A minimal physical model for curvotaxis driven by curved protein complexes at the cell's leading edge.

Cells often migrate on curved surfaces inside the body, such as curved tissues, blood vessels, or highly curved protrusions of other cells. Recent in vitro experiments provide clear evidence that motile cells are affected by the curvature of the substrate on which they migrate, preferring certain curvatures to others, termed "curvotaxis." The origin and underlying mechanism that gives rise to this curvature sensitivity are not well understood. Here, we employ a "minimal cell" model which is composed of a vesicle that contains curved membrane protein complexes, that exert protrusive forces on the membrane (representing the pressure due to actin polymerization). This minimal-cell model gives rise to spontaneous emergence of a motile phenotype, driven by a lamellipodia-like leading edge. By systematically screening the behavior of this model on different types of curved substrates (sinusoidal, cylinder, and tube), we show that minimal ingredients and energy terms capture the experimental data. The model recovers the observed migration on the sinusoidal substrate, where cells move along the grooves (minima), while avoiding motion along the ridges. In addition, the model predicts the tendency of cells to migrate circumferentially on convex substrates and axially on concave ones. Both of these predictions are verified experimentally, on several cell types. Altogether, our results identify the minimization of membrane-substrate adhesion energy and binding energy between the membrane protein complexes as key players of curvotaxis in cell migration.

Prestress and Area Compressibility of Actin Cortices Determine the Viscoelastic Response of Living Cells.

Shape, dynamics, and viscoelastic properties of eukaryotic cells are primarily governed by a thin, reversibly cross-linked actomyosin cortex located directly beneath the plasma membrane. We obtain time-dependent rheological responses of fibroblasts and MDCK II cells from deformation-relaxation curves using an atomic force microscope to access the dependence of cortex fluidity on prestress. We introduce a viscoelastic model that treats the cell as a composite shell and assumes that relaxation of the cortex follows a power law giving access to cortical prestress, area-compressibility modulus, and the power law exponent (fluidity). Cortex fluidity is modulated by interfering with myosin activity. We find that the power law exponent of the cell cortex decreases with increasing intrinsic prestress and area-compressibility modulus, in accordance with previous finding for isolated actin networks subject to external stress. Extrapolation to zero tension returns the theoretically predicted power law exponent for transiently cross-linked polymer networks. In contrast to the widely used Hertzian mechanics, our model provides viscoelastic parameters independent of indenter geometry and compression velocity.

Adhesion of Epithelial Cells to PNIPAm Treated Surfaces for Temperature-Controlled Cell-Sheet Harvesting.

Stimuli responsive polymer coatings are a common motive for designing surfaces for cell biological applications. In the present study, we have characterized temperature dependent adhesive properties of poly(N-isopropylacrylamide) (PNIPAm) microgel coated surfaces (PMS) using various atomic force microscopy based approaches. We imaged and quantified the material properties of PMS upon a temperature switch using quantitative AFM imaging but also employed single-cell force spectroscopy (SCFS) before and after decreasing the temperature to assess the forces and work of initial adhesion between cells and PMS. We performed a detailed analysis of steps in the force-distance curves. Finally, we applied colloid probe atomic force microscopy (CP-AFM) to analyze the adhesive properties of two major components of the extracellular matrix to PMS under temperature control, namely collagen I and fibronectin. In combination with confocal imaging, we could show that these two ECM components differ in their detachment properties from PNIPAm microgel films upon cell harvesting, and thus gained a deeper understanding of cell-sheet maturation and harvesting process and the involved partial ECM dissolution.

Novel micropatterning technique reveals dependence of cell-substrate adhesion and migration of social amoebas on parental strain, development, and fluorescent markers.

