For rod-shaped bacterial model organisms, the division plane is defined by the geometry of the cell. However, for Neisseria gonorrhoeae, a coccoid organism that most commonly exists as a diplococcus and that possesses genes coding for rod-based cell division systems, the relationship between cell geometry and division is unclear. Here, we characterized the organization of N. gonorrhoeae division using a combination of fluorescent probes, genetics, and time-lapse microscopy. We found that the planes of successive cell divisions are orthogonal and temporally overlapping, thereby maintaining diplococcal morphology. Division takes place perpendicular to a long axis in each coccus. In keeping with the ParABS and the MinCDE systems reading the more pronounced long axis of rod-shaped bacteria, in the coccoid N. gonorrhoeae, ParB segregates along this long axis and cells lacking minCDE suffer severe morphological consequences, including an inability to perform orthogonal division and aberrant assembly of the division plane at the cell poles. Taken together, this stresses the central role of even slight dimensional asymmetry as a general organizational principle in coccoid bacterial cell division.

Abstract The first step of cell division is deforming the planar cell membrane inward towards the cytoplasm. As deforming membranes is energetically costly, biology has developed various protein systems to accomplish this task. The mechanisms providing the force to deform bacterial membranes to initiate division remain unknown. In vivo studies have shown the condensation of FtsZ filaments into a sharp ring is required to initiate cell division, an observation mirrored in vitro with FtsZ filaments encapsulated inside liposomes. Similarly, the force for membrane deformation in many eukaryotic deforming systems arises from the local crowding of proteins on the membrane surface. As any membrane deforming system works against the membrane tension, here we modulated the amount of lipid synthesis and thus membrane tension in Bacillus subtilis to examine: 1) if the condensation of FtsZ filaments by FtsZ bundling proteins serves to overcome the cellular membrane tension to deform the membrane inward and 2) how changes to the membrane tension affect the subsequent invagination of the septum. First, we developed methods to simultaneously measure and modulate membrane tension in live cells. Next, we determined how altering the membrane tension affected the cell’s ability to initiate division with reduced levels of FtsZ bundling proteins. While cells depleted of 2 FtsZ bundling proteins were unable to divide, reducing membrane tension to a given threshold restored their ability to initiate division. Likewise, cells with intermediate levels of FtsZ bundling proteins required a lesser decrease in membrane tension to initiate division. We also found that reductions in membrane tension increase the rate of Z ring constriction, with the constriction rate scaling linearly with the membrane tension. Interestingly, while the constriction rate in wild-type B. subtilis is limited by FtsZ treadmilling, the rate of constriction becomes independent of FtsZ’s treadmilling rate when membrane tension is reduced. These experiments give two major insights: First, the filament condensation caused by FtsZ bundling proteins works to overcome membrane tension and deform the membrane inward to initiate division. Second, the rate of septal constriction is limited by membrane tension, suggesting that membrane fluctuations at the tip of the growing septa limit the rate of cell wall synthesis. Finally, our measurements allow the estimation of several physical values of cell division, such as the force required to bend the membrane, but also that the cell membrane provides only 0.1%, a small amount of surface tension relative to the entire cell envelope, indicating 99.9% of the pressure drop occurs across the cell wall. These calculations also indicate that cell division occurs via comparatively very small membrane tension fluctuations relative to the high turgor pressure that exists across the entire cell envelope.

