Tissue rheology in embryonic organization

Tissue morphogenesis in multicellular organisms is brought about by spatiotemporal coordination of mechanical and chemical signals. Extensive work on how mechanical forces together with the well‐established morphogen signalling pathways can actively shape living tissues has revealed evolutionary conserved mechanochemical features of embryonic development. More recently, attention has been drawn to the description of tissue material properties and how they can influence certain morphogenetic processes. Interestingly, besides the role of tissue material properties in determining how much tissues deform in response to force application, there is increasing theoretical and experimental evidence, suggesting that tissue material properties can abruptly and drastically change in development. These changes resemble phase transitions, pointing at the intriguing possibility that important morphogenetic processes in development, such as symmetry breaking and self‐organization, might be mediated by tissue phase transitions. In this review, we summarize recent findings on the regulation and role of tissue material properties in the context of the developing embryo. We posit that abrupt changes of tissue rheological properties may have important implications in maintaining the balance between robustness and adaptability during embryonic development

Impact of substrate topology, chemical stimuli and Janus nanoparticles on cellular properties

Cellular behavior is influenced by many biochemical but also physical factors in the direct cellular environment. Thereby, cells not only react to external cues, the interaction between cells and their environment is also dependent on the properties of the cell itself. The endocytosis of nanoparticles for example depends on the intermolecular forces between plasma membrane and particle as well as on the mechanical properties of the membrane. In the first part of this thesis I focus on the interaction between inorganic Janus nanoparticles, a new type of nanomaterials, which possess amphiphilic properties, and model membranes. In coarse grain simulations it has been demostrated that incubation of membranes with these particles lead either to pore formation in the lipid bilayer or to tubulation and vesiculation by long-range attractive interaction between particles bound to the membrane. Conducting surface plasmon resonance spectroscopy experiments I show that the binding energy of the used inorganic Janus particles to a solid supported monolayer could be sufficient to induce tubulation of tension-free membranes but is to small to provide the energy necessary to form a vesicle. This result is confirmed by fluorescence microscopic examination of giant unilamellar vesicles serving as a model system for the plasmamembrane, which were treated with Janus particles. Vesicles incubated with Janus particles show inwards directed membrane tubes, while incubation of vesicles with isotropic control particles had no effect on the membrane or could be attributed to an osmotic gradient. However, uptake experiments into living cells and cytotoxicity assays show no obvious difference between spherical particles and Janus particles, which hints for a negligible contribution of nanoparticle-induced tubulation or vesiculation to cellular uptake of nanoparticles and cytotoxicity. On the one hand mechanical properties of the cell influence the interaction between the cell and its environment. On the other hand, mechanical properties of cells change in response to environmental cues. Therefore, in the next part, atomic force microscopy-based microrheology is used to measure frequency-dependent mechanical properties of cells in different conditions. Fixation of cells with different chemical fixatives and transformation of epithelial cells to mesenchymal cells lead to more solid-like mechanical properties, while interaction with the actin cytoskeleton lead to more fluid-like properties. A comparison between malignant cells and non-malignant cells shows that malignant cells are more fluid-like compared to their non-malignant counterparts. Furthermore, the influence of substrate topology on cellular mechanics and cytoskeletal arrangement is examined. Changing physical properties of the substrate such as stiffness or topography has been shown to affect plenty of cellular processes like migration, proliferation, morphology or differentiation. Here, I investigate the impact of porous substrates on cellular morphology, cytoskeletal organization and elasticity in the context of confluent epithelial monolayers. I found that cells eventually self-organize to match the geometry of the pore pattern and remodel their actin cytoskeleton to reinforce their adhesion zone. Cells fluidize with increasing pore size up to 2 µm but eventually become stiffer if grown on very large pores up to 5 µm. The adhesion of cells to substrates is further researched by application of metal-induced energy transfer fluorescence lifetime imaging, which is used for the first time for this purpose. The fluorescence lifetime of a fluorophore in proximity to a metal layer is a function of the distance between fluorophore and metal layer. Applying a quantitative model of this interaction facilitates locating the fluorophore with nanometer precision in the axial direction up to 200 nm above the metal layer. By staining of the plasmamembrane I was able to image to basal membrane of three different cell lines and follow spreading of cells with high axial resolution. The introduced method is not restricted to measurement of cell/substrate distance and can be used for applications, which necessitate axial nanometer resolution in a range up to 200 nm

Biomechanical Characterization of Endothelial Cells Exposed to Shear Stress Using Acoustic Force Spectroscopy.

