A microdevice to locally electroporate embryos with high efficiency and reduced cell damage.

The ability to follow and modify cell behaviour with accurate spatiotemporal resolution is a prerequisite to study morphogenesis in developing organisms. Electroporation, the delivery of exogenous molecules into targeted cell populations through electric permeation of the plasma membrane, has been used with this aim in different model systems. However, current localised electroporation strategies suffer from insufficient reproducibility and mediocre survival when applied to small and delicate organisms such as early post-implantation mouse embryos. We introduce here a microdevice to achieve localised electroporation with high efficiency and reduced cell damage. In silico simulations using a simple electrical model of mouse embryos indicated that a dielectric guide-based design would improve on existing alternatives. Such a device was microfabricated and its capacities tested by targeting the distal visceral endoderm (DVE), a migrating cell population essential for anterior-posterior axis establishment. Transfection was efficiently and reproducibly restricted to fewer than four visceral endoderm cells without compromising cell behaviour and embryo survival. Combining targeted mosaic expression of fluorescent markers with live imaging in transgenic embryos revealed that, like leading DVE cells, non-leading ones send long basal projections and intercalate during their migration. Finally, we show that the use of our microsystem can be extended to a variety of embryological contexts, from preimplantation stages to organ explants. Hence, we have experimentally validated an approach delivering a tailor-made tool for the study of morphogenesis in the mouse embryo. Furthermore, we have delineated a comprehensive strategy for the development of ad hoc electroporation devices.

Peptidic ligands to control the three-dimensional self-assembly of quantum rods in aqueous media.

The use of peptidic ligands is validated as a generic chemical platform allowing one to finely control the organization in solid phase of semiconductor nanorods originally dispersed in an aqueous media. An original method to generate, on a macroscopic scale and with the desired geometry, three-dimensional supracrystals composed of quantum rods is introduced. In a first step, nanorods are transferred in an aqueous phase thanks to the substitution of the original capping layer by peptidic ligands. Infrared and nuclear magnetic resonance spectroscopy data prove that the exchange is complete; fluorescence spectroscopy demonstrates that the emitter optical properties are not significantly altered; electrophoresis and dynamic light scattering experiments assess the good colloidal stability of the resulting aqueous suspension. In a second step, water evaporation in a microstructured environment yields superstructures with a chosen geometry and in which nanorods obey a smectic B arrangement, as shown by electron microscopy. Incidentally, bulk drying in a capillary tube generates a similar local order, as evidenced by small angle X-ray scattering.

Spatiotemporal control of microtubule nucleation and assembly using magnetic nanoparticles.

Decisions on the fate of cells and their functions are dictated by the spatiotemporal dynamics of molecular signalling networks. However, techniques to examine the dynamics of these intracellular processes remain limited. Here, we show that magnetic nanoparticles conjugated with key regulatory proteins can artificially control, in time and space, the Ran/RCC1 signalling pathway that regulates the cell cytoskeleton. In the presence of a magnetic field, RanGTP proteins conjugated to superparamagnetic nanoparticles can induce microtubule fibres to assemble into asymmetric arrays of polarized fibres in Xenopus laevis egg extracts. The orientation of the fibres is dictated by the direction of the magnetic force. When we locally concentrated nanoparticles conjugated with the upstream guanine nucleotide exchange factor RCC1, the assembly of microtubule fibres could be induced over a greater range of distances than RanGTP particles. The method shows how bioactive nanoparticles can be used to engineer signalling networks and spatial self-organization inside a cell environment.

Magnetic control of protein spatial patterning to direct microtubule self-assembly.

Living systems offer attractive strategies to generate nanoscale structures because of their innate functional properties such as the dynamic assembly of ordered nanometer fibers, the generation of mechanical forces, or the directional transport mediated by molecular motors. The design of hybrid systems, capable of interfacing artificial building blocks with biomolecules, may be a key step toward the rational design of nanoscale devices and materials. Here, we have designed a bottom-up approach to organize cytoskeletal elements in space using the self-assembly properties of magnetic nanoparticles conjugated to signaling proteins involved in microtubule nucleation. We show that magnetic nanoparticles conjugated to signaling proteins involved in microtubule nucleation can control the positioning of microtubule assembly. Under a magnetic field, a self-organized pattern of biofunctionalized nanoparticles is formed and leads to the nucleation of a periodical network of microtubules in Xenopus laevis egg extract. Our method shows how bioactive nanoparticles can generate a biochemically active pattern upon magnetic actuation, which triggers the spatial organization of nonequilibrium biological structures.

Three-dimensional self-assembling of gold nanorods with controlled macroscopic shape and local smectic B order.

We describe a method of controlled evaporation on a textured substrate for self-assembling and shaping gold-nanorod-based materials. Tridimensional wall features are formed over areas as large as several square millimeters. Furthermore, analyses by small-angle X-ray scattering and scanning electron microscopy techniques demonstrate that colloids are locally ordered as a smectic B phase. Such crystallization is in fact possible because we could finely adjust the nanoparticle charge, knowledge that additionally enables tuning the lattice parameters. In the future, the type of ordered self-assemblies of gold nanorods we have prepared could be used for amplifying optical signals.