ABSTRACT Across the domains of life, actin homologs are integral components of many essential processes, such as DNA segregation, cell division, and cell shape determination. Archaeal genomes, like those of bacteria and eukaryotes, also encode actin homologs, but much less is known about these proteins’ in vivo dynamics and cellular functions. We identified and characterized the function and dynamics of Salactin, an actin homolog in the hypersaline archaeon Halobacterium salinarum . Live-cell time-lapse imaging revealed that Salactin forms dynamically unstable filaments that grow and shrink out of the cell poles. Like other dynamically unstable polymers, Salactin monomers are added at the growing filament end, and its ATP-bound critical concentration is substantially lower than the ADP-bound form. When H. salinarum’s chromosomal copy number becomes limiting under low-phosphate growth conditions, cells lacking Salactin show perturbed DNA distributions. Taken together, we propose that Salactin is part of a previously unknown chromosomal segregation apparatus required during low-ploidy conditions. IMPORTANCE Protein filaments play important roles in many biological processes. We discovered an actin homolog in halophilic archaea, which we call Salactin. Just like the filaments that segregate DNA in eukaryotes, Salactin grows out of the cell poles towards the middle, and then quickly depolymerizes, a behavior known as dynamic instability. Furthermore, we see that Salactin affects the distribution of DNA in daughter cells when cells are grown in low-phosphate media, suggesting Salactin filaments might be involved in segregating DNA when the cell has only a few copies of the chromosome.

Angewandte Chemie International EditionVolume 60, Issue 9 p. 4385-4405 Graphical AbstractFree Access Graphical Abstract: Angew. Chem. Int. Ed. 9/2021 First published: 16 February 2021 https://doi.org/10.1002/anie.202180911AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Volume60, Issue9February 23, 2021Pages 4385-4405 RelatedInformation

A universal gain-of-function approach for the spatiotemporal control of protein activity is highly desirable when reconstituting biological modules in vitro. Here we used orthogonal translation with a photocaged amino acid to map and elucidate molecular mechanisms in the self-organization of the prokaryotic filamentous cell-division protein (FtsZ) that is highly relevant for the assembly of the division ring in bacteria. We masked a tyrosine residue of FtsZ by site-specific incorporation of a photocaged tyrosine analogue. While the mutant still shows self-assembly into filaments, dynamic self-organization into ring patterns can no longer be observed. UV-mediated uncaging revealed that tyrosine 222 is essential for the regulation of the protein's GTPase activity, self-organization, and treadmilling dynamics. Thus, the light-mediated assembly of functional protein modules appears to be a promising minimal-regulation strategy for building up molecular complexity towards a minimal cell.

FtsZ is a key component in bacterial cell division, being the primary protein of the presumably contractile Z ring. In vivo and in vitro, it shows two distinctive features that could so far, however, not be mechanistically linked: self-organization into directionally treadmilling vortices on solid supported membranes, and shape deformation of flexible liposomes. In cells, circumferential treadmilling of FtsZ was shown to recruit septum-building enzymes, but an active force production remains elusive. To gain mechanistic understanding of FtsZ dependent membrane deformations and constriction, we design an in vitro assay based on soft lipid tubes pulled from FtsZ decorated giant lipid vesicles (GUVs) by optical tweezers. FtsZ filaments actively transform these tubes into spring-like structures, where GTPase activity promotes spring compression. Operating the optical tweezers in lateral vibration mode and assigning spring constants to FtsZ coated tubes, the directional forces that FtsZ-YFP-mts rings exert upon GTP hydrolysis can be estimated to be in the pN range. They are sufficient to induce membrane budding with constricting necks on both, giant vesicles and E.coli cells devoid of their cell walls. We hypothesize that these forces result from torsional stress in a GTPase activity dependent manner.

Cortical actomyosin flows, among other mechanisms, scale up spontaneous symmetry breaking and thus play pivotal roles in cell differentiation, division, and motility. According to many model systems, myosin motor-induced local contractions of initially isotropic actomyosin cortices are nucleation points for generating cortical flows. However, the positive feedback mechanisms by which spontaneous contractions can be amplified towards large-scale directed flows remain mostly speculative. To investigate such a process on spherical surfaces, we reconstituted and confined initially isotropic minimal actomyosin cortices to the interfaces of emulsion droplets. The presence of ATP leads to myosin-induced local contractions that self-organize and amplify into directed large-scale actomyosin flows. By combining our experiments with theory, we found that the feedback mechanism leading to a coordinated directional motion of actomyosin clusters can be described as asymmetric cluster vibrations, caused by intrinsic non-isotropic ATP consumption with spatial confinement. We identified fingerprints of vibrational states as the basis of directed motions by tracking individual actomyosin clusters. These vibrations may represent a generic key driver of directed actomyosin flows under spatial confinement in vitro and in living systems.

