5 research outputs found
Utilisation de nanoparticules magnétiques pour perturber la localisation spatiotemporelle de protéines de signalisation
An increasing number of studies highlight the importance of signaling localization. We developed methods to perturb this localization using magnetic nanoparticles. Proteins of interest are grafted on magnetic nanoparticles, allowing to magnetically localize them. We first propose a new method to engineer directly a spatial gradient of signaling protein concentration within in cell extract droplets using super-paramagnetic nanoparticles. We observed a link between a spatial asymmetry in biochemical cues and microtubules aster positional information. Our assay provides a bottom-up approach to examine the minimum ingredients generating polarization and symmetry breaking within cells. We then examined the possibility to magnetically perturb endosomes position in HeLa cell. We found the experimental conditions to achieve this goal. Finally, we used directly cytoskeleton elements as actin filament to trigger asymmetrically confined signaling proteins and trigger microtubule assembly, in cell extract droplets. More generally, these results show how symmetry breaking within cells can be induced and studied using magnetic nanoparticles and biophysical tools.De plus en plus dâĂ©tudes soulignent lâimportance de la localisation intracellulaire des voies de signalisation. Nous avons dĂ©veloppĂ© des mĂ©thodes permettant de perturber cette localisation Ă lâaide de nanoparticules magnĂ©tiques. Ces derniĂšres sont fonctionnalisĂ©es avec les protĂ©ines dâintĂ©rĂȘts et deviennent ainsi un vecteur permettant de contrĂŽler la localisation de la signalisation. Nous avons tout dâabord appliquĂ© cette mĂ©thode dans un systĂšme modĂšle, des gouttes dâextrait cellulaire de XĂ©nope, dans lesquelles nous avons crĂ©Ă© artificiellement un gradient de protĂ©ines de signalisation Ă lâaide de nanoparticules magnĂ©tiques. Nous avons mis en Ă©vidence lâinfluence dâune asymĂ©trie biochimique sur la localisation dâasters de microtubules. Dans un deuxiĂšme temps nous avons examinĂ© la possibilitĂ© dâappliquer cette mĂ©thode dans des cellules HeLa adhĂ©rentes, pour perturber la localisation dâendosomes de signalisation rendus magnĂ©tiques. Nous avons cherchĂ© Ă optimiser les conditions expĂ©rimentales nĂ©cessaires pour contrĂŽler la position dâendosomes de signalisation magnĂ©tiques Enfin, un troisiĂšme projet dont les rĂ©sultats prĂ©liminaires sont prĂ©sentĂ©s dans cette thĂšse, a consistĂ© Ă utiliser un actuateur, non plus magnĂ©tique, mais biologique pour confiner une cascade de signalisation. Plus prĂ©cisĂ©ment la contraction dâun rĂ©seau dâactine confinĂ© dans des gouttes dâextrait cellulaire est utilisĂ©e pour localiser des protĂ©ines de signalisation. Ces rĂ©sultats dĂ©montrent lâintĂ©rĂȘt de nanoparticules magnĂ©tiques pour induire et Ă©tudier des phĂ©nomĂšnes de brisures de symĂ©tries dans des environnements biologique
Use of magnetic nanoparticles to pertub the spatiotemporal localization of signaling proteins
De plus en plus dâĂ©tudes soulignent lâimportance de la localisation intracellulaire des voies de signalisation. Nous avons dĂ©veloppĂ© des mĂ©thodes permettant de perturber cette localisation Ă lâaide de nanoparticules magnĂ©tiques. Ces derniĂšres sont fonctionnalisĂ©es avec les protĂ©ines dâintĂ©rĂȘts et deviennent ainsi un vecteur permettant de contrĂŽler la localisation de la signalisation. Nous avons tout dâabord appliquĂ© cette mĂ©thode dans un systĂšme modĂšle, des gouttes dâextrait cellulaire de XĂ©nope, dans lesquelles nous avons crĂ©Ă© artificiellement un gradient de protĂ©ines de signalisation Ă lâaide de nanoparticules magnĂ©tiques. Nous avons mis en Ă©vidence lâinfluence dâune asymĂ©trie biochimique sur la localisation dâasters de microtubules. Dans un deuxiĂšme temps nous avons examinĂ© la possibilitĂ© dâappliquer cette mĂ©thode dans des cellules HeLa adhĂ©rentes, pour perturber la localisation dâendosomes de signalisation rendus magnĂ©tiques. Nous avons cherchĂ© Ă optimiser les conditions expĂ©rimentales nĂ©cessaires pour contrĂŽler la position dâendosomes de signalisation magnĂ©tiques Enfin, un troisiĂšme projet dont les rĂ©sultats prĂ©liminaires sont prĂ©sentĂ©s dans cette thĂšse, a consistĂ© Ă utiliser un actuateur, non