Characterizing mechanical properties of cells is important for understanding many cellular processes, such as cell movement, shape, and growth, as well as adaptation to changing environments. In this study, we explore the mechanical properties of endothelial cells that form the biological barrier lining blood vessels, whose dysfunction leads to development of many cardiovascular disorders. Stiffness of living endothelial cells was determined by Acoustic Force Spectroscopy (AFS), by pull parallel multiple functionalized microspheres located at the cell-cell periphery. The unique configuration of the acoustic microfluidic channel allowed us to develop a long-term dynamic culture protocol exposing cells to laminar flow for up to 48 h, with shear stresses in the physiological range (i.e., 6 dyn/cm2). Two different Endothelial cells lines, Human Aortic Endothelial Cells (HAECs) and Human Umbilical Vein Endothelial Cells (HUVECs), were investigated to show the potential of this tool to capture the change in cellular mechanical properties during maturation of a confluent endothelial monolayer. Immunofluorescence microscopy was exploited to follow actin filament rearrangement and junction formation over time. For both cell types we found that the application of shear-stress promotes the typical phenotype of a mature endothelium expressing a linear pattern of VE-cadherin at the cell-cell border and actin filament rearrangement along the perimeter of Endothelial cells. A staircase-like sequence of increasing force steps, ranging from 186 pN to 3.5 nN, was then applied in a single measurement revealing the force-dependent apparent stiffness of the membrane cortex in the kPa range. We also found that beads attached to cells cultured under dynamic conditions were harder to displace than cells cultured under static conditions, showing a stiffer membrane cortex at cell periphery. All together these results demonstrate that the AFS can identify changes in cell mechanics based on force measurements of adherent cells under conditions mimicking their native microenvironment, thus revealing the shear stress dependence of the mechanical properties of neighboring endothelial cells

Mechanical fluidity of fully suspended biological cells

Mechanical characteristics of single biological cells are used to identify and possibly leverage interesting differences among cells or cell populations. Fluidity---hysteresivity normalized to the extremes of an elastic solid or a viscous liquid---can be extracted from, and compared among, multiple rheological measurements of cells: creep compliance vs. time, complex modulus vs. frequency, and phase lag vs. frequency. With multiple strategies available for acquisition of this nondimensional property, fluidity may serve as a useful and robust parameter for distinguishing cell populations, and for understanding the physical origins of deformability in soft matter. Here, for three disparate eukaryotic cell types deformed in the suspended state via optical stretching, we examine the dependence of fluidity on chemical and environmental influences around a time scale of 1 s. We find that fluidity estimates are consistent in the time and the frequency domains under a structural damping (power-law or fractional derivative)model, but not under an equivalent-complexity lumpedcomponent (spring-dashpot) model; the latter predicts spurious time constants. Although fluidity is suppressed by chemical crosslinking, we find that adenosine triphosphate (ATP) depletion in the cell does not measurably alter the parameter, and thus conclude that active ATP-driven events are not a crucial enabler of fluidity during linear viscoelastic deformation of a suspended cell. Finally, by using the capacity of optical stretching to produce near-instantaneous increases in cell temperature, we establish that fluidity increases with temperature---now measured in a fully suspended, sortable cell without the complicating factor of cell-substratum adhesion

Spectral domain phase microscopy for local measurements of cytoskeletal rheology in single cells

We present spectral domain phase microscopy (SDPM) as a new tool for measurements at the cellular scale. SDPM is a functional extension of spectral domain optical coherence tomography that allows for the detection of cellular motions and dynamics with nanometer-scale sensitivity in real time. Our goal was to use SDPM to investigate the mechanical properties of the cytoskeleton of MCF-7 cells. Magnetic tweezers were designed to apply a vertical force to ligand-coated magnetic beads attached to integrin receptors on the cell surfaces. SDPM was used to resolve cell surface motions induced by the applied stresses. The cytoskeletal response to an applied force is shown for both normal cells and those with compromised actin networks due to treatment with Cytochalasin D. The cell response data were fit to several models for cytoskeletal rheology, including one- and two-exponential mechanical models, as well as a power law. Finally, we correlated displacement measurements to physical characteristics of individual cells to better compare properties across many cells, reducing the coefficient of variation of extracted model parameters by up to 50%

Elasticity spectra as a tool to investigate actin cortex mechanics

Background: The mechanical properties of single living cells have proven to be a powerful marker of the cell physiological state. The use of nanoindentation-based single cell force spectroscopy provided a wealth of information on the elasticity of cells, which is still largely to be exploited. The simplest model to describe cell mechanics is to treat them as a homogeneous elastic material and describe it in terms of the Young’s modulus. Beside its simplicity, this approach proved to be extremely informative, allowing to assess the potential of this physical indicator towards high throughput phenotyping in diagnostic and prognostic applications. Results: Here we propose an extension of this analysis to explicitly account for the properties of the actin cortex. We present a method, the Elasticity Spectra, to calculate the apparent stiffness of the cell as a function of the indentation depth and we suggest a simple phenomenological approach to measure the thickness and stiffness of the actin cortex, in addition to the standard Young’s modulus. Conclusions: The Elasticity Spectra approach is tested and validated on a set of cells treated with cytoskeleton-affecting drugs, showing the potential to extend the current representation of cell mechanics, without introducing a detailed and complex description of the intracellular structure

Giardia lamblia growth in viscoelastic fluids

Giardia lamblia is a single-celled protozoan parasite that when ingested, causes diarrheal disease and infects 33% of people in developing countries. Previous studies observe Giardia in water-like fluids, but Giardia\u27s infectious environment consists of viscoelastic mucus in the small intestine. Therefore, Giardia was cultured in viscoelastic fluids, and its population growth was observed in vitro. To create shear-thinning viscoelastic fluids, 0.2% and 0.4% long-chain polyacrylamide (LCPAM) was added to cell culture media. Giardia was cultured in control media, 0.2% LCPAM, and 0.4% LCPAM, and population growth was quantitatively determined over time. Increasing LCPAM concentration resulted in a solution with higher viscosity and elasticity. Experimental results suggest that Giardia growth is delayed in more viscoelastic fluids, but the population adjusts to the viscoelastic environments over time