Dissecting cell membrane tension dynamics and its effect on Piezo1-mediated cellular mechanosensitivity using force-controlled nanopipettes.

The dynamics of cellular membrane tension and its role in mechanosensing, which is the ability of cells to respond to physical stimuli, remain incompletely understood, mainly due to the lack of appropriate tools. Here, we report a force-controlled nanopipette-based method that combines fluidic force microscopy with fluorescence imaging for precise manipulation of the cellular membrane tension while monitoring the impact on single-cell mechanosensitivity. The force-controlled nanopipette enables control of the indentation force imposed on the cell cortex as well as of the aspiration pressure applied to the plasma membrane. We show that this setup can be used to concurrently monitor the activation of Piezo1 mechanosensitive ion channels via calcium imaging. Moreover, the spatiotemporal behavior of the tension propagation is assessed with the fluorescent membrane tension probe Flipper-TR, and further dissected using molecular dynamics modeling. Finally, we demonstrate that aspiration and indentation act independently on the cellular mechanobiological machinery, that indentation induces a local pre-tension in the membrane, and that membrane tension stays confined by links to the cytoskeleton.

Biophysical Reviews' Topical issue: The 7th Nanoengineering for Mechanobiology Symposium 2024 Camogli, Genoa, Italy.

This Commentary describes an open call for submissions to the upcoming Biophysical Reviews' Issue Focus: The 7th Nanoengineering for Mechanobiology (Genova, Italy). The submission deadline is August 1st of 2024. Interested parties are requested to make contact with the Issue Focus editors prior to submission.

Metal Cross-Linked Supramolecular Gel Noodles: Structural Insights and Antibacterial Assessment.

Achieving precise control over gelator alignment and morphology is crucial for crafting tailored materials and supramolecular structures with distinct properties. We successfully aligned the self-assembled micelles formed by a functionalized dipeptide 2NapFF into long 1-D "gel noodles" by cross-linking with divalent metal chlorides. We identify the most effective cross-linker for alignment, enhancing mechanical stability, and imparting functional properties. Our study shows that Group 2 metal ions are particularly suited for creating mechanically robust yet flexible gel noodles because of their ionic and nondirectional bonding with carboxylate groups. In contrast, the covalent nature and high directional bonds of d-block metal ions with carboxylates tend to disrupt the self-assembly of 2NapFF. Furthermore, the 2NapFF-Cu noodles demonstrated selective antibacterial activity, indicating that the potent antibacterial property of the copper(II) ion is preserved within the cross-linked system. By merging insights into molecular alignment, gel extrusion processing, and integrating specific functionalities, we illustrate how the versatility of dipeptide-based gels can be utilized in creating next-generation soft materials.

Nanoscatterer-Assisted Fluorescence Amplification Technique.

Weak fluorescence signals, which are important in research and applications, are often masked by the background. Different amplification techniques are actively investigated. Here, a broadband, geometry-independent and flexible feedback scheme based on the random scattering of dielectric nanoparticles allows the amplification of a fluorescence signal by partial trapping of the radiation within the sample volume. Amplification of up to a factor of 40 is experimentally demonstrated in ultrapure water with dispersed TiO2 nanoparticles (30 to 50 nm in diameter) and fluorescein dye at 200 µmol concentration (pumped with 5 ns long, 3 mJ laser pulses at 490 nm). The measurements show a measurable reduction in linewidth at the emission peak, indicating that feedback-induced stimulated emission contributes to the large gain observed.

Multi-disciplinary Insights from the First European Forum on Visceral Myopathy 2022 Meeting.

