Water-mediated structuring of bone apatite

International audienceIt is well known that organic molecules from the vertebrate extracellular matrix of calcifying tissues are essential in structuring the apatite mineral. Here, we show that water also plays a structuring role. By using solid-state nuclear magnetic resonance, wide-angle X-ray scattering and cryogenic transmission electron microscopy to characterize the structure and organization of crystalline and biomimetic apatite nanoparticles as well as intact bone samples, we demonstrate that water orients apatite crystals through an amorphous calcium phosphate-like layer that coats the crystalline core of bone apatite. This disordered layer is reminiscent of those found around the crystalline core of calcified biominerals in various natural composite materials in vivo. This work provides an extended local model of bone biomineralization

Vimentin intermediate filament assembly is a reversible process

Déposé dans BioRxiv le 6 avril 2022Networks of intermediate filaments (IF) need to constantly reorganize to fulfill their functions at different locations within the cell. IF assembly results from end-to-end annealing, which is commonly assumed to be irreversible. By contrast, the mechanisms involved in IF disassembly are far less understood. IF fragmentation has however been observed in many cell types, and it has been suggested that it could be associated with post-translational modifications. In this article, we investigate the contribution of filament fragmentation in the assembly dynamics of type III vimentin IF using a combination of in vitro reconstitution, fluorescence imaging, and theoretical modeling. We first show that vimentin assembly at low concentrations results in an equilibrium between filament annealing and fragmentation at time ≥ 24 h. At higher concentration, entanglements kinetically trap the system out of equilibrium, and we show that this trapping is reversible upon dilution. Taking into account both fragmentation and entanglement, we estimated that the mean bond breaking time was ∼18 hours Finally, we provide direct evidence through dual color imaging that filament fragmentation and annealing coexist during assembly. By showing that IF fragmentation can occur without cofactors or post-translational-modifications, our study provides a physical understanding of the IF length regulation. Significance Statement Vimentin intermediate filaments are a key component of the cytoskeleton and are involved in many cellular functions, such as the regulation of cell shape, migration and division. These functions require cytoskeletal filaments to simultaneously assemble and disassemble throughout the life of the cell. While the mechanisms of intermediate filament assembly have been widely studied, the minimal ingredients underpinning their disassembly are not understood. Here, we demonstrate that vimentin constantly disassembles through filament breakage without the assistance of any other protein or post-translational modification, contrary to common wisdom. Our findings suggest that the dynamic cytoskeletal steady-states observed in cells could be largely shaped by simple physical effects linked to the reversible association of vimentin subunits, combined with dramatic kinetic trapping effects that hinder network reorganization as soon as the filaments become too dense and too long

Fragmentation and Entanglement Limit Vimentin Intermediate Filament Assembly

International audienceNetworks of intermediate filaments (IFs) need to constantly reorganize to fulfil their functions at different locations within the cell. The mechanism of IF assembly is well described and involves filament end-to-end annealing. By contrast, the mechanisms involved in IF disassembly are far less understood. In vitro, IFs are assumed to be very stable and their disassembly negligible. IF fragmentation has been observed in many cell types, but it has been suggested to be associated with active processes such as IF post-translational modifications. In this article, we uncover the contribution of filament spontaneous fragmentation in the assembly dynamics of type III vimentin IF using a combination of in vitro reconstitution probed by fluorescence imaging and theoretical modeling. We first show that vimentin assembly at low concentrations results in an equilibrium between filament annealing and fragmentation at times ≥24 h. At higher concentrations, entanglements kinetically trap the system out of equilibrium, and we show that this trapping is reversible upon dilution. Taking into account both fragmentation and entanglement, we estimate that the mean bond breaking time is ∼18 h. This translates into a mean breaking time of ∼5 h for a 1-μm-long filament, which is a relevant timescale for IF reorganization in live cells. Finally, we provide direct evidence through dual-color imaging that filament fragmentation and annealing coexist during assembly. By showing that IF fragmentation can occur without cofactors or posttranslational modifications, our study provides new insights into the physical understanding of the IF length regulation

Synergistic role of nucleotides and lipids for the self-assembly of Shs1 septin oligomers

