Towards bottom-up reconstitution of a functional FtsZ-based cell division machinery

Merino Salomón, Adrián (2023): Towards bottom-up reconstitution of a functional FtsZ-based cell division machinery. Dissertation, LMU München: Fakultät für Biologie

Abstract

Synthetic biology aims at the understanding of living organisms through an engineering perspective, with the goal of improving or creating new biological systems. The prospect of building a synthetic cell focuses on producing life from basic elements by combining synthetic and/or organic cellular components in a bottom-up manner. To create a synthetic cell, the minimal functions of life are required and cell-free synthetic biology offers a suitable framework for understanding biological processes outside the inherently noisy environment of cells. A synthetic cell is expected to exhibit characteristics of a living cell, such as fundamental metabolism, proliferation, and communication. The bottom-up approach utilizes a wide range of in vitro tools/technologies such as biomimetic membranes, protein reconstitution, cell-free expression reactions, and microfluidics. As tools, they enable the thorough characterization of functional modules such as metabolism, replication, and cell division. The ultimate goal is to integrate these modules to construct a predictable, customizable, and controllable entity. Among the functional modules of living organisms, cell division stands out as a hallmark feature. The machinery of division has evolved into a highly organized set of proteins with the aim of accurately splitting a mother cell into two daughter cells, while preserving the genetic information and cellular integrity. In the case of bacteria, and more concretely Escherichia coli, cell division is mediated by the divisome, a contractile ring consisting of a multiprotein complex that precisely assembles at midcell. At the center of this machinery is the essential FtsZ protein, which is able to polymerize and form the FtsZ-ring. This ring is key to the process, serving as a scaffold for the divisome and driving the division process. However, the molecular details of how the ring is functionally assembled, stabilized, and positioned are still not well understood. Therefore, the aim of this thesis is to develop and expand the knowledge about the molecular mechanism of the FtsZ-ring assembly and its function as a potential primary component in the minimal division machinery of synthetic cells. To this end, and following a bottom-up approach, we conducted assays based on the in vitro reconstitution of FtsZ in cellular mimic environments using lipid vesicles. This allows the characterization of FtsZ’s behavior and functionalities in environments that are similar to a potential synthetic cell. Firstly, we designed a microfluidic device to deform lipid vesicles into bacterial rod-shaped compartments to analyze the effect of different geometries and membrane tension on FtsZ. We found that FtsZ filaments align with the shorter axis of the rod-shaped vesicles and reorganize into cone-like structures when the membrane tension is lowered, causing membrane deformations. This suggests that there is a geometry and tension-dependent mechanism in the assembly of FtsZ structures on membranes. Secondly, we designed an in vitro reconstitution assay based on soft lipid tubes pulled from FtsZ-decorated vesicles using optical tweezers. We observed the transformation of lipid tubes into 3D spring-like structures, where the GTPase activity of FtsZ drives spring compression likely through torsional stress. This allowed us to gain mechanistic insights into the molecular dynamics behind the force generated by FtsZ filaments. Thirdly, we studied the spatiotemporal localization of the division ring by co-reconstituting FtsZ inside lipid vesicles with the MinCDE system, which is involved in positioning the divisome in vivo, and FtsA, the natural tether of FtsZ to the membrane. We achieved the assembly, placement, and onset of constriction of a minimal division ring inside lipid vesicles using two different approaches: purified components or cell-free expression of the MinCDE, FtsA, and FtsZ proteins. This represents a significant advance towards the in vitro reconstitution of functional modules in a synthetic cell and expands our understanding of the molecular mechanism underlying the spatiotemporal organization of the FtsZ-ring. Lastly, we employed biochemical studies combined with cryo-ET visualization to characterize the stabilization of the division ring and the crosslinking of FtsZ filaments by ZapD, a protein known as one of the stabilizers of the divisome. We observed the formation of toroidal structures in solution that are assembled by short FtsZ filaments connected by ZapD and have bacterial size. Their characterization in 3D brings valuable structural information about the FtsZ-ring and its functional stabilization, which is important for its further reconstitution in minimal systems. In conclusion, this thesis provides important insights into the molecular dynamics of the central protein of division in E. coli and most bacteria, addressing its activity on the membrane, mechanism of force constriction, spatiotemporal localization and stabilization of the FtsZ-ring. Furthermore, we demonstrate significant advancements towards the implementation of FtsZ-based division systems in minimal synthetic cells using a bottom-up approach.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Bottom-up Synthetic Biology, Synthetic Cell, Bacterial Division, Escherichia Coli, FtsZ, Protein Reconstitution
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 11. Oktober 2023
• 1. Bericht­erstatter:in: Cordes, Thorben