Mathieu Bennet

A vacuole-like compartment concentrates a disordered calcium phase in a key coccolithophorid alga

Coccoliths are calcitic particles produced inside the cells of unicellular marine algae known as coccolithophores. They are abundant components of sea-floor carbonates, and the stoichiometry of calcium to other elements in fossil coccoliths is widely used to infer past environmental conditions. Here we study cryo-preserved cells of the dominant coccolithophore Emiliania huxleyi using state-of-the-art nanoscale imaging and spectroscopy. We identify a compartment, distinct from the coccolith-producing compartment, filled with high concentrations of a disordered form of calcium. Co-localized with calcium are high concentrations of phosphorus and minor concentrations of other cations. The amounts of calcium stored in this reservoir seem to be dynamic and at a certain stage the compartment is in direct contact with the coccolith-producing vesicle, suggesting an active role in coccolith formation. Our findings provide insights into calcium accumulation in this important calcifying organism.

On the pathway of mineral deposition in larval zebrafish caudal fin bone

A poorly understood aspect of bone biomineralization concerns the mechanisms whereby ions are sequestered from the environment, concentrated, and deposited in the extracellular matrix. In this study, we follow mineral deposition in the caudal fin of the zebrafish larva in vivo. Using fluorescence and cryo-SEM-microscopy, in combination with Raman and XRF spectroscopy, we detect the presence of intracellular mineral particles located between bones, and in close association with blood vessels. Calcium-rich particles are also located away from the mineralized bone, and these are also in close association with blood vessels. These observations challenge the view that mineral formation is restricted to osteoblast cells juxtaposed to bone, or to the extracellular matrix. Our results, derived from observations performed in living animals, contribute a new perspective to the comprehensive mechanism of bone formation in vertebrates, from the blood to the bone. More broadly, these findings may shed light on bone mineralization processes in other vertebrates, including humans.

Simultaneous Raman microspectroscopy and fluorescence imaging of bone mineralization in living zebrafish larvae.

Confocal Raman microspectroscopy and fluorescence imaging are two well-established methods providing functional insight into the extracellular matrix and into living cells and tissues, respectively, down to single molecule detection. In living tissues, however, cells and extracellular matrix coexist and interact. To acquire information on this cell-matrix interaction, we developed a technique for colocalized, correlative multispectral tissue analysis by implementing high-sensitivity, wide-field fluorescence imaging on a confocal Raman microscope. As a proof of principle, we study early stages of bone formation in the zebrafish (Danio rerio) larvae because the zebrafish has emerged as a model organism to study vertebrate development. The newly formed bones were stained using a calcium fluorescent marker and the maturation process was imaged and chemically characterized in vivo. Results obtained from early stages of mineral deposition in the zebrafish fin bone unequivocally show the presence of hydrogen phosphate containing mineral phases in addition to the carbonated apatite mineral. The approach developed here opens significant opportunities in molecular imaging of metabolic activities, intracellular sensing, and trafficking as well as in vivo exploration of cell-tissue interfaces under (patho-)physiological conditions.

Selecting for function: solution synthesis of magnetic nanopropellers.

We show that we can select magnetically steerable nanopropellers from a set of carbon coated aggregates of magnetic nanoparticles using weak homogeneous rotating magnetic fields. The carbon coating can be functionalized, enabling a wide range of applications. Despite their arbitrary shape, all nanostructures propel parallel to the vector of rotation of the magnetic field. We use a simple theoretical model to find experimental conditions to select nanopropellers which are predominantly smaller than previously published ones.

