Biology subjects

Rivas, G.

Publications and source records attributed to Rivas, G..

8 recordsLinked to original sources

bioRxiv2024

Bacterial cell cycle regulator MatP forms biomolecular condensates and directly interacts with cell division protein FtsZ

An increasing number of proteins involved in bacterial cell cycle events have been recently shown to undergo phase separation. The resulting biomolecular condensates play an important role in cell cycle protein function and may be involved in development of persister cells tolerant to antibiotics. Here we report that the E. coli chromosomal Ter macrodomain organizer MatP, a division site selection protein implicated in the coordination of chromosome segregation with cell division, forms biomolecular condensates in cytomimetic systems. These condensates are favored by crowding and preferentially localize at the membrane of microfluidics droplets, a behavior probably mediated by MatP-lipid binding. Condensates are negatively regulated and partially dislodged from the membrane by DNA sequences recognized by MatP (matS), which partition into them. Unexpectedly, MatP condensation is enhanced by FtsZ, a core component of the division machinery previously described to undergo phase separation. Our biophysical analyses uncover a direct interaction between the two proteins, disrupted by matS sequences. This binding might have implications for FtsZ ring positioning at mid-cell by the Ter linkage, which comprises MatP and two other proteins that bridge the canonical MatP/FtsZ interaction. FtsZ/MatP condensates interconvert with bundles in response to GTP addition, providing additional levels of regulation. Consistent with discrete foci reported in cells, MatP biomolecular condensates may facilitate MatPs role in chromosome organization and spatiotemporal regulation of cytokinesis and DNA segregation. Moreover, sequestration of MatP in these membraneless compartments, with or without FtsZ, could promote cell entry into dormant states that are able to survive antibiotic treatments.

biochemistry↗

bioRxiv2023

Crosslinking by ZapD drives the assembly of short, discontinuous FtsZ filaments into ring-like structures in solution

Bacterial cell division relies on the Z ring, a cytoskeletal structure that acts as a scaffold for the assembly of the divisome. To date, the detailed mechanisms underlying the assembly and stabilization of the Z ring remain elusive. This study highlights the role of the FtsZ-associated protein (Zap) ZapD in the assembly and stabilization of Z-ring-like structures via filament crosslinking. Using cryo-electron tomography and biochemical analysis, we show that, at equimolar concentrations of ZapD and FtsZ, ZapD induces the formation of toroidal structures composed of short, curved FtsZ filaments that are crosslinked vertically, but also laterally and diagonally. At higher concentrations of ZapD, regularly spaced ZapD dimers crosslink FtsZ filaments from above, resulting in the formation of straight bundles. Despite the simplicity of this reconstituted system, these findings provide valuable insights into the structural organization and stabilization of the Z ring by Zap proteins in bacterial cells, revealing the key role of optimal crosslinking density and geometry in enabling filament curvature and ring formation.

biochemistry↗

bioRxiv2020

Assembly of bacterial cell division protein FtsZ into dynamic biomolecular condensates

Biomolecular condensation through phase separation may be a novel mechanism to regulate bacterial processes, including cell division. Previous work revealed FtsZ, a protein essential for cytokinesis in most bacteria, and the E. coli division site selection factor SlmA form FtsZ*SlmA biomolecular condensates. The absence of condensates composed solely of FtsZ under the conditions used in that study suggested this mechanism was restricted to nucleoid occlusion or SlmA-containing bacteria. Here we report that FtsZ alone can demix into condensates in bulk and when encapsulated in synthetic cell-like systems. Condensate assembly depends on FtsZ being in the GDP-bound state and on crowding conditions that promote its oligomerization. FtsZ condensates are dynamic and gradually convert into FtsZ filaments upon GTP addition. Notably, FtsZ lacking its C-terminal disordered region, a structural element likely to favor biomolecular condensation, also forms condensates, albeit less efficiently. The inherent tendency of FtsZ to form condensates susceptible to modulation by physiological factors, including binding partners, suggests that such mechanisms may play a more general role in bacterial cell division than initially envisioned.

biochemistry↗

bioRxiv2020

Cell-free biogenesis of bacterial division proto-rings that can constrict liposomes

A major challenge towards the realization of an autonomous synthetic cell resides in the encoding of a division machinery in a genetic programme. A key event in the bacterial cell cycle is the assembly of cytoskeletal proteins into a ring that 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 membrane and generate budding vesicles, a phenotype that has not been reported before. 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 provide new insights on the self-organization of basic cytoskeletal elements involved in bacterial division. Moreover, they demonstrate that gene-directed protein synthesis and assembly of membrane-constricting FtsZ-rings can be combined in a liposome-based artificial cell.

synthetic biology↗

bioRxiv2018

FtsZ assembles the bacterial cell division machinery by a diffusion-and-capture mechanism

The mechanism of bacterial cell division is largely unknown. The protein machinery performing cell division is organized by FtsZ, a tubulin-homolog that forms treadmilling filaments at the cell division site. Treadmilling is thought to actively move proteins around the cell thereby distributing peptidoglycan synthesis to make two new cell poles. To understand this process, we reconstituted part of the bacterial cell division machinery using the purified components FtsZ, FtsA and truncated transmembrane proteins essential for cell division. We found that membrane-bound cytosolic peptides of FtsN and FtsQ co-migrated with treadmilling FtsZ-FtsA filaments. Remarkably, rather than moving in a directed fashion, individual peptides followed FtsZ filaments by a diffusion-and-capture mechanism. Our work provides a mechanism for how the Z-ring dynamically recruits divisome proteins and highlights the importance of transient interactions for the self-organization of complex biological structures. We propose that this mechanism is used more widely to organize and transmit spatiotemporal information in living cells.\n\nOne Sentence SummaryFtsZ treadmilling assembles bacterial division machinery by diffusion-and-capture mechanism.

biochemistry↗

bioRxiv2018

The bacterial DNA binding protein MatP involved in linking the nucleoid terminal domain to the divisome at midcell interacts with lipid membranes

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 MatP, when encapsulated inside microdroplets generated by microfluidics and giant vesicles, 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 bio-layer interferometry assays. 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 interaction of MatP with lipids may modulate its spatiotemporal localization and its recognition of other ligands.\n\nIMPORTANCEThe 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 comprising 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.

biochemistry↗

bioRxiv2018

Escherichia coli ZipA organizes FtsZ polymers into dynamic ring-like protofilament structures

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 nm 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.\n\nIMPORTANCEBacterial cells use a membrane-attached ring of proteins to mark and guide formation of a division septum at mid-cell 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 only observed 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.

microbiology↗

bioRxiv2018

Bacterial division FtsZ forms liquid condensates with nucleoid-associated Z-ring inhibitor SlmA

Macromolecular condensation resulting from biologically regulated liquid-liquid phase transitions is emerging as a mechanism to organize the intracellular space in eukaryotic systems, with broad implications in cell physiology and pathology. Here we show that FtsZ, central element of the division ring in most bacteria, forms condensates when in complex with SlmA, the protein preventing septal ring assembly nearby the chromosome in E. coli. The formation of condensates is promoted by crowding and enhanced by sequence-specific binding of SlmA to DNA. These structures are dynamic and FtsZ within them remains active for GTP-triggered fiber formation. Their location is sensitive to compartmentalization and to the presence of a membrane boundary in microfluidics-based cell mimetic systems, likely affecting their reactivity. We propose that reversible condensation may play a role in the modulation of FtsZ assembly and/or location by SlmA and, hence, in the regulation of ring stability, constituting a singular example of a prokaryotic nucleoprotein complex exhibiting this kind of phase transition.

biochemistry↗