BACTERIAL CELL DIVISION IN-SIGHT: A SUPER-RESOLUTION STUDY OF THE ESCHERICHIA COLI DIVISOME
Embargo until
2014-12-01
Date
2013-10-09
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Publisher
Johns Hopkins University
Abstract
The polymerization of FtsZ into an annular structure (Z-ring) at midcell is the first recognized step in E. coli cell division, and is required for the recruitment of the remaining essential division proteins. Further studies suggest that the Z-ring itself may provide the force that drives constriction. Despite its importance, the in-vivo structure of the Z-ring remains unknown.
Given the small size of bacteria, visualizing a cytoplasmic structure within an intact cell is technically challenging. Conventional fluorescence microscopy lacks the resolution needed for molecular insight, while electron microscopy fails to unambiguously identify subcellular structures in whole bacteria. To circumvent these challenges, we applied a single molecule-based super-resolution imaging technique, photoactivated localization microscopy (PALM), and determined the in-vivo structure of the Z-ring with a resolution of ~30nm. We found that in contrast to the homogenous, ring-like structure expected from previous studies, the Z-ring adopted a heterogeneous configuration with dimensions indicative of a loose, multi-layered protofilament arrangement.
To further investigate the structural composition of the Z-ring, we determined the effect of two FtsZ-associated proteins, ZapA and ZapB, on the assembly dynamics and structure of the Z-ring. In cells deleted of zapA or zapB, we observed abnormal septa and highly dynamic FtsZ structures that resolve into disordered arrangements of FtsZ clusters via PALM. Quantitative analyses found these clusters were larger and contained more molecules than a single FtsZ protofilament, and likely represented a distinct polymeric species that is inherent to the assembly pathway of the Z-ring. Our results suggest that ZapA/B function to promote the alignment of FtsZ clusters into a ring-like arrangement and further propose that the FtsZ cluster, not the protofilament, is the basic structural unit of the Z-ring. Complimentary studies employing a variety of two-color and three-dimensional high-resolution imaging methods were applied to elucidate the spatial arrangement of FtsZ, ZapA and ZapB. Our results are consistent with a layered model whereby ZapB forms a stable, cytoplasmic raft upon which clusters of FtsZ protofilaments are anchored through ZapA.
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Keywords
FtsZ-ring, ZapA, ZapB, division