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FtsZ is an essential and central protein for cell division in most bacteria. Because of its ability to organize into dynamic polymers at the cell membrane and recruit other protein partners to form a “divisome”, FtsZ is a leading target in the quest for new antibacterial compounds. Strategies to potentially arrest the essential and tightly regulated cell division process include perturbing FtsZ’s ability to interact with itself and other divisome proteins. Here, we discuss the available methodologies to screen for and characterize those interactions. In addition to assays that measure protein-ligand interactions in solution, we also discuss the use of minimal membrane systems and cell-like compartments to better approximate the native bacterial cell environment and hence provide a more accurate assessment of a candidate compound’s potential in vivo effect. We particularly focus on ways to measure and inhibit under-explored interactions between FtsZ and partner proteins. Finally, we discuss recent evidence that FtsZ forms biomolecular condensates in vitro, and the potential implications of these assemblies in bacterial resistance to antibiotic treatment.

Hereditary transthyretin amyloidosis (ATTR) is an autosomal dominant disease characterized by the extracellular deposition of the transport protein transthyretin (TTR) as amyloid fibrils. Despite the progress achieved in recent years, understanding why different TTR residue substitutions lead to different clinical manifestations remains elusive. Here, we studied the molecular basis of disease-causing missense mutations affecting residues R34 and K35. R34G and K35T variants cause vitreous amyloidosis, whereas R34T and K35N mutations result in amyloid polyneuropathy and restrictive cardiomyopathy. All variants are more sensitive to pH-induced dissociation and amyloid formation than the wild-type (WT)-TTR counterpart, specifically in the variants deposited in the eyes amyloid formation occurs close to physiological pHs. Chemical denaturation experiments indicate that all the mutants are less stable than WT-TTR, with the vitreous amyloidosis variants, R34G and K35T, being highly destabilized. Sequence-induced stabilization of the dimer–dimer interface with T119M rendered tetramers containing R34G or K35T mutations resistant to pH-induced aggregation. Because R34 and K35 are among the residues more distant to the TTR interface, their impact in this region is therefore theorized to occur at long range. The crystal structures of double mutants, R34G/T119M and K35T/T119M, together with molecular dynamics simulations indicate that their strong destabilizing effect is initiated locally at the BC loop, increasing its flexibility in a mutation-dependent manner. Overall, the present findings help us to understand the sequence-dynamic-structural mechanistic details of TTR amyloid aggregation triggered by R34 and K35 variants and to link the degree of mutation-induced conformational flexibility to protein aggregation propensity.

A major challenge towards the realization of an autonomous synthetic cell resides in the encoding of a division machinery in a genetic programme. In the bacterial cell cycle, the assembly of cytoskeletal proteins into a ring defines the division site. At the onset of the formation of the Escherichia coli divisome, a proto-ring consisting of FtsZ and its membrane-recruiting proteins takes place. Here, we show that FtsA-FtsZ ring-like structures driven by cell-free gene expression can be reconstituted on planar membranes and inside liposome compartments. Such cytoskeletal structures are found to constrict the liposome, generating elongated membrane necks and budding vesicles. Additional expression of the FtsZ cross-linker protein ZapA yields more rigid FtsZ bundles that attach to the membrane but fail to produce budding spots or necks in liposomes. These results demonstrate that gene-directed protein synthesis and assembly of membrane-constricting FtsZ-rings can be combined in a liposome-based artificial cell.

