Organization of FtsZ filaments in the bacterial division ring measured from polarized fluorescence microscopy

Abstract

Cytokinesis in bacteria is accomplished by a ring-shaped cell-division complex (the Z-ring). The primary component of the Z-ring is FtsZ, a filamentous tubulin homolog that serves as a scaffold for the recruitment of other cell-division-related proteins. FtsZ forms filaments and bundles. In the cell, it has been suggested that FtsZ filaments form the arcs of the ring and are aligned in the cell-circumferential direction. Using polarized fluorescence microscopy in live Escherichia coli cells, we measure the structural organization of FtsZ filaments in the Z-ring. The data suggest a disordered organization: a substantial portion of FtsZ filaments are aligned in the cell-axis direction. FtsZ organization in the Z-ring also appears to depend on the bacterial species. Taken together, the unique arrangement of FtsZ suggests novel unexplored mechanisms in bacterial cell division.

Figure 1

Polarized fluorescence measurement of purified FtsZ in vitro. (A and B) Polarized fluorescence images (A) and electron microscopy images (B) are shown for small and large FtsZ bundles. The electron microscopy images show parallel protofilaments of FtsZ bundled together. Fluorescence images are obtained when the linear polarizer is parallel and perpendicular to the x axis. α is the angle between the bundle and the x axis. (C) The largest polarization anisotropy, P = I_|| − I₋)/(I_||+ I₋), occurs when the angle between the bundle and the x axis is 90°. Large bundles of FtsZ show a stronger anisotropy than do small protofilament bundles. Error bars correspond to the mean ± SE. C-terminal YFP large bundles (174 samples) and small bundles (92 samples), and N-terminal GFP small bundles (64 samples), all show a similar orientational alignment (see also Fig. S2). These results indicate that the fluorophore dipole is roughly parallel to the bundle. To see this figure in color, go online.

Figure 2

PFM of freely diffusing GFP in bacterial cells. (A) Side view of E. coli cells expressing free GFP (213 cells included). As the angle between the cell axis and the lab axis, α, changes, the images show an intrinsic polarization anisotropy. The reason for this is unclear; it is likely the result of birefringence of biomaterials such as the peptidoglycan cell wall. The results can be used to derive an anisotropy factor, C, which can be used to correct the intensity from fluorophores attached to FtsZ (see text and Supporting Material). (B) C. crescentus cells with freely diffusing YFP show a similar intrinsic anisotropy (133 cells included). To see this figure in color, go online.

Figure 3

PFM of FtsZ filaments in vivo. (A) Fluorescence images are collected from a side view (with cells lying flat). To obtain 3D information, emitted fluorescence intensities are collected at different planes across the Z-ring. (B) Fluorescence images obtained at the top of the ring (plane a in A) for the polarizer in the vertical and horizontal directions. α is the angle between the Z-ring and the x axis. (C–E) Corrected polarization anisotropy as a function of α for E. coli cells with different FtsZ fluorophore labels. Here, blue and green halves of FtsZ are the C- and N-terminal ends, respectively. Measurements were made for 284 FtsZ-YFP (C), 206 FtsZ-GFP (D), and 56 GFP-FtsZ (E) cells. Error bars correspond to the mean ± SE. Fluorescence was collected at two imaging planes, a and b, which show a similar degree of anisotropy. As a negative control, corrected polarization anisotropy from cells expressing free GFP is also shown. The blue line is a quantitative fit to the data using a distribution of filament orientations, which indicates a disordered organization of FtsZ filaments. (F and G) Corrected polarization anisotropy as a function of α for C. crescentus cells expressing FtsZ-YFP, with 87 cells included. The blue line is a quantitative fit to the data, which indicate a circumferential alignment of FtsZ filaments. To see this figure in color, go online.

Figure 4

PFM of FtsZ in cross section. (A) Cells with FtsZ-YFP are made to stand vertically with respect to the microscope stage. The emitted light is collected from the Z-ring. (B) Images obtained when the polarizer is oriented in the x and y directions. Plot is based on images of 38 cells. Arrows denote scale bars of 1 μm. (C) The polarization anisotropy as a function of the angle around the ring in the xy plane. Error bars correspond to the mean ± SE. To see this figure in color, go online.

Figure 5

Pictorial representation of FtsZ filament organization in E. coli and C. crescentus. The angular orientation of filaments is generated from probability distributions fitted to experimental data (see the Supporting Material). The results indicate that FtsZ filaments are disorganized in E. coli but aligned circumferentially in C. crescentus. For E. coli, a Z-ring organization where regions of the ring are circumferential and toward the edge is axial could also explain our data (inset). Higher-resolution studies are needed to distinguish between these models. To see this figure in color, go online.