Cell swelling and cytoskeletal disruption are known to be secondary effects of cell membrane permeabilization induced by nanosecond pulsed electric fields (nsPEFs). In this study, we used healthy and cancer urothelial cells to investigate the role of Ca2+ influx on cytoskeleton remodeling and morphological changes of cells following exposure. A train of 200 nsPEFs (300 ns pulse duration, 10 Hz), delivered via contact electrodes, effectively permeabilized the cell membrane in an isosmotic physiological solution. Subsequent shrinkage of the actin cortex and a reduction in actin fluorescence were observed only in the presence of extracellular Ca2+. In its absence, no significant changes in the phalloidin-stained actin cortex were detected. Time-lapse imaging using scanning ion conductance microscopy (SICM) revealed that a significantly greater and more immediate increase in projected cell area and cell volume occurred after nsPEFs exposure in a solution containing Ca2+ compared to a solution without Ca2+. These findings demonstrate that Ca2+ is a key driver of actin cytoskeleton disintegration and morphological changes following membrane permeabilization with nsPEFs.

Intracellular pathogens, such as Listeria monocytogenes (LM), manipulate host cells to spread from the initial infection site to distant organs through the bloodstream. For that, LM hijacks mononuclear phagocytes to traverse vascular endothelial cell (EC) linings, but how transmigration is regulated by ECs is poorly understood. Here, we show that LM infection profoundly alters EC biomechanical responses to macrophages (MΦs). Videomicroscopy revealed that EC-MΦ contact induces EC polarization, alignment, and reduced motility. However, only interactions with uninfected MΦs increased EC traction and monolayer stresses and barrier integrity. This biomechanical response is largely contact-dependent and significantly attenuated during infection, thus contributing to the enhanced rate of LM-infected MΦ transmigration. Consistently, in the zebrafish model, infection increased endothelial permeability and phagocyte extravasation. These findings reveal that LM infection overrides MΦ-induced endothelial barrier strengthening to promote pathogen dissemination, a biomechanical strategy that could be harnessed for infection control.

Platelets are small, anucleate cells critical for hemostasis and thrombosis. Within platelet plugs and narrow capillaries, they often encounter spatially confining microenvironments. To examine how confinement influences platelet properties, we employed microcontact printing to generate fibrinogen micropatterns of varying sizes and shapes. Platelets cultured on these micropatterns adapted both their morphology and mechanical characteristics. Scanning ion conductance microscopy revealed changes in area, aspect ratio, and height, while fluorescence microscopy showed F-actin redistribution toward the periphery. Confinement reduced platelet stiffness in a size-dependent but shape-independent manner. To explore the role of intracellular signaling, we examined cyclic guanosine monophosphate (cGMP), a key inhibitor of platelet activation, adhesion, and aggregation. Treatment with a cGMP analog preserved F-actin redistribution but prevented stiffness changes in response to confinement, indicating that cGMP inhibits stiffness modulation without affecting cytoskeletal reorganization.

A cyclic guanosine monophosphate (cGMP) signaling pathway composed of the extracellular ligand C-type natriuretic peptide (CNP), the transmembrane natriuretic peptide receptor 2 (Npr2), and the cGMP-dependent protein kinase I (cGKI) regulates axon bifurcation of embryonic dorsal root ganglion (DRG) neurons in mice. Despite the importance of this process for the development of neuronal connectivity, the underlying mechanisms are only partially understood. Axon bifurcation requires an orchestrated rearrangement of the cytoskeleton in growth cones, the highly motile structures at axon tips. In this study, we explored the effects of cGMP signaling on growth cones in fixed and living DRG explant cultures obtained from mouse embryos. The cytoskeletal organization and stiffness of growth cones was examined by fluorescence microscopy and scanning ion conductance microscopy (SICM). Activation of cGMP signaling by CNP or the membrane-permeable cGMP analog 8-Bromo-cGMP reduced growth cone and axon shaft stiffness. Experiments with DRG neurons from Npr2 knockout (KO) mice confirmed that the anti-stiffness effect of CNP was Npr2-dependent. Pharmacological disruption of the cytoskeleton revealed that growth cone stiffness was determined by F-actin content. Activation of cGMP signaling reduced F-actin content in growth cones. Next, we studied the mechanism of cGMP-mediated cytoskeletal remodeling in growth cones. Genetic deletion of vasodilator-stimulated phosphoprotein (Vasp), a phosphorylation target of cGKI that regulates actin polymerization, did not impair cGMP-induced reduction of growth cone and axon shaft stiffness in vitro and axon bifurcation in vivo. Since growth cone dynamics is also regulated by the intracellular Ca2+ concentration, we performed simultaneous imaging of cGMP and Ca2+ in living growth cones. CNP-induced cGMP elevations suppressed ATP-induced Ca2+ transients in wild-type growth cones, but not in cGKI-deficient growth cones. In summary, this study indicates that the CNP-Npr2-cGMP-cGKI axis in DRG neurons controls Ca2+ signaling, remodeling of the actin cytoskeleton, and growth cone mechanics. Thereby, it might contribute to regulating axonal branching.

