ABSTRACT
The extent of the influence that molecular curvature plays on the self-assembly of supramolecular polymers remains an open question in the field. We began addressing this fundamental question with the introduction of "carpyridines", which are saddle-shaped monomers that can associate with one another through π-π interactions and in which the rotational and translational movements are restricted. The topography displayed by the monomers led, previously, to the assembly of highly ordered 2D materials even in the absence of strong directional interactions such as hydrogen bonding. Here, we introduce a simple strategy to gain control over the dimensionality of the formed structures yielding classical unidimensional polymers. These have been characterized using well-established protocols allowing us to determine and confirm the self-assembly mechanism of both fibers and sheets. The calculated interaction energies are significantly higher than expected for flexible self-assembling units lacking classical "strong" non-covalent interactions. The versatility of this supramolecular unit to assemble into either supramolecular fibers or 2D sheets with strong association energies highlights remarkably well the potential and importance of molecular shape for the design of supramolecular materials and the applications thereof.
ABSTRACT
During bacterial division, polymers of the tubulin-like GTPase FtsZ assemble at midcell to form the cytokinetic Z-ring, which coordinates peptidoglycan (PG) remodeling and envelope constriction. Curvature of FtsZ filaments promotes membrane deformation in vitro, but its role in division in vivo remains undefined. Inside cells, FtsZ directs PG insertion at the division plane, though it is unclear how FtsZ structure and dynamics are mechanistically coupled to PG metabolism. Here we study FzlA, a division protein that stabilizes highly curved FtsZ filaments, as a tool for assessing the contribution of FtsZ filament curvature to constriction. We show that in Caulobacter crescentus, FzlA must bind to FtsZ for division to occur and that FzlA-mediated FtsZ curvature is correlated with efficient division. We observed that FzlA influences constriction rate, and that this activity is associated with its ability to bind and curve FtsZ polymers. Further, we found that a slowly constricting fzlA mutant strain develops 'pointy' poles, suggesting that FzlA influences the relative contributions of radial versus longitudinal PG insertion at the septum. These findings implicate FzlA as a critical coordinator of envelope constriction through its interaction with FtsZ and suggest a functional link between FtsZ curvature and efficient constriction in C. crescentus.
Subject(s)
Bacterial Proteins/metabolism , Caulobacter crescentus/physiology , Cell Cycle Proteins/metabolism , Cell Division/physiology , Cytoskeletal Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Caulobacter crescentus/cytology , Caulobacter crescentus/genetics , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Division/genetics , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Cell Wall/metabolism , Cell Wall/ultrastructure , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/genetics , GTP Phosphohydrolases/metabolism , Gene Library , Peptidoglycan/metabolism , Protein Binding/genetics , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs/geneticsABSTRACT
The effect of phytantriol (PT)-based liquid-crystalline nanoparticles, cubosomes, on the lipid bilayer membranes has been investigated using the combined Langmuir-Blodgett/Langmuir-Schaefer (LB-LS) technique to form an h-1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) monolayer at the air-water interface and transfer the lipid bilayer onto the Au(111) substrate. Changes of the compression isotherms confirmed incorporation of cubosomes dispersed in the subphase into the h-DMPC monolayer at the air-water interface. The photon polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) measurements of the gold electrode covered by the transferred DMPC bilayer showed for the first time how the incorporation of cubosome material affects the orientation and conformation of lipid molecules in the membrane. Exposure to cubosomes affected the packing of d54-DMPC bilayers and introduced disorder of chains by increasing the contribution of gauche conformation. The decrease of the tilt angle of the acyl chains of adsorbed DMPC in the whole range of potentials applied to the gold electrode confirmed that incorporation of cubosome material results in a more tightly packed bilayer. The presence of phytantriol molecules within the d63-DMPC matrix was confirmed by PM-IRRAS studies of the PT-related bands. The LB and PM-IRRAS studies demonstrated in a convincing way that PT-based cubosomes change the organization of model lipid layers leading to structural changes of the membranes which have to be taken into consideration when PT-cubosomes are employed as drug carriers.
