High throughput 3D super-resolution microscopy reveals Caulobacter crescentus in vivo Z-ring organization

Abstract

We created a high-throughput modality of photoactivated localization microscopy (PALM) that enables automated 3D PALM imaging of hundreds of synchronized bacteria during all stages of the cell cycle. We used high-throughput PALM to investigate the nanoscale organization of the bacterial cell division protein FtsZ in live Caulobacter crescentus. We observed that FtsZ predominantly localizes as a patchy midcell band, and only rarely as a continuous ring, supporting a model of "Z-ring" organization whereby FtsZ protofilaments are randomly distributed within the band and interact only weakly. We found evidence for a previously unidentified period of rapid ring contraction in the final stages of the cell cycle. We also found that DNA damage resulted in production of high-density continuous Z-rings, which may obstruct cytokinesis. Our results provide a detailed quantitative picture of in vivo Z-ring organization.

Keywords: SOS response; bacterial cytoskeleton.

Fig. 1.

Localization and hypothesized structure of the Z-ring. (A) Microscale localization of FtsZ (in green) during the C. crescentus cell cycle. (B) The patchy band model proposes a scattered band of FtsZ protofilaments without lateral contacts. (C) The lateral interaction model proposes (i) a single ring or (ii) a compressed helix stabilized by lateral interactions between individual protofilaments. Transitions between states B and C might also occur: FtsZ protofilaments could initially form a patchy band, but a transition to a continuous ring might be required for constriction.

Fig. 2.

The 3D structure of the Z-ring throughout the C. crescentus cell cycle. (A) FtsZ axial localization relative to cell center as a function of time for 275 cells. Color map ranges from white (lowest density) to blue and then red (highest density). (B–E) Representative PALM images of FtsZ localization throughout the cell cycle, in xy (Left) and in zd cross-section (Right), where d is lateral distance from cell midplane in transformed xy coordinates, which removes the effect of cell curvature. Arrow indicates region shown in z cross-section. Yellow line indicates automatically determined cell outline. Corresponding phase-contrast images are shown in SI Appendix, Fig. S3A. (Scale bars, 500 nm.)

Fig. 3.

FtsZ predominantly forms patches or incomplete rings. (A) (i–v) Three-dimensional volume reconstruction of midcell FtsZ localization for five separate bacteria in the early predivisional stage of cell cycle. Complete rings (v) are much less common than the other morphologies shown. (B) (i–v) Midcell FtsZ localization for five bacteria in mid-/late predivisional stage of cell cycle. Corresponding 2D cross-section images are shown in SI Appendix, Fig. S6. Corresponding phase-contrast images are shown in SI Appendix, Fig. S3. A, ii and iv, and B, ii, correspond to the cells shown in Fig. 2 C, i and ii, and D, i, respectively.

Fig. 4.

Quantification of Z-ring contraction and morphology. (A) Semiautomated classification of Z-ring morphology as a function of the cell cycle (n = 275). (B–D) Z-ring diameter (B; n = 263), axial thickness (C: n = 263), and radial thickness (D; n = 141) as a function of the cell cycle. Because radial thickness could not be measured for the spot morphology, spots are not included in D. Ring contraction rate (B) is 0.8 nm/min. Mean axial thickness (C) is 71 nm. Mean radial thickness (D) is 64 nm. Gray circles indicate polar spots; yellow circles indicate midcell spots; green circles indicate patchy Z-rings; purple circles indicate incomplete Z-rings; orange circles indicate complete Z-rings; red line indicate fitted ring contraction rate. Orange shading indicates values below spatial resolution of measurement: axial and radial thickness, 35 nm (xy); diameter, 120 nm (z).

Fig. 5.

Model of Z-ring organization, including information from HTPALM measurements. (A) (i) During the late stalked/early predivisional (PD) cell-cycle stage, the Z-ring assembles at midcell. Sparsely distributed noninteracting protofilaments are randomly distributed around the circumference of the inner membrane, forming a patchy band. (ii) As the cell cycle progresses (late PD), Z-ring radius decreases. Reduced radius means randomly distributed protofilaments are more likely to overlap circumferentially. (iii) At the end of the late PD stage, a period of rapid Z-ring contraction occurs, coincident with or followed by scission. (B) During DNA damage-induced cytokinesis arrest, many FtsZ protofilaments assemble at midcell to form a high-density Z-ring, which may obstruct cytokinesis.