Chiral vortex dynamics on membranes is an intrinsic property of FtsZ, driven by GTP hydrolysis

Abstract

The primary protein of the bacterial Z ring guiding cell division, FtsZ, has recently been shown to engage in intriguing self-organization together with one of its natural membrane anchors, FtsA. When co-reconstituted on flat supported membranes, these proteins assemble into dynamic chiral vortices whose diameters resemble the cell circumference.These dynamics are due to treadmilling polar FtsZ filaments, supposedly destabilized by the co-polymerizing membrane adaptor FtsA, thus catalysing their turnover. Here we show that FtsA is in fact dispensable and that the phenomenon is an intrinsic property of FtsZ alone when supplemented with a membrane anchor. The emergence of these chiral dynamic patterns is critically dependent on GTP concentration and FtsZ surface densities, in agreement with theoretical predictions. The interplay of membrane tethering, GTP binding, and hydrolysis promotes both, the assembly and the destabilization of FtsZ polymers, leading to the observed treadmilling dynamics. Notably, the vortex chirality is defined by the position of the membrane targeting sequence (mts) and can be inverted when attaching it to the opposite end of FtsZ. This reveals a so far unknown vectorial character of these cytomotive filaments, comprising three orthogonal directions: Filament polarity, curvature, and membrane attachment.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

Species- and C-terminal linker-dependent variations in the dynamic behavior of FtsZ on membranesin vitro

“…In the current study, we have described an in vitro reconstitution approach for observing FtsZ polymerization on planar SLBs, which, in addition to providing high spatial and temporal resolution, enables control of reaction conditions. As a validation of our approach, we demonstrate the reconstitution of His 6 - Ec FtsZ-venus-MTS polymers into dynamic patterns (Figure 1B) that are in agreement with the results of prior reconstitution efforts using E. coli FtsZ on SLBs (Arumugam et al , 2012; Loose and Mitchison, 2014; Arumugam et al , 2014; Ramirez et al , 2016). Unlike for E. coli FtsZ or Ec His 6 -FtsZ-venus-MTS on SLB, we never observed stable patterns of dynamic filament bundles for C. crescentus FtsZ on SLBs.…”

“…To compare our reconstitution approach to previously published studies on FtsZ polymers on SLBs, we first examined structures formed by E. coli FtsZ with the YFP derivative venus and MTS fused to its C-terminus in tandem and replacing the CTC ( Ec His 6 -FtsZ-venus-MTS) (Osawa et al , 2008). This is modeled after Ec FtsZ-YFP-MTS which has been used in the past to observe E. coli FtsZ polymerization on membranes, within vesicles as well as on planar SLB (Osawa et al , 2008; Osawa et al , 2009; Osawa and Erickson, 2011; Arumugam et al , 2012; Osawa and Erickson, 2013; Loose and Mitchison, 2014; Arumugam et al , 2014; Ramirez et al , 2016). When we flowed in 2 μM Ec His 6 -FtsZ-venus-MTS premixed with 2 mM GTP for 30 minutes, we observed dynamic assembly of fluorescent clusters on the membrane (Figure1B, Movie 1.1.).…”

“…In contrast, the emergent bundles of Ec FtsZ at steady state extend longer than 2 μm, dimensions not reported in cells for E. coli FtsZ. While the patterns formed by E. coli FtsZ protofilaments provide insights into the effects of constraining gently curved dynamic filaments to a flat and fluid surface (Ramirez et al , 2016), their relevance to understanding FtsZ assembly in vivo might thus be limited.…”

“…It is unclear which of the structures of FtsZ polymers observed in vitro are physiologically relevant in cells, especially in the context of attachment to the membrane. Efforts to observe dynamic assembly of FtsZ polymers on a membrane have been limited to E. coli FtsZ (Arumugam et al , 2012; Loose and Mitchison, 2014; Arumugam et al , 2014; Ramirez et al , 2016). In particular, purified E. coli FtsZ assembles into large, dynamic bundles of treadmilling protofilaments when anchored to a membrane either using an artificial membrane targeting sequence (MTS) or membrane-anchoring proteins such as FtsA and observed by total internal reflection fluorescence microscopy (TIRFM) in vitro (Loose and Mitchison, 2014;Ramirez et al , 2016).…”

“…Here, we have developed an in vitro TIRFM-based assay to image FtsZ polymers anchored to supported lipid bilayers (SLB) through an artificial membrane tethering sequence peptide (MTS) from E. coli MinD (Osawa et al , 2008). Unlike prior reconstitution studies of FtsZ polymerization on membrane constrained within wells placed on coverslips (Loose and Mitchison, 2014; Arumugam et al , 2014; Ramirez et al , 2016), we adapted a system to allow for rapid depletion and repletion experiments in a controlled environment using flow cells (Vecchiarelli et al , 2016). Using our flow cell setup, we observe that whereas E. coli FtsZ assembles into large, dynamic bundles, C. crescentus FtsZ assembles into smaller dynamic clusters under identical in vitro conditions.…”