FtsZ condensates: an in vitro electron microscopy study

Abstract

In vivo cell division protein FtsZ from E. coli forms rings and spirals which have only been observed by low resolution light microscopy. We show that these suprastructures are likely formed by molecular crowding which is a predominant factor in prokaryotic cells and enhances the weak lateral bonds between proto-filaments. Although FtsZ assembles into single proto-filaments in dilute aqueous buffer, with crowding agents above a critical concentration, it forms polymorphic supramolecular structures including rings and toroids (with multiple protofilaments) about 200 nm in diameter, similar in appearance to DNA toroids, and helices with pitches of several hundred nm as well as long, linear bundles. Helices resemble those observed in vivo, whereas the rings and toroids may represent a novel energy minimized state of FtsZ, at a later stage of Z-ring constriction. We shed light on the molecular arrangement of FtsZ filaments within these suprastructures using high resolution electron microscopy.

FIGURE 1

Representative images of FtsZ filaments in the presence of various amounts of crowding agents, nucleotides, and KMg buffer. Results were pH independent (shown here pH 7.7, which is to the internal pH of E. coli in vivo). The appearance of the structures was similar with different crowding agents used, only the concentrations to induce the condensation phenomena differed between crowding agents. (a) FtsZ filaments below a critical concentration of crowding agent were mainly single filaments. Shown are FtsZ-GTP filaments in the presence of 0.4% MC highlighted as dotted lines, scale bar 100 nm. (b) Above the critical concentration, the equilibrium was shifted to rings consisting of several individual FtsZ filaments with an average diameter of about 220 nm. Shown are FtsZ-GTP filaments in the presence of 1.6% MC, scale bar 500 nm. (c) Higher crowding agent concentrations (shown here FtsZ-GMPPNP in the presence of 8% PVA) condensed the structures into well defined toroids, scale bar 100 nm. (d) A closer look at the architecture of rings, which just started to condense above the critical concentration (shown at 1% MC). Individual filaments which form lateral contacts to neighboring filaments can be seen and the ends of individual filaments are marked with an arrow. Most filaments appeared to be between 400 and 800 nm long. Most rings observed consisted of single filaments, scale bar 100 nm.

FIGURE 2

Although the ring and toroid-like structures were predominant in all cases in KMg buffers in the presence of crowding agents above c₀, other structures were also observed. These structures could be observed with GTP or nonhydrolysable nucleotides and were independent of the type of crowding agent used. Shown here are representative images of other FtsZ-GTP suprastrutures (a) long multistranded helices with a pitch of about 300 nm and amplitude of about 200 nm (shown in the presence of 1.6% MC), scale bar 200 nm. (b) Long straight 3-D bundles (8% PVA), scale bar 200 nm. The outer filament layer of bundles or toroids was clearly visible when zooming in on some areas. (d) Shows the surface filaments of an FtsZ bundle. Note that the filaments do not appear to be straight proto-filaments; rather they seem to be twisted, scale bar 5 nm. This is reflected by typical optical diffraction patterns of bundles (c) which give rise to two major reflections. An equatorial peak at about 6.8 nm arising from the side by side packing of FtsZ filaments and an off meridional reflection at about 4.4 nm, arising from the subunits in the filament forming a narrow spiral.

FIGURE 3

Shown are the effects of EDTA on the formation of FtsZ suprastructures. KEDTA buffer was used together with crowding agents above c₀. (a) At pH to 6.6, only helical filaments were observed both with GTP and GMPPNP shown here FtsZ-GTP in the presence of 2% MC, scale bar 500 nm. (b) Closer views showed that the helices formed under these conditions were remarkably regular with a very long pitch around 780 nm and an amplitude of about 260 nm, scale bar 500 nm. (c) The architecture of these helical suprastructures becomes apparent when visualizing partially condensed spirals which were more common at low salt. Several µm long FtsZ filaments span the length of the helices and form lateral contacts with their neighbors, scale bar 100 nm.

FIGURE 4

Effect of sodium ions on the formation of FtsZ suprastructures. (a) In the presence of sodium and magnesium ions (NaMg buffer at both pH 6.6 and 7.7) and crowding agents above c₀, the majority species observed were helices. Both GTP and GMPPNP gave similar structures; the average pitch was around 390 nm with amplitude around 130 nm. Shown here FtsZ-GTP, 2% MC, pH 7.7 scale bar 500 nm. (b) Replacing Mg²⁺ ions by EDTA led to the formation of large spindle shaped suprastructures. These appeared independently of pH and for both GTP and GMPPNP. Shown here FtsZ-GTP, 2% MC, pH 6.6, scale bar 1 µm. (c) Closer inspection of these structures revealed that they are planar waves with a pitch of about 330 nm and an amplitude of around 130 nm, scale bar 200 nm. (d) Planar waves could be up to several µm wide and very regular, scale bar 200 nm.

FIGURE 5

FtsZ condensates formed by cations differed from those under crowed conditions. (a) Under low salt conditions and in the presence of magnesium ions and Hexammine Cobalt mostly 2-D crystals formed (scale bar 100 nm), which gave rise to a characteristic set of diffraction spots (insert, scale bar 5 nm). (b) Shows the averaged filtered projection structure with individual protofilaments running parallel to the long axis of the crystal, scale bar 10 nm. (c–e) In the presence of EDTA and Hexammine Cobalt, different condensates formed depending on the bound nucleotide. (c) In the presence of GDP, mainly large tubes formed, scale bar 100 nm, which gave an optical diffraction pattern with sharp spots (insert, scale bar 5 nm). (d) In the presence of GTP, long helical ribbons formed, whereas in the presence of GMPPNP, (e) helical tubes were the predominant species, scale bars 200 nm.

FIGURE 6

In the presence of other cations, FtsZ formed long rafts, thin bundles, or rolled sheets. (a) With BaCl₂, the FtsZ suprastructures gave rise to a well-defined diffraction pattern shown in (b) A typical optical transform. Here, two clear reflections are visible, an off meridional reflection at about 45 Å and a weak meridional reflection at 42 Å, indicated by the arrows. (c) Spermine induced suprastructures where similar in appearance to those formed by BaCl₂, yet did not show a clear diffraction pattern, scale bars 200 nm.