Anna Pietuch

Toxicity of gold-nanoparticles: Synergistic effects of shape and surface functionalization on micromotility of epithelial cells

Abstract Nanoparticle exposure is monitored by a combination of two label-free and non-invasive biosensor devices which detect cellular shape and viscoelasticity (quartz crystal microbalance), cell motility and the dynamics of epithelial cell-cell contacts (electric cell-substrate impedance sensing). With these tools we have studied the impact of nanoparticle shape on cellular physiology. Gold (Au) nanoparticles coated with CTAB were synthesized and studied in two distinct shapes: Spheres with a diameter of (43 ± 4) nm and rods with a size of (38 ± 7) nm × (17 ± 3) nm. Dose-response experiments were accompanied by conventional cytotoxicity tests as well as fluorescence and dark-field microscopy to visualize the intracellular particle distribution. We found that spherical gold nanoparticles with identical surface functionalization are generally more toxic and more efficiently ingested than rod-shaped particles. We largely attribute the higher toxicity of CTAB-coated spheres as compared to rod-shaped particles to a higher release of toxic CTAB upon intracellular aggregation.

Mechanics of spreading cells probed by atomic force microscopy

Cellular adhesion and motility are fundamental processes in biological systems such as morphogenesis and tissue homeostasis. During these processes, cells heavily rely on the ability to deform and supply plasma membrane from pre-existing membrane reservoirs, allowing the cell to cope with substantial morphological changes. While morphological changes during single cell adhesion and spreading are well characterized, the accompanying alterations in cellular mechanics are scarcely addressed. Using the atomic force microscope, we measured changes in cortical and plasma membrane mechanics during the transition from early adhesion to a fully spread cell. During the initial adhesion step, we found that tremendous changes occur in cortical and membrane tension as well as in membrane area. Monitoring the spreading progress by means of force measurements over 2.5 h reveals that cortical and membrane tension become constant at the expense of excess membrane area. This was confirmed by fluorescence microscopy, which shows a rougher plasma membrane of cells in suspension compared with spread ones, allowing the cell to draw excess membrane from reservoirs such as invaginations or protrusions while attaching to the substrate and forming a first contact zone. Concretely, we found that cell spreading is initiated by a transient drop in tension, which is compensated by a decrease in excess area. Finally, all mechanical parameters become almost constant although morphological changes continue. Our study shows how a single cell responds to alterations in membrane tension by adjusting its overall membrane area. Interference with cytoskeletal integrity, membrane tension and excess surface area by administration of corresponding small molecular inhibitors leads to perturbations of the spreading process.

Membrane tension homeostasis of epithelial cells through surface area regulation in response to osmotic stress

Osmotic stress poses one of the most fundamental challenges to living cells. Particularly, the largely inextensible plasma membrane of eukaryotic cells easily ruptures under in-plane tension calling for sophisticated strategies to readily respond to osmotic stress. We describe how epithelial cells react and adapt mechanically to the exposure to hypotonic and hypertonic solutions in the context of a confluent monolayer. Site-specific indentation experiments in conjunction with tether pulling on individual cells have been carried out with an atomic force microscope to reveal spatio-temporal changes in membrane tension and surface area. We found that cells compensate for an increase in lateral tension due to hypoosmotic stress by sacrificing excess of membrane area stored in protrusions and invaginations such as microvilli and caveolae. At mild hypotonic conditions lateral tension increases partly compensated by surface are regulation, i.e. the cell sacrifices some of its membrane reservoirs. A loss of membrane-actin contacts occurs upon exposure to stronger hypotonic solutions giving rise to a drop in lateral tension. Tension release recovers on longer time scales by an increasing endocytosis, which efficiently removes excess membrane from the apical side to restore the initial pre-stress. Hypertonic solutions lead to shrinkage of cells and collapse of the apical membrane onto the cortex. Exposure to distilled water leads to stiffening of cells due to removal of excess surface area and tension increase due to elevated osmotic pressure across the plasma membrane.

