Defining elastic fiber interactions by molecular fishing: an affinity purification and mass spectrometry approach.

Deciphering interacting networks of the extracellular matrix is a major challenge. We describe an affinity purification and mass spectrometry strategy that has provided new insights into the molecular interactions of elastic fibers, essential extracellular assemblies that provide elastic recoil in dynamic tissues. Using cell culture models, we defined primary and secondary elastic fiber interaction networks by identifying molecular interactions with the elastic fiber molecules fibrillin-1, MAGP-1, fibulin-5, and lysyl oxidase. The sensitivity and validity of our method was confirmed by identification of known interactions with the bait proteins. Our study revealed novel extracellular protein interactions with elastic fiber molecules and delineated secondary interacting networks with fibronectin and heparan sulfate-associated molecules. This strategy is a novel approach to define the macromolecular interactions that sustain complex extracellular matrix assemblies and to gain insights into how they are integrated into their surrounding matrix.

FtsZ polymer-bundling by the Escherichia coli ZapA orthologue, YgfE, involves a conformational change in bound GTP.

Cell division is a fundamental process for both eukaryotic and prokaryotic cells. In bacteria, cell division is driven by a dynamic, ring-shaped, cytoskeletal element (the Z-ring) made up of polymers of the tubulin-like protein FtsZ. It is thought that lateral associations between FtsZ polymers are important for function of the Z-ring in vivo, and that these interactions are regulated by accessory cell division proteins such as ZipA, EzrA and ZapA. We demonstrate that the putative Escherichia coli ZapA orthologue, YgfE, exists in a dimer/tetramer equilibrium in solution, binds to FtsZ polymers, strongly promotes FtsZ polymer bundling and is a potent inhibitor of the FtsZ GTPase activity. We use linear dichroism, a technique that allows structure analysis of molecules within linear polymers, to reveal a specific conformational change in GTP bound to FtsZ polymers, upon bundling by YgfE. We show that the consequences of FtsZ polymer bundling by YgfE and divalent cations are very similar in terms of GTPase activity, bundle morphology and GTP orientation and therefore propose that this conformational change in bound GTP reveals a general mechanism of FtsZ bundling.

New temperature-sensitive alleles of ftsZ in Escherichia coli.

We isolated five new temperature-sensitive alleles of the essential cell division gene ftsZ in Escherichia coli, using P1-mediated, localized mutagenesis. The five resulting single amino acid changes (Gly109-->Ser109 for ftsZ6460, Ala129-->Thr129 for ftsZ972, Val157-->Met157 for ftsZ2066, Pro203-->Leu203 for ftsZ9124, and Ala239-->Val239 for ftsZ2863) are distributed throughout the FtsZ core region, and all confer a lethal cell division block at the nonpermissive temperature of 42 degrees C. In each case the division block is associated with loss of Z-ring formation such that fewer than 2% of cells show Z rings at 42 degrees C. The ftsZ9124 and ftsZ6460 mutations are of particular interest since both result in abnormal Z-ring formation at 30 degrees C and therefore cause significant defects in FtsZ polymerization, even at the permissive temperature. Neither purified FtsZ9124 nor purified FtsZ6460 exhibited polymerization when it was assayed by light scattering or electron microscopy, even in the presence of calcium or DEAE-dextran. Hence, both mutations also cause defects in FtsZ polymerization in vitro. Interestingly, FtsZ9124 has detectable GTPase activity, although the activity is significantly reduced compared to that of the wild-type FtsZ protein. We demonstrate here that unlike expression of ftsZ84, multicopy expression of the ftsZ6460, ftsZ972, and ftsZ9124 alleles does not complement the respective lethalities at the nonpermissive temperature. In addition, all five new mutant FtsZ proteins are stable at 42 degrees C. Therefore, the novel isolates carrying single ftsZ(Ts) point mutations, which are the only such strains obtained since isolation of the classical ftsZ84 mutation, offer significant opportunities for further genetic characterization of FtsZ and its role in cell division.

FtsZ fiber bundling is triggered by a conformational change in bound GTP.

Polymer formation by the essential FtsZ protein plays a crucial role in the cytokinesis of most prokaryotes. Lateral associations between these FtsZ polymers to form bundles or sheets are widely predicted to be extremely important for FtsZ function in vivo. We have carried out a study in vitro of FtsZ polymer formation and bundling using linear dichroism (LD) to assess structural properties of the polymers. We demonstrate proof-of-principle experiments to show that LD can be used as a technique to follow FtsZ polymerization, and we present the LD spectra of FtsZ polymers. Our subsequent examination of FtsZ polymer bundling induced by calcium reveals a substantial increase in the LD signal indicative of increased polymer length and rigidity. We also detect a specific conformational change in the guanine moiety associated with bundling, whereas the conformation and configuration of the FtsZ monomers within the polymer remain largely unchanged. We demonstrate that other divalent cations can induce this conformational change in FtsZ-bound GTP coincident with polymer bundling. Therefore, we present "flipping" of the guanine moiety in FtsZ-bound GTP as a mechanism that explains the link between reduced GTPase activity, increased polymer stability, and polymer bundling.

Dynamic FtsZ polymerization is sensitive to the GTP to GDP ratio and can be maintained at steady state using a GTP-regeneration system.

In vitro polymerization of the essential bacterial cell division protein FtsZ, in the presence of GTP, is rapid and transient due to its efficient binding and hydrolysis of GTP. In contrast, the in vivo polymeric FtsZ structure which drives cell division - the Z-ring - is present in cells for extended periods of time whilst undergoing constant turnover of FtsZ. It is demonstrated that dynamic polymerization of Escherichia coli FtsZ in vitro is sensitive to the ratio of GTP to GDP concentration. Increase of GDP concentration in the presence of a constant GTP concentration reduces both the duration of FtsZ polymerization and the initial light-scattering maximum which occurs upon addition of GTP. It is also demonstrated that by use of a GTP-regeneration system, polymers of FtsZ can be maintained in a steady state for up to 85 min, while preserving their dynamic properties. The authors therefore present the use of a GTP-regeneration system for FtsZ polymerization as an assay more representative of the in vivo situation, where FtsZ polymers are subject to a constant, relatively high GTP to GDP ratio.