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.bacterial cytoskeleton | SOS response

Defining the rate-limiting processes of bacterial cytokinesis

“…The combined mean Z-ring width (along the cell long axis) of 99 ± 3 nm (n = 168; Fig. 1C) is consistent with previous measurements of 60-120 nm in E. coli (19,48,59) and Caulobacter crescentus (53,60). Note that dimension measurements reported here have been deconvolved from the achieved resolution to facilitate comparison across different experiments (61) (Materials and Methods); see SI Appendix, Table S1 for apparent dimensions before deconvolution.…”

“…We showed that the Z-ring is not a smooth structure but instead comprises clusters of FtsZ protofilaments that loosely associate into a 3D bundle (19,48). This heterogeneous morphology has also been observed with several different fluorescent protein tags (19), in different bacterial species (51)(52)(53)(54), by immuno-superresolution imaging targeting native FtsZ (19,54), by superresolution imaging of FtsZ-binding proteins (19,54), and in an early electron cryotomography (ECT) study (37). Here, we further demonstrate that an FtsZ-GFP fusion colocalizes with native FtsZ in midcell clusters in two-color superresolution imaging using antibodies against native FtsZ and GFP (SI Appendix, Fig.…”

“…These studies support a smooth, continuous Z-ring organization. However, Z-ring discontinuity has been observed in many bacterial species (19,37,48,(51)(52)(53)(54), and incomplete Z-rings have been shown to lead to cell wall indentation (110), suggesting that ring completion is not required for Z-ring function. We suggest that the continuous and discontinuous organizations reflect different dynamic states of the Z-ring modulated by FtsZ's GTPase activity, which is known to promote the fracture of long FtsZ polymers (111) and to modulate FtsZ turnover dynamics (82).…”

Quantitative evaluation of software packages for single-molecule localization microscopy

“…For this study, to ensure that each software was properly tuned to our simulated database, npg we encouraged the developers themselves to run their own software, guided by the training data. To alleviate the difficulty of presetting parameters, we suggest that developers incorporate self-calibration capabilities or dynamically tune the parameters 26 . We predict that this will be one of the key factors determining the success of future software 14 .…”

Lessons from bacterial homolog of tubulin, FtsZ for microtubule dynamics

“…up of loosely arranged irregular patches of protofilaments, which gives it an overall discontinuous structure (Li et al 2007, Holden et al 2014. Following studies using highresolution fluorescence microscopy techniques revealed that such was the case in many other bacterial species (Strauss et al 2012, Rowlett & Margolin 2014, Jacq et al 2015.…”