Protection of the chromosome from scission by the division machinery during cytokinesis is critical for bacterial survival and fitness. This is achieved by nucleoid occlusion, which, in conjunction with other mechanisms, ensures formation of the division ring at midcell. In Escherichia coli, this mechanism is mediated by SlmA, a specific DNA binding protein that antagonizes assembly of the central division protein FtsZ into a productive ring in the vicinity of the chromosome. Here, we provide evidence supporting direct interaction of SlmA with lipid membranes, tuned by its binding partners FtsZ and SlmA binding sites (SBS) on chromosomal DNA. Reconstructions in minimal membrane systems that mimic cellular environments show that SlmA binds to lipid-coated microbeads or locates at the edge of microfluidic-generated microdroplets, inside which the protein is encapsulated. DNA fragments containing SBS sequences do not seem to be recruited to the membrane by SlmA but instead compete with SlmA's ability to bind lipids. The interaction of SlmA with FtsZ modulates this behavior, ultimately triggering membrane localization of the SBS sequences alongside the two proteins. The ability of SlmA to bind lipids uncovered in this work extends the interaction network of this multivalent regulator beyond its well-known protein and nucleic acid recognition, which may have implications in the overall spatiotemporal control of division ring assembly.IMPORTANCE Successful bacterial proliferation relies on the spatial and temporal precision of cytokinesis and its regulation by systems that protect the integrity of the nucleoid. In Escherichia coli, one of these protectors is SlmA protein, which binds to specific DNA sites around the nucleoid and helps to shield the nucleoid from inappropriate bisection by the cell division septum. Here, we discovered that SlmA not only interacts with the nucleoid and septum-associated cell division proteins but also binds directly to cytomimetic lipid mem

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.

Here we have summarized several strategies to reconstruct complexes containing the FtsZ protein, a central element of the cell division machinery in most bacteria, and to test their functional organization in minimal membrane systems and cell-like containers, as vesicles and droplets produced by microfluidics. These synthetic systems have been devised to mimic elements of the intracellular complexity, as excluded volume effects due to natural crowding, and macromolecular condensation resulting from biologically regulated liquid-liquid phase separation, in media of known and controllable composition. This integrative approach has allowed to demonstrate that macromolecular phase separation and crowding may also help to dynamically organize FtsZ in the intracellular space thus modulating its functional reactivity in cell division.

Most bacteria accomplish cell division with the help of a dynamic protein complex called the divisome, which spans the cell envelope in the plane of division. Assembly and activation of this machinery are coordinated by the tubulin-related GTPase FtsZ, which was found to form treadmilling filaments on supported bilayers in vitro1, as well as in live cells, in which filaments circle around the cell division site2,3. Treadmilling of FtsZ is thought to actively move proteins around the division septum, thereby distributing peptidoglycan synthesis and coordinating the inward growth of the septum to form the new poles of the daughter cells4. However, the molecular mechanisms underlying this function are largely unknown. Here, to study how FtsZ polymerization dynamics are coupled to downstream proteins, we reconstituted part of the bacterial cell division machinery using its purified components FtsZ, FtsA and truncated transmembrane proteins essential for cell division. We found that the membrane-bound cytosolic peptides of FtsN and FtsQ co-migrated with treadmilling FtsZ–FtsA filaments, but despite their directed collective behaviour, individual peptides showed random motion and transient confinement. Our work suggests that divisome proteins follow treadmilling FtsZ filaments by a diffusion-and-capture mechanism, which can give rise to a moving zone of signalling activity at the division site.