ABSTRACT
In bacteria and archaea, the most widespread cell division system is based on the tubulin homologue FtsZ protein, whose filaments form the cytokinetic Z-ring. FtsZ filaments are tethered to the membrane by anchors such as FtsA and SepF and are regulated by accessory proteins. One such set of proteins is responsible for Z-ring's spatiotemporal regulation, essential for the production of two equal-sized daughter cells. Here, we describe how our still partial understanding of the FtsZ-based cell division process has been progressed by visualising near-atomic structures of Z-rings and complexes that control Z-ring positioning in cells, most notably the MinCDE and Noc systems that act by negatively regulating FtsZ filaments. We summarise available data and how they inform mechanistic models for the cell division process.
Subject(s)
Bacteria/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/metabolism , Cell Division , CytoskeletonABSTRACT
FtsA is an early component of the Z-ring, the structure that divides most bacteria, formed by tubulin-like FtsZ. FtsA belongs to the actin family of proteins, showing an unusual subdomain architecture. Here we reconstitute the tethering of FtsZ to the membrane via FtsA's C-terminal amphipathic helix in vitro using Thermotoga maritima proteins. A crystal structure of the FtsA:FtsZ interaction reveals 16 amino acids of the FtsZ tail bound to subdomain 2B of FtsA. The same structure and a second crystal form of FtsA reveal that FtsA forms actin-like protofilaments with a repeat of 48 Å. The identical repeat is observed when FtsA is polymerized using a lipid monolayer surface and FtsAs from three organisms form polymers in cells when overexpressed, as observed by electron cryotomography. Mutants that disrupt polymerization also show an elongated cell division phenotype in a temperature-sensitive FtsA background, demonstrating the importance of filament formation for FtsA's function in the Z-ring.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Thermotoga maritima/chemistry , Thermotoga maritima/metabolism , Crystallography, X-Ray , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/metabolism , Microscopy, Electron , Models, Biological , Models, Molecular , Protein Binding , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, QuaternaryABSTRACT
Amyloid-ß (Aß) assemblies have been shown to bind to lipid bilayers. This can disrupt membrane integrity and cause a loss of cellular homeostasis, that triggers a cascade of events leading to Alzheimer's disease. However, molecular mechanisms of Aß cytotoxicity and how the different assembly forms interact with the membrane remain enigmatic. Here we use cryo-electron tomography (cryoET) to obtain three-dimensional nano-scale images of various Aß assembly types and their interaction with liposomes. Aß oligomers and curvilinear protofibrils bind extensively to the lipid vesicles, inserting and carpeting the upper-leaflet of the bilayer. Aß oligomers concentrate at the interface of vesicles and form a network of Aß-linked liposomes, while crucially, monomeric and fibrillar Aß have relatively little impact on the membrane. Changes to lipid membrane composition highlight a significant role for GM1-ganglioside in promoting Aß-membrane interactions. The different effects of Aß assembly forms observed align with the highlighted cytotoxicity reported for Aß oligomers. The wide-scale incorporation of Aß oligomers and curvilinear protofibrils into the lipid bilayer suggests a mechanism by which membrane integrity is lost.
ABSTRACT
Osteoblasts orchestrate bone formation through the secretion of type I collagen and other constituents of the matrix on which hydroxyapatite crystals mineralize. Here, we show that TENT5A, whose mutations were found in congenital bone disease osteogenesis imperfecta patients, is a cytoplasmic poly(A) polymerase playing a crucial role in regulating bone mineralization. Direct RNA sequencing revealed that TENT5A is induced during osteoblast differentiation and polyadenylates mRNAs encoding Col1α1, Col1α2, and other secreted proteins involved in osteogenesis, increasing their expression. We postulate that TENT5A, possibly together with its paralog TENT5C, is responsible for the wave of cytoplasmic polyadenylation of mRNAs encoding secreted proteins occurring during bone mineralization. Importantly, the Tent5a knockout (KO) mouse line displays bone fragility and skeletal hypomineralization phenotype resulting from quantitative and qualitative collagen defects. Thus, we report a biologically relevant posttranscriptional regulator of collagen production and, more generally, bone formation.