FtsZ is a key component in bacterial cell division, being the primary protein of the presumably contractile Z ring. In vivo and in vitro , it shows two distinctive features that could so far however not be mechanistically linked: self-organization into directionally treadmilling vortices on solid supported membranes, and shape deformation of flexible liposomes. In cells, circumferential treadmilling of FtsZ was shown to recruit septum-building enzymes, but an active force production remains elusive. To gain mechanistic understanding of FtsZ dependent membrane deformations and constriction, we designed an in vitro assay based on soft lipid tubes pulled from FtsZ decorated giant lipid vesicles (GUVs) by optical tweezers. FtsZ actively transformed these tubes into spring-like structures, where GTPase activity promoted spring compression. Operating the optical tweezers in lateral vibration mode and assigning spring constants to FtsZ coated tubes, we found that FtsZ rings indeed exerts 0.14 – 1.09 pN forces upon GTP hydrolysis, through torsional stress induced by bidirectional treadmilling. These directional forces could further be demonstrated to induce membrane budding with constricting necks on both, giant vesicles and E.coli cells devoid of their cell walls. ### Competing Interest Statement The authors have declared no competing interest.

FtsZ is a key component in bacterial cell division, being the primary protein of the presumably contractile Z ring. In vivo and in vitro, it shows two distinctive features that could so far however not be mechanistically linked: self-organization into directionally treadmilling vortices on solid supported membranes, and shape deformation of flexible liposomes. In cells, circumferential treadmilling of FtsZ was shown to recruit septum-building enzymes, but an active force production remains elusive. To gain mechanistic understanding of FtsZ dependent membrane deformations and constriction, we designed an in vitro assay based on soft lipid tubes pulled from FtsZ decorated giant lipid vesicles (GUVs) by optical tweezers. FtsZ actively transformed these tubes into spring-like structures, where GTPase activity promoted spring compression. Operating the optical tweezers in lateral vibration mode and assigning spring constants to FtsZ coated tubes, we found that FtsZ rings indeed exerts 0.14 – 1.09 pN forces upon GTP hydrolysis, through torsional stress induced by bidirectional treadmilling. These directional forces could further be demonstrated to induce membrane budding with constricting necks on both, giant vesicles and E.coli cells devoid of their cell walls.

FtsZ is a key component in bacterial cell division, being the primary protein of the presumably contractile Z ring. Reconstituted in vitro, it shows two distinctive features that could so far however not be mechanistically linked: self-organization into directionally treadmilling vortices on solid supported membranes, and shape deformation of flexible liposomes. In cells, circumferential treadmilling of FtsZ was shown to recruit septum-building enzymes, but an active force production remains elusive. To determine direct contributions of FtsZ to membrane constriction, we designed a novel in vitro assay based on soft lipid tubes pulled from FtsZ decorated giant lipid vesicles (GUVs) by optical tweezers. FtsZ actively transformed these tubes into spring-like structures, where GTPase activity promoted spring compression. Operating the optical tweezers in lateral vibration mode and assigning spring constants to FtsZ coated tubes, we found that that FtsZ indeed exerts pN forces upon GTP hydrolysis, through torsional stress induced by bidirectional treadmilling.

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.

Cortical actomyosin flows play pivotal roles in cell motility, cell division and animal morphogenesis. According to many model systems, myosin motor induced local contractions are key for generating cortical flows. However, the original mechanism how large-scale directed flows emerge from local motor activity in an apparently isotropic cortex is unknown. We reconstituted and confined minimal actomyosin cortices to the interfaces of emulsion droplets. The presence of ATP leads to myosin-induced cortical contractions that self-organize into directed flow-like actomyosin motions. By combining our experiments with theory, we found that the large-scale directional motion of actomyosin clusters emerges from individual asymmetric cluster vibrations, caused by intrinsic non-isotropic ATP consumption, in conjunction with spherical confinement. By tracking individual actomyosin clusters, we identified fingerprints of vibrational states as the basis of directed motions. These vibrations may represent a generic key driver of directed actomyosin flows under spatial confinement in vitro and in living systems.

