Mathieu Morel

Asymmetric redistribution of GABA receptors during GABA gradient sensing by nerve growth cones analyzed by single quantum dot imaging

During development of the nervous system, the tip of a growing axon, the growth cone (GC), must respond accurately to stimuli that direct its growth. This axonal navigation depends on extracellular concentration gradients of numerous guidance cues, including GABA. GCs can detect even weak directional signals, yet the mechanisms underlying this sensitivity remain unclear. Past studies in other eukaryotic chemotactic systems have pointed to the role of the spatial reorganization of the transduction pathway in their sensitive response. Here we have developed a single-molecule assay to observe individual GABAA receptors (GABAARs) in the plasma membrane of nerve GCs subjected to directional stimuli. We report that in the presence of an external GABA gradient GABAARs redistribute asymmetrically across the GC toward the gradient source. Single-particle tracking of GABAARs shows that the redistribution results from transient interactions between the receptors and the microtubules. Moreover, the relocalization is accompanied by an enhancement in the asymmetry of intracellular calcium concentration. Altogether, our results reveal a microtubule-dependent polarized reorganization of chemoreceptors at the cell surface and suggest that this polarization serves as an amplification step in GABA gradient sensing by nerve GCs.

Light-Directed Particle Patterning by Evaporative Optical Marangoni Assembly

Controlled particle deposition on surfaces is crucial for both exploiting collective properties of particles and their integration into devices. Most available methods depend on intrinsic properties of either the substrate or the particles to be deposited making them difficult to apply to complex, naturally occurring or industrial formulations. Here we describe a new strategy to pattern particles from an evaporating drop, regardless of inherent particle characteristics and suspension composition. We use light to generate Marangoni surface stresses resulting in flow patterns that accumulate particles at predefined positions. Using projected images, we generate a broad variety of complex patterns, including multiple spots, lines and letters. Strikingly, this method, which we call evaporative optical Marangoni assembly (eOMA), allows us to pattern particles regardless of their size or surface properties, in model suspensions as well as in complex, real-world formulations such as commercial coffee.

Protein Adsorption and Reorganization on Nanoparticles Probed by the Coffee-Ring Effect: Application to Single Point Mutation Detection

The coffee-ring effect denotes the accumulation of particles at the edge of an evaporating sessile drop pinned on a substrate. Because it can be detected by simple visual inspection, this ubiquitous phenomenon can be envisioned as a robust and cost-effective diagnostic tool. Toward this direction, here we systematically analyze the deposit morphology of drying drops containing polystyrene particles of different surface properties with various proteins (bovine serum albumin (BSA) and different forms of hemoglobin). We show that deposit patterns reveal information on both the adsorption of proteins onto particles and their reorganization following adsorption. By combining pattern analysis with adsorption isotherm and zeta potential measurements, we show that the suppression of the coffee-ring effect and the formation of a disk-shaped pattern is primarily associated with particle neutralization by protein adsorption. However, our findings also suggest that protein reorganization following adsorption can dramatically invert this tendency. Exposure of hydrophobic (respectively charged) residues can lead to disk (respectively ring) deposit morphologies independently of the global particle charge. Surface tension measurements and microscopic observations of the evaporating drops show that the determinant factor of the deposit morphology is the accumulation of particles at the liquid/gas interface during evaporation. This general behavior opens the possibility to probe protein adsorption and reorganization on particles by the analysis of the deposit patterns, the formation of a disk being the robust signature of particles rendered hydrophobic by protein adsorption. We show that this method is sensitive enough to detect a single point mutation in a protein, as demonstrated here by the distinct patterns formed by human native hemoglobin h-HbA and its mutant form h-HbS, which is responsible for sickle cell anemia.

