Albrecht Ott

Microplates: a new tool for manipulation and mechanical perturbation of individual cells

We present a new type of microinstrument allowing manipulation and mechanical perturbation of individual cells under an optical microscope. These instruments, which we call microplates, are pulled from rectangular glass bars. They have flat tips, typically 2 microm thick x 20 microm wide, whose specific shape and stiffness can be adjusted through the pulling protocol. After appropriate chemical treatment, microplates can support cell adhesion and/or spreading. Rigid microplates are used to hold cells, whereas more flexible ones serve as stress sensors, i.e. their deflexion is used to probe forces in the range of 1-1000 nN. The main advantages of microplates are their simple geometry and surface properties, and their ability to provide mechanical measurements. In this methodological paper, we give details about microplate preparation and adhesiveness, manipulation set-up, force calibration, and image analysis. Several manipulations have already been carried out on fibroblasts, including uniaxial deformation, micropipet aspiration of adherent cells, and cell-substrate separation. Our results to date provide new insights into the morphology, mechanical properties, and adhesive resistance of cells. Many future applications can be envisaged.

ACTING ON ACTIN : THE ELECTRIC MOTILITY ASSAY

Abstract We have developed a novel technique which allows one to direct the two dimensional motion of actin filaments on a myosin coated sheet using a weak electric field parallel to the plane of motion. The filament velocity can be increased or decreased, and even reversed, as a function of orientation and strength of the field. PMMA (poly(methylmethacrylate)) gratings, which act as rails for actin, allow one for the first time to explore three quadrants of the force velocity diagram. We discuss effective friction, duty ratio and stall force at different myosin densities. A discontinuity in the velocity force relationship suggests the existence of dynamical phase transition.

Measurement of the elasticity of the actin tail of Listeria monocytogenes

Abstract We report biophysical experiments performed on the bacterium Listeria monocytogenes, a model system to study actin-based motility. Using optical tweezers and electrophoresis experiments, we find that the bacterium is firmly attached to its tail, and we demonstrate that the tail responds as an elastic gel when deformed. We have measured its elastic modulus at a value of 103–104 Pa, which is 10 times higher than the rigidity of the eukaryotic cytoplasm. These results demonstrate that the bacterium and its tail form a very robust system, consistent with the steadyness of the motion observed in vivo. We propose an elastic model for the propulsion mechanism which takes into account the connection and thus the interaction between the actin filaments. It provides a generic description of the various aspects of actin-tail based movements.

Filament Assembly from Profilin-Actin

Profilin plays a major role in the assembly of actin filament at the barbed ends. The thermodynamic and kinetic parameters for barbed end assembly from profilin-actin have been measured turbidimetrically. Filament growth from profilin-actin requires MgATP to be bound to actin. No assembly is observed from profilin-CaATP-actin. The rate constant for association of profilin-actin to barbed ends is 30% lower than that of actin, and the critical concentration for F-actin assembly from profilin-actin units is 0.3 μm under physiological ionic conditions. Barbed ends grow from profilin-actin with an ADP-Pi cap. Profilin does not cap the barbed ends and is not detectably incorporated into filaments. The EDC-cross-linked profilin-actin complex (PAcov) both copolymerizes with F-actin and undergoes spontaneous self-assembly, following a nucleation-growth process characterized by a critical concentration of 0.2 μm under physiological conditions. The PAcovpolymer is a helical filament that displays the same diffraction pattern as F-actin, with layer lines at 6 and 36 nm. The PAcov filaments bound phalloidin with the same kinetics as F-actin, bound myosin subfragment-1, and supported actin-activated ATPase of myosin subfragment-1, but they did not translocate in vitro along myosin-coated glass surfaces. These results are discussed in light of the current models of actin structure.

COMPARISON OF THE MECHANICAL PROPERTIES OF NORMAL AND TRANSFORMED FIBROBLASTS

In order to achieve coordinated migration through extracellular matrix and endothelial barriers during metastasis, cancer cells must be endowed with specific structural and adhesive properties. In this context, comparison of the mechanical properties of transformed versus normal cells, on which little quantitative information is available, was the focus of this study. Normal human dermal fibroblasts and their SV40-transformed counterparts were analyzed using various manipulations. First, micropipet aspiration of suspended cells allowed calculation of a cortical tension (similar for normal and transformed cells), and an apparent viscosity (30% lower for transformed than for normal fibroblasts); in addition, transformed fibroblasts exhibited a more fragile surface than their normal counterparts. Second, tangential ultracentrifugation of adherent cells demonstrated cellular elongation in the direction of the centrifugal field and the existence of critical forces for cell detachment, around 10(-7) N: these were 1.6-fold greater for normal than for transformed cells. Finally, examination of the wrinkle patterns formed by cells plated on a deformable polydimethylsiloxane substrate, plus analysis of cell retraction caused by ATP treatment following detergent permeabilization showed that normal fibroblasts exhibited much more contractility than their transformed counterparts, which we characterized by a cell contraction rate. Such quantitative parameters which reveal differences in the mechanical behavior of normal and transformed cells may be used in the future as new markers of oncogenic transformation.