BACKGROUND/AIMS:To explore the feasibility and effectiveness of Tumor Destructive Mechanical Impulse (TMI) treatment of solid tumors, biomechanical preconditions for subsequent computational simulation of focused shock wave propagation within cells and tissue are investigated. This innovative "soft" approach is different from the FDA-approved high intensity focused ultrasound (HIFU)-based histotripsy, and from electrical Tumor Treating Fields (TTFs). METHODS:Atomic force microscopy investigation for cell mechanics, multiple parametric computational simulations for focused shock wave propagation, technical TMI generator and applicator design, light- and electron-microscopic evaluation of treatment effects on tumor cells and tissue. RESULTS:Individual tumor cell evaluation of physical properties as basis for multiple parametric simulations determine the optimal treatment parameters (total energy required, energy flux density, shock wave frequency) and applicator positions; design flexibility of applicator devices for extra- and intracorporeal treatment. CONCLUSION:The fundamental feasibility, effectiveness and reliability of TMI treatment of solid tumors were proven, providing a reliable theoretical basis for the broadly applicable translation into clinical practice.

The stiffness and the volume of human platelets change under various conditions, affecting their function and viability. Although the influence of platelet volume on platelet function in health and disease has been extensively studied, the relationship between volume and stiffness - in contrast to many other cell types - remains unexplored for platelets, probably due to the difficulty in measuring platelet mechanics as platelets tend to activate under stress. Here, we investigate the relationship between platelet volume and stiffness using scanning ion conductance microscopy (SICM). SICM can image the topography and therefore quantify the volume as well as measure the mechanical properties of living cells under physiological conditions with submicrometer resolution. We found a link between platelet stiffness and volume changes caused by water efflux/influx due to osmotic compression/expansion at the single cell level. With increasing platelet volume, the stiffness decreased and vice versa. We then confirmed this inverse relationship by measurements of platelets during two additional, physiologically highly relevant situations: The dynamic spreading of platelets on a surface and platelets subjected to a spatial confinement, where a decrease in volume was also accompanied by an increase in stiffness and platelets subjected to spatial confinement showed a significantly larger volume compared to unconfined platelets, with a correspondingly lower stiffness, respectively. In conclusion, our SICM analysis revealed a universal, inverse correlation between platelet stiffness and volume change, opening up new perspectives in platelet research.

Epithelial cells respond to infection with the intracellular bacterial pathogen Listeria monocytogenes by altering their mechanics to promote collective infected cell extrusion (CICE) and limit infection spread across cell monolayers. However, the underlying biochemical pathways remain elusive. Here, using in vitro (epithelial monolayers) and in vivo (zebrafish larvae) models of infection with L. monocytogenes or Shigella flexneri, we explored the role of extracellular-signal-regulated kinase (ERK) activity waves in coordinating the mechanical battle between infected and surrounder uninfected cells that leads to CICE. We discovered that when ERK waves are suppressed, cells fail to exhibit alterations in cell shape and kinematics associated with CICE and behave more like quiescent uninfected monolayers. In particular, uninfected cells surrounding infection foci are unable to polarize, reinforce their monolayer stresses, and promote CICE. Our findings reveal that crosstalk between ERK waves and cell mechanics is key to collective elimination of large domains of infected cells.