Cell-substrate adhesion of the social amoeba Dictyostelium discoideum, a model organism often used for the study of chemotaxis, is non-specific and does not involve focal adhesion complexes. Therefore, micropatterned substrates where adherent Dictyostelium cells are constrained to designated microscopic regions are difficult to make. Here we present a micropatterning technique for Dictyostelium cells that relies on coating the substrate with an ∼1µm thick layer of polyethylene glycol (PEG) gel. We show that, when plated on a substrate with narrow parallel stripes of PEG-gel and glass, Dictyostelium cells nearly exclusive adhere to and migrate along the glass stripes, thus providing a model system to study one-dimensional migration of amoeboid cells. Surprisingly, we find substantial differences in the adhesion to PEG-gel and glass stripes between vegetative and developed cells and between two different axenic laboratory strains of Dictyostelium, AX2 and AX4. Even more surprisingly, we find that the distribution of Dictyostelium cells between PEG-gel and glass stripes is significantly affected by the expression of several fluorescent protein markers of the cytoskeleton. We carry out atomic force microscopy based single cell force spectroscopy measurements that confirm that the force of adhesion to PEG-gel substrate can be significantly different between vegetative and developed cells, AX2 and AX4 cells, and cells with and without fluorescent markers. Thus, the choice of parental background, the degree of development, and the expression of fluorescent protein markers can all have a profound effect on cell-substrate adhesion and should be considered when comparing migration of cells and when designing micropatterned substrates.

Adhesion strategies of Dictyostelium discoideum- a force spectroscopy study.

Biological adhesion is essential for all motile cells and generally limits locomotion to suitably functionalized substrates displaying a compatible surface chemistry. However, organisms that face vastly varying environmental challenges require a different strategy. The model organism Dictyostelium discoideum (D.d.), a slime mould dwelling in the soil, faces the challenge of overcoming variable chemistry by employing the fundamental forces of colloid science. To understand the origin of D.d. adhesion, we realized and modified a variety of conditions for the amoeba comprising the absence and presence of the specific adhesion protein Substrate Adhesion A (sadA), glycolytic degradation, ionic strength, surface hydrophobicity and strength of van der Waals interactions by generating tailored model substrates. By employing AFM-based single cell force spectroscopy we could show that experimental force curves upon retraction exhibit two regimes. The first part up to the critical adhesion force can be described in terms of a continuum model, while the second regime of the curve beyond the critical adhesion force is governed by stochastic unbinding of individual binding partners and bond clusters. We found that D.d. relies on adhesive interactions based on EDL-DLVO (Electrical Double Layer-Derjaguin-Landau-Verwey-Overbeek) forces and contributions from the glycocalix and specialized adhesion molecules like sadA. This versatile mechanism allows the cells to adhere to a large variety of natural surfaces under various conditions.

Adhesion forces and cortical tension couple cell proliferation and differentiation to drive epidermal stratification.

To establish and maintain organ structure and function, tissues need to balance stem cell proliferation and differentiation rates and coordinate cell fate with position. By quantifying and modelling tissue stress and deformation in the mammalian epidermis, we find that this balance is coordinated through local mechanical forces generated by cell division and delamination. Proliferation within the basal stem/progenitor layer, which displays features of a jammed, solid-like state, leads to crowding, thereby locally distorting cell shape and stress distribution. The resulting decrease in cortical tension and increased cell-cell adhesion trigger differentiation and subsequent delamination, reinstating basal cell layer density. After delamination, cells establish a high-tension state as they increase myosin II activity and convert to E-cadherin-dominated adhesion, thereby reinforcing the boundary between basal and suprabasal layers. Our results uncover how biomechanical signalling integrates single-cell behaviours to couple proliferation, cell fate and positioning to generate a multilayered tissue.

Variability and Order in Cytoskeletal Dynamics of Motile Amoeboid Cells.

The chemotactic motion of eukaryotic cells such as leukocytes or metastatic cancer cells relies on membrane protrusions driven by the polymerization and depolymerization of actin. Here we show that the response of the actin system to a receptor stimulus is subject to a threshold value that varies strongly from cell to cell. Above the threshold, we observe pronounced cell-to-cell variability in the response amplitude. The polymerization time, however, is almost constant over the entire range of response amplitudes, while the depolymerization time increases with increasing amplitude. We show that cell-to-cell variability in the response amplitude correlates with the amount of Arp2/3, a protein that enhances actin polymerization. A time-delayed feedback model for the cortical actin concentration is consistent with all our observations and confirms the role of Arp2/3 in the observed cell-to-cell variability. Taken together, our observations highlight robust regulation of the actin response that enables a reliable timing of cell movement.