plus magnĂ©tique, mais biologique pour confiner une cascade de signalisation. Plus prĂ©cisĂ©ment la contraction dâun rĂ©seau dâactine confinĂ© dans des gouttes dâextrait cellulaire est utilisĂ©e pour localiser des protĂ©ines de signalisation. Ces rĂ©sultats dĂ©montrent lâintĂ©rĂȘt de nanoparticules magnĂ©tiques pour induire et Ă©tudier des phĂ©nomĂšnes de brisures de symĂ©tries dans des environnements biologiquesAn increasing number of studies highlight the importance of signaling localization. We developed methods to perturb this localization using magnetic nanoparticles. Proteins of interest are grafted on magnetic nanoparticles, allowing to magnetically localize them. We first propose a new method to engineer directly a spatial gradient of signaling protein concentration within in cell extract droplets using super-paramagnetic nanoparticles. We observed a link between a spatial asymmetry in biochemical cues and microtubules aster positional information. Our assay provides a bottom-up approach to examine the minimum ingredients generating polarization and symmetry breaking within cells. We then examined the possibility to magnetically perturb endosomes position in HeLa cell. We found the experimental conditions to achieve this goal. Finally, we used directly cytoskeleton elements as actin filament to trigger asymmetrically confined signaling proteins and trigger microtubule assembly, in cell extract droplets. More generally, these results show how symmetry breaking within cells can be induced and studied using magnetic nanoparticles and biophysical tools
Detachment and fracture of cellular aggregates
International audienceThe dynamics of cellular adhesion and deadhesion, which play key roles in many cellular processes, have most often been studied at the scale of single bonds or single cells. However, multicellular adhesion and deadhesion are also central processes in tissue mechanics, morphogenesis, and pathophysiology, where collective tissue phenomena may introduce additional effects that are absent at the single-cell level. In this paper we present experiments on the adhesion of cellular aggregates and a laboratory model system to study tissue mechanics. We introduce a technique to measure the forces and energies involved in the detachment of an aggregate from a substrate (which can be viewed as a cellular tack assay) and in the fracture between two partially fused aggregates, as a function of the adhesion time, the pulling speed, and the cadherin density at the cell surface. We develop a model based on polymer physics to interpret the observations. We identify a significant contribution to the adhesion energy of viscous dissipation mechanisms present at the tissue scale that are absent at the single-cell level, as well as a significant effect of the speed at which the separation force is applied
Optical Magnetometry of Single Biocompatible Micromagnets for Quantitative Magnetogenetic and Magnetomechanical Assays
The
mechanical manipulation of magnetic nanoparticles is a powerful
approach to probing and actuating biological processes in living systems.
Implementing this technique in high-throughput assays can be achieved
using biocompatible micromagnet arrays. However, the magnetic properties
of these arrays are usually indirectly inferred from simulations or
Stokes drag measurements, leaving unresolved questions about the actual
profile of the magnetic fields at the micrometer scale and the exact
magnetic forces that are applied. Here, we exploit the magnetic field
sensitivity of nitrogen-vacancy color centers in diamond to map the
3D stray magnetic field produced by a single soft ferromagnetic microstructure.
By combining this wide-field optical magnetometry technique with magneto-optic
Kerr effect microscopy, we fully analyze the properties of the micromagnets,
including their magnetization saturation and their size-dependent
magnetic susceptibility. We further show that the high magnetic field
gradients produced by the micromagnets, greater than 10<sup>4</sup> T·m<sup>â1</sup> under an applied magnetic field of
about 100 mT, enables the manipulation of magnetic nanoparticles smaller
than 10 nm inside living cells. This work paves the way for quantitative
and parallelized experiments in magnetogenetics and magnetomechanics
in cell biology