Amplification and Temporal Filtering during Gradient Sensing by Nerve Growth Cones Probed with a Microfluidic Assay

Nerve growth cones (GCs) are chemical sensors that convert graded extracellular cues into oriented axonal motion. To ensure a sensitive and robust response to directional signals in complex and dynamic chemical landscapes, GCs are presumably able to amplify and filter external information. How these processing tasks are performed remains however poorly known. Here, we probe the signal-processing capabilities of single GCs during γ-Aminobutyric acid (GABA) directional sensing with a shear-free microfluidic assay that enables systematic measurements of the GC output response to variable input gradients. By measuring at the single molecule level the polarization of GABA(A) chemoreceptors at the GC membrane, as a function of the external GABA gradient, we find that GCs act as i), signal amplifiers over a narrow range of concentrations, and ii), low-pass temporal filters with a cutoff frequency independent of stimuli conditions. With computational modeling, we determine that these systems-level properties arise at a molecular level from the saturable occupancy response and the lateral dynamics of GABA(A) receptors.

Photodependent Melting of Unmodified DNA Using a Photosensitive Intercalator: A New and Generic Tool for Photoreversible Assembly of DNA Nanostructures at Constant Temperature

External control of DNA melting and hybridization, a key step in bio- and DNA nanotechnology, is commonly achieved with temperature. The use of light to direct this process is a challenging alternative, which has been only possible with a DNA modification, such as covalent grafting or mismatch introduction, so far. Here we describe the first photocontrol of DNA melting that relies on the addition of a molecule that noncovalently interacts with unmodified DNA and affects its melting properties in a photoreversible and highly robust manner, without any prerequisite in the length or sequence of the target DNA molecule. We synthesize azobenzene-containing guanidinium derivatives and show that a bivalent molecule with a conformation-dependent binding mode, AzoDiGua, strongly increases the melting temperature (Tm) of DNA under dark conditions because its trans isomer intercalates in the DNA double helix. Upon UV irradiation at 365 nm, the trans-cis isomerization induced the ejection of AzoDiGua from the intercalation binding sites, resulting in a decrease in Tm up to 18 °C. This illumination-dependent Tm shift is observed on many types of DNA, from self-complementary single-stranded or double-stranded oligonucleotides to long genomic duplex DNA molecules. Finally, we show that simply adding AzoDiGua allows us to photoreversibly control the assembly/disassembly of a DNA nanostructure at constant temperature, as demonstrated here with a self-hybridized DNA hairpin. We anticipate that this strategy will be the key ingredient in a new and generic way of placing DNA-based bio- and nanotechnologies under dynamic control by light.

Cellular heterogeneity mediates inherent sensitivity-specificity tradeoff in cancer targeting by synthetic circuits.

Significance The recent advance in the use of viral vectors for gene delivery, combined with the design of synthetic gene circuits to diagnose and target cells, brings opportunities for effective treatment of cancer. So far, gene circuits have been considered logical devices capable of discriminating normal from malignant cells as discrete states, ignoring cellular heterogeneity in cancer expression markers. We addressed the inherent limitations heterogeneity imposes on the precision of targeting circuits. Using molecular parameters to control circuit gain amplification and threshold, we show an inherent tradeoff emerges between specificity and sensitivity. In light of this tradeoff, the molecular optimization of targeting circuits will be an important step for effective implementation of personalized gene therapy. Synthetic gene circuits are emerging as a versatile means to target cancer with enhanced specificity by combinatorial integration of multiple expression markers. Such circuits must also be tuned to be highly sensitive because escape of even a few cells might be detrimental. However, the error rates of decision-making circuits in light of cellular variability in gene expression have so far remained unexplored. Here, we measure the single-cell response function of a tunable logic AND gate acting on two promoters in heterogeneous cell populations. Our analysis reveals an inherent tradeoff between specificity and sensitivity that is controlled by the AND gate amplification gain and activation threshold. We implement a tumor-mimicking cell-culture model of cancer cells emerging in a background of normal ones, and show that molecular parameters of the synthetic circuits control specificity and sensitivity in a killing assay. This suggests that, beyond the inherent tradeoff, synthetic circuits operating in a heterogeneous environment could be optimized to efficiently target malignant state with minimal loss of specificity.