[EN]Systems biochemistry of macromolecular interactions involved in theregulation of the division ring stability in bacteria.Escherichia coli cell division is mediated by the divisome, a contractile ringconsisting of a multiprotein complex that accurately assembles at midcell (corresponding to the long axis midpoint of this bacillus) toward the end of the cellcycle. The first step in the formation of this division machinery is the assembly ofthe so-called Z-ring, a dynamic macromolecular structure involving the polymerization of the conserved protein FtsZ that serves as a scaffold for the recruitment of the rest of division proteins. Correct spatiotemporal assembly of theZ-ring is critical for the generation of viable daughter cells, since deviations in its positioning might result in catastrophic outcomes such as chromosome bisectionor generation of anucleate cells. For this reason, E. coli relies on dedicate dpositioning mechanisms to ensure proper formation of the Z-ring at midcell, being the canonical ones the Min system and nucleoid occlusion, which act by blocking the assembly of the Z-ring at undesired locations. The Min system establishes an oscillation pattern that prevents Z-ring formation at the cell poles while allowing its assembly in the cell center. For its part, nucleoid occlusion inhibits Z-ring assembly in the vicinity of the chromosome through direct interaction of the SlmA protein, bound to specific DNA sequences (SBS) in the chromosome, with FtsZ. Besides, the concerted action of these two negative regulatory mechanisms is complemented by the Ter linkage, a protein network comprising ZapA, ZapB and the DNA-binding protein MatP that reinforces the formation of the Z-ring at midcell. This regulated assembly of the Z-ring takes place in a highly crowded environment, with a large fraction of the cell volume occupied by macromolecules. It is well known that crowding affects the reactivity of biochemical interactions, primarily through the excluded-vo, Interestingly, examination of conditions, within the physiological ranges described in E. coli, revealed that FtsZ can reversibly undergo biomolecular condensation on its own under conditions known to promote its oligomerization tendency (chapter 2). Condensates formed by FtsZ in the absence of SlmA were still dynamic and evolved toward fibers after GTP addition. Besides, encapsulation of FtsZ inside cytomimetic containers led to incipient formation of condensates in conditions under which no condensates were detected in bulk, suggesting that confinement and/or the presence of a lipid membrane influence their formation. Finally, experiments with an FtsZ mutant lacking the C-terminal unstructured domain demonstrated that this region of FtsZ was not strictly required for condensation albeit it likely enhances the overall process. The last two chapters encompass the identification, analysis and discussion of the lipid interaction of the DNA-binding proteins SlmA (chapter 3) and MatP (chapter 4). These studies were performed using different minimal membrane systems following an orthogonal approach that included biochemical, biophysical and imaging methods. SlmA was found to bind either to the lipid mixture matching the composition in the E. coli inner membrane (EcL), which contains negatively charged components, or to phosphatidylcholine (PC), a neutral phospholipid (chapter 3). For the two types of compositions, the affinity of the interaction was found to be greatly enhanced at low ionic strength but weakly affected above a certain threshold, suggesting that both electrostatic and hydrophobic forces play a role in the membrane binding activity of SlmA. Evaluation of this interaction in the presence of the specific SBS sequences recognized by the protein showed that they compete with the lipids for binding to SlmA. Intriguingly, the presence of FtsZ radically changed this behavior, promoting the recruitment of SBS sequences to the membrane by SlmA. Protein-lipid intera, [ES]Bioquímica de sistemas de interacciones macromoleculares implicadas en la regulación de la estabilidad del anillo de división en bacterias En Escherichia coli la división se encuentra mediada por el divisoma, un anillo contráctil consistente en un complejo multiproteico posicionado con gran precisión en el centro de la célula (correspondiente al punto medio del eje largo de este bacilo) hacia el final del ciclo celular. El primer paso en la formación de esta maquinaria de división es el ensamblaje del denominado anillo Z, una estructura macromolecular dinámica que implica la polimerización de la proteína conservada FtsZ y que actúa como plataforma sobre la que se incorporan el resto de proteínas de división. El correcto posicionamiento espacial y temporal del anillo Z es crítico para la generación de células hijas viables, dado que desviaciones en la adecuada localización del mismo pueden conllevar consecuencias catastróficas para la bacteria como la bisección del cromosoma o la generación de células anucleadas. Por este motivo, E. coli cuenta con mecanismos de posicionamiento que aseguran la formación del anillo en el centro celular, de entre los cuales el sistema Min y el sistema de oclusión por nucleoide son los más estudiados. El sistema Min establece un patrón de oscilaciones que impide la formación del anillo en los polos celulares a la vez que permite su ensamblaje en el centro de la célula. Por su parte, el sistema de oclusión por nucleoide inhibe la formación del anillo Z en las proximidades del cromosoma bacteriano a través de la interacción directa de la proteína SlmA, unida a secuencias específicas de ADN (SBS) en el cromosoma, con FtsZ. Además, la acción conjunta de estos dos mecanismos reguladores negativos está complementada por el anclaje Ter, consistente en una red multiproteica en la que participan las proteínas ZapA, ZapB y la proteína de unión a ADN MatP que refuerza la formación del anillo Z en posiciones centrales. Este ensamblaje regulado, El objetivo de la presente tesis doctoral es contribuir al conocimiento de los mecanismos involucrados en la división bacteriana y, particularmente, en el posicionamiento del anillo de división a través de la regulación coordinada del ensamblaje, localización y distribución de la proteína central de división FtsZ. Específicamente, el propósito de esta tesis es analizar la influencia de la aglomeración macromolecular y de la presencia de membranas y microambientes sobre FtsZ, así como sobre SlmA y MatP, las proteínas de unión a ADN implicadas en la regulación del ensamblaje del anillo Z. Para ello, se han analizado interacciones proteína-proteína, proteína-ADN y proteína-lípido siguiendo un enfoque que incluye el uso de métodos bioquímicos y biofísicos, así como de reconstitución en ambientes citomiméticos. En los dos primeros capítulos de esta tesis se describe el ensamblaje mediante separación de fases de condensados formados por FtsZ en presencia (capítulo 1) o ausencia (capítulo 2) de la proteína efectora del sistema de oclusión por nucleoide, SlmA, bajo condiciones de aglomeración. La reconstitución de FtsZ (en ausencia de GTP) y SlmA, unida a su secuencia específica SBS, resultó en la formación de estructuras micrométricas compatibles con condensados líquidos (capítulo 1) que fueron visualizados mediante microscopía confocal. Este hecho fue confirmado a través de ensayos de turbidez. Además, se observó que la formación de estas estructuras se encontraba favorecida a baja fuerza iónica y a concentración creciente de agente aglomerante y de proteína., Se demostró que estos ensamblados eran dinámicos y reversibles, cualidades distintivas de los condensados biomoleculares, dada su capacidad para intercambiar proteína con la fase externa (comprobada mediante ensayos de captura) y la conservación de las propiedades de polimerización/despolimerización dependiente de GTP de FtsZ. El impacto de la presencia de diferentes microambientes, imitando a los que se encuentran en el interior celular, se exploró mediante el uso de sistemas de separación de fase líquido-líquido formados por dos agentes aglomerantes no relacionados que se separan en dos fases a altas concentraciones, generando compartimentos. Los condensados reconstituidos en estos medios multifásicos se distribuían asimétricamente entre estos compartimentos localizándose principalmente cerca de la superficie lipídica cuando fueron encapsulados en contenedores citomiméticos. Una exploración más exhaustiva abarcando un mayor número de condiciones, dentro del rango fisiológico descrito para E. coli, reveló que FtsZ puede formar reversiblemente condensados biomoleculares por sí misma en condiciones que promueven su oligomerización (capítulo 2). Los condensados formados por FtsZ en ausencia de SlmA resultaron ser dinámicos y capaces de evolucionar hacia fibras tras la adición de GTP. Además, se observó que la encapsulación de FtsZ en contenedores citomiméticos daba lugar a la formación incipiente de condensados en condiciones bajo las que no se detectaron en sistemas abiertos, sugiriendo una posible influencia del confinamiento o de la presencia de la membrana lipídica en su formación. Finalmente, ensayos con un mutante de FtsZ que carece del dominio no estructurado C-terminal demostraron que esta región de la proteína no es estrictamente necesaria para la formación de condensados, aunque probablemente participa potenciando el proceso global. Los dos últimos capítulos de esta tesis comprenden la identificación, análisis y discusión de la interacción de las proteínas d