Membrane tension increases fusion efficiency of model membranes in the presence of SNAREs.

The large gap in time scales between membrane fusion occurring in biological systems during neurotransmitter release and fusion observed between model membranes has provoked speculations over a large number of possible factors that might explain this discrepancy. One possible reason is an elevated lateral membrane tension present in the presynaptic membrane. We investigated the tension-dependency of fusion using model membranes equipped with a minimal fusion machinery consisting of syntaxin 1, synaptobrevin and SNAP 25. Two different strategies were realized; one based on supported bilayers and the other one employing sessile giant liposomes. In the first approach, isolated patches of planar bilayers derived from giant unilamellar vesicles containing syntaxin 1 and preassembled SNAP 25 (ΔN-complex) were deposited on a dilatable PDMS sheet. In a second approach, lateral membrane tension was controlled through the adhesion of intact giant unilamellar vesicles on a functionalized surface. In both approaches fusion efficiency increases considerably with lateral tension and we identified a threshold tension of 3.4 mN m-1, at which the number of fusion events is increased substantially.

Shear force-based genetic screen reveals negative regulators of cell adhesion and protrusive activity.

The model organism Dictyostelium discoideum has greatly facilitated our understanding of the signal transduction and cytoskeletal pathways that govern cell motility. Cell-substrate adhesion is downstream of many migratory and chemotaxis signaling events. Dictyostelium cells lacking the tumor suppressor PTEN show strongly impaired migratory activity and adhere strongly to their substrates. We reasoned that other regulators of migration could be obtained through a screen for overly adhesive mutants. A screen of restriction enzyme-mediated integration mutagenized cells yielded numerous mutants with the desired phenotypes, and the insertion sites in 18 of the strains were mapped. These regulators of adhesion and motility mutants have increased adhesion and decreased motility. Characterization of seven strains demonstrated decreased directed migration, flatness, increased filamentous actin-based protrusions, and increased signal transduction network activity. Many of the genes share homology to human genes and demonstrate the diverse array of cellular networks that function in adhesion and migration.

How tetraspanins shape endothelial and leukocyte nano-architecture during inflammation.

Tetraspanins are ubiquitous membrane proteins that induce local membrane curvature and hence co-ordinate cell-to-cell contacts. This review highlights their role in inflammation, which requires control of the nano-architecture of attachment sites between endothelial cells and leukocytes. The active role of endothelial cells in preparing for transmigration of leukocytes and determining the severity of an inflammation is often underscored. A clear hint to endothelial pre-activation is their ability to protrude clustered adhesion proteins upward prior to leukocyte contact. The elevation of molecular adhesive platforms toward the blood stream is crucially dependent on tetraspanins. In addition, leukocytes require tetraspanins for their activation. The example of the B-cell receptor is referenced in some detail here, since it provides deeper insights into the receptor-coreceptor interplay. To lift the role of tetraspanins from an abstract model of inflammation toward a player of clinical significance, two pathologies are analyzed for the known contributions of tetraspanins. The recent publication of the first crystal structure of a full-length tetraspanin revealed a cholesterol-binding site, which provides a strong link to the pathophysiological condition of atherosclerosis. Dysregulation of the inflammatory cascade in autoimmune diseases by endothelial cells is exemplified by the involvement of tetraspanins in multiple sclerosis.

Noisy Oscillations in the Actin Cytoskeleton of Chemotactic Amoeba.

Biological systems with their complex biochemical networks are known to be intrinsically noisy. Here we investigate the dynamics of actin polymerization of amoeboid cells, which are close to the onset of oscillations. We show that the large phenotypic variability in the polymerization dynamics can be accurately captured by a generic nonlinear oscillator model in the presence of noise. We determine the relative role of the noise with a single dimensionless, experimentally accessible parameter, thus providing a quantitative description of the variability in a population of cells. Our approach, which rests on a generic description of a system close to a Hopf bifurcation and includes the effect of noise, can characterize the dynamics of a large class of noisy systems close to an oscillatory instability.

