Biology subjects

Fabry, B.

Publications and source records attributed to Fabry, B..

27 records · Page 2Linked to original sources

bioRxiv2021

Stress relaxation amplitude of hydrogels determines migration, proliferation, and morphology of cells in 3-D

The viscoelastic behavior of hydrogel matrices sensitively influences the cell behavior in 3D culture and biofabricated tissue model systems. Previous reports have demonstrated that cells tend to adhere, spread, migrate and proliferate better in hydrogels with pronounced stress relaxation. However, it is currently unknown if cells respond more sensitively to the amplitude of stress relaxation, or to the relaxation time constant. To test this, we compare the behavior of fibroblasts cultured for up to 10 days in alginate and oxidized alginate hydrogels with similar Youngs moduli but diverging stress relaxation behavior. We find that fibroblasts elongate, migrate and proliferate better in hydrogels that display a higher stress relaxation amplitude. By contrast, the cells response to the relaxation time constant was less pronounced and less consistent. Together, these data suggest that it is foremost the stress relaxation amplitude of the matrix that determines the ability of cells to locally penetrate and remodel the matrix, which subsequently leads to better spreading, faster migration, and higher cell proliferation. We conclude that the stress relaxation amplitude is a central design parameter for optimizing cell behavior in 3-D hydrogels.

biophysics↗

bioRxiv2021

Biologging of emperor penguins - attachment techniques and associated deployment performance

An increasing number of marine animals are equipped with biologgers, to study their physiology, behaviour and ecology, often for conservation purposes. To minimise the impacts of biologgers on the animals welfare, the Refinement principle from the Three Rs framework (Replacement, Reduction, Refinement) urges to continuously test and evaluate new and updated biologging protocols. Here, we propose alternative and promising techniques for emperor penguin (Aptenodytes forsteri) capture and on-site logger deployment that aim to mitigate the potential negative impacts of logger deployment on these birds. We equipped adult emperor penguins for short-term (GPS, Time-Depth Recorder (TDR)) and long-term (i.e. planned for one year) deployments (ARGOS platforms, TDR), as well as juvenile emperor penguins for long-term deployments (ARGOS platforms) in the Weddell Sea area where they had not yet been studied. We describe and qualitatively evaluate our protocols for the attachment of biologgers on-site at the colony, the capture of the animals and the recovery of the devices after deployment. We report unprecedented recaptures of long-term equipped adult emperor penguins (50% of equipped individuals recaptured after 290 days). Our data demonstrate that the traditional technique of long-term attachment by gluing the biologgers directly to the back feathers is detrimental to the birds. It causes excessive feather breakage and the loss of the devices at an early stage. We therefore propose an alternative method of attachment for back-mounted devices. This technique led to successful year-round deployments on 37.5% of the equipped juveniles. Finally, we also disclose the first deployments of leg-bracelet mounted TDRs on emperor penguins. Our findings highlight the importance of monitoring potential impacts of biologger deployments on the animals and the need to remain critical towards established and new protocols.

ecology↗

bioRxiv2021

Juvenile emperor penguin range calls for extended conservation measures in the Southern Ocean

To protect the unique Southern Ocean biodiversity, conservation measures like marine protected areas (MPAs) are implemented based on the known habitat distribution of ecologically important species. However, distribution models focus on adults, neglecting that immatures animals can inhabit vastly different areas. Here, we show that current conservation efforts in the Southern Ocean are insufficient for ensuring the protection of the highly mobile Emperor penguin. We find that juveniles spend [~]90% of their time outside the boundaries of proposed and existing MPAs, and that their distribution extends far beyond (> 1500 km) the species extent of occurrence as defined by the International Union for Conservation of Nature. We argue that strategic conservation plans for Emperor penguin and long-lived ecologically important species must consider the dynamic habitat range of all age classes.

ecology↗

bioRxiv2020

Plectin Ensures Intestinal Epithelial Integrity and Protects Colon Against Colitis