In most bacteria, the early step of septum formation implies the association of soluble FtsZ polymers to the cytoplasmic membrane. ZipA, together with FtsA, provide membrane tethering to FtsZ in Escherichia coli, forming a dynamic proto-ring that serves as an assembly scaffold for the remaining elements of the divisome. Despite their importance for bacterial cell division, multivalent interactions between proto-ring elements at membrane surfaces remain poorly characterized in quantitative terms. Here we measured the binding of FtsZ to ZipA incorporated in supported lipid bilayers at controlled densities by using a combination of biophysical surface-sensitive techniques (quartz crystal microbalance and spectroscopic ellipsometry) and analyzed how ZipA density and FtsZ concentration control the state of assembly of FtsZ. We find that ZipA attachment enables FtsZ-GMPCPP (where GMPCPP is a GTP analogue with reduced hydrolysis) to assemble in several distinct ways, that is: (i) two-dimensional polymerization at the membrane, and (ii) three-dimensional polymerization from the membrane into the solution phase where this may be associated with the formation of higher-order complexes. In these processes, ZipA is required to enrich FtsZ at the surface but the FtsZ bulk concentration defines which morphology is being formed. Moreover, we report a strong effect of the nucleotide (GDP vs GMPCPP/GTP) on the kinetics of ZipA-association/dissociation of FtsZ. These results provide insights on the mode of interaction of proto-ring elements in minimal membrane systems and contribute to complete our understanding of the initial events of bacterial division.

