Coiled coil cytoskeleton in cell architecture and osmotic stress response in Streptomyces

Abstract: Popular Abstract in English Antonie van Leeuwenhoek is known as the father of microbiology, because he made the first microscopic observation of bacteria in human history, published in 1683. For the naked human eyes, bacterial cells are too small to observe, and the development of microscopy techniques has since then been fundamental for examining bacterial cells. Recent advances in visualizing tools, including uses of fluorescent tags, super-resolution microscopy, and cryo-electron tomography, have even enabled us to scrutinize the inside of bacterial cells. Our understanding of bacterial cellular organization has remarkably progressed in the last 2 decades. It turned out that bacteria have a very elaborate cellular organization, and that not only the temporal, but also spatial orchestration of different cellular processes is essential for the viability of cells. Thus, the once prevailing notion of ‘bacteria as bags of randomly diffusing enzymes and chemicals’ was completely wrong. But, how do bacteria, lacking any membrane bound-organelles, achieve the intricate cellular organization in their cytoplasm? Bacterial cytoskeletons mediate spatial organization of proteins. Bacteria were shown to possess counterparts of major eukaryotic cytoskeleton components, such as the actin homologue MreB or the tubulin homologue FtsZ, but bacteria-specific cytoskeletal proteins with no eukaryotic counterparts have also been identified. There have been numerous studies to characterize bacterial cytoskeletons using in vivo, biochemistry or structural biology tools. However, how bacterial cytoskeletons are interacting with each other in the cytoplasm has not been well explored in bacteria. One of the aims of this thesis is to expose the biological significance of interplays between cytoskeletal proteins in our model system. This thesis concerns one type of cytoskeletal proteins, so-called coiled coil proteins that contribute to spatial organization of other proteins by acting as scaffolds, and can also provide mechanical strength to cells. We have particularly focused on bacterial intermediate filament-like proteins that display similar properties to metazoan intermediate filament (IF) proteins. IFs, actin microfilaments and microtubules constitute the three main cytoskeletal systems of metazoan cells. Used organism in this thesis is Steptomyces, a soil-dwelling mycelial bacterium belonging to the phylum Actinobacteria that is well known for production of a large variety of secondary metabolites, including antibiotics. However, my study exploited Streptomyces as a model organism in cell biology. Streptomyces has a unique mode of growth, it grows both by extending the tips of multicellular hyphae, and by forming new branches. It also exhibits a truly complex life cycle (Figure. 1), offering a genetically tractable system to learn fundamental processes such as cell polarity or regulated differentiation. Our group previously reported that a coiled coil protein called FilP (filament-forming protein) in Streptomyces, has several properties of IF proteins. For example, FilP forms filaments without any cofactors in vitro, and assembles into cytoskeletal structures in vivo that contribute to the rigidity and elasticity of Streptomyces hyphae. An important function of animal IFs, such as keratins in the skin cells, is to give mechanical strength to the cells and support their integrity in stress conditions. Studies of human IFs, however, are technically challenging because they need to be studied in differentiated animal cells. Further investigation of FilP properties in a genetically and cell-biologically tractable Streptomyces might thus be useful in understanding the basic molecular mechanisms of human IFs. The investigation of coiled coil cytoskeletons was also extended to studying the cellular responses to hyper-osmotic stress in Streptomyces. Our motivations for the study were to understand 1) what are the biological roles of coiled coil cytoskeletons during osmotic stress in Streptomyces, 2) if turgor is a driving force in Streptomyces cell elongation, as suggested for rod-shaped bacteria and shown in several eukaryotic tip-growing organisms. The first chapter (Chapter I) of the thesis gives a general background on the topics of coiled coil proteins, Streptomyces apical growth, Streptomyces coiled coil cytoskeletons, and osmotic stress responses in bacteria. In Chapter II, my own research will be presented and discussed. The main focus is to elucidate the mechanism how intrinsically non-dynamic FilP behaves dynamically in hyphae, and the biological function of FilP as a bona fide cytoskeleton. These two will be discussed in relation to interactions between FilP and DivIVA, in vitro polymerization of FilP network, and polarisome complex of Streptomyces hyphae. At the end, our new finding in Streptomyces cellular response to osmotic stress will be discussed.