Influence of magnetic fields on magneto-aerotaxis

The response of cells to changes in their physico-chemical micro-environment is essential to their survival. For example, bacterial magnetotaxis uses the Earths magnetic field together with chemical sensing to help microorganisms move towards favoured habitats. The studies of such complex responses are lacking a method that permits the simultaneous mapping of the chemical environment and the response of the organisms, and the ability to generate a controlled physiological magnetic field. We have thus developed a multi-modal microscopy platform that fulfils these requirements. Using simultaneous fluorescence and high-speed imaging in conjunction with diffusion and aerotactic models, we characterized the magneto- aerotaxis of Magnetospirillum gryphiswaldense. We assessed the influence of the magnetic field (orientation; strength) on the formation and the dynamic of a micro-aerotactic band (size, dynamic, position). As previously described by models of magnetotaxis, the application of a magnetic field pointing towards the anoxic zone of an oxygen gradient results in an enhanced aerotaxis even down to Earths magnetic field strength. We found that neither a ten-fold increase of the field strength nor a tilt of 45° resulted in a significant change of the aerotactic efficiency. However, when the field strength is zeroed or when the field angle is tilted to 90°, the magneto-aerotaxis efficiency is drastically reduced. The classical model of magneto-aerotaxis assumes a response proportional to the cosine of the angle difference between the directions of the oxygen gradient and that of the magnetic field. Our experimental evidence however shows that this behaviour is more complex than assumed in this model, thus opening up new avenues for research.

Interaction of Proteins Associated with the Magnetosome Assembly in Magnetotactic Bacteria As Revealed by Two-Hybrid Two-Photon Excitation Fluorescence Lifetime Imaging Microscopy Förster Resonance Energy Transfer

Bacteria have recently revealed an unexpectedly complex level of intracellular organization. Magnetotactic bacteria represent a unique class of such organization through the presence of their magnetosome organelles, which are organized along the magnetosome filament. Although the role of individual magnetosomes-associated proteins has started to be unraveled, their interaction has not been addressed with current state-of-the-art optical microscopy techniques, effectively leaving models of the magnetotactic bacteria protein assembly arguable. Here we report on the use of FLIM-FRET to assess the interaction of MamK (actin-like protein) and MamJ, two magnetosome membrane associated proteins essential to the assembly of magnetosomes in a chain. We used a host organism (E. coli) to express eGFP_MamJ and MamK_mCherry, the latest expectedly forming a filament. We found that in the presence of MamK the fluorescence of eGFP_MamJ is distributed along the MamK filament. FRET analysis using the fluorescence lifetime of the donor, eGFP, revealed a spatial proximity of MamK_mCherry and eGFP_MamJ typical of a stable physical interaction between two proteins. Our study effectively led to the reconstruction of part of the magnetotactic apparatus in vivo.

Probing the Mechanical Properties of Magnetosome Chains in Living Magnetotactic Bacteria

The mechanical properties of cytoskeletal networks are intimately involved in determining how forces and cellular processes are generated, directed, and transmitted in living cells. However, determining the mechanical properties of subcellular molecular complexes in vivo has proven to be difficult. Here, we combine in vivo measurements by optical microscopy, X-ray diffraction, and transmission electron microscopy with theoretical modeling to decipher the mechanical properties of the magnetosome chain system encountered in magnetotactic bacteria. We exploit the magnetic properties of the endogenous intracellular nanoparticles to apply a force on the filament-connector pair involved in the backbone formation and stabilization. We show that the magnetosome chain can be broken by the application of external field strength higher than 30 mT and suggest that this originates from the rupture of the magnetosome connector MamJ. In addition, we calculate that the biological determinants can withstand in vivo a force of 25 pN. This quantitative understanding provides insights for the design of functional materials such as actuators and sensors using cellular components.

