All cells spatially organize their interiors, and this arrangement is necessary for cell viability. Until recently, it was believed that only eukaryotic cells spatially segregate their components. However, it is becoming increasingly clear that bacteria also assemble their proteins into complex patterns. In eukaryotic cells, spatial organization arises from membrane bound organelles as well as motor transport proteins which can move cargos within the cell. To date, there are no known motor transport proteins in bacteria and most microbes lack membrane bound organelles, so it remains a mystery how bacterial spatial organization emerges.
In hind-sight it is not surprising that bacteria also exhibit complex spatial organization considering much of what we have learned about the basic processes that take place in all cells, such as transcription and translation was first discovered in prokaryotic cells. Perhaps the fundamental principles that govern spatial organization in prokaryotic cells may be applicable in eukaryotic cells as well. In addition, bacteria are attractive model organism for spatial organization studies because they are genetically tractable, grow quickly and much biochemical and structural data is known about them.
A powerful tool for observing spatial organization in cells is the fluorescence microscope. By specifically tagging a protein of interest with a fluorescent probe, it is possible to examine how proteins organize and dynamically assemble inside cells. A significant disadvantage of this technology is its spatial resolution (approximately 250 nm laterally and 500 nm axially). This limitation on resolution causes closely spaced proteins to look blurred making it difficult to observe the fine structure within the complexes. This resolution limit is especially problematic within small cells such as bacteria.
With the recent invention of new optical microscopies, we now can surpass the existing limits of fluorescence imaging. In some cases, we can now see individual proteins inside of large complexes or observe structures with ten times the resolution of conventional imaging. These techniques are known as super-resolution microscopes.
In this dissertation, I use super-resolution microscopes to understand how a model microbe, Escherichia coli, assembles complex protein structures. I focus on two spatially organized systems, the chemotaxis network and the cell division machinery. These assembly mechanisms could be general mechanisms for protein assembly in all organisms. I also characterize new fluorescent probes for use in multiple super-resolution imaging modalities and discuss the practicalities of using different super-resolution microscopes.
The chemotaxis network in E. coli is the best understood signal transduction network in biology. Chemotaxis receptors cluster into complexes of thousands of proteins located at the cell poles and are used to move bacteria towards favorable stimuli in the environment. In these dense clusters, the receptors can bind each other and communicate to filter out noise and amplify weak signals. It is surprising that chemotaxis receptors are spatially segregated and the mechanism for polar localization of these complexes remains unclear. Using data from PALM images, we develop a model to understand how bacteria organize their receptors into large clusters. The model, stochastic cluster nucleation, is surprising in that is generates micron-scale periodic patterns without the need for accessory proteins to provide scaffolding or active transport. This model may be a general mechanism that cells utilize to organize small and large complexes of proteins.
During cell division, E. coli must elongate, replicate its DNA and position its components properly prior to binary fission. Prior to septum formation, a ubiquitous protein called FtsZ, assembles into a ring at mid-cell (Z-ring) which constricts during cell division and recruits the remaining proteins necessary for cytokinesis. Though many details have been revealed about FtsZ, the detailed in vivo structure of the Z-ring is not well understood, and many questions remain about how ring constriction occurs. Using multiple super-resolution imaging modalities, in combination with conventional time-lapse fluorescence imaging, we show that the Z-ring does not form a long uniform filament around the circumference of the bacterium. We detail how this structure changes during division and how removal of proteins that help to position FtsZ affects the Z-ring as it proceeds through cytokinesis. Ultimately we present a simple model for Z-ring constriction during division.