International audienceBudding yeast septins are essential for cell division and polarity. Septins assemble as palindromic linear octameric complexes. The function and ultra-structural organization of septins are finely governed by their molecular polymorphism. In particular, in budding yeast, the end subunit can stand either as Shs1 or Cdc11. We have dissected, here, for the first time, the behavior of the Shs1 protomer bound to membranes at nanometer resolution, in complex with the other septins. Using electron microscopy, we have shown that on membranes, Shs1 protomers self-assemble into rings, bundles, filaments or two-dimensional gauzes. Using a set of specific mutants we have demonstrated a synergistic role of both nucleotides and lipids for the organization and oligomerization of budding yeast septins. Besides, cryo-electron tomography assays show that vesicles are deformed by the interaction between Shs1 oligomers and lipids. The Shs1-Shs1 interface is stabilized by the presence of phosphoinositides, allowing the visualization of micro-metric long filaments formed by Shs1 protomers. In addition, molecular modeling experiments have revealed a potential molecular mechanism regarding the selectivity of septin subunits for phosphoinositide lipids

Unusual, pH-induced, self-assembly of sophorolipid biosurfactants

An increasing need exists for simple, bioderived, nontoxic, and up-scalable compounds with stimuli-responsive properties. Acidic sophorolipids (SL) are glucose-based biosurfactants derived from the yeast broth of Candida bombicola (teleomorph: Starmerella bombicola). The specific design of this molecule, a sophorose head with a free end-COON group at the end of the alkyl chain, makes it a potentially interesting pH-responsive compound. We have specifically investigated this assumption using a combination of small angle neutron scattering (SANS), transmission electron microscopy under cryogenic conditions (Cryo-TEM), and nuclear magnetic resonance (NMR) techniques and found a strong dependence of SL self-assembly on the degree of ionization, alpha, of the COOH group at concentration values as low as Sand 03 wt %. At least three regimes can be identified where the supramolecular behavior of SL is unexpectedly different: (1) at low alpha values, self-assembly is driven by concentration, C and micelles are mainly identified as nonionic objects whose curvature decreases (sphere-to-rod) with C; (2) at mid alpha values, the formation of COO- groups introduces negative charges at the micellar surface inducing an increase in curvature (rod-to-sphere transition). Repulsive electrostatic long-range Interactions appear at this stage. In both regimes 1 and 2, the cross-section radius of the micelles is below 25 angstrom. This behavior is concentration independent. (3) At alpha = 1, individual micelles seem to favor the formation of large netlike tubular aggregates whose size is above 100 nm. Such a complex behavior is very unique as it is generally not observed for common alkyl-based surfactants in concentration ranges below 5-10 wt %

CEMOVIS on a pathogen: analysis of Bacillus anthracis spores.

International audienceUnder conditions of starvation, bacteria of Bacillus ssp. are able to form a highly structured cell type, the dormant spore. When the environment presents more favourable conditions, the spore starts to germinate, which will lead to the release of the vegetative form in the life cycle, the bacillus. For Bacillus anthracis, the aetiological agent of anthrax, germination is normally linked to host uptake and represents an important step in the onset of anthrax disease. Morphological studies analysing the organization of the spore and the changes during germination at the electron microscopy level were only previously performed with techniques relying on fixation with aldehydes and osmium, and subsequent dehydration, which can produce artefacts. In the present study, we describe the morphology of dormant spores using CEMOVIS (Cryo-Electron Microscopy of Vitreous Sections). Biosafety measures do not permit freezing of native spores of B. anthracis without chemical fixation. To study the influence of aldehyde fixation on the ultrastructure of the spore, we chose to analyse spores of the closely related non-pathogen Bacillus cereus T. For none of the investigated structures could we find a difference in morphology induced by aldehyde fixation compared with the native preparations for CEMOVIS. This result legitimizes work with aldehyde-fixed spores from B. anthracis. Using CEMOVIS, we describe two new structures present in the spore: a rectangular structure, which connects the BclA filaments with the basal layer of the exosporium, and a repetitive structure, which can be found in the terminal layer of the coat. We studied the morphological changes of the spore during germination. After outgrowth of the bacillus, coat and exosporium stay associated, and the layered organization of the coat, as well as the repetitive structure within it, remain unchanged

