The speed of FtsZ treadmilling is tightly regulated by membrane binding.

As one of the key elements in bacterial cell division, the cytoskeletal protein FtsZ appears to be highly involved in circumferential treadmilling along the inner membrane, yielding circular vortices when transferred to flat membranes. However, it remains unclear how a membrane-targeted protein can produce these dynamics. Here, we dissect the roles of membrane binding, GTPase activity, and the unstructured C-terminal linker on the treadmilling of a chimera FtsZ protein through in vitro reconstitution of different FtsZ-YFP-mts variants on supported membranes. In summary, our results suggest substantial robustness of dynamic vortex formation, where only significant mutations, resulting in abolished membrane binding or compromised lateral interactions, are detrimental for the generation of treadmilling rings. In addition to GTPase activity, which directly affects treadmilling dynamics, we found a striking correlation of membrane binding with treadmilling speed as a result of changing the MTS on our chimera proteins. This discovery leads to the hypothesis that the in vivo existence of two alternative tether proteins for FtsZ could be a mechanism for controlling FtsZ treadmilling.

Plasmonic Nanosensors Reveal a Height Dependence of MinDE Protein Oscillations on Membrane Features.

Single-particle plasmon spectroscopy has become a standard technique to detect and quantify the presence of unlabeled macromolecules. Here, we extend this method to determine their exact distance from the plasmon sensors with sub-nanometer resolution by systematically varying the sensing range into the surrounding by adjusting the size of the plasmonic nanoparticles. We improved current single-particle plasmon spectroscopy to record continuously for hours the scattering spectra of thousands of nanoparticles of different sizes simultaneously with 1.8 s time resolution. We apply this technique to study the interaction dynamics of bacterial Min proteins with supported lipid membranes of different composition. Our experiments reveal a surprisingly flexible operating mode of the Min proteins: In the presence of cardiolipin and membrane curvature induced by nanoparticles, the protein oscillation occurs on top of a stationary MinD patch. Our results reveal the need to consider membrane composition and local curvature as important parameters to quantitatively understand the Min protein system and could be extrapolated to other macromolecular systems. Our label-free method is generally easily implementable and well suited to measure distances of interacting biological macromolecules.

Treadmilling analysis reveals new insights into dynamic FtsZ ring architecture.

FtsZ, the primary protein of the bacterial Z ring guiding cell division, has been recently shown to engage in intriguing treadmilling dynamics along the circumference of the division plane. When coreconstituted in vitro with FtsA, one of its natural membrane anchors, on flat supported membranes, these proteins assemble into dynamic chiral vortices compatible with treadmilling of curved polar filaments. Replacing FtsA by a membrane-targeting sequence (mts) to FtsZ, we have discovered conditions for the formation of dynamic rings, showing that the phenomenon is intrinsic to FtsZ. Ring formation is only observed for a narrow range of protein concentrations at the bilayer, which is highly modulated by free Mg2+ and depends upon guanosine triphosphate (GTP) hydrolysis. Interestingly, the direction of rotation can be reversed by switching the mts from the C-terminus to the N-terminus of the protein, implying that the filament attachment must have a perpendicular component to both curvature and polarity. Remarkably, this chirality switch concurs with previously shown inward or outward membrane deformations by the respective FtsZ mutants. Our results lead us to suggest an intrinsic helicity of FtsZ filaments with more than one direction of curvature, supporting earlier hypotheses and experimental evidence.

FtsZ Polymers Tethered to the Membrane by ZipA Are Susceptible to Spatial Regulation by Min Waves.

Bacterial cell division is driven by an FtsZ ring in which the FtsZ protein localizes at mid-cell and recruits other proteins, forming a divisome. In Escherichia coli, the first molecular assembly of the divisome, the proto-ring, is formed by the association of FtsZ polymers to the cytoplasmic membrane through the membrane-tethering FtsA and ZipA proteins. The MinCDE system plays a major role in the site selection of the division ring because these proteins oscillate from pole to pole in such a way that the concentration of the FtsZ-ring inhibitor, MinC, is minimal at the cell center, thus favoring FtsZ assembly in this region. We show that MinCDE drives the formation of waves of FtsZ polymers associated to bilayers by ZipA, which propagate as antiphase patterns with respect to those of Min as revealed by confocal fluorescence microscopy. The emergence of these FtsZ waves results from the displacement of FtsZ polymers from the vicinity of the membrane by MinCD, which efficiently competes with ZipA for the C-terminal region of FtsZ, a central hub for multiple interactions that are essential for division. The coupling between FtsZ polymers and Min is enhanced at higher surface densities of ZipA or in the presence of crowding agents that favor the accumulation of FtsZ polymers near the membrane. The association of FtsZ polymers to the membrane modifies the response of FtsZ to Min, and comigrating Min-FtsZ waves are observed when FtsZ is free in solution and not attached to the membrane by ZipA. Taken together, our findings show that the dynamic Min patterns modulate the spatial distribution of FtsZ polymers in controlled minimal membranes. We propose that ZipA plays an important role in mid-cell recruitment of FtsZ orchestrated by MinCDE.

Giant vesicles: a powerful tool to reconstruct bacterial division assemblies in cell-like compartments.

The use of artificial lipid membranes, structured as giant unilamellar vesicles (GUVs), provides the opportunity to investigate membrane-associated biological processes under defined experimental conditions. Due to their large size, they are uniquely adapted to investigate the properties and organization (in time and space) of macromolecular complexes incorporated in the vesicle interior by imaging and micro-spectroscopic techniques. Experimental methods to produce giant vesicles and to encapsulate proteins inside them are here reviewed. Previous experimental work to reconstitute elements of the bacterial division machinery in these membrane-like systems is summarized. Future challenges towards reconstructing minimal divisome assemblies in giant vesicles as cytomimetic containers are discussed.