Sub-micron light-guided protein localization and super-resolution microscopy for studying division and signaling in bacteria
My interest is in understanding symmetry breaking and its relation with cell signaling and differentiation on multiple temporal and spatial scales. The main model system in my lab is the sporulation process of the bacteria Bacillus subtilis. Using it we will study differentiation decisions, symmetry breaking in cell division and signaling across a septum.
1. Light guided systems for studying signaling and molecular assembly in living cells at the cellular and sub-cellular resolution
Several systems have been developed recently for using light to guide gene expression and protein localization in living cells. We will use these systems to explore aspects of differentiation and protein localization during sporulation.
Division septum assembly and site-selection
Bacterial cytokinesis (division) is guided by the assembly of a membrane bound ‘ring’ of the tubulin-like protein ftsZ. The subcellular localization of this ring is guided by a complex network of inhibitors that restrict its localization. During growth, a single cytokinetic ring is usually restricted to the middle of the bacteria. Upon sporulation, two rings are formed at the two distal ends of the bacteria but only one of them continues through cytokinesis while the other disassembles. We will explore the re-localization of the ring and the symmetry breaking event it goes through by re-designing the membrane binding protein that link the ftsZ filaments to the membrane to allow membrane localization by light. This will allow us to guide the position of localization and the number of localization sites at a diffraction limiting resolution of 300nm. We will combine this technique with time-lapse microscopy of fluorescently tagged ftsZ to monitor the assembly and dissociation dynamics, the interaction between assembly sites and the dependence of the system on various proteins that modify the preferred localization.
Sporulation decision making
The decision to sporulate is guided by a complex regulatory system, involving many feedbacks at the cellular and inter-cellular levels. We will use a light-activated gene expression system to control gene expression in time and space in order to understand the basic logic of this system and the way it guides symmetry breaking on the multi-cellular level.
2.Super-resolution techniques for studying complex formation across septa
Asymmetric septation during sporulation is followed by the differentiation of the two cells, which is guided by signaling between them across the septa. This system serves as a simple model to analyze direct interaction between cells. Membranal proteins involved in cross-septal signaling are characterized by asymmetric distribution on the two sides, by coupled formation of complexes on the two sides and by the formation of fine-resolution patterns. Most of these characteristics cannot be examined by regular light-microscopy because of the nanometric scale of the system. We will use STORM (stochastic optical reconstruction microscopy) super-resolution techniques to reconstruct the signaling apparatus at the 10nm resolution and analyze their dependence on interaction and signaling. While a one-color STORM is sufficient to analyze the interdependence of localization across the septa, two colors are needed to resolve co-localization of proteins within the compartment and such methods will be explored.