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.

Antimicrobial peptides are small amphiphilic proteins found in animals and plants as essential components of the innate immune system and whose function is to control bacterial infectious activity. In order to accomplish their function, antimicrobial peptides use different mechanisms of action which have been deeply studied in view of their potential exploitation to treat antibiotic-resistant bacterial infections. One of the main mechanisms of action of these peptides is the disruption of the bacterial membrane through pore formation, which, in some cases, takes place via a monomer to oligomer cooperative transition. Previous studies have shown that lipid composition, and the presence of exogenous components, such as cholesterol in model membranes or carotenoids in bacteria, can affect the potency of distinct antimicrobial peptides. At the same time, considering the membrane as a two-dimensional material, it has been shown that membrane composition defines its mechanical properties which might be relevant in many membrane-related processes. Nevertheless, the correlation between the mechanical properties of the membrane and antimicrobial peptide potency has not been considered according to the importance it deserves. The relevance of these mechanical properties in membrane deformation due to peptide insertion is reviewed here for different types of pores in order to elucidate if indeed membrane composition affects antimicrobial peptide activity by modulation of the mechanical properties of the membrane. This would also provide a better understanding of the mechanisms used by bacteria to overcome antimicrobial peptide activity.

We explore whether changes in fatty acid composition in bacterial membrane models lead to inhibition of amphiphilic antimicrobial peptide-(AMPs) through the modulation of the membrane elastic properties. Specifically, we examined whether changes in the unsaturated/saturated fatty acid ratio, which is controlled by bacteria, inhibit AMP function. In phosphatidylglycerol (PG) lipid bilayers we observe a 10-fold increase in resistance to Magainin-2 (MAG) in POPG (16:0/18:1) vs. DPPG (16:0/16:0) bilayers and a 20-fold increase in resistance in DOPG (18:1/18:1) vs. DPPG bilayers, as measured by calcein release at 45 C (where lipids are in the liquid-crystalline phase). By analyzing the leakage kinetics, we find that the activation energy for pore formation in DPPG/POPG mixtures increases linearly with POPG content, confirming that unsaturation is energetically unfavorable for pore formation. Laurdan polarization (GP) measurements correlate well with MAG resistance, suggesting that head group spacing is an indicator of resistance (increased head-group spacing could allow for more peptides to partition into the head group region, instead of becoming inserted into the bilayer as bilayer-spanning monomers). The changes in potency cannot be explained solely by the increased head group spacing, however. We therefore performed leakage experiments using known modifiers of membrane elastic properties (Triton-X, capsaicin) that both reduce bilayer stiffness but cause opposite changes in curvature. Bilayer softening plays a role in decreasing MAG's potency, whereas the curvature alters only the leakage kinetics, not potency. Based on previous studies, we propose that membrane softening somehow makes it more difficult for MAG to reach a critical concentration necessary to produce pore formation⎯maybe because the energetics of lateral association among bilayer-spanning MAG monomers favors the monomers in softer bilayers. Unsaturated lipids may therefore influence MAG potency by modulating membrane stiffness.

