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
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:79229 |
| Date | 20 May 2022 |
| Creators | Yan, Victoria Tianjing |
| Contributors | Alberti, Simon, Grill, Stephan, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | info:eu-repo/grantAgreement/European Research Commission/Horizon2020/742712//Chiral Morphogenesis - Physical Mechanisms of Actomyosin-Based Left/Right Symmetry Breaking in Biological Systems/CHIMO |
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