Tension Monitoring during Epithelial-to-Mesenchymal Transition Links the Switch of Phenotype to Expression of Moesin and Cadherins in NMuMG Cells

Structural alterations during epithelial-to-mesenchymal transition (EMT) pose a substantial challenge to the mechanical response of cells and are supposed to be key parameters for an increased malignancy during metastasis. Herein, we report that during EMT, apical tension of the epithelial cell line NMuMG is controlled by cell-cell contacts and the architecture of the underlying actin structures reflecting the mechanistic interplay between cellular structure and mechanics. Using force spectroscopy we find that tension in NMuMG cells slightly increases 24 h after EMT induction, whereas upon reaching the final mesenchymal-like state characterized by a complete loss of intercellular junctions and a concerted down-regulation of the adherens junction protein E-cadherin, the overall tension becomes similar to that of solitary adherent cells and fibroblasts. Interestingly, the contribution of the actin cytoskeleton on apical tension increases significantly upon EMT induction, most likely due to the formation of stable and highly contractile stress fibers which dominate the elastic properties of the cells after the transition. The structural alterations lead to the formation of single, highly motile cells rendering apical tension a good indicator for the cellular state during phenotype switching. In summary, our study paves the way towards a more profound understanding of cellular mechanics governing fundamental morphological programs such as the EMT.

Ezrin is a Major Regulator of Membrane Tension in Epithelial Cells

Plasma membrane tension is responsible for a variety of cellular functions such as motility, cell division, and endocytosis. Since membrane tension is dominated by the attachment of the actin cortex to the inner leaflet of the plasma membrane, we investigated the importance of ezrin, a major cross-linker of the membrane-cytoskeleton interface, for cellular mechanics of confluent MDCK II cells. For this purpose, we carried out ezrin depletion experiments and also enhanced the number of active ezrin molecules at the interface. Mechanical properties were assessed by force indentation experiments followed by membrane tether extraction. PIP2 micelles were injected into individual living cells to reinforce the linkage between plasma membrane and actin-cortex, while weakening of this connection was reached by ezrin siRNA and administration of the inhibitors neomycin and NSC 668394, respectively. We observed substantial stiffening of cells and an increase in membrane tension after addition of PIP2 micelles. In contrast, reduction of active ezrin led to a decrease of membrane tension accompanied by loss of excess surface area, increase in cortical tension, remodelling of actin cytoskeleton, and reduction of cell height. The data confirm the importance of the ezrin-mediated connection between plasma membrane and cortex for cellular mechanics and cell morphology.

Elastic properties of cells in the context of confluent cell monolayers: impact of tension and surface area regulation

Epithelial cells usually form a dense continuous cobblestone-like sheet that is frequently exposed to a variety of mechanical challenges encompassing osmotic stress and external forces. The response to external forces was investigated and the question of how individual polar epithelial cells organized in confluent monolayers respond to pharmaceutical stimuli targeting the key players of cellular mechanics was answered. In particular, we ask how epithelial cells respond to changes in cortical and membrane tension by surface area regulation if challenged by diverse chemical and mechanical cues. Here, a tension-based model is used that allows capturing the relevant modes of cell deformation. Together with independent measurements of membrane tension, cortical tension and excess surface area of confluent MDCK II cells it is possible to draw a mechanistic picture of how confluent cells respond to mechanical stimuli in general. Changes in tension are provoked by external stimuli directed towards the contractile actomyosin cortex (cytochalasin D, blebbistatin), and changes in the excess surface area are produced by cholesterol extraction (methyl-β-cyclodextrin) or inhibition of dynamin (dynasore). A combination of AFM-indentation experiments with membrane–tether pulling at the same position allowed us to simultaneously monitor changes in membrane tension, cortical tension and excess surface area. Generally, we observed that removing or producing excess surface area of the plasma membrane readily adjusts membrane tension that is pivotal for the mechanical response of confluent cells. We found that isolated apical membranes from confluent MDCK II monolayers display similar mechanical properties as the apical side of living MDCK II cells in a confluent monolayer confirming that membrane mechanics in conjunction with cytoskeletal adhesion dominates the elastic response of confluent epithelial cells at large strain.

