Biology subjects

Betz, T.

Publications and source records attributed to Betz, T..

24 records · Page 2Linked to original sources

bioRxiv2020

Microchip based microrheology via Acoustic Force Spectroscopy shows that endothelial cell mechanics follows a fractional viscoelastic model

Active microrheology is one of the main methods to determine the mechanical properties of cells and tissue, and the modelling of the viscoelastic properties of cells and tissue is under heavy debate with many competing approaches. Most experimental methods of active microrheology such as optical tweezers or atomic force microscopy based approaches rely on single cell measurements, and thus suffer from a low throughput. Here, we present a novel method for cell based microrheology using acoustic forces which allows multiplexed measurements of several cells in parallel. Acoustic Force Spectroscopy (AFS) is used to generate multi-oscillatory forces in the range of pN-nN on particles attached to primary human umbilical vein endothelial cells (HUVEC) cultivated inside a microfluidic chip. While the AFS was introduced as a single-molecule technique to measure mechanochemical properties of biomolecules, we exploit the AFS to measure the dynamic viscoelastic properties of cells exposed to different conditions, such as flow shear stresses or drug injections. By controlling the force and measuring the position of the particle, the complex shear modulus G*(ω) can be measured continuously over several hours. The resulting power-law shear moduli are consistent with fractional viscoelastic models. In our experiments we confirm a decrease in shear modulus after perturbing the actin cytoskeleton via cytochalasin B. This effect was reversible after washing out the drug. Although these measurements are possible, we provide critical information regarding the AFS as a measurement tool showing its capabilities and limitations. A key result is that for performing viscoelastic measurements with the AFS, a thorough calibration and careful data analysis is crucial, for which we provide protocols and guidelines.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2020

Global and local tension measurements in biomimetic skeletal muscle tissues reveals early mechanical homeostasis

The mechanical properties and tension of muscle tissue are tightly related to proper skeletal muscle function, which makes experimental access to the biomechanics of muscle tissue development a key requirement to advance our understanding of muscle function and development. Recently developed elastic in vitro culture chambers allow for raising 3D muscle tissue under controlled conditions and measurements of tissue force generation. However, these chambers are inherently incompatible with high resolution microscopy limiting their usability to global force measurements, and preventing the exploitation of modern fluorescence based investigation methods for live and dynamic measurements. Here we present a new chamber design pairing global force measurements, quantified from post deflection, with local tension measurements obtained from elastic hydrogel beads embedded in the muscle tissue. High resolution 3D video microscopy of engineered muscle development, enabled by the new chamber, shows an early mechanical tissue homeostasis that remains stable in spite of continued myotube maturation.Competing Interest StatementTB and AH have filed a patent for the presented chamber system.View Full Text

biophysics↗

bioRxiv2019

Cortical cell stiffness is independent of substrate mechanics

Cortical stiffness is an important cellular property that changes during migration, adhesion, and growth. Previous atomic force microscopy (AFM) indentation measurements of cells cultured on deformable substrates suggested that cells adapt their stiffness to that of their surroundings. Here we show that the force applied by AFM onto cells results in a significant deformation of the underlying substrate if it is softer than the cells. This soft substrate effect leads to an underestimation of a cells elastic modulus when analyzing data using a standard Hertz model, as confirmed by finite element modelling (FEM) and AFM measurements of calibrated polyacrylamide beads, microglial cells, and fibroblasts. To account for this substrate deformation, we developed the composite cell-substrate model (CoCS model). Correcting for the substrate indentation revealed that cortical cell stiffness is largely independent of substrate mechanics, which has significant implications for our interpretation of many physiological and pathological processes.

biophysics↗

bioRxiv2019

Actin accumulates nesprin-2 at the front of the nucleus during confined cell migration

The mechanisms by which cells exert forces on their nuclei to migrate through openings smaller than the nuclear diameter remain unclear. In microfluidic devices, the hourglass shape of the nucleus and its strain patterns as it translocates through narrow constrictions suggest pulling forces. We use CRISPR/Cas9 to fluorescently label nesprin-2 giant, a protein that links the cytoskeleton to the interior of the nucleus. We demonstrate that nesprin-2 giant accumulates at the front of the nucleus during nuclear deformation through narrow constrictions, independently of the nuclear lamina. We find that nesprins are more mobile than lamin A/C, at time scales similar to that of the accumulation. Using artificial constructs, we show that the actin-binding domain of nesprin-2 is necessary and sufficient to generate this accumulation, and that microtubules are not necessary. Actin filaments are organized in a barrel structure around the moving nucleus in the direction of movement, suggesting that this structure is responsible for redistribution of nesprins towards the front of the nucleus. Two-photon ablation and the use of drugs inhibiting the cytoskeleton demonstrate a pulling force on the nucleus from the front of the cell that is dependent on formin and actomyosin contractility. This elastic recoil is significantly reduced when nesprins are reduced at the nuclear envelope. We thus show that actin redistributes nesprin-2 giant towards the front of the nucleus and contributes to pulling the nucleus through narrow constrictions, in concert with myosin.

cell biology↗

bioRxiv2019

Actin modulates shape and mechanics of tubular membranes

The actin cytoskeleton shapes cells and also organizes internal membranous compartments. In particular, it interacts with membranes in intracellular transport of material in mammalian cells, yeast or plant cells. Tubular membrane intermediates, pulled along microtubule tracks, are involved during these processes, and destabilize into vesicles. While the role of actin in this destabilization process is still debated, literature also provide examples of membranous structures stabilization by actin. To directly address this apparent contradiction, we mimic the geometry of tubular intermediates with preformed membrane tubes. The growth of an actin sleeve at the tube surface is monitored spatio-temporally. Depending on network cohesiveness, actin is able to stabilize, or maintain membrane tubes under pulling. Indeed, on a single tube, thicker portions correlate with the presence of actin. Such structures relax over several minutes, and may provide enough time and curvature geometries for other proteins to act on tube stability.

biophysics↗

bioRxiv2019

Centering based on active diffusion in mouse oocytes is non-specific

The mechanism for nucleus centering in mouse oocytes results from a gradient of actin-positive vesicles. By microinjecting oil droplets and fluorescent beads, we analyze the consequences of the gradient of activity on transport of exogenous tracer particles of different sizes. We also use optical tweezers to probe rheological properties of the cytoplasm. We find that the gradient activity induces a general centering force, akin to an effective pressure gradient, leading to centering of oil droplets with velocities comparable to nuclear ones. High temporal resolution measurements reveal that passive particles, larger than 1{micro}m, experience the activity gradient by a biased diffusion towards the cell center. Unexpectedly, this general and size dependent mechanism is maintained in Meiosis I but contrasted by a further process that specifically off-centers the spindle. These antagonizing processes depend on myosin activity, thus we reconcile how the same molecular actors can have two opposite functions (centering versus off-centering).

cell biology↗