Division ring formation at midcell is controlled by various mechanisms in Escherichia coli, one of them being the linkage between the chromosomal Ter macrodomain and the Z-ring mediated by MatP, a DNA binding protein that organizes this macrodomain and contributes to the prevention of premature chromosome segregation. Here we show that, during cell division, just before splitting the daughter cells, MatP seems to localize close to the cytoplasmic membrane, suggesting that this protein might interact with lipids. To test this hypothesis, we investigated MatP interaction with lipids in vitro. We found that, when encapsulated inside vesicles and microdroplets generated by microfluidics, MatP accumulates at phospholipid bilayers and monolayers matching the lipid composition in the E. coli inner membrane. MatP binding to lipids was independently confirmed using lipid-coated microbeads and biolayer interferometry assays, which suggested that the recognition is mainly hydrophobic. Interaction of MatP with the lipid membranes also occurs in the presence of the DNA sequences specifically targeted by the protein, but there is no evidence of ternary membrane/protein/DNA complexes. We propose that the association of MatP with lipids may modulate its spatiotemporal localization and its recognition of other ligands., IMPORTANCE The division of an E. coli cell into two daughter cells with equal genomic information and similar size requires duplication and segregation of the chromosome and subsequent scission of the envelope by a protein ring, the Z-ring. MatP is a DNA binding protein that contributes both to the positioning of the Z-ring at midcell and the temporal control of nucleoid segregation. Our integrated in vivo and in vitro analysis provides evidence that MatP can interact with lipid membranes reproducing the phospholipid mixture in the E. coli inner membrane, without concomitant recruitment of the short DNA sequences specifically targeted by MatP. This observation strongly suggests that the membrane may play a role in the regulation of the function and localization of MatP, which could be relevant for the coordination of the two fundamental processes in which this protein participates, nucleoid segregation and cell division.

Protein modification by lipid derived reactive species or lipoxidation is increased during oxidative stress, a common feature observed in many pathological conditions. Biochemical and functional consequences of lipoxidation include changes in the conformation and assembly of the target proteins, altered recognition of ligands and/or cofactors, changes in the interactions with DNA or protein-protein interactions, modifications in membrane partitioning and binding and/or subcellular localization. These changes may impact, directly or indirectly, signaling pathways involved in the activation of cell defense mechanisms, but when these are overwhelmed they may lead to pathological outcomes. Mass spectrometry provides state of the art approaches for the identification and characterization of lipoxidized proteins/residues and the modifying species. Nevertheless, understanding the complexity of the functional effects of protein lipoxidation requires the use of additional methodologies. Herein, biochemical and biophysical methods used to detect and measure functional effects of protein lipoxidation at different levels of complexity, from in vitro and reconstituted cell-like systems to cells, are reviewed, focusing especially on macromolecular interactions. Knowledge generated through innovative and complementary technologies will contribute to comprehend the role of lipoxidation in pathophysiology and, ultimately, its potential as target for therapeutic intervention.

