The dual role of MamB in magnetosome membrane assembly and magnetite biomineralization

Magnetospirillum gryphiswaldense MSR‐1 synthesizes membrane‐enclosed magnetite (Fe$_3$O$_4$) nanoparticles, magnetosomes, for magnetotaxis. Formation of these organelles involves a complex process comprising key steps which are governed by specific magnetosome‐associated proteins. MamB, a cation diffusion facilitator (CDF) family member has been implicated in magnetosome‐directed iron transport. However, deletion mutagenesis studies revealed that MamB is essential for the formation of magnetosome membrane vesicles, but its precise role remains elusive. In this study, we employed a multi‐disciplinary approach to define the role of MamB during magnetosome formation. Using site‐directed mutagenesis complemented by structural analyses, fluorescence microscopy and cryo‐electron tomography, we show that MamB is most likely an active magnetosome‐directed transporter serving two distinct, yet essential functions. First, MamB initiates magnetosome vesicle formation in a transport‐independent process, probably by serving as a landmark protein. Second, MamB transport activity is required for magnetite nucleation. Furthermore, by determining the crystal structure of the MamB cytosolic C‐terminal domain, we also provide mechanistic insight into transport regulation. Additionally, we present evidence that magnetosome vesicle growth and chain formation are independent of magnetite nucleation and magnetic interactions respectively. Together, our data provide novel insight into the role of the key bifunctional magnetosome protein MamB, and the early steps of magnetosome formation

Interaction des composants de la paroi cellulaire végétale : vers un système de modèle bio-inspiré

The goal of this work was to develop an in vitro model of the plant primary cell wall. A bottom up approach was chosen for the rational design of 2D and 3D constructs made of a lipid membrane, cellulose nano crystals (CNCs) and xyloglucan (XG). First, the interaction between the building blocks was probed using light scattering, isothermal titration calorimetry, quartz crystal microbalance and electron microscopy, revealing firstly the electrostatic nature of the interaction between CNCs and a lipidic membrane and secondly, specific interaction between CNCs and XG in a precise stoichiometric ratio. Then, the optimal parameters from the interaction studies were used to obtain 2D and 3D structures by depositing alternating layers of CNCs and XG on flat substrates (multilayered films) and giant unilamellar vesicles (GUVs). A linear growth of the films was revealed by atomic force microscopy (AFM) experiments while the response of decorated vesicles to osmotic shocks lead to their buckling due to the rigidification of the lipid membrane. Finally, the mechanical properties of the constructs were characterized using AFM indentation, revealing a Young's modulus of few hundred kPa, similarly to what is observed for real plant cell walls.L'objectif de ce travail était de développer un modèle in vitro de la paroi primaire des plantes. Une approche ascendante a été choisie pour la conception rationnelle de constructions 2D et 3D faites d'une membrane lipidique, de nanocristaux de cellulose (CNC) et de xyloglucane (XG). Tout d'abord, l'interaction entre les blocs de base a été examinée à l'aide de la diffusion de la lumière, la calorimétrie de titrage isotherme, la microbalance à quartz et la microscopie électronique, révélant tout d'abord la nature électrostatique de l'interaction entre les CNC et une membrane lipidique ainsi qu'une interaction spécifique entre les CNC et XG dans un rapport stoechiométrique précis. Par la suite, les paramètres optimisés des études d'interaction ont été utilisés pour obtenir des structures 2D et 3D en déposant des couches alternées de CNC et XG sur des substrats plats (films multicouches) et des vésicules unilamellaires géantes (GUV). Une croissance linéaire des films a été révélée par les expériences de microscopie à force atomique (AFM), tandis que la réponse des vésicules décorées aux chocs osmotiques conduit à leur flambage en raison de la rigidification de la membrane lipidique. Enfin, les propriétés mécaniques des constructions ont été caractérisées en utilisant l’AFM par indentation, révélant un module d'Young de quelques centaines de kPa, similaire à celui observé pour de vraies parois cellulaires végétales

Interaction of plant cell wall building blocks : towards a bioinspired model system

L'objectif de ce travail était de développer un modèle in vitro de la paroi primaire des plantes. Une approche ascendante a été choisie pour la conception rationnelle de constructions 2D et 3D faites d'une membrane lipidique, de nanocristaux de cellulose (CNC) et de xyloglucane (XG). Tout d'abord, l'interaction entre les blocs de base a été examinée à l'aide de la diffusion de la lumière, la calorimétrie de titrage isotherme, la microbalance à quartz et la microscopie électronique, révélant tout d'abord la nature électrostatique de l'interaction entre les CNC et une membrane lipidique ainsi qu'une interaction spécifique entre les CNC et XG dans un rapport stoechiométrique précis. Par la suite, les paramètres optimisés des études d'interaction ont été utilisés pour obtenir des structures 2D et 3D en déposant des couches alternées de CNC et XG sur des substrats plats (films multicouches) et des vésicules unilamellaires géantes (GUV). Une croissance linéaire des films a été révélée par les expériences de microscopie à force atomique (AFM), tandis que la réponse des vésicules décorées aux chocs osmotiques conduit à leur flambage en raison de la rigidification de la membrane lipidique. Enfin, les propriétés mécaniques des constructions ont été caractérisées en utilisant l’AFM par indentation, révélant un module d'Young de quelques centaines de kPa, similaire à celui observé pour de vraies parois cellulaires végétales.The goal of this work was to develop an in vitro model of the plant primary cell wall. A bottom up approach was chosen for the rational design of 2D and 3D constructs made of a lipid membrane, cellulose nano crystals (CNCs) and xyloglucan (XG). First, the interaction between the building blocks was probed using light scattering, isothermal titration calorimetry, quartz crystal microbalance and electron microscopy, revealing firstly the electrostatic nature of the interaction between CNCs and a lipidic membrane and secondly, specific interaction between CNCs and XG in a precise stoichiometric ratio. Then, the optimal parameters from the interaction studies were used to obtain 2D and 3D structures by depositing alternating layers of CNCs and XG on flat substrates (multilayered films) and giant unilamellar vesicles (GUVs). A linear growth of the films was revealed by atomic force microscopy (AFM) experiments while the response of decorated vesicles to osmotic shocks lead to their buckling due to the rigidification of the lipid membrane. Finally, the mechanical properties of the constructs were characterized using AFM indentation, revealing a Young's modulus of few hundred kPa, similarly to what is observed for real plant cell walls