Plectin, a highly versatile cytolinker protein, provides tissues with mechanical stability through the integration of intermediate filaments (IFs) with cell junctions. Here, we hypothesize that plectin-controlled cytoarchitecture is a critical determinant of the intestinal barrier function and homeostasis. Mice lacking plectin in intestinal epithelial cells (IEC; Ple{Delta}IEC) spontaneously developed colitis characterized by extensive detachment of IECs from the basement membrane (BM), increased intestinal permeability, and inflammatory lesions. Moreover, plectin expression was reduced in colons of ulcerative colitis (UC) patients and negatively correlated with the severity of colitis. Mechanistically, plectin deficiency in IECs led to aberrant keratin filament (KF) network organization and formation of dysfunctional hemidesmosomes (HDs) and intercellular junctions. In addition, the hemidesmosomal 6{beta}4 integrin (Itg) receptor showed attenuated association with KFs, and protein profiling revealed prominent downregulation of junctional constituents. Consistent with effects of plectin loss in the intestinal epithelium, plectin-deficient IECs exhibited remarkably reduced mechanical stability and limited adhesion capacity in vitro. Feeding mice with a low-residue liquid diet that reduced mechanical stress and antibiotic treatment successfully mitigated epithelial damage in the Ple{Delta}IEC colon.

molecular biology↗

bioRxiv2020

pyTFM: A tool for Traction Force and Monolayer Stress Microscopy

Cellular force generation and force transduction are of fundamental importance for numerous biological processes and can be studied with the methods of Traction Force Microscopy (TFM) and Monolayer Stress Microscopy. Traction Force Microscopy and Monolayer Stress Microscopy solve the inverse problem of reconstructing cell-matrix tractions and inter- and intra-cellular stresses from the measured cell force-induced deformations of an adhesive substrate with known elasticity. Although several laboratories have developed software for Traction Force Microscopy and Monolayer Stress Microscopy computations, there is currently no software package available that allows non-expert users to perform a full evaluation of such experiments. Here we present pyTFM, a tool to perform Traction Force Microscopy and Monolayer Stress Microscopy on single cells, cell patches and cell layers grown in a 2-dimensional environment. pyTFM was optimized for ease-of-use; it is open-source and well documented (hosted at https://pytfm.readthedocs.io/) including usage examples and explanations of the theoretical background. pyTFM can be used as a standalone Python package or as an add-on to the image annotation tool ClickPoints. In combination with the ClickPoints environment, pyTFM allows the user to set all necessary analysis parameters, select regions of interest, examine the input data and intermediary results, and calculate a wide range of parameters describing forces, stresses, and their distribution. The Monolayer Stress Microscopy implementation in pyTFM allows for the analysis of small cell patches and single cells; we analyze the accuracy and performance of Traction Force Microscopy and Monolayer Stress Microscopy algorithms using synthetic and experimental data from epithelial cell patches.

biophysics↗

bioRxiv2019

High-force magnetic tweezers with hysteresis-free force feedback

Magnetic tweezers based on solenoids with iron alloy cores are widely used to apply large forces (~100 nN) onto micron-sized (~5 m) superparamagnetic particles for mechanical manipulation or microrheological measurements at the cellular and molecular level. The precision of magnetic tweezers, however, is limited by the magnetic hysteresis of the core material, especially for time-varying force protocols. Here, we eliminate magnetic hysteresis by a feedback control of the magnetic induction, which we measure with a Hall sensor mounted to the distal end of the solenoid core. We find that the generated force depends on the induction according to a power-law relationship, and on the bead-tip distance according to a stretched exponential relationship. Together, both relationships allow for an accurate force calibration and precise force feedback with only 3 calibration parameters. We apply our method to measure the force-dependence of the viscoelastic and plastic properties of fibroblasts using a protocol with stepwise increasing and decreasing forces. We find that soft cells show an increasing stiffness but decreasing plasticity at higher forces, indicating a pronounced stress stiffening of the cytoskeleton. By contrast, stiff cells show no stress stiffening but an increasing plasticity at higher forces. These findings indicate profound differences between soft and stiff cells regarding their protection mechanisms against external mechanical stress. In summary, our method increases the precision, simplifies the handling and extends the applicability of magnetic tweezers. SIGNIFICANCEMagnetic tweezers are widely used, versatile tools to investigate the mechanical behavior of cells or to measure the strength of receptor-ligand bonds. A limitation of existing high-force magnetic tweezer setups, however, is caused by the magnetic hysteresis of the tweezer core material. This magnetic hysteresis requires that the tweezer core must be de-magnetized (de-Gaussed) prior to each measurement, and that flexible force protocols with decreasing forces are not possible. We describe how these limitations can be overcome with a force feedback though direct magnetic field measurement. We demonstrate the applicability of our setup by investigating the visco-elastic and plastic deformations of fibroblasts to forces of different amplitudes.

