Abstract
FtsZ is the main regulator of bacterial cell division. It has been implicated in acting as a scaffolding protein for other division proteins, a force generator during constriction, and more recently, as an active regulator of septal cell wall production. FtsZ assembles into a heterogeneous structure coined the Z-ring due to its resemblance to a ring confined by the midcell geometry. Here, to establish a framework for examining geometrical influences on proper Z-ring assembly and dynamics, we sculpted Escherichia coli cells into unnatural shapes using division- and cell wall-specific inhibitors in a micro-fabrication scheme. This approach allowed us to examine FtsZ behavior in engineered Z-squares and Z-hearts. We use stimulated emission depletion (STED) nanoscopy to show that FtsZ clusters in sculpted cells maintain the same dimensions as their wild-type counterparts. Based on our results, we propose that the underlying membrane geometry is not a deciding factor for FtsZ cluster maintenance and dynamics in vivo.
Highlights
FtsZ is the main regulator of bacterial cell division
In vitro data have shown that FtsZ can self-organize into swirling rings on supported bilayers when unconfined by geometrical constraints[21], but we were curious as to whether geometrical changes to cell shape would influence Z-ring formation and dynamics, as this would result in a better understanding of FtsZ behavior in live cells
We trapped the cells in a vertical position in micron-sized holes that were produced in agarose pads using silica micron pillar arrays[14] (Fig. 1b, Supplementary Figure 3), and imaged the cells using super-resolution time-gated STimulated Emission Depletion nanoscopy
Summary
FtsZ is the main regulator of bacterial cell division. It has been implicated in acting as a scaffolding protein for other division proteins, a force generator during constriction, and more recently, as an active regulator of septal cell wall production. To establish a framework for examining geometrical influences on proper Z-ring assembly and dynamics, we sculpted Escherichia coli cells into unnatural shapes using division- and cell wall-specific inhibitors in a micro-fabrication scheme. This approach allowed us to examine FtsZ behavior in engineered Z-squares and Z-hearts. Functioning as a recruitment base, the proto-ring components enlist the remaining essential division proteins to form a mature divisome[5] As soon as it is fully assembled, the divisome starts to constrict the cell envelope by reshaping the septal geometry, leading to sequential closure of the inner and outer membranes[8,9,10]. We show that FtsZ formation and dynamics are independent of cell shape and membrane curvature
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