Magnetic Actuation of Drops and Liquid Marbles Using a Deformable Paramagnetic Liquid Substrate

Abstract The magnetic actuation of deposited drops has mainly relied on volume forces exerted on the liquid to be transported, which is poorly efficient with conventional diamagnetic liquids such as water and oil, unless magnetosensitive particles are added. Herein, we describe a new and additive‐free way to magnetically control the motion of discrete liquid entities. Our strategy consists of using a paramagnetic liquid as a deformable substrate to direct, using a magnet, the motion of various floating liquid entities, ranging from naked drops to liquid marbles. A broad variety of liquids, including diamagnetic (water, oil) and nonmagnetic ones, can be efficiently transported using the moderate magnetic field (ca. 50 mT) produced by a small permanent magnet. Complex trajectories can be achieved in a reliable manner and multiplexing potential is demonstrated through on‐demand drop fusion. Our paramagnetofluidic method advantageously works without any complex equipment or electric power, in phase with the necessary development of robust and low‐cost analytical and diagnostic fluidic devices.

Evaporative Optical Marangoni Assembly: Tailoring the Three-Dimensional Morphology of Individual Deposits of Nanoparticles from Sessile Drops

We have recently devised the evaporative optical Marangoni assembly (eOMA), a novel and versatile interfacial flow-based method for directing the deposition of colloidal nanoparticles (NPs) on solid substrates from evaporating sessile drops along desired patterns using shaped UV light. Here, we focus on a fixed UV spot irradiation resulting in a cylinder-like deposit of assembled particles and show how the geometrical features of the single deposit can be tailored in three dimensions by simply adjusting the optical conditions or the sample composition, in a quantitative and reproducible manner. Sessile drops containing cationic NPs and a photosensitive surfactant at various concentrations are allowed to evaporate under a single UV beam with a diameter much smaller than that of the drop. After complete evaporation, the geometrical characteristics of the NP deposits are precisely assessed using optical profilometry. We show that both the volume and the radial size of the light-directed NP deposit can be adjusted by varying the diameter or the intensity of the UV beam or alternatively by changing the concentration of the photosensitive surfactant. Notably, in all these cases, the deposits display an almost constant median height corresponding to a few layers of particles. Moreover, both the radial and the axial extent of the patterns are tuned by changing the NP concentration. These results are explained by the correlation among the strength of Marangoni flow, the particle trapping efficiency, and the volume of the deposit, and by the role of evaporation-driven flow in strongly controlling the deposit height. Finally, we extend the versatility of eOMA by demonstrating that NPs down to 30 nm in diameter can be effectively patterned on glass or polymeric substrates.

Intramolecularly Protein-Crosslinked DNA Gels: New Biohybrid Nanomaterials with Controllable Size and Catalytic Activity

DNA micro- and nanogels-small-sized hydrogels made of a crosslinked DNA backbone-constitute new promising materials, but their functions have mainly been limited to those brought by DNA. Here a new way is described to prepare sub-micrometer-sized DNA gels of controllable crosslinking density that are able to embed novel functions, such as an enzymatic activity. It consists of using proteins, instead of traditional base-pairing assembly or covalent approaches, to form crosslinks inside individual DNA molecules, resulting in structures referred to as intramolecularly protein-crosslinked DNA gels (IPDGs). It is first shown that the addition of streptavidin to biotinylated T4DNA results in the successful formation of thermally stable IPDGs with a controllable crosslinking density, forming structures ranging from elongated to raspberry-shaped and pearl-necklace-like morphologies. Using reversible DNA condensation strategies, this paper shows that the gels can be reversibly actuated at a low crosslinking density, or further stabilized when they are highly crosslinked. Finally, by using streptavidin-protein conjugates, IPDGs with various enzymes are successfully functionalized. It is demonstrated that the enzymes keep their catalytic activity upon their incorporation into the gels, opening perspectives ranging from biotechnologies (e.g., enzyme manipulation) to nanomedicine (e.g., vectorization).