Towards understanding the assembly mechanisms of the Z-ring in Archaea and Bacteria

Life on Earth emerged when biomacromolecules were membrane-enclosed in a confined space where many essential chemical reactions were more likely to happen and thereby accelerate evolution. These kinds of membranes separated internal reactions from the outside chaos while staying flexible so that those primordial cells can move, adopt their shape and, most importantly, propagate. Such membrane plasticity still remains a defining feature of all modern cell types. This remarkable ability to change their shape is most prominently observed during their propagation (i.e., cell division). Throughout division, a cell undergoes drastic change in its shape, usually at the middle of the cell, pulling the two opposite membrane sides inward, closer to each other, and, finally, culminating in pinching off to separate the cell into two daughter cells. To achieve this, a cell needs to employ a protein machinery, usually termed divisome, that can coordinate all necessary intracellular processes with membrane remodelling and synthesis of other extracellular structures that decorate a cell. The focus of this dissertation is a membrane-remodelling FtsZ system that is present across all domains of life. FtsZ forms filaments that further self-organize into ring-like structures at the cell septum and together with other division proteins perform cell envelope synthesis and constriction. However, there are still knowledge gaps in our mechanistic understanding of division in both archaea and bacteria. My work presented in this dissertation centres around a simple yet not well understood question: How is the divisome positioned correctly at the mid-cell? To achieve the proper positioning, the divisome needs to (i) be recruited to the mid-cell and (ii) localized orthogonally to the long cell axis. I tackle these processes in two different systems by applying an in vitro biochemical bottom-up reconstitution approach. I use purified components of Haloferax volcanii and Escherichia coli divisome to explore how divisome is recruited to the mid-cell in archaea and how the Z-ring positions orthogonally to the long cell axis in bacteria, respectively. Firstly, I collaborate with archaeal cell and structural biologists to explore the assembly of early division proteins in two FtsZ-containing archaeon H. volcanii, a standard model system for understudied archaeal organisms. I particularly address the hierarchy of interactions that allow a tripartite complex formation (SepF-CdpB1-CdpB2) and how the hierarchy of interactions ultimately leads to the recruitment of FtsZ filaments to the septum. This part of work has been published in (Nußbaum et al., 2024). In collaboration with evolutionary biologists, I shed light on ancient features that archaeal divisome has retained to this day and also speculate on a property that it might have lost during the course of evolution. Next, I switch my attention to E. coli divisome. Particularly, I address the FtsZ’s intrinsic biophysical property that drives the Z-ring diameter, and thereby the perpendicular orientation of the Z-ring to the long cell axis based on suggested membrane curvature sensing mechanism (Vanhille-Campos et al., 2024). This property allows formation of different Z-ring diameters that match the variety of cell diameters present in prokaryotes. The results showcase that the distribution of charged amino acids in the intrinsically disordered linker at the C-terminus (CTL) of FtsZ is the major determining factor of Z-ring diameter with inter-CTL interactions as an underlying mechanism. Finally, I thoroughly explain the methodology I used to address the abovementioned projects, and I finish with a discussion on how early archaeal divisome assembly and curvature sensing mechanism in bacteria, at first sight unrelated topics, are interconnected and important groundwork for both fundamental and translational research.