During changes in temperature, bacterial membranes present broad but cooperative lipid chain-melting events, where the membrane transitions from a solid-ordered state into a liquid-disordered state. For example, in Staphylococcus aureus, this melting event occurs at 15C. This transition has important implications. When temperature decreases near to the lipid melting event, cell division is inhibited. Gram-positive bacteria present cold-shock response mechanisms that shift the melting event to lower temperatures by varying the saturated fatty acids/unsaturated fatty acids ratio (SFA/UFA). It thus appears that the bacteria adaptatively strives to minimize the solid-ordered phase. However, the solid-ordered phase is not completely detrimental to cell viability. Recently we have shown that the solid-ordered phase induces resistance to PLA2-IIA, an innate human antibacterial agent that acts by disrupting membrane integrity. Therefore, we propose that adaptative modulation of the SFA/UFA ratio of bacterial membranes may alter the physical activity of antimicrobial agents that disrupt membrane integrity. To corroborate this, we studied, with the use of model systems, the activity of two antimicrobial peptides Magainin-2 and Novocidin (AMPs). Based on fluorescence spectroscopy, we measured calcein leakage potency on POPG (unsaturated) / DPPG (saturated) large unilamellar vesicles (LUVs). As we modified the SFA/UFA ratio we observe as expected a shift in the phase transition from ordered to disordered phase. This leads to 1) changes in the peptide needed to induce 50% leakage, and 2) changes in the leakage kinetic rate constant. Furthermore, we relate this kinetic rate to the energetics of a two state model in order to quantify the changes in the activation energy required for AMPs to perturb the bilayer as a function of the SFA/UFA ratio. We relate this change in activation energy to levels of lipid packing by measuring Laurdan generalized polarization.

There is a wide discussion regarding the origin and function of lipid domains in human cells. An example in which lipid domains may play a functional role is the plasma membrane of human platelets. Platelets are susceptible to chilling, activating when the temperature falls below 20 °C. This limitation represents a crucial issue in terms of storage of platelets. It has been previously shown that cold-induced platelet activation is correlated with the formation of macroscopic lipid domains during cooling. This is a result of the fact that platelets present low cholesterol content (15 mol%), which results in the presence of a cooperative lipid melting transition centered at 15°C. This transition is responsible for the formation of the macroscopic lipid domains during cooling. Human secretory phopholipase A2 type IIA (sPLA2-IIA) catalyses the hydrolysis of the sn-2 ester bond in glycerolipids to produce fatty acids and lysolipids. Recently we have shown that its activity is triggered by the local enrichment of anionic lipids in fluid domains during phase coexistence. Since human platelets expose their anionic lipid content during chilling, we propose that PLA2-IIA is triggered during this process, and that the activity of the enzyme on the fluid domains induces an overall increase in rigidity in the platelet plasma membrane due to an increase in the proportion of ordered lipids. We present a combined FTIR and Laurdan fluorescence spectroscopy study on the thermotropic phase behavior of human platelets, and show that during cold-induced activation membrane rigidity increases. We present results on how the activity of sPLA2-IIA is regulated by the thermotropic phase behavior of the platelet plasma membrane after platelets have been artificially activated through sonication. We show that PLA2-IIA activity increases the rigidity of these activated platelet plasma membranes.

There has been ample debate on whether cell membranes can present macroscopic lipid domains as predicted by three-component phase diagrams obtained by fluorescence microscopy. Several groups have argued that membrane proteins and interactions with the cytoskeleton inhibit the formation of large domains. In contrast, some polarizable cells do show large regions with qualitative differences in lipid fluidity. It is important to ask more precisely, based on the current phase diagrams, under what conditions would large domains be expected to form in cells. In this work we study the thermotropic phase behavior of the platelet plasma membrane by FTIR, and compare it to a POPC/Sphingomyelin/Cholesterol model representing the outer leaflet composition. We find that this model closely reflects the platelet phase behavior. Previous work has shown that the platelet plasma membrane presents inhomogeneous distribution of Dil18:0 at 24 degrees C, but not at 37 degrees C, which suggests the formation of macroscopic lipid domains at low temperatures. We show by fluorescence microscopy, and by comparison with published phase diagrams, that the outer leaflet model system enters the macroscopic domain region only at the lower temperature. In addition, the low cholesterol content in platelets (similar to 15 mol%), appears to be crucial for the formation of large domains during cooling. (c) 2009 Elsevier B.V. All rights reserved.