Thermal properties of ruthenium alkylidene-polymerized dicyclopentadiene

Differential scanning calorimetry (DSC) analysis of ring opening methatesis polymerization (ROMP) derived polydicyclopentadiene (PDCPD) revealed an unexpected thermal behavior. A recurring exothermic signal can be observed in the DSC analysis after an elapsed time period. This exothermic signal was found to be proportional to the resting period and was accompanied by a constant increase in the glass-transition temperature. We hypothesize that a relaxation mechanism within the cross-linked scaffold, together with a long-lived stable ruthenium alkylidene species are responsible for the observed phenomenon

Optical sectioning for reflection interference microscopy

Abstract Reflection Interference Contrast Microscopy (RICM, also known as interference reflection microscopy) and related techniques have become of wide interest to the biophysical, soft matter and biochemistry communities owing to their exquisite sensitivity for characterising thin films or individual nanoscopic objects adsorbed onto surfaces, or for monitoring cell-substrate interactions. Over the recent years, striking progresses have been made to improve the sensitivity and the quantitative analysis of RICM. Its use in more complex environments, with spurious reflections stemming from a variety of structures in the sample, remains however challenging. In this paper, we demonstrate two methods for adding optical sectioning capabilities to a RICM setup: line confocal detection, and structured illumination microscopy. We characterise experimentally the axial resolution and demonstrate its use for the quantitative imaging of complex biological and biomimetic samples: cellular membranes, thin organic films, surface biofunctionalization. We then discuss the benefits of each method and provide guidelines to arbitrate between sectioning and signal-to-noise ratio. Finally, we provide a detailed description of our experimental setup and a home-written image acquisition and processing software that should allow the interested reader to duplicate such a setup on a home-built or commercial microscope

Light-Controlled Hierarchical Self-Assembly of Polyelectrolytes and Supramolecular Polymers

Dynamic control over supramolecular interactions using various stimuli continues to drive the development of smart materials. We describe here the extension of dynamic self-assembly to a self-assembled hierarchical structure. A peptide amphiphile (PA) was designed with a photocleavable nitrobenzyl ester component such that it would undergo a sphere-to-cylinder transition upon irradiation, as confirmed by cryogenic transmission electron microscopy and small-angle X-ray scattering (SAXS). The photocleavable PA was then tested in the formation of a macroscopic sac made through a complex hierarchical self-assembly process between PA and hyaluronic acid. The microstructure of the resulting sac has previously been noted to depend dramatically on the geometry of the PA nanostructure. Photolysis of the PA solution during sac formation led to a sac microstructure that displayed characteristics of sacs made with both cylinder-forming PAs and sphere-forming PAs, as measured by scanning electron microscopy and SAXS

pH-Sensitive Interactions between Cellulose Nanocrystals and DOPC Liposomes

The interaction of 1,2 dioleolyl-<i>sn</i>-glycero-3-phosphatidylcholine (DOPC) vesicles with cellulose nanocrystals (CNCs) using several complementary techniques. Dynamic light scattering, zeta-potential, cryo-transmission electron microscopy and isothermal titration calorimetry (ITC) analyses confirmed the formation of pH-dependent CNC–liposome complexes. ITC was used to characterize the thermodynamic properties of this interaction. Positive values of enthalpy were found at pH lower than 5 where the charge sign of the constituents was opposite. The association was more pronounced at lower pH, as indicated by the higher values of association constant. We suggest that the positive enthalpy is derived from the release of counterions from the particle hydration shell during the association and that the charge of the vesicles plays a significant role in this interaction

Building an Artificial Plant Cell Wall on a Lipid Bilayer by Assembling Polysaccharides and Engineered Proteins

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Disease-Homologous Mutation in the Cation Diffusion Facilitator Protein MamM Causes Single-Domain Structural Loss and Signifies Its Importance

Cation diffusion facilitators (CDF) are highly conserved, metal ion efflux transporters that maintain divalent transition metal cation homeostasis. Most CDF proteins contain two domains, the cation transporting transmembrane domain and the regulatory cytoplasmic C-terminal domain (CTD). MamM is a magnetosome-associated CDF protein essential for the biomineralization of magnetic iron-oxide particles in magnetotactic bacteria. To investigate the structure-function relationship of CDF cytoplasmic domains, we characterized a MamM M250P mutation that is synonymous with the disease-related mutation L349P of the human CDF protein ZnT-10. Our results show that the M250P exchange in MamM causes severe structural changes in its CTD resulting in abnormal reduced function. Our in vivo, in vitro and in silico studies indicate that the CTD fold is critical for CDF proteins’ proper function and support the previously suggested role of the CDF cytoplasmic domain as a CDF regulatory element. Based on our results, we also suggest a mechanism for the effects of the ZnT-10 L349P mutation in human