Nanopores provide controlled nanoconfinement that can be used to induce localized chemical reactions. Here, we present a nanopore exhibiting memory in the frequency of ion current oscillations induced by the dynamic formation and removal of nanoprecipitates within the pore volume. We find that the onset and characteristics of these current oscillations depend on the direction of the voltage scan, with memory effects evidenced in the frequency of switching between high and low conductance states and the probability of the pore to be in the open state. We have also emulated conductive synaptic switching behavior by applying voltage pulses and demonstrated an ability for the system to exhibit long-term potentiation (LTP) and long-term depression (LTD) that mimic learning and memory of synapses. A hypothesis is presented stating that the memory effects arise from the delayed formation and clearing of nanoprecipitates due to spatial-temporal asymmetry as well as from long-term variations in the effective surface charge. We propose a model in which precipitate formation is limited by the cation arrival rate. Our delayed logistic expression successfully recreates steady-state and oscillatory features in the transmembrane current. Nanopores with memory encoded in the frequency of ion current oscillations emulate how the brain stores information, open the possibility to achieve high-dimensional ionic memory, and move beyond the hysteresis in average conductance of ionic memristors.

Platelets have long been established as a safeguard of our vascular system. Recently, haptotactic platelet migration has been discovered as a part of the immune response. In addition, platelets exhibit mechanosensing properties, changing their behavior in response to the stiffness of the underlying substrate. However, the influence of substrate stiffness on platelet migration behavior remains elusive. Here, we investigated the migration of platelets on fibrinogen-coated polydimethylsiloxane (PDMS) substrates with different stiffnesses. Using phase-contrast and fluorescence microscopy as well as a deep-learning neural network, we tracked single migrating platelets and measured their migration distance and velocity. We found that platelets migrated on stiff PDMS substrates (E = 2 MPa), while they did not migrate on soft PDMS substrates (E = 5 kPa). Platelets migrated also on PDMS substrates with intermediate stiffness (E = 100 kPa), but their velocity and the fraction of migrating platelets were diminished compared to platelets on stiff PDMS substrates. The straightness of platelet migration, however, was not significantly influenced by substrate stiffness. We used scanning ion conductance microscopy (SICM) to image the three-dimensional shape of migrating platelets, finding that platelets on soft substrates did not show the polarization and shape change associated with migration. Furthermore, the fibrinogen density gradient, which is generated by migrating platelets, was reduced for platelets on soft substrates. Our work demonstrates that substrate stiffness, and thus platelet mechanosensing, influences platelet migration. Substrate stiffness for optimal platelet migration is quite high (>100 kPa) in comparison to other cell types, with possible implications on platelet behavior in inflammatory and injured tissue. STATEMENT OF SIGNIFICANCE: Platelets can feel and react to the stiffness of their surroundings - a process called mechanosensation. Additionally, platelets migrate via substrate-bound fibrinogen as part of the innate immune response during injury or inflammation. It has been shown that the migration of immune cells is influenced by the stiffness of the underlying substrate, but the effect of substrate stiffness on the migration of platelets has not yet been investigated. Using differently stiff substrates made from PDMS, we show that substrate stiffness affects platelet migration. Stiff substrates facilitate fast and frequent platelet migration with a strong platelet shape anisotropy and a strong fibrinogen removal while soft substrates inhibit platelet migration. These findings highlight the influence of the stiffness of the surrounding tissue on the platelet immune response, possibly enhancing platelet migration in inflamed tissue.