Visceral myopathy is a rare, life-threatening disease linked to identified genetic mutations in 60% of cases. Mostly due to the dearth of knowledge regarding its pathogenesis, effective treatments are lacking. The disease is most commonly diagnosed in children with recurrent or persistent disabling episodes of functional intestinal obstruction, which can be life threatening, often requiring long-term parenteral or specialized enteral nutritional support. Although these interventions are undisputedly life-saving as they allow affected individuals to avoid malnutrition and related complications, they also seriously compromise their quality of life and can carry the risk of sepsis and thrombosis. Animal models for visceral myopathy, which could be crucial for advancing the scientific knowledge of this condition, are scarce. Clearly, a collaborative network is needed to develop research plans to clarify genotype-phenotype correlations and unravel molecular mechanisms to provide targeted therapeutic strategies. This paper represents a summary report of the first 'European Forum on Visceral Myopathy'. This forum was attended by an international interdisciplinary working group that met to better understand visceral myopathy and foster interaction among scientists actively involved in the field and clinicians who specialize in care of people with visceral myopathy.

The Impact of Experimental Conditions on Cell Mechanics as Measured with Nanoindentation.

The evaluation of cell elasticity is becoming increasingly significant, since it is now known that it impacts physiological mechanisms, such as stem cell differentiation and embryogenesis, as well as pathological processes, such as cancer invasiveness and endothelial senescence. However, the results of single-cell mechanical measurements vary considerably, not only due to systematic instrumental errors but also due to the dynamic and non-homogenous nature of the sample. In this work, relying on Chiaro nanoindenter (Optics11Life), we characterized in depth the nanoindentation experimental procedure, in order to highlight whether and how experimental conditions could affect measurements of living cell stiffness. We demonstrated that the procedure can be quite insensitive to technical replicates and that several biological conditions, such as cell confluency, starvation and passage, significantly impact the results. Experiments should be designed to maximally avoid inhomogeneous scenarios to avoid divergences in the measured phenotype.

Patient's dermal fibroblasts as disease markers for visceral myopathy.

Visceral myopathy (VSCM) is a rare genetic disease, orphan of pharmacological therapy. VSCM diagnosis is not always straightforward due to symptomatology similarities with mitochondrial or neuronal forms of intestinal pseudo-obstruction. The most prevalent form of VSCM is associates with variants in the gene ACTG2, encoding the protein gamma-2 actin. Overall, VSCM is a mechano-biological disorder, in which different genetic variants lead to similar alterations to the contractile phenotype of enteric smooth muscles, resulting in the emergence of life-threatening symptoms. In this work we analyzed the morpho-mechanical phenotype of human dermal fibroblasts from patients affected with VSCM, demonstrating that they retain a clear signature of the disease when compared with different controls. We evaluated several biophysical traits of fibroblasts, and we show that a measure of cellular traction forces can be used as a non-specific biomarker of the disease. We propose that a simple assay based on traction forces could be designed to provide a valuable support for clinical decision or pre-clinical research.

Nanotopography reveals metabolites that maintain the immunomodulatory phenotype of mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are multipotent progenitor cells that are of considerable clinical potential in transplantation and anti-inflammatory therapies due to their capacity for tissue repair and immunomodulation. However, MSCs rapidly differentiate once in culture, making their large-scale expansion for use in immunomodulatory therapies challenging. Although the differentiation mechanisms of MSCs have been extensively investigated using materials, little is known about how materials can influence paracrine activities of MSCs. Here, we show that nanotopography can control the immunomodulatory capacity of MSCs through decreased intracellular tension and increasing oxidative glycolysis. We use nanotopography to identify bioactive metabolites that modulate intracellular tension, growth and immunomodulatory phenotype of MSCs in standard culture and during larger scale cell manufacture. Our findings demonstrate an effective route to support large-scale expansion of functional MSCs for therapeutic purposes.

Cell shape and tension alter focal adhesion structure.

Cells are not only anchored to the extracellular matrix via the focal adhesion complex, the focal adhesion complex also serves as a sensor for force transduction. How tension influences the structure of focal adhesions is not well understood. Here, we analyse the effect of tension on the location of key focal adhesion proteins, namely vinculin, paxillin and actin. We use micropatterning on gold surfaces to manipulate the cell shape, to create focal adhesions at specific cell areas, and to perform metal-induced energy transfer (MIET) measurements on the patterned cells. MIET resolves the different protein locations with respