Structure and dynamics of filaments formed by bacterial cell division protein ftsz on flat surfaces

Resumen

Irruption of structural biology during the 1950s, establishing the atomic structure of nucleic acids and proteins, constituted a turning point in the knowledge of living matter. However, understanding the mechanisms underlying cell processes requires not only an atomistic description of their isolated components but also knowledge about how they interact and assemble to carry out their functions. Nowadays it is well known that a multitude of dynamic processes occur simultaneously inside a cell. Genetic expression and regulation, energy generation, tracking of biomolecules or cell division, among others, are essential to ensure cell viability. In this thesis we have investigated the self-assembly of FtsZ, a main protein in prokaryotic cell division machinery. In vivo, FtsZ polymerizes on the inner face of bacterial cytoplasmic membrane forming a dynamic ring (the Z-ring) able to generate, at least partially, the force necessary for cell division. In vitro, FtsZ retains its capacity to form dynamic laments in the presence of GTP. This PhD work is focused on understanding the dynamics of FtsZ from its simplest form, i.e. isolated laments, to the formation of higher order structures such aslament aggregates and highly interconnected networks. Previous works have proposed models for FtsZ laments from dynamic and structural experimental data obtained separately with spectroscopic techniques and electron microscopy (EM). In contrast, we have used an atomic force microscope (AFM) to obtain simultaneously the structure and dynamics of FtsZ laments adsorbed on at surfaces. This microscopy is able to acquire high resolution images discerning the structure of FtsZ single protolaments in real time under buer solution. The rich structural polymorphism and dynamic behavior observed experimentally were used to formulate theoretical models in terms of interactions between FtsZ monomers. Finally, statistical analysis and computational simulations revealed key quantitative aspects of the dynamic behavior of FtsZ laments. The thesis is structured as followed: Part I is of a general introduction describing brie y the bacterial cell division process (chapter 1) and the instrumentation used in the project (chapter 2). Part II reports the structure and dynamics of FtsZ laments on mica: from single isolated protolaments (chapter 3) to the collective behavior of many laments (chapter 4). Part III describes the formation of lament networks when FtsZ polymerizes on lipid bilayers (chapter 5). At the end general conclusions were include to integrate and complete the whole work. In chapter 3 the study of single isolated FtsZ aments revealed the relationship between FtsZ GTPase activity and protein surface mobility with its depolymerization mechanism. We conclude that rupture events of the polymeric bonds between FtsZ monomers occur at random locations along the lament and their frequencies correlates with GTP hydrolysis rate. Protein surface mobility and nucleotide turnover were also identied as relevant for reannealing of polymeric bonds after GTP hydrolysis. Chapter 4 looks at the collective behavior of many laments in terms of FtsZ monomer-monomer interactions. Quantitative values for longitudinal bond energy, polymer curvature and exibility, and lateral attraction were given. These four parameters were found as the essential traits to account for the structure and dynamics of FtsZ aggregates observed experimentally. In chapter 5, we found that the polymerization of FtsZ on lipid bilayers leads to the formation of highly interconnected networks. The main dynamic features of FtsZ laments, rupture and reannealing of longitudinal bonds and lateral attraction, were still present in the meshes formed. Lipid composition of the bilayer was also found to modulate the structure of the networks, aecting their curvature, height, and connectivity. To conclude, I want to remark that this work has been a multidisciplinary eort involving biologists, physical-chemists, and theoretical physicists. The diferent contributions necessary during the project are described as followed: Protein purifcation and characterization of their biochemical activity 1. Experimental design. Surface sample preparations and membrane model system reconstitution. AFM and QCM measurements. Image and data analysis Modeling of experimental results and implementation of computational tools for Monte Carlo (MC) and Languevin simulations. Single protolaments segmentation and statistical data analysis