Modular engineering of self-division in synthetic cells

Reverte López, María (2025): Modular engineering of self-division in synthetic cells. Dissertation, LMU München: Fakultät für Biologie

Abstract

Bottom-up synthetic biology is an interdisciplinary area of research working towards one grand goal: building an artificial cell from scratch. To achieve such feat, this discipline employs a forward-engineering strategy based on mimicking life's fundamental principles. Accordingly, researchers from diverse backgrounds apply a modular assembly approach, recapitulating essential cellular processes into independent operating modules within giant unilamellar vesicles (GUVs) as in vitro minimal cell models. Each of these synthetic modules, composed of molecular building blocks, confers the lipid-based vesicles with specific functions, enabling the construction of minimal cellular systems through their combinatorial integration. However, one of the major challenges posed by this building strategy is the efficient integration of various modules within vesicles to yield a functioning reconstituted system exhibiting the desired properties. Without thorough module characterization and careful adjustment of design variables, our reconstituted minimal cells can display non-functional attributes and behaviours due to incompatibilities. Despite these challenges, a critical endeavour in the field of synthetic biology is the development of a minimal division machinery to confer artificial cells with the ability to split into two identical daughter cells. One of the approaches working towards this goal focuses on assembling a eukaryotic-inspired synthetic division machinery in the form of a contractile actomyosin ring. Although preliminary studies employing a minimal set of proteins have shown promising results, in vitro reconstituted actomyosin rings fail to effectively transmit their contractile forces to the vesicle membrane due to a lack of spatiotemporal control. In animal cells, precise equatorial positioning of actomyosin rings is crucial for cleavage furrow formation and symmetric division. Nevertheless, recapitulating this highly regulated and convoluted process in vitro within GUVs is currently unattainable. A simpler mechanism for achieving mid-cell positioning of in vitro actomyosin rings has yet to be established. To address this challenge, we have further characterized and exploited a bacterial protein system as a positioning module: the Escherichia coli MinDE system. This reaction-diffusion system self-organizes on membranes through ATP-driven attachment-detachment cycles, forming dynamic and quasi-stationary patterns. While its in vivo role involves inhibiting the formation of the bacterial division ring at the cell poles via their lateral (pole-to-pole) oscillations, the MinDE system has shown an unexpected new function in vitro: the spatiotemporal control of diffusible, membrane-bound cargo via diffusiophoretic transport. Consequently, to characterize its positioning capabilities and exploit them for other biotechnological applications, we first evaluated the MinDE system as a versatile patterning tool for complex 3D structures like microrobots and microcarriers for drug delivery. Employing two-photon lithography, we 3D-printed microswimmer-like robotic structures and demonstrated that Min proteins can spatiotemporal control biomolecules of varying nature on their lipid-coated surfaces. In addition, we showed that the patterning capabilities of Min proteins extend to free-standing membranes and achieved the quasi-stationary positioning of cargo on the inner leaflet of vesicular microcarrier systems. After demonstrating the robustness of the MinDE system as a synthetic positioning module, we then leveraged it to tackle our primary challenge: the spatiotemporal control of actomyosin rings at the equator of GUVs for the synthetic division of these minimal cell models. Through the in vitro co-reconstitution of the actomyosin contraction module with the MinDE system under optimized encapsulation conditions, we demonstrated that integrating both modules yields the effective MinDE-driven positioning of actomyosin rings at mid-cell, which generated the sustained equatorial deformation of vesicles. Intriguingly, we also showed that the synergistic effect of Min oscillations and contractile actomyosin networks leads to the emergence of unexpected behaviours, such as the formation of dynamic bleb-like outward protrusions in vesicles and the remodelling of phase-separated lipid domains. Taken together, the experimental research presented in this thesis provides strong evidence that the MinDE system can be employed as a robust positioning module not only for the eukaryotic-based division of synthetic cells, but also for other biomedically relevant applications such as the functionalization of microfabricated devices and microcarrier systems. Moreover, this work offers new insights into the synergistic effects arising from the integration of well-defined functional modules, as well as the mechanistic aspects of the MinDE system that remain unclear and will require further scrutiny and characterization. Hence, by demonstrating the successful in vitro integration of synthetic modules, this thesis advances synthetic biology efforts towards the long-term goal of building an artificial cell capable of autonomous self-division.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Synthetic Biology, Biomimetics, Cytoskeletal Proteins, Min proteins
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 6. August 2025
• 1. Bericht­erstatter:in: Leonhardt, Heinrich