BACKGROUND & PURPOSE:Arterial stiffness, or loss of elastic compliance in large arteries, is an independent precursor of cardiovascular disease (CVD) [1] and dementia [2] for which currently there are no targeted therapies. We previously discovered that decreases in NO-sensitive guanylyl cyclase (NO-GC), the NO receptor which synthesizes cGMP, and in its target vasodilator-stimulated phosphoprotein (pVASPS239), lead to increased cytoskeletal actin polymerization in vascular smooth muscle cells (VSMCs) contributing to increased arterial stiffness [3]. In the current study, we tested whether activating NO-GC with an NO-GC activator (cinaciguat) modulates pVASPS239 and cytoskeletal actin polymerization in VSMCs, thereby preventing obesity-induced arterial stiffness. EXPERIMENTAL APPROACH & KEY RESULTS:Cinaciguat administration (5 mg/kg) to high fat, high sucrose diet (HFHS)-fed mice, our established model of arterial stiffness [4], (1) decreased pulse wave velocity, the in vivo index of arterial stiffness, without affecting blood pressure; (2) increased aortic pVASPS239 levels; and (3) decreased actin polymerization, measured as ratio of filamentous (F) to globular (G) actin, compared to vehicle administration. In cultured VSMCs, cinaciguat (10 μmol/L) increased pVASPS239 levels and decreased the F/G actin ratio at baseline and after stimulation with the cytokine tumor necrosis factor α (TNFα), which we previously showed is significantly increased in the aorta of HFHS-fed mice [4-6]. These effects were abrogated in aortas and VSMCs from mice with smooth muscle-specific cGKI deletion (cGKISMKO), while being mimicked by a cell-permeable cGMP analog (8-Br-cGMP), which also decreased VSMC stiffness in vitro. CONCLUSIONS & IMPLICATIONS:Collectively, our data strongly support the notion that pharmacological NO-GC activation would be beneficial in decreasing obesity-associated arterial stiffness by decreasing VSMC cytoskeletal actin hyper-polymerization. If translated to humans, NO-GC activators could become a viable approach to clinically treat arterial stiffness, which remains an unmet medical need.

Plasma membrane integrity is vital for cell viability, yet its controlled disruption enables targeted delivery of therapeutic agents. Here, we examined membrane durability and repair capacity in normal and malignant urothelial cells using short, high-voltage nanosecond pulses. Pulses were applied to monolayer cultures, spheroids, and patient-derived organoids. Plasma membrane permeability was assessed via YO-PRO-1 dye uptake, and mechanical effects of permeabilization were analyzed using atomic force microscopy. Urothelial cancer cells exhibited nearly fourfold higher dye uptake than non-malignant cells, along with more pronounced osmotic swelling and loss of cellular stiffness. Membrane resealing in cancer cells was delayed and exhibited stronger dependence on extracellular Ca²⁺. The higher susceptibility of urothelial cells was correlated with their larger size, which enable them to reach the electroporation threshold at lower electric fields. These findings highlight key differences in membrane vulnerability and repair dynamics, providing foundation for the development of membrane-targeted therapies for urothelial cancer.

The scanning ion conductance microscope (SICM) is an emerging imaging technique for the investigation of delicate samples on the nanometer scale in liquid environments using ion current through a glass nanopipette. In recent years, the SICM has been increasingly applied to mechanical measurements, typically using a microfluidic flow in the nanopipette induced by hydrostatic pressure. Here, we introduce the use of electroosmotic flow (EOF) in mechanical SICM measurements. We show that the EOF in small SICM nanopipettes is comparable to the flow induced by commonly applied hydrostatic pressures. We quantify the electroosmotic mobility, which is a central parameter of EOF but strongly depends on experimental conditions, by measuring the streaming current independent of nanopipette geometry. Using decane microdroplets, we show that both EOF and hydrostatic pressure can be used to mechanically probe elastic samples on the nanometer scale. We then develop a numerical model to quantify the stiffness and the Young's modulus of elastic samples using EOF. Finally, we use EOF to map the Young's modulus of living cells, which gives similar results to the hydrostatic pressure method. We thereby demonstrate that EOF can be used to quantitatively probe sample stiffness with the SICM.

There has been a great amount of interest in nanopores as the basis for sensors and templates for preparation of biomimetic channels as well as model systems to understand transport...

