Probing for Binding Regions of the FtsZ Protein Surface through Site-Directed Insertions: Discovery of Fully Functional FtsZ-Fluorescent Proteins

FtsZ, a bacterial tubulin homologue, is a cytoskeletal protein that assembles into protofilaments that are one subunit thick. These protofilaments assemble further to form a “Z ring” at the center of prokaryotic cells. The Z ring generates a constriction force on the inner membrane and also serves as a scaffold to recruit cell wall remodeling proteins for complete cell division in vivo. One model of the Z ring proposes that protofilaments associate via lateral bonds to form ribbons; however, lateral bonds are still only hypothetical. To explore potential lateral bonding sites, we probed the surface of Escherichia coli FtsZ by inserting either small peptides or whole fluorescent proteins (FPs). Among the four lateral surfaces on FtsZ protofilaments, we obtained inserts on the front and back surfaces that were functional for cell division. We concluded that these faces are not sites of essential interactions. Inserts at two sites, G124 and R174, located on the left and right surfaces, completely blocked function, and these sites were identified as possible sites for essential lateral interactions. However, the insert at R174 did not interfere with association of protofilaments into sheets and bundles in vitro. Another goal was to find a location within FtsZ that supported insertion of FP reporter proteins while allowing the FtsZ-FPs to function as the sole source of FtsZ. We discovered one internal site, G55-Q56, where several different FPs could be inserted without impairing function. These FtsZ-FPs may provide advances for imaging Z-ring structure by superresolution techniques. IMPORTANCE One model for the Z-ring structure proposes that protofilaments are assembled into ribbons by lateral bonds between FtsZ subunits. Our study excluded the involvement of the front and back faces of the protofilament in essential interactions in vivo but pointed to two potential lateral bond sites, on the right and left sides. We also identified an FtsZ loop where various fluorescent proteins could be inserted without blocking function; these FtsZ-FPs functioned as the sole source of FtsZ. This advance provides improved tools for all fluorescence imaging of the Z ring and may be especially important for superresolution imaging

Reaction-diffusion kinetics on lattice at the microscopic scale

Lattice-based stochastic simulators are commonly used to study biological reaction-diffusion processes. Some of these schemes that are based on the reaction-diffusion master equation (RDME), can simulate for extended spatial and temporal scales but cannot directly account for the microscopic effects in the cell such as volume exclusion and diffusion-influenced reactions. Nonetheless, schemes based on the high-resolution microscopic lattice method (MLM) can directly simulate these effects by representing each finite-sized molecule explicitly as a random walker on fine lattice voxels. The theory and consistency of MLM in simulating diffusion-influenced reactions have not been clarified in detail. Here, we examine MLM in solving diffusion-influenced reactions in 3D space by employing the Spatiocyte simulation scheme. Applying the random walk theory, we construct the general theoretical framework underlying the method and obtain analytical expressions for the total rebinding probability and the effective reaction rate. By matching Collins-Kimball and lattice-based rate constants, we obtained the exact expressions to determine the reaction acceptance probability and voxel size. We found that the size of voxel should be about 2% larger than the molecule. MLM is validated by numerical simulations, showing good agreement with the off-lattice particle-based method, eGFRD. MLM run time is more than an order of magnitude faster than eGFRD when diffusing macromolecules with typical concentrations in the cell. MLM also showed good agreements with eGFRD and mean-field models in case studies of two basic motifs of intracellular signaling, the protein production-degradation process and the dual phosphorylation cycle. Moreover, when a reaction compartment is populated with volume-excluding obstacles, MLM captures the non-classical reaction kinetics caused by anomalous diffusion of reacting molecules

Asymmetric function theory

The classical theory of symmetric functions has a central position in algebraic combinatorics, bridging aspects of representation theory, combinatorics, and enumerative geometry. More recently, this theory has been fruitfully extended to the larger ring of quasisymmetric functions, with corresponding applications. Here, we survey recent work extending this theory further to general asymmetric polynomials.Comment: 36 pages, 8 figures, 1 table. Written for the proceedings of the Schubert calculus conference in Guangzhou, Nov. 201

Production of Gas Bubbles in Reduced Gravity Environments

In a wide variety of applications such as waste water treatment, biological reactors, gas-liquid reactors, blood oxygenation, purification of liquids, etc., it is necessary to produce small bubbles in liquids. Since gravity plays an essential role in currently available techniques, the adaptation of these applications to space requires the development of new tools. Under normal gravity, bubbles are typically generated by forcing gas through an orifice in a liquid. When a growing bubble becomes large enough, the buoyancy dominates the surface tension force causing it to detach from the orifice. In space, the process is quite different and the bubble may remain attached to the orifice indefinitely. The most practical approach to simulating gravity seems to be imposing an ambient flow to force bubbles out of the orifice. In this paper, we are interested in the effect of an imposed flow in 0 and 1 g. Specifically, we investigate the process of bubble formation subject to a parallel and a cross flow. In the case of parallel flow, we have a hypodermic needle in a tube from which bubbles can be produced. On the other hand, the cross flow condition is established by forcing bubbles through an orifice on a wall in a shear flow. The first series of experiments have been performed under normal gravity conditions and the working fluid was water. A high quality microgravity facility has been used for the second type and silicone oil is used as the host liquid

Endothelin XIII

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