Critical centrifugal forces induce adhesion rupture or structural reorganization in cultured cells

Cultured epithelial cells were exposed to accelerations ranging from 9,000 to 70,000g for time periods of 5, 15, or 60 min, by centrifugation in a direction tangential to their plastic substrate. Three regimes describe the cellular response: (1) Cell morphology and density remain unaltered at forces below a threshold of about 10(-7) N; (2) Between this critical force and a second threshold of about 1.5 10(-7)N, the number of adherent cells decreases exponentially with time and acceleration, with no alteration of cell morphology. This behavior can be modeled by a constant probability of detaching and by an exponential distribution of cell-to-substrate adhesive forces; (3) Past the second threshold, cells that are still adherent exhibit elongated morphologies, the degree of elongation increasing linearly with the force. The fact that cells lose their vinculin-rich focal contacts past the first threshold and that cells cultured on gelatin-coated plastic show an increased resistance to detachment suggests a rupture of cell-to-substrate adhesions upon centrifugation. Immunofluorescent labeling of cells for actin and tubulin shows a reorganization of the cytoskeleton upon centrifugation, and treatment of cells with the drugs cytochalasin D and nocodazole demonstrates that cytoskeletal elements are actively involved in the structural deformation of cells past the second acceleration threshold, microtubules and microfilaments paying antagonistic roles.

Single cell mechanics: stress stiffening and kinematic hardening.

Cell mechanical properties are fundamental to the organism but remain poorly understood. We report a comprehensive phenomenological framework for the complex rheology of single fibroblast cells: a superposition of elastic stiffening and viscoplastic kinematic hardening. Despite the complexity of the living cell, its mechanical properties can be cast into simple, well-defined rules. Our results reveal the key role of crosslink slippage in determining mechanical cell strength and robustness.

Physico-chemical foundations underpinning microarray and next-generation sequencing experiments

Hybridization of nucleic acids on solid surfaces is a key process involved in high-throughput technologies such as microarrays and, in some cases, next-generation sequencing (NGS). A physical understanding of the hybridization process helps to determine the accuracy of these technologies. The goal of a widespread research program is to develop reliable transformations between the raw signals reported by the technologies and individual molecular concentrations from an ensemble of nucleic acids. This research has inputs from many areas, from bioinformatics and biostatistics, to theoretical and experimental biochemistry and biophysics, to computer simulations. A group of leading researchers met in Ploen Germany in 2011 to discuss present knowledge and limitations of our physico-chemical understanding of high-throughput nucleic acid technologies. This meeting inspired us to write this summary, which provides an overview of the state-of-the-art approaches based on physico-chemical foundation to modeling of the nucleic acids hybridization process on solid surfaces. In addition, practical application of current knowledge is emphasized.

Shear rheology of a cell monolayer

We report a systematic investigation of the mechanical properties of fibroblast cells using a novel cell monolayer rheology (CMR) technique. The new technique provides quantitative rheological parameters averaged over ~106 cells making the experiments highly reproducible. Using this method, we are able to explore a broad range of cell responses not accessible using other present day techniques. We perform harmonic oscillation experiments and step shear or step stress experiments to reveal different viscoelastic regimes. The evolution of the live cells under externally imposed cyclic loading and unloading is also studied. Remarkably, the initially nonlinear response becomes linear at long timescales as well as at large amplitudes. Within the explored rates, nonlinear behaviour is only revealed by the effect of a nonzero average stress on the response to small, fast deformations. When the cell cytoskeletal crosslinks are made permanent using a fixing agent, the large amplitude linear response disappears and the cells exhibit a stress stiffening response instead. This result shows that the dynamic nature of the cross-links and/or filaments is responsible for the linear stress-strain response seen under large deformations. We rule out the involvement of myosin motors in this using the inhibitor drug blebbistatin. These experiments provide a broad framework for understanding the mechanical responses of the cortical actin cytoskeleton of fibroblasts to different imposed mechanical stimuli.

Elastohydrodynamic study of actin filaments using fluorescence microscopy

We probed the bending of actin subject to external forcing and viscous drag. Single actin filaments were moved perpendicular to their long axis in an oscillatory way by means of an optically tweezed latex bead attached to one end of the filaments. Shapes of these polymers were observed by epifluorescence microscopy. They were found to be in agreement with predictions of semiflexible polymer theory and slender-body hydrodynamics. A persistence length of 7.460.2 mm could be extracted. @S1063-651X~97!50808-7# PACS number~s!: 87.10.1e, 83.10.Nn, 42.62.2b