Platelets are small blood cells involved in hemostasis and wound healing. After activation, platelets interact with their surrounding environment and respond to biochemical and mechanical stimuli by mechanosensitive and haptotactic mechanisms. We used microcontact printing (μCP) to mimic the physiological conditions and limited space in small blood vessels in vitro. With μCP, we created 4-μm-wide fibrinogen lines to provide a spatially confined spreading space for platelets. We then let platelets adhere and spread on these lines while imaging them with optical microscopy and scanning ion conductance microscopy (SICM). Confined platelets showed significantly altered morphology, spreading dynamics, and mechanics compared with control platelets. Altered mechanical properties of confined platelets revealed reorganization of the actin cytoskeleton and the formation of regions of increased elastic modulus at the edges of the fibrinogen lines. Our results indicate that spatial confinement affects platelet mechanics and morphology on a subcellular level.

Background Platelets are anucleate cells that play an important role in wound closure following vessel injury. Maintaining a constant platelet volume is critical for platelet function. For example, water-induced swelling can promote procoagulant activity and initiate thrombosis. However, techniques for measuring changes in platelet volume such as light transmittance or impedance techniques have inherent limitations as they only allow qualitative measurements or do not work on the single-cell level. Methods Here, we introduce high-speed scanning ion conductance microscopy (HS-SICM) as a new platform for studying volume regulation mechanisms of individual platelets. We optimized HS-SICM to quantitatively image the morphology of adherent platelets as a function of time at scanning speeds up to 7 seconds per frame and with 0.1 fL precision. Results We demonstrate that HS-SICM can quantitatively measure the rapid swelling of individual platelets after a hypotonic shock and the following regulatory volume decrease (RVD). We found that the RVD of thrombin-, ADP-, and collagen-activated platelets was significantly reduced compared with nonactivated platelets. Applying the Boyle-van't Hoff relationship allowed us to extract the nonosmotic volume and volume fraction on a single-platelet level. Activation by thrombin or ADP, but not by collagen, resulted in a decrease of the nonosmotic volume, likely due to a release reaction, leaving the total volume unaffected. Conclusion This work shows that HS-SICM is a versatile tool for resolving rapid morphological changes and volume dynamics of adherent living platelets.

Cell stiffness is regulated by dynamic interaction between ras-related C3 botulinum toxin substrate 1 (Rac1) and p21 protein-activated kinase 1 (PAK1) proteins, besides other biochemical and molecular regulators. In this study, we investigated how the Placental Growth Factor (PlGF) changes endometrial mechanics by modifying the actin cytoskeleton at the maternal interface. We explored the global effects of PlGF in endometrial stromal cells (EnSCs) using the concerted approach of proteomics, atomic force microscopy (AFM), and electrical impedance spectroscopy (EIS). Proteomic analysis shows PlGF upregulated RhoGTPases activating proteins and extracellular matrix organization-associated proteins in EnSCs. Rac1 and PAK1 transcript levels, activity, and actin polymerization were significantly increased with PlGF treatment. AFM further revealed an increase in cell stiffness with PlGF treatment. The additive effect of PlGF on actin polymerization was suppressed with siRNA-mediated inhibition of Rac1, PAK1, and WAVE2. Interestingly, the increase in cell stiffness by PlGF treatment was pharmacologically reversed with pravastatin, resulting in improved trophoblast cell invasion. Taken together, aberrant PlGF levels in the endometrium can contribute to an altered pre-pregnancy maternal microenvironment and offer a unifying explanation for the pathological changes observed in conditions such as pre-eclampsia (PE). High levels of placental growth factor can result in a stiffer microenvironment milieu impeding trophoblast invasion of endometrial cells prior to pregnancy, providing insight into the pathogenesis of pre-eclampsia.

Platelet activation plays a critical role in thrombosis and hemostasis. Several pathophysiological situations lead to hemolysis, resulting in the liberation of free ferric iron-containing hemin. Hemin has been shown to activate platelets and induce thrombo-inflammation. Classical antiplatelet therapy failed to prevent hemin-induced platelet activation. Thus, the aim of the present study was to characterize the mechanism of hemin-induced platelet death (ferroptosis). We evaluated the in vitro effect of hemin on platelet activation, signaling, oxylipins, and plasma membrane destruction using light transmission aggregometry, ex vivo thrombus formation, multiparametric flow cytometry, micro-UHPLC mass spectrometry for oxylipin profiling, and scanning ion conductance microscopy (SICM). We found that hemin induces platelet cell death indicated by increased ROS levels, phosphatidyl serine (PS) exposure, and loss of mitochondrial membrane potential (ΔΨm). Further, hemin causes lipid peroxidation and generation of distinct oxylipins, which strongly affects plasma membrane integrity leading to generation of platelet-derived microvesicles. Interestingly, hemin-dependent platelet death (ferroptosis) is specifically regulated by the subtilisin-like proprotein convertase furin. In summary, platelet undergo a non-apoptotic cell death mediated by furin. Inhibition of furin may offer a therapeutic strategy to control hemin-induced thrombosis and thrombo-inflammation at a site of hemolysis.