Cell-substrate impedance fluctuations of single amoeboid cells encode cell-shape and adhesion dynamics.

We show systematic electrical impedance measurements of single motile cells on microelectrodes. Wild-type cells and mutant strains were studied that differ in their cell-substrate adhesion strength. We recorded the projected cell area by time-lapse microscopy and observed irregular oscillations of the cell shape. These oscillations were correlated with long-term variations in the impedance signal. Superposed to these long-term trends, we observed fluctuations in the impedance signal. Their magnitude clearly correlated with the adhesion strength, suggesting that strongly adherent cells display more dynamic cell-substrate interactions.

The effect of surface charge on nonspecific uptake and cytotoxicity of CdSe/ZnS core/shell quantum dots.

In this work, cytotoxicity and cellular impedance response was compared for CdSe/ZnS core/shell quantum dots (QDs) with positively charged cysteamine-QDs, negatively charged dihydrolipoic acid-QDs and zwitterionic D-penicillamine-QDs exposed to canine kidney MDCKII cells. Pretreatment of cells with pharmacological inhibitors suggested that the uptake of nanoparticles was largely due to receptor-independent pathways or spontaneous entry for carboxylated and zwitterionic QDs, while for amine-functionalized particles involvement of cholesterol-enriched membrane domains is conceivable. Cysteamine-QDs were found to be the least cytotoxic, while D-penicillamine-QDs reduced the mitochondrial activity of MDCKII by 20-25%. Although the cell vitality appeared unaffected (assessed from the changes in mitochondrial activity using a classical MTS assay after 24 h of exposure), the binding of QDs to the cellular interior and their movement across cytoskeletal filaments (captured and characterized by single-particle tracking), was shown to compromise the integrity of the cytoskeletal and plasma membrane dynamics, as evidenced by electric cell-substrate impedance sensing.

Mechanical properties of MDCK II cells exposed to gold nanorods.

BACKGROUND: The impact of gold nanoparticles on cell viability has been extensively studied in the past. Size, shape and surface functionalization including opsonization of gold particles ranging from a few nanometers to hundreds of nanometers are among the most crucial parameters that have been focussed on. Cytoxicity of nanomaterial has been assessed by common cytotoxicity assays targeting enzymatic activity such as LDH, MTT and ECIS. So far, however, less attention has been paid to the mechanical parameters of cells exposed to gold particles, which is an important reporter on the cellular response to external stimuli. RESULTS: Mechanical properties of confluent MDCK II cells exposed to gold nanorods as a function of surface functionalization and concentration have been explored by atomic force microscopy and quartz crystal microbalance measurements in combination with fluorescence and dark-field microscopy. CONCLUSION: We found that cells exposed to CTAB coated gold nanorods display a concentration-dependent stiffening that cannot be explained by the presence of CTAB alone. The stiffening results presumably from endocytosis of particles removing excess membrane area from the cell's surface. Another aspect could be the collapse of the plasma membrane on the actin cortex. Particles coated with PEG do not show a significant change in elastic properties. This observation is consistent with QCM measurements that show a considerable drop in frequency upon administration of CTAB coated rods suggesting an increase in acoustic load corresponding to a larger stiffness (storage modulus).

Scanning x-ray nanodiffraction on Dictyostelium discoideum.

We have performed scanning x-ray nanobeam diffraction experiments on single cells of the amoeba Dictyostelium discoideum. Cells have been investigated in 1), freeze-dried, 2), frozen-hydrated (vitrified), and 3), initially alive states. The spatially resolved small-angle x-ray scattering signal shows characteristic streaklike patterns in reciprocal space, which we attribute to fiber bundles of the actomyosin network. From the intensity distributions, an anisotropy parameter can be derived that indicates pronounced local variations within the cell. In addition to nanobeam small-angle x-ray scattering, we have evaluated the x-ray differential phase contrast in view of the projected electron density. Different experimental aspects of the x-ray experiment, sample preparation, and data analysis are discussed. Finally, the x-ray results are correlated with optical microscopy (differential phase contrast and confocal microscopy of mutant strains with fluorescently labeled actin and myosin II), which have been carried out in live and fixed states, including optical microscopy under cryogenic conditions.