biophysics↗

bioRxiv2019

Cryopreservation impairs cytotoxicity and migration of NK cells in 3-D tissue: Implications for cancer immunotherapy

Natural killer (NK) cells are important effector cells in the immune response to cancer. Clinical trials on adoptively transferred NK cells in patients with solid tumors, however, have thus far been unsuccessful. As NK cells need to pass stringent safety evaluation for clinical use, the cells are cryopreserved to bridge the necessary evaluation time. While a degranulation assay confirms the ability of cryopreserved NK cells to kill target cells, we find a significant decrease of cytotoxicity after cryopreservation in a chromium release assay. We complement these standard assays with measurements of NK cell motility and cytotoxicity in 3-dimensional (3-D) collagen gels that serve as a substitute for connective tissue. We find a 5.6 fold decrease of cytotoxicity after cryopreservation and establish that this is mainly caused by a 6-fold decrease in the fraction of motile NK cells. These findings may explain the persistent failure of NK cell therapy in patients with solid tumors and highlight the crucial role of a 3-D environment for testing NK cell function.\n\nSynopsisCryopreservation of natural killer (NK) cells dramatically impairs their motility and cytotoxicity in tissue. This finding may explain the persistent failure of clinical trials in which NK cell therapy is used for treating solid tumors.

immunology↗

bioRxiv2019

Measurement of skeletal muscle fiber contractility with high-speed traction microscopy

We describe a technique for simultaneous quantification of the contractile forces and cytosolic calcium dynamics of muscle fibers embedded in three-dimensional biopolymer gels. We derive a scaling law for linear elastic matrices such as basement membrane extract hydrogels (Matrigel) that allows us to measure contractile force from the shape of the relaxed and contracted muscle cell and the Youngs modulus of the matrix, without further knowledge of the matrix deformations surrounding the cell and without performing computationally intensive inverse force reconstruction algorithms. We apply our method to isolated mouse flexor digitorum brevis (FDB) fibers that are embedded in 10 mg/ml Matrigel. Upon electrical stimulation, individual FDB fibers show twitch forces of 0.37 {micro}N {+/-} 0.15 {micro}N and tetanic forces (100 Hz stimulation frequency) of 2.38 {micro}N {+/-} 0.71 {micro}N, corresponding to a tension of 0.44 kPa {+/-} 0.25 kPa and 2.53 kPa {+/-} 1.17 kPa, respectively. Contractile forces of FDB fibers increase in response to caffeine and the troponin-calcium-stabilizer Tirasemtiv, similar to responses measured in whole muscle. From simultaneous high-speed measurements of cell length changes and cytosolic calcium concentration using confocal line scanning at a frequency of 2048 Hz, we show that twitch and tetanic force responses to electric pulses follow the low-pass filtered calcium signal. In summary, we present a technically simple high speed and high throughput method for measuring contractile forces and cytosolic calcium dynamics of single muscle fibers. We expect that our method will help to reduce preparation time, costs, and the number of sacrificed animals needed for experiments such as drug testing.\n\nStatement of significanceWe describe a high speed, high throughput method for the simultaneous measurement of contractile force and cytoplasmic calcium dynamics following electrical pulse stimulation of muscle fibers embedded in a 3-dimensional biopolymer matrix. In contrast to the classical approach of attaching muscle fibers to a force-transducer, our method allows for a highly efficient, parallel analysis of large numbers of fibers under different treatment conditions.

bioengineering↗

bioRxiv2019

Collective forces of tumor spheroids in three-dimensional biopolymer networks

We describe a method for quantifying the contractile forces that tumor spheroids collectively exert on highly nonlinear three-dimensional collagen networks. While three-dimensional traction force microscopy for single cells in a nonlinear matrix is computationally complex due to the variable cell shape, here we exploit the spherical symmetry of tumor spheroids to derive a scale-invariant relationship between spheroid contractility and the surrounding matrix deformations. This relationship allows us to directly translate the magnitude of matrix deformations to the total contractility of arbitrarily sized spheroids. We show that collective forces of tumor spheroids reflect the contractility of individual cells for up to 1h after seeding, while collective forces on longer time-scales are guided by mechanical feedback from the extracellular matrix.

biophysics↗