Enzymatic Digestion of Single DNA Molecules Anchored on Nanogold-Modified Surfaces

To study enzyme–DNA interactions at single molecular level, both the attachment points and the immediate surroundings of surfaces must be carefully considered such that they do not compromise the structural information and biological properties of the sample under investigation. The present work demonstrates the feasibility of enzymatic digestion of single DNA molecules attached to nanoparticle-modified surfaces. With Nanogold linking DNA to the mica surface by electrostatic interactions, advantageous conditions with fewer effects on the length and topography of DNA are obtained, and an appropriate environment for the activities of DNA is created. We demonstrate that by using Dip-Pen Nanolithography, individual DNA molecules attached to modified mica surfaces can be efficiently digested by DNase I

Fluorescence Quenching of Alpha-Fetoprotein by Gold Nanoparticles: Effect of Dielectric Shell on Non-Radiative Decay

Fluorescence quenching spectrometry was applied to study the interactions between gold colloidal nanoparticles and alpha-fetoprotein (AFP). Experimental results show that the gold nanoparticles can quench the fluorescence emission of adsorbed AFP effectively. Furthermore, the intensity of fluorescence emission peak decreases monotonously with the increasing gold nanoparticles content. A mechanism based on surface plasmon resonance–induced non-radiative decay was investigated to illuminate the effect of a dielectric shell on the fluorescence quenching ability of gold nanoparticles. The calculation results show that the increasing dielectric shell thickness may improve the monochromaticity of fluorescence quenching. However, high energy transfer efficiency can be obtained within a wide wavelength band by coating a thinner dielectric shell

Transverse fluctuation analysis of single extended DNA molecules

We observed fluctuations of elongated DNA molecules by fluorescence microscopy. The molecules are fixed at both ends and undulate. Mode analysis of the thermally excited undulations of the labeled DNA molecules gives access to the spectral density of the amplitude fluctuations. From these measurements we estimate the tension acting on the DNA molecules. We found the forces to be within the entropic elasticity range of a typical DNA molecule (measured on the single-molecule level)

Triggering signaling pathways using F-actin self-organization

International audienceThe spatiotemporal organization of proteins within cells is essential for cell fate behavior. Although it is known that the cytoskeleton is vital for numerous cellular functions, it remains unclear how cytoskeletal activity can shape and control signaling pathways in space and time throughout the cell cytoplasm. Here we show that F-actin self-organization can trigger signaling pathways by engineering two novel properties of the microfilament self-organization: (1) the confinement of signaling proteins and (2) their scaffolding along actin polymers. Using in vitro reconstitutions of cellular functions, we found that both the confinement of nanoparticle-based signaling platforms powered by F-actin contractility and the scaffolding of engineered signaling proteins along actin microfilaments can drive a signaling switch. Using Ran-dependent microtubule nucleation, we found that F-actin dynamics promotes the robust assembly of microtubules. Our in vitro assay is a first step towards the development of novel bottom-up strategies to decipher the interplay between cytoskeleton spatial organization and signaling pathway activity

Effects of Confinement on the Self-Organization of Microtubules and Motors

SummaryThe regulation of the cytoskeleton is essential for the proper organization and function of eukaryotic cells. For instance, radial arrays of microtubules (MTs), called asters, determine the intracellular localization of organelles [1, 2]. Asters can be generated through either MT organizing center (MTOC)-dependent regulation or self-organization processes [1, 3, 4]. In vivo, this occurs within the cell boundaries. How the properties of these boundaries affect MT organization is unknown. To approach this question, we studied the organization of microtubules inside droplets of eukaryotic cellular extracts with varying sizes and elastic properties. Our results show that the size of the droplet determined the final steady-state MT organization, which changed from symmetric asters to asymmetric semi-asters and, finally, to cortical bundles. A simple physical model recapitulated these results, identifying the main physical parameters of the transitions. The use of vesicles with more elastic boundaries resulted in very different morphologies of microtubule structures, such as asymmetrical semi-asters, “Y-branching” organizations, cortical-like bundles, “rackets,” and bundled organizations. Our results highlight the importance of taking into account the physical characteristics of the cellular confinement to understand the formation of cytoskeleton structures in vivo

Effet du confinement sur l'organisation des microtubules

Communication oral

Engineering Spatial Gradients of Signaling Proteins Using Magnetic Nanoparticles

Intracellular biochemical reactions are often localized in space and time, inducing gradients of enzymatic activity that may play decisive roles in determining cell’s fate and functions. However, the techniques available to examine such enzymatic gradients of activity remain limited. Here, we propose a new method to engineer a spatial gradient of signaling protein concentration within <i>Xenopus</i> egg extracts using superparamagnetic nanoparticles. We show that, upon the application of a magnetic field, a concentration gradient of nanoparticles with a tunable length extension is established within confined egg extracts. We then conjugate the nanoparticles to RanGTP, a small G-protein controlling microtubule assembly. We found that the generation of an artificial gradient of Ran-nanoparticles modifies the spatial positioning of microtubule assemblies. Furthermore, the spatial control of the level of Ran concentration allows us to correlate the local fold increase in Ran-nanoparticle concentration with the spatial positioning of the microtubule-asters. Our assay provides a bottom-up approach to examine the minimum ingredients generating polarization and symmetry breaking within cells. More generally, these results show how magnetic nanoparticles and magnetogenetic tools can be used to control the spatiotemporal dynamics of signaling pathways