Formation of silicones mediated by the sponge enzyme silicatein-α

The sponge-restricted enzyme silicatein-α catalyzes in vivo silica formation from monomeric silicon compounds from sea water (i.e. silicic acid) and plays the pivotal role during synthesis of the siliceous sponge spicules. Recombinant silicatein-α, which was cloned from the demosponge Suberites domuncula (phylum Porifera), is shown to catalyze in vitro condensation of alkoxy silanes during a phase transfer reaction at neutral pH and ambient temperature to yield silicones like the straight-chained polydimethylsiloxane (PDMS). The reported condensation reaction is considered to be the first description of an enzymatically enhanced organometallic condensation reaction.

In situ Synthesis of Lipopeptides as Versatile Receptors for the Specific Binding of Nanoparticles and Liposomes to Solid-Supported Membranes

A detailed study of the in situ coupling of small peptides such as CGGH6 (H6) and CGWK8 (K8) to maleimide functionalized phospholipid bilayers is presented. Individually addressable microstructured membranes are employed to unequivocally probe the conjugation. The in situ coupling of peptides via a terminal cysteine moiety to maleimide functionalized phospholipids is shown to be a convenient and versatile way to selectively fabricate peptide-modified phospholipid bilayers serving as specific receptor platforms for functionalized vesicles and nanoparticles. Specific binding of functional vesicles to the peptide-modified bilayers is achieved by either histidine complexation with Ni-NTA-DOGS containing vesicles or electrostatic interaction between positively charged oligolysine bearing lipopeptides and negatively charged POPC/POPG vesicles. Peptide receptors are also found to be easily accessible from the aqueous phase and not buried within the membrane interior.

Phosphatidylinositol 4,5-Bisphosphate Alters the Number of Attachment Sites between Ezrin and Actin Filaments A COLLOIDAL PROBE STUDY

Background: Ezrin can establish a dynamic linkage between plasma membrane and cytoskeleton. Results: The individual bond strength between ezrin and F-actin is small, but the number of attachment sites is significantly altered by phosphatidylinositol 4,5-bisphosphate (PIP2). Conclusion: PIP2 activates ezrin to establish multiple weak ezrin/F-actin interactions. Significance: Plasma membrane tension is maintained by ezrin/F-actin interactions. Direct linkage between the plasma membrane and the actin cytoskeleton is controlled by the protein ezrin, a member of the ezrin-radixin-moesin protein family. To function as a membrane-cytoskeleton linker, ezrin needs to be activated in a process that involves binding of ezrin to phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphorylation of a conserved threonine residue. Here, we used colloidal probe microscopy to quantitatively analyze the interaction between ezrin and F-actin as a function of these activating factors. We show that the measured individual unbinding forces between ezrin and F-actin are independent of the activating parameters, in the range of approximately 50 piconewtons. However, the cumulative adhesion energy greatly increases in the presence of PIP2 demonstrating that a larger number of bonds between ezrin and F-actin has formed. In contrast, the phosphorylation state, represented by phosphor-mimetic mutants of ezrin, only plays a minor role in the activation process. These results are in line with in vivo experiments demonstrating that an increase in PIP2 concentration recruits more ezrin to the apical plasma membrane of polarized cells and significantly increases the membrane tension serving as a measure of the adhesion sites between the plasma membrane and the F-actin network.

Cell Adhesion to Ordered Pores: Consequences for Cellular Elasticity

The adhesion of MDCK II cells to porous and non-porous silicon substrates has been investigated by means of fluorescence and atomic force microscopy. The MDCK II cell density and the average height of the cells were increased on porous silicon substrates with regular 1.2 μm pores as compared to flat, non-porous surfaces. In addition, we found a substantially reduced actin cytoskeleton within confluent cells cultured on the macroporous substrate compared to flat surfaces. The perturbation of the cytoskeleton relates to a significantly reduced expression of integrins on the porous area. The loss of stress fibers and cortical actin is accompanied by a dramatically reduced Youngs modulus of 0.15 kPa compared to 6 kPa on flat surfaces as revealed by site-specific force–indentation experiments.