Cell size and actin architecture determine force generation in optogenetically activated cells.

Adherent cells use actomyosin contractility to generate mechanical force and to sense the physical properties of their environment, with dramatic consequences for migration, division, differentiation, and fate. However, the organization of the actomyosin system within cells is highly variable, with its assembly and function being controlled by small GTPases from the Rho family. To understand better how activation of these regulators translates into cell-scale force generation in the context of different physical environments, here we combine recent advances in non-neuronal optogenetics with micropatterning and traction force microscopy on soft elastic substrates. We find that, after whole-cell RhoA activation by the CRY2/CIBN optogenetic system with a short pulse of 100 ms, single cells contract on a minute timescale in proportion to their original traction force, before returning to their original tension setpoint with near perfect precision, on a longer timescale of several minutes. To decouple the biochemical and mechanical elements of this response, we introduce a mathematical model that is parametrized by fits to the dynamics of the substrate deformation energy. We find that the RhoA response builds up quickly on a timescale of 20 s, but decays slowly on a timescale of 50 s. The larger the cells and the more polarized their actin cytoskeleton, the more substrate deformation energy is generated. RhoA activation starts to saturate if optogenetic pulse length exceeds 50 ms, revealing the intrinsic limits of biochemical activation. Together our results suggest that adherent cells establish tensional homeostasis by the RhoA system, but that the setpoint and the dynamics around it are strongly determined by cell size and the architecture of the actin cytoskeleton, which both are controlled by the extracellular environment.

Bio-functionalization of silicon carbide nanostructures for SiC nanowire-based sensors realization.

The bio-functionalization process consisting in grafting desoxyribo nucleic acid via aminopropyl-triethoxysilane is performed on several kinds of silicon carbide nanostructures. Prior, the organic layer is characterized on planar surface with fluorescence microscopy and X-ray photoelectron spectroscopy. Then, the functionalization is performed on two kinds of nanopillar arrays. One is composed of top-down SiC nanopillars with a wide pitch of 5 microm while the other one is a dense array (pitch: 200 nm) of core-shell Si-SiC nanowires obtained by carburization of silicon nanowires. Depending on both the pillar morphology and the pitch, different results in term of DNA surface coverages are obtained, as seen from fluorescence microscopy images. Particularly, in the case of the wide pitch array, it has been shown that the DNA molecules are located all along the nanopillars. To achieve a DNA sensor based on a nanowire-field effect transistor, the functionalization must be conducted on a single SiC nanowire or nanopillar that constitutes the channel of the field effect transistor. The localization of the functionalization in a small area around the nanostructures guarantees high performances to the sensor. In this aim, the functionalization process is combined with common microelectronics techniques of lithography and lift-off. The DNA immobilization is investigated by fluorescence microscopy and atomic force microscopy.

In vivo measurement of human brain elasticity using a light aspiration device.

The brain deformation that occurs during neurosurgery is a serious issue impacting the patient "safety" as well as the invasiveness of the brain surgery. Model-driven compensation is a realistic and efficient solution to solve this problem. However, a vital issue is the lack of reliable and easily obtainable patient-specific mechanical characteristics of the brain which, according to clinicians' experience, can vary considerably. We designed an aspiration device that is able to meet the very rigorous sterilization and handling process imposed during surgery, and especially neurosurgery. The device, which has no electronic component, is simple, light and can be considered as an ancillary instrument. The deformation of the aspirated tissue is imaged via a mirror using an external camera. This paper describes the experimental setup as well as its use during a specific neurosurgery. The experimental data was used to calibrate a continuous model. We show that we were able to extract an in vivo constitutive law of the brain elasticity: thus for the first time, measurements are carried out per-operatively on the patient, just before the resection of the brain parenchyma. This paper discloses the results of a difficult experiment and provide for the first time in vivo data on human brain elasticity. The results point out the softness as well as the highly non-linear behavior of the brain tissue.

Theoretical analysis of the adaptive contractile behaviour of a single cardiomyocyte cultured on elastic substrates with varying stiffness.

In vivo, cardiomyocytes interact with surrounding extracellular matrix while performing periodically a contractile behaviour, which is the main determinant of heart performance. As extracellular substrates with easily tunable stiffness properties, polyacrylamide gels (PAGs) provide valuable flexible media for studying in vitro the dynamical behaviour of cardiomyocytes responding to stiffness variations of their surrounding environment. We propose in this paper an original mechano-chemical model of the cardiac cell contraction that sheds light on the adaptive response of cardiomyocytes evidenced recently in the experiments of Qin et al. [2007. Dynamical stress characterization and energy evaluation of single cardiac myocyte actuating on flexible substrate. Biochem. Biophys. Res. Commun. 360, 352-356]. The model links the amplitude of the extracellular PAGs strain fields to the spatio-temporal variation of the intracellular stresses in every part of the cell during the sarcomeres contraction-relaxation. In a continuum mechanics framework, we derived a unified description of the sarcomere-length dependence of intracellular active stress and of its control by anisotropic calcium diffusion and autocatalytic calcium release from the sarcoplasmic reticulum. Taking benefit of our previous work on the characterization of mechanical properties of PAGs with varying stiffness, we were thus able to evaluate the active intracellular stress exerted by the cardiomyocyte on flexible PAGs with different and known Young's moduli. Interestingly, we were able to explain the intriguing increase of maximal cellular stress observed experimentally when substrate stiffness is increased. By providing an evaluation of the whole-field cell stresses and strains, this integrative approach of cardiomyocyte contraction provides a reliable basis for further analysis of additional cooperativity and mechanotransduction mechanisms involved in cell contractility regulation, notably in physiological and pathological situations where modifications of cardiac performance are linked to varied stiffness of the cardiomyocytes environment.

A light sterilizable pipette device for the in vivo estimation of human soft tissues constitutive laws.

This paper introduces a new light device for the in vivo estimation of human soft tissues constitutive laws. It consists of an aspiration pipette able to meet the very severe sterilization and handling issues imposed during surgery. The simplicity of the device, free of any electronic circuitry, allows using it as an ancillary instrument. The deformation of the aspired tissue is imaged via a mirror using an external camera. The paper describes the experimental setup as well as the protocol that should be used during surgery. First feasibility measurements are shown for human tongue and forearm skin.

Influences of adhesion area and biological sample size on the estimation of Young's modulus and Poisson's ratio assessed by micropipette aspiration technique.

The micropipette aspiration experiment remains a widely used micromanipulation technique for quantifying the mechanical properties of biological samples. Our study extends previous results by investigating the influence of sample size and adhesion area on the mechanical response of compressible thin biological samples. We thus defined a nonlinear relationship between aspirated length, Young's modulus, Poisson's ratio and sample thickness which allowed us to develop an original experimental protocol for simultaneous quantification of the Poisson's ratio and Young's modulus of adherent samples. We first validated our method by characterizing mechanical properties of polyacrylamide gels with tunable stiffness. We then considered application of these results to the quantification of cell elasticity, focusing on the influence of cell adhesion area onto the measured apparent cell stiffness.