Cell substratum adhesion during early development of Dictyostelium discoideum.

Vegetative and developed amoebae of Dictyostelium discoideum gain traction and move rapidly on a wide range of substrata without forming focal adhesions. We used two independent assays to quantify cell-substrate adhesion in mutants and in wild-type cells as a function of development. Using a microfluidic device that generates a range of hydrodynamic shear stress, we found that substratum adhesion decreases at least 10 fold during the first 6 hr of development of wild type cells. This result was confirmed using a single-cell assay in which cells were attached to the cantilever of an atomic force probe and allowed to adhere to untreated glass surfaces before being retracted. Both of these assays showed that the decrease in substratum adhesion was dependent on the cAMP receptor CAR1 which triggers development. Vegetative cells missing talin as the result of a mutation in talA exhibited slightly reduced adhesive properties compared to vegetative wild-type cells. In sharp contrast to wild-type cells, however, these talA mutant cells did not show further reduction of adhesion during development such that after 5 hr of development they were significantly more adhesive than developed wild type cells. In addition, both assays showed that substrate adhesion was reduced in 0 hr cells when the actin cytoskeleton was disrupted by latrunculin. Consistent with previous observations, substrate adhesion was also reduced in 0 hr cells lacking the membrane proteins SadA or SibA as the result of mutations in sadA or sibA. However, there was no difference in the adhesion properties between wild type AX3 cells and these mutant cells after 6 hr of development, suggesting that neither SibA nor SadA play an essential role in substratum adhesion during aggregation. Our results provide a quantitative framework for further studies of cell substratum adhesion in Dictyostelium.

Modeling self-organized spatio-temporal patterns of PIP3 and PTEN during spontaneous cell polarization.

During spontaneous cell polarization of Dictyostelium discoideum cells, phosphatidylinositol (3,4,5)-triphoshpate (PIP3) and PTEN (phosphatase tensin homolog) have been identified as key signaling molecules which govern the process of polarization in a self-organized manner. Recent experiments have quantified the spatio-temporal dynamics of these signaling components. Surprisingly, it was found that membrane-bound PTEN can be either in a high or low state, that PIP3 waves were initiated in areas lacking PTEN through an excitable mechanism, and that PIP3 was degraded even though the PTEN concentration remained low. Here we develop a reaction-diffusion model that aims to explain these experimental findings. Our model contains bistable dynamics for PTEN, excitable dynamics for PIP3, and postulates the existence of two species of PTEN with different dephosphorylation rates. We show that our model is able to produce results that are in good qualitative agreement with the experiments, suggesting that our reaction-diffusion model underlies the self-organized spatio-temporal patterns observed in experiments.

Mammalian cell growth on gold nanoparticle-decorated substrates is influenced by the nanoparticle coating.

In this work, we study epithelial cell growth on substrates decorated with gold nanorods that are functionalized either with a positively charged cytotoxic surfactant or with a biocompatible polymer exhibiting one of two different end groups, resulting in a neutral or negative surface charge of the particle. Upon observation of cell growth for three days by live cell imaging using optical dark field microscopy, it was found that all particles supported cell adhesion while no directed cell migration and no significant particle internalization occurred. Concerning cell adhesion and spreading as compared to cell growth on bare substrates after 3 days of incubation, a reduction by 45% and 95%, respectively, for the surfactant particle coating was observed, whereas the amino-terminated polymer induced a reduction by 30% and 40%, respectively, which is absent for the carboxy-terminated polymer. Furthermore, interface-sensitive impedance spectroscopy (electric cell-substrate impedance sensing, ECIS) was employed in order to investigate the micromotility of cells added to substrates decorated with various amounts of surfactant-coated particles. A surface density of 65 particles/µm(2) (which corresponds to 0.5% of surface coverage with nanoparticles) diminishes micromotion by 25% as compared to bare substrates after 35 hours of incubation. We conclude that the surface coating of the gold nanorods, which were applied to the basolateral side of the cells, has a recognizable influence on the growth behavior and thus the coating should be carefully selected for biomedical applications of nanoparticles.