Subject(s)
Calcification, Physiologic/genetics , Osteoblasts/metabolism , Osteogenesis Imperfecta/genetics , Osteogenesis/genetics , Polynucleotide Adenylyltransferase/genetics , RNA, Messenger/genetics , Animals , Cell Differentiation , Collagen Type I/genetics , Collagen Type I/metabolism , Collagen Type I, alpha 1 Chain/genetics , Collagen Type I, alpha 1 Chain/metabolism , Disease Models, Animal , Eye Proteins/genetics , Eye Proteins/metabolism , Female , Gene Expression Regulation, Developmental , Humans , Male , Mice , Mice, Knockout , Nerve Growth Factors/genetics , Nerve Growth Factors/metabolism , Nucleotidyltransferases/genetics , Nucleotidyltransferases/metabolism , Osteoblasts/pathology , Osteogenesis Imperfecta/metabolism , Osteogenesis Imperfecta/pathology , Osteonectin/genetics , Osteonectin/metabolism , Polyadenylation , Polynucleotide Adenylyltransferase/metabolism , Protein Isoforms/deficiency , Protein Isoforms/genetics , RNA, Messenger/metabolism , Sequence Analysis, RNA , Serpins/genetics , Serpins/metabolism , Signal TransductionABSTRACT
Cubosome nanocarriers are promising biomimetic drug delivery systems used in particular for highly toxic drugs in cases where decreasing unwanted side effects is especially important. The properties of electrode supported lipid bilayer prepared by the combined Langmuir-Blodgett and Langmuir-Schaefer techniques were studied using electrochemical techniques following exposure of the film - covered electrode to a solution containing phytantriol - based cubosomes. The inclusion of the carrier in the model membrane under different experimental conditions was probed and the modifications induced in the lipid organization were for the first time inferred by quantitative analysis of the responses of cyclic voltammetry (CV), AC voltammetry and Electrochemical Impedance Spectroscopy (EIS) as well as blocking assays using a redox probe in the solution. Exposure of a preformed DMPC bilayer to cubosome solution resulted in the improved barrier properties of the film reflecting disintegration of cubosomes and formation of additional phytantriol/Pluronic F-108 polymer layer on the top of the DMPC bilayer. On the other hand, formation of the layer in the presence of cubosomes in the subphase lead to an increased capacitance of the film since penetration of the lipid layers by the cubosomal phytantriol increased the porosity of the film.
Subject(s)
Drug Carriers/chemistry , Gold/chemistry , Lipid Bilayers/chemistry , Nanostructures/chemistry , Electrochemistry , Electrodes , Fatty Alcohols/chemistry , Liquid Crystals/chemistry , PorosityABSTRACT
Contractile injection systems (CISs) mediate cell-cell interactions by a phage tail-like apparatus. Their conserved mechanism relies on the anchoring of the proximal end of a sheath-tube module to a membrane, followed by contraction of the sheath towards the attachment site and ejection of the inner tube. Here we reveal a major variation of the CIS mechanism in the type six secretion system (T6SS) of enteroaggregative Escherichia coli (EAEC). We show that both ends of the sheath-tube module are attached to opposite sides of the cell, enabling the structure to contract in two opposite directions. The protein TssA1 mediates the interaction of the distal end with the cell envelope, the termination of tail elongation, and non-canonical contraction towards the distal end. We provide a framework for the molecular processes at the T6SS distal end. Further research will address whether bidirectional contraction allows for bidirectional effector secretion. The unrecognized concept of non-canonical contractions could be relevant to biofilms of the human intestine.