Biologically controlled synthesis and assembly of magnetite nanoparticles

Magnetite nanoparticles have size- and shape-dependent magnetic properties. In addition, assemblies of magnetite nanoparticles forming one-dimensional nanostructures have magnetic properties distinct from zero-dimensional or non-organized materials due to strong uniaxial shape anisotropy. However, assemblies of free-standing magnetic nanoparticles tend to collapse and form closed-ring structures rather than chains in order to minimize their energy. Magnetotactic bacteria, ubiquitous microorganisms, have the capability to mineralize magnetite nanoparticles, the so-called magnetosomes, and to direct their assembly in stable chains via biological macromolecules. In this contribution, the synthesis and assembly of biological magnetite to obtain functional magnetic dipoles in magnetotactic bacteria are presented, with a focus on the assembly. We present tomographic reconstructions based on cryo-FIB sectioning and SEM imaging of a magnetotactic bacterium to exemplify that the magnetosome chain is indeed a paradigm of a 1D magnetic nanostructure, based on the assembly of several individual particles. We show that the biological forces are a major player in the formation of the magnetosome chain. Finally, we demonstrate by super resolution fluorescence microscopy that MamK, a protein of the actin family necessary to form the chain backbone in the bacteria, forms a bundle of filaments that are not only found in the vicinity of the magnetosome chain but are widespread within the cytoplasm, illustrating the dynamic localization of the protein within the cells. These very simple microorganisms have thus much to teach us with regards to controlling the design of functional 1D magnetic nanoassembly.

Materials science: Magnetic nanoparticles line up

Certain bacteria contain strings of magnetic nanoparticles and therefore align with magnetic fields. Inspired by these natural structures, researchers have now fabricated synthetic one-dimensional arrays of such particles.

Positioning the Flagellum at the Center of a Dividing Cell To Combine Bacterial Division with Magnetic Polarity

ABSTRACT Faithful replication of all structural features is a sine qua non condition for the success of bacterial reproduction by binary fission. For some species, a key challenge is to replicate and organize structures with multiple polarities. Polarly flagellated magnetotactic bacteria are the prime example of organisms dealing with such a dichotomy; they have the challenge of bequeathing two types of polarities to their daughter cells: magnetic and flagellar polarities. Indeed, these microorganisms align and move in the Earths magnetic field using an intracellular chain of nano-magnets that imparts a magnetic dipole to the cell. The paradox is that, after division occurs in cells, if the new flagellum is positioned opposite to the old pole devoid of a flagellum during cell division, the two daughter cells will have opposite magnetic polarities with respect to the positions of their flagella. Here we show that magnetotactic bacteria of the class Gammaproteobacteria pragmatically solve this problem by synthesizing a new flagellum at the division site. In addition, we model this particular structural inheritance during cell division. This finding opens up new questions regarding the molecular aspects of the new division mechanism, the way other polarly flagellated magnetotactic bacteria control the rotational direction of their flagella, and the positioning of organelles. IMPORTANCE Magnetotactic bacteria produce chains of magnetic nanoparticles that endow the cells with a magnetic dipole, a “compass” used for navigation. This feature, however, also drastically complicates cellular division in the case of polarly flagellated bacteria. In this case, the bacteria have to pass on to their daughter cells two types of cellular polarities simultaneously, their magnetic polarity and the polarity of their motility apparatus. We show here that magnetotactic bacteria of the Gammaproteobacteria class pragmatically solve this problem by synthesizing the new flagellum at the division site, a division scheme never observed so far in bacteria. Even though the molecular mechanisms behind this scheme cannot be resolved at the moment due to the lack of genetic tools, this discovery provides a new window into the organizational complexity of simple organisms. Magnetotactic bacteria produce chains of magnetic nanoparticles that endow the cells with a magnetic dipole, a “compass” used for navigation. This feature, however, also drastically complicates cellular division in the case of polarly flagellated bacteria. In this case, the bacteria have to pass on to their daughter cells two types of cellular polarities simultaneously, their magnetic polarity and the polarity of their motility apparatus. We show here that magnetotactic bacteria of the Gammaproteobacteria class pragmatically solve this problem by synthesizing the new flagellum at the division site, a division scheme never observed so far in bacteria. Even though the molecular mechanisms behind this scheme cannot be resolved at the moment due to the lack of genetic tools, this discovery provides a new window into the organizational complexity of simple organisms.