is a type 1 transmembrane receptor known as the anthrax toxin receptor 2 (ANTXR2). While it is documented that the cell surface receptor CMG2 mediates anthrax toxin entry into the cell via endocytosis, the physiological role of CMG2 is not well understood. Others have suggested that CMG2 may have a role in mediating ECM homeostasis and angiogenesis. Additionally, both anthrax protective antigen (PA) and a furin protease-resistant mutant, PASSSR, inhibit corneal neovascularization in a mouse model, and interestingly PASSSR has a greater affinity to CMG2 receptor. PASSSRalso has a more potent antiangiogenic effect than wild-type PA. However, a mechanism for PASSSR inhibition of the putative CMG2 role in angiogenesis is not yet elucidated. The experimental results in this thesis provide evidence that CMG2 is the key receptor for regulating adhesion, migration, and actin dynamics in cells, and 200-pM PASSSR inhibits cell adhesion, migration, and actin localization at the cell leading edge. Furthermore, we observed that PASSSR remains bound to CMG2 under acidic conditions similar to the lysosome (pH 4). This observation suggests that the PASSSR-CMG2 complex remains intact following internalization and traffic to lysosomes, different from previous reports for PA, which likely results in CMG2 recycling. Together, these results suggest that following PASSSR treatment, CMG2 traffics to the lysosome for degradation; hence, we predict fewer CMG2 receptors are available at the cell surface to function in their native role in signaling angiogenic processes such as adhesion and chemotaxis towards vascular growth factors.