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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.

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. INTRODUCTIONThe FtsZ protein has a fundamental role in bacterial cell division , is highly conserved throughout the eubacteria and appears to be required for division of chloroplasts, some archaea and some mitochondria (Beech et al., 2000;Vitha et al., 2001;Wang & Lutkenhaus, 1996). FtsZ is a cytoplasmic protein which, at a particular stage in the Escherichia coli cell cycle, locates to the cell centre, forming a polymeric ring (the Z-ring) around the inner circumference of the cytoplasmic membrane Bi & Lutkenhaus, 1991;Dai & Lutkenhaus, 1991; Den Blaauwen et al., 1999;Pla et al., 1991). Invagination of the division septum then follows the shape of the Z-ring as it reduces in diameter until septation is complete Bi & Lutkenhaus, 1991. FtsZ is strongly related to a/b-tubulin in terms of threedimensional structure (Lowe & Amos, 1998), the possession of GTPase activity (de Boer et al., 1992;Mukherjee et al., 1993;RayChaudhuri & Park, 1992) and the ability to polymerize in a nucleotide-dependent manner in vitro (Erickson et al., 1996;. Indeed, FtsZ forms a variety of polymeric structures in vitro depending on experimental conditions (Erickson et al., 1996;Lowe & Amos, 1999, 2000Yu & Margolin, 1997); however, all of these represent different arrangements of linear protofilaments.There have been multiple studies of the dynamics of FtsZ polymerization in vitro. In the presence of GDP (Rivas et al., 2000(Rivas et al., , 2001 Sossong et al., 1999) or in the presence of GTP under particular conditions where only single protofilaments are formed (Romberg et al., 2001), polymerization is proposed to be isodesmic. Under conditions where FtsZ forms more complex polymers, polymerization and the GTPase activity are co-operative (Caplan & Erickson, 2003;Mukherjee & Lutkenhaus, 1999;White et al., 2000).The technique of right-angled light-scattering has proved useful for real-time monitoring of FtsZ polymerization (Mingorance et al., 2001;...

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 (Gly 109 3Ser 109 for ftsZ6460, Ala 129 3Thr 129 for ftsZ972, Val 157 3Met 157 for ftsZ2066, Pro 203 3Leu 203 for ftsZ9124, and Ala 239 3 Val 239 for ftsZ2863) are distributed throughout the FtsZ core region, and all confer a lethal cell division block at the nonpermissive temperature of 42°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°C. The ftsZ9124 and ftsZ6460 mutations are of particular interest since both result in abnormal Z-ring formation at 30°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°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.The FtsZ protein of Escherichia coli is essential throughout the process of cell division (3,11,29,33,48,49). Following the segregation of daughter chromosomes, FtsZ polymerizes around the inner circumference at midcell to form the Z ring (9). At least nine proteins (all of which are essential for cell division) are recruited to the division site in an FtsZ-dependent fashion (12, 13), and the Z ring then leads this division apparatus inward, ultimately allowing separation of daughter cells. Division proteins ZipA and FtsA are recruited to the Z ring by direct interaction with FtsZ, and the recruitment of at least one of these proteins is essential for productive Z-ring formation (21,23,25,31,32,45,56,59). All other division proteins are thought to localize to the Z ring in a hierarchical fashion (that is, the localization is dependent on the prior presence of other cell division proteins). Therefore, all proteins which make up the divisome ultimately depend on the presence of the Z ring for their localization to the septum.The timing of the arrival at the division site of these proteins is thought to reflect the order in which they are involved during cell division (4,13,22,30). An exception is FtsW, which has been ascribed either an early (10, 24) or ...

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.