A Novel Type of Polyhedral Viruses Infecting Hyperthermophilic Archaea

International audienceEncapsidation of genetic material into polyhedral particles is one of the most common structural solutions employed by viruses infecting hosts in all three domains of life. Here, we describe a new virus of hyperthermophilic archaea, Sulfolobus polyhedral virus 1 (SPV1), which condenses its circular double-stranded DNA genome in a manner not previously observed for other known viruses. The genome complexed with virion proteins is wound up sinusoidally into a spherical coil which is surrounded by an envelope and further encased by an outer polyhedral capsid apparently composed of the 20-kDa virion protein. Lipids selectively acquired from the pool of host lipids are integral constituents of the virion. None of the major virion proteins of SPV1 show similarity to structural proteins of known viruses. However, minor structural proteins, which are predicted to mediate host recognition, are shared with other hyperthermophilic archaeal viruses infecting members of the order Sulfolobales The SPV1 genome consists of 20,222 bp and contains 45 open reading frames, only one-fifth of which could be functionally annotated.IMPORTANCE Viruses infecting hyperthermophilic archaea display a remarkable morphological diversity, often presenting architectural solutions not employed by known viruses of bacteria and eukaryotes. Here we present the isolation and characterization of Sulfolobus polyhedral virus 1, which condenses its genome into a unique spherical coil. Due to the original genomic and architectural features of SPV1, the virus should be considered a representative of a new viral family, "Portogloboviridae.

Surface charge of acidic sophorolipid micelles: effect of base and time

International audienceAcidic sophorolipids, SL–COOH, bio-derived glycolipids, are known to form micelles whose interactions vary as a function of pH. Upon partial ionization of the COOH group, intermicellar interactions take place. Here, we explore the nature of these interactions by using small angle neutron scattering (SANS) on SL–COOH solutions to which increasing amounts of NaOH are added. The effect of the nature of the base is also explored by replacing NaOH with aqueous NH3, KOH and Ca(OH)2. Time effects up to 36 days are also discussed. All SANS data have been successfully fitted using an appropriate model of core–shell prolate ellipsoids of revolution with an interaction potential, U(r), which combines hard-sphere and screened Coulomb (described by a repulsive Yukawa potential) potentials. Modelling quantifies the effect of the base in terms of micellar size, effective surface charge and interfacial hydration, thus showing the possibility of tuning them at will

Structure of the DP1–DP2 PolD complex bound with DNA and its implications for the evolutionary history of DNA and RNA polymerases

PolD is an archaeal replicative DNA polymerase (DNAP) made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2). Recently, we reported the individual crystal structures of the DP1 and DP2 catalytic cores, thereby revealing that PolD is an atypical DNAP that has all functional properties of a replicative DNAP but with the catalytic core of an RNA polymerase (RNAP). We now report the DNA-bound cryo–electron microscopy (cryo-EM) structure of the heterodimeric DP1–DP2 PolD complex from Pyrococcus abyssi, revealing a unique DNA-binding site. Comparison of PolD and RNAPs extends their structural similarities and brings to light the minimal catalytic core shared by all cellular transcriptases. Finally, elucidating the structure of the PolD DP1–DP2 interface, which is conserved in all eukaryotic replicative DNAPs, clarifies their evolutionary relationships with PolD and sheds light on the domain acquisition and exchange mechanism that occurred during the evolution of the eukaryotic replisome

Molecular organization and mechanics of single vimentin filaments revealed by super-resolution imaging

Intermediate filaments (IF) are involved in key cellular functions including polarization, migration, and protection against large deformations. These functions are related to their remarkable ability to extend without breaking, a capacity that should be determined by the molecular organization of subunits within filaments. However, this structure-mechanics relationship remains poorly understood at the molecular level. Here, using super-resolution microscopy (SRM), we show that vimentin filaments exhibit a~49 nm axial repeat both in cells and in vitro. As unit-length-filaments (ULFs) precursors were measured at~59 nm, this demonstrates a partial overlap of ULFs during filament assembly. Using an SRM-compatible stretching device, we also provide evidence that the extensibility of vimentin is due to the unfolding of its subunits and not to their sliding, thus establishing a direct link between the structural organization and its mechanical properties. Overall, our results pave the way for future studies of IF assembly, mechanical and structural properties in cells