Subject(s)
Cell Communication , Escherichia coli/metabolism , Type VI Secretion Systems/physiology , Cryoelectron Microscopy , Escherichia coli/genetics , Escherichia coli/ultrastructure , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/physiology , Models, BiologicalABSTRACT
The need for bacteria to interact with their environment has driven the evolution of elaborate secretion systems. By virtue of their function, secretion systems are macromolecular complexes associated with the cell envelope and therefore inherently difficult to study by conventional structural biology techniques. Cryo-electron microscopy (cryoEM) has become an invaluable technique to study large membrane-embedded complexes and led to major advances in the mechanistic understanding of secretion systems. CryoEM comprises of two main modalities, namely single particle analysis and tomography. Here, we review how detailed structures retrieved by single particle analysis combine elegantly with tomography experiments in which the secretion systems are observed in their native cellular context.
Subject(s)
Bacterial Secretion Systems , Cryoelectron Microscopy , Cryoelectron Microscopy/methods , Macromolecular Substances/chemistry , Macromolecular Substances/metabolism , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/metabolism , Models, BiologicalABSTRACT
MreB is essential for rod shape in many bacteria. Membrane-associated MreB filaments move around the rod circumference, helping to insert cell wall in the radial direction to reinforce rod shape. To understand how oriented MreB motion arises, we altered the shape of Bacillus subtilis. MreB motion is isotropic in round cells, and orientation is restored when rod shape is externally imposed. Stationary filaments orient within protoplasts, and purified MreB tubulates liposomes in vitro, orienting within tubes. Together, this demonstrates MreB orients along the greatest principal membrane curvature, a conclusion supported with biophysical modeling. We observed that spherical cells regenerate into rods in a local, self-reinforcing manner: rapidly propagating rods emerge from small bulges, exhibiting oriented MreB motion. We propose that the coupling of MreB filament alignment to shape-reinforcing peptidoglycan synthesis creates a locally-acting, self-organizing mechanism allowing the rapid establishment and stable maintenance of emergent rod shape.
Subject(s)
Bacillus subtilis/cytology , Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , Cell Membrane/metabolism , Cell Wall/metabolism , Peptidoglycan/metabolism , Protein TransportABSTRACT
Membrane constriction is a prerequisite for cell division. The most common membrane constriction system in prokaryotes is based on the tubulin homologue FtsZ, whose filaments in E. coli are anchored to the membrane by FtsA and enable the formation of the Z-ring and divisome. The precise architecture of the FtsZ ring has remained enigmatic. In this study, we report three-dimensional arrangements of FtsZ and FtsA filaments in C. crescentus and E. coli cells and inside constricting liposomes by means of electron cryomicroscopy and cryotomography. In vivo and in vitro, the Z-ring is composed of a small, single-layered band of filaments parallel to the membrane, creating a continuous ring through lateral filament contacts. Visualisation of the in vitro reconstituted constrictions as well as a complete tracing of the helical paths of the filaments with a molecular model favour a mechanism of FtsZ-based membrane constriction that is likely to be accompanied by filament sliding.
Subject(s)
Bacterial Proteins/chemistry , Caulobacter crescentus/ultrastructure , Cell Division , Cell Membrane/ultrastructure , Cytoskeletal Proteins/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/ultrastructure , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Caulobacter crescentus/metabolism , Cell Membrane/metabolism , Cryoelectron Microscopy , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Expression , Liposomes/chemistry , Liposomes/ultrastructure , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
The divisome and elongasome are bacterial protein complexes responsible for peptidoglycan (PG) synthesis during cell division and elongation, respectively. We review several lines of evidence, arguing for a shared evolutionary past of the divisome and elongasome. Both integrate closely related penicillin-binding proteins (PBPs) for PG synthesis, use proteins of the RodA/FtsW (SEDS, shape, elongation, division and sporulation) family for Lipid II export and interact with MraY/Mur proteins for Lipid II synthesis. It was recently shown that the actin-like protein FtsA of the divisome polymerises on membranes, adding another parallel, since membrane-associated filaments of the bacterial actin MreB guide the elongasome. Given these similarities, it seems plausible to conclude that the elongasome is a modified version of the divisome, without the membrane-constricting FtsZ-ring and its associated machinery on the inside.