The arrangement of solvent molecules and ions at solid-liquid interfaces determines electrochemical properties that are important in separations platforms, sensing technologies, and energy-storage systems. Here we show that single glass and polymer pores in contact with propylene carbonate (PC) solutions of LiClO4 exhibit an effective surface potential that is modulated by the enantiomeric excess of the solvent. In particular, electrochemical and electrokinetic measurements of ionic transport through glass pipettes and polymer pores reveal that the effective surface potential is significantly lower in solutions prepared using enantiomerically pure PC than in solutions prepared using racemic PC. Both pore systems became positively charged in all racemic solutions examined in the range of LiClO4 concentrations between 1 mM and 100 mM, whereas solutions in (R)-(+)-PC induced a positive surface potential only at concentrations above similar to 5 mM. The effective surface potential is quantified through asymmetry in current-voltage curves and zeta-potential measurements. Vibrational sum-frequency-generation experiments on LiClO4 solutions in racemic and enantiomerically pure PC indicate that the surface lipid-bilayer-like region in the former is more strongly organized than in the latter, dictating the favorable positions for lithium and perchlorate ions in each case. The more ordered molecular packing in the racemic liquid leads to accumulation of lithium ions on the outside of the bilayer, creating a higher effective positive charge. Our results highlight the extreme sensitivity of the interfacial potential on molecular organization of the solvent, and the relatively unexplored role that chirality can play in electrokinetic phenomena.

Cancer affects the mechanical properties of tissue. Therefore, elastography techniques can be used to differentiate cancerous from healthy tissue. Due to probe size and restricted handling, most elastography techniques are not applicable in minimally invasive surgery (MIS). Established techniques such as endoscopic ultrasound elastography measure under undefined boundary conditions, making the determination of quantitative mechanical properties challenging. Water flow elastography (WaFE) has recently been introduced for application in MIS. Here, we present an improved WaFE measurement method in which the probe attaches itself to the sample with a small suction pressure. This leads to defined boundary conditions, allowing for a quantitative determination of the Young's modulus of tissue. To facilitate fast measurements, we developed a correction model for the hydrodynamic resistance and the fluid inertia of the tubing. We used WaFE for ex vivo measurements on human bladders and found a significantly larger Young's modulus for cancerous vs. healthy tissue. We determined the optimal classification threshold for the Young's modulus to be 8 kPa and found that WaFE can differentiate between cancerous and healthy tissue with a sensitivity of 0.96 and a specificity of 1. Our results underline that WaFE can be a helpful differentiating tool in MIS.

Imaging and probing liquid-liquid interfaces at the micro- and nanoscale are of high relevance, for example, in materials science, surface chemistry, and microfluidics. However, existing imaging techniques are limited in resolution, average over large sample areas, or interact with the sample. Here, we present a method to quantify the shape, stiffness, and interface tension of liquid droplets with the scanning ion conductance microscope (SICM), providing submicrometer resolution and the ability to perform noncontact mechanical measurements. We show that we can accurately image the three-dimensional shape of micrometer-sized liquid droplets made of, for example, decane, hexane, or different oils. We then introduce numerical models to quantitatively obtain their stiffness and interface tension from SICM data. We verified our method by measuring the interface tension of decane droplets changing under the influence of surfactants at different concentrations. Finally, we use SICM to resolve the dissolution dynamics of decane droplets, showing that droplet shape exhibits different dissolution modes and stiffness continuously increases while the interface tension remains constant. We thereby demonstrate that SICM is a useful method to investigate liquid-liquid interfaces on the microscale with applications in materials or life sciences.