An Analysis of FtsZ Assembly Using Small Angle X-ray Scattering and Electron Microscopy

Abstract

Small angle X-ray scattering (SAXS) was used for the first time to study the self-assembly of the bacterial cell division protein, FtsZ, with three different additives: calcium chloride, monosodium glutamate and DEAE-dextran hydrochloride in solution. The SAXS data were analyzed assuming a model form factor and also by a model-independent analysis using the pair distance distribution function. Transmission electron microscopy (TEM) was used for direct observation of the FtsZ filaments. By sectioning and negative staining with glow discharged grids, very high bundling as well as low bundling polymers were observed under different assembly conditions. FtsZ polymers formed different structures in the presence of different additives and these additives were found to increase the bundling of FtsZ protofilaments by different mechanisms. The combined use of SAXS and TEM provided us a significant insight of the assembly of FtsZ and microstructures of the assembled FtsZ polymers.

ZipA Binds to FtsZ with High Affinity and Enhances the Stability of FtsZ Protofilaments

“…Then, 1 mM MgCl 2 and 1 mM GTP were added to the reaction mixtures and polymerized for 10 min at 37°C. The samples were taken on carbon coated grids and negatively stained using uranyl acetate as described earlier [24], [25]. The grids were observed under TECHNAI G 2 transmission electron microscope.…”

Cationic lipid enhances assembly of bacterial cell division protein FtsZ: A possible role of bacterial membrane in FtsZ assembly dynamics

“…After addition of 1 mM GTP, the polymerization reaction was initiated by immediately transferring the mixture to 37 • C. After 15 min of assembly, the samples were taken onto a carbon coated grid and further stained with 2% uranyl acetate and observed under FEI Tecnai-G 2 transmission electron microscope as described previously [17,34].…”

“…Several other proteins preferentially interact with FtsZ to promote Z-ring stabilization or destabilization [13][14][15][16]. FtsZ polymerization is also influenced by small molecules and macromolecular crowding [17][18][19][20]. Recently, the lipids in the bacterial cell membrane were shown to play a role in directing the FtsZ assembly at a particular position (centre) by virtue of the interaction between lipids and cell division proteins [21].…”

“…Several cations were reported to modulate FtsZ assembly in vitro and strongly enhanced the bundling of FtsZ protofilaments [17,25,26]. Cations exert their polymer-promoting action by shielding repulsive anionic charges on FtsZ.…”

“…Cations exert their polymer-promoting action by shielding repulsive anionic charges on FtsZ. FtsZ bundles differ in length, width and shape depending on the nature of inducers [17]. Consequently, the effect of counterions has become an important consideration in understanding FtsZ assembly.…”