Macromolecular condensation resulting from biologically regulated liquid-liquid phase separation is emerging as a mechanism to organize intracellular space in eukaryotes, with broad implications for cell physiology and pathology. Despite their small size, bacterial cells are also organized by proteins such as FtsZ, a tubulin homolog that assembles into a ring structure precisely at the cell midpoint and is required for cytokinesis. Here, we demonstrate that FtsZ can form crowding-induced condensates, reminiscent of those observed for eukaryotic proteins. Formation of these FtsZ-rich droplets occurs when FtsZ is bound to SlmA, a spatial regulator of FtsZ that antagonizes polymerization, while also binding to specific sites on chromosomal DNA. The resulting condensates are dynamic, allowing FtsZ to undergo GTP-driven assembly to form protein fibers. They are sensitive to compartmentalization and to the presence of a membrane boundary in cell mimetic systems. This is a novel example of a bacterial nucleoprotein complex exhibiting condensation into liquid droplets, suggesting that phase separation may also play a functional role in the spatiotemporal organization of essential bacterial processes.

Single-particle plasmon spectroscopy has become a standard technique todetect and quantify the presence of unlabeled macromolecules. Here, we extend thismethod to determine their exact distance from the plasmon sensors with sub-nanometerresolution by systematically varying the sensing range into the surrounding by adjustingthe size of the plasmonic nanoparticles. We improved current single-particle plasmonspectroscopy to record continuously for hours the scattering spectra of thousands ofnanoparticles of different sizes simultaneously with 1.8 s time resolution. We apply thistechnique to study the interaction dynamics of bacterial Min proteins with supportedlipid membranes of different composition. Our experiments reveal a surprisinglyflexibleoperating mode of the Min proteins: In the presence of cardiolipin and membranecurvature induced by nanoparticles, the protein oscillation occurs on top of a stationaryMinD patch. Our results reveal the need to consider membrane composition and localcurvature as important parameters to quantitatively understand the Min protein system and could be extrapolated to othermacromolecular systems. Our label-free method is generally easily implementable and well suited to measure distances ofinteracting biological macromolecules

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.

ZipA is an essential cell division protein in Escherichia coli. Together with FtsA, ZipA tethers dynamic polymers of FtsZ to the cytoplasmic membrane, and these polymers are required to guide synthesis of the cell division septum. This dynamic behavior of FtsZ has been reconstituted on planar lipid surfaces in vitro, visible as GTP-dependent chiral vortices several hundred nanometers in diameter, when anchored by FtsA or when fused to an artificial membrane binding domain. However, these dynamics largely vanish when ZipA is used to tether FtsZ polymers to lipids at high surface densities. This, along with some in vitro studies in solution, has led to the prevailing notion that ZipA reduces FtsZ dynamics by enhancing bundling of FtsZ filaments. Here, we show that this is not the case. When lower, more physiological levels of the soluble, cytoplasmic domain of ZipA (sZipA) were attached to lipids, FtsZ assembled into highly dynamic vortices similar to those assembled with FtsA or other membrane anchors. Notably, at either high or low surface densities, ZipA did not stimulate lateral interactions between FtsZ protofilaments. We also used E. coli mutants that are either deficient or proficient in FtsZ bundling to provide evidence that ZipA does not directly promote bundling of FtsZ filaments in vivo. Together, our results suggest that ZipA does not dampen FtsZ dynamics as previously thought, and instead may act as a passive membrane attachment for FtsZ filaments as they treadmill., IMPORTANCE: Bacterial cells use a membrane-attached ring of proteins to mark and guide formation of a division septum at midcell that forms a wall separating the two daughter cells and allows cells to divide. The key protein in this ring is FtsZ, a homolog of tubulin that forms dynamic polymers. Here, we use electron microscopy and confocal fluorescence imaging to show that one of the proteins required to attach FtsZ polymers to the membrane during E. coli cell division, ZipA, can promote dynamic swirls of FtsZ on a lipid surface in vitro. Importantly, these swirls are observed only when ZipA is present at low, physiologically relevant surface densities. Although ZipA has been thought to enhance bundling of FtsZ polymers, we find little evidence for bundling in vitro. In addition, we present several lines of in vivo evidence indicating that ZipA does not act to directly bundle FtsZ polymers.

A major challenge towards the realization of an autonomous synthetic cell resides in the encoding of a division machinery in a genetic programme. In the bacterial cell cycle, the assembly of cytoskeletal proteins into a ring defines the division site. At the onset of the formation of the Escherichia coli divisome, a proto-ring consisting of FtsZ and its membrane-recruiting proteins takes place. Here, we show that FtsA-FtsZ ring-like structures driven by cell-free gene expression can be reconstituted on planar membranes and inside liposome compartments. Such cytoskeletal structures are found to constrict the liposome, generating elongated membrane necks and budding vesicles. Additional expression of the FtsZ cross-linker protein ZapA yields more rigid FtsZ bundles that attach to the membrane but fail to produce budding spots or necks in liposomes. These results demonstrate that gene-directed protein synthesis and assembly of membrane-constricting FtsZ-rings can be combined in a liposome-based artificial cell., BN/Christophe Danelon Lab, Education and Student Affairs, BN/Technici en Analisten

The intermediate filament protein vimentin constitutes a critical sensor for electrophilic and oxidative stress. We previously showed that vimentin interacts with zinc, which affects its assembly and redox sensing. Here, we used vimentin wt and C328S, an oxidation-resistant mutant showing improved NaCl-induced polymerization, to assess the impact of zinc on soluble and polymerized vimentin by light scattering and electron microscopy. Zinc acts as a switch, reversibly inducing the formation of vimentin oligomeric species. High zinc concentrations elicit optically-detectable vimentin structures with a characteristic morphology depending on the support. These effects also occur in vimentin C328S, but are not mimicked by magnesium. Treatment of vimentin with micromolar ZnCl2 induces fibril-like particles that do not assemble into filaments, but form aggregates upon subsequent addition of NaCl. In contrast, when added to NaCl-polymerized vimentin, zinc increases the diameter or induces lateral association of vimentin wt filaments. Remarkably, these effects are absent or attenuated in vimentin C328S filaments. Therefore, the zinc-vimentin interaction depends on the chemical environment and on the assembly state of the protein, leading to atypical polymerization of soluble vimentin, likely through electrostatic interactions, or to broadening and lateral association of preformed filaments through mechanisms requiring the cysteine residue. Thus, the impact of zinc on vimentin assembly and redox regulation is envisaged.

There is growing interest in analyzing the effect of microenvironments, which may be mimicked through liquid-liquid phase separation (LLPS), on the reactivity of biological macromolecules. We report the encapsulation by microfluidics of the division protein FtsZ and a LLPS system inside microdroplets and their conversion into permeable vesicles (allowing ligand uptake), with higher yield, homogeneity and biomolecular compatibility than those previously described.

Biochemistry is at the core of constructive bottom-up approaches in synthetic biologyaiming to generate a minimal cell. Any sort of cellular organization depends on com-partmentalization of macromolecular assemblies and machineries within lipid, oramphiphilic lipid-like, vesicles. Following the increasing interest in the use of liposomesas cytomimetic devices, here we discuss our achievements toward using liposomes,especially giant unilamellar vesicles, as containers to reconstitute the amyloidogenicaggregation of the cytotoxic prionoid (prion-like protein) RepA-WH1. We address rele-vant issues as the importance of lipid composition and the effect or cofactors on assem-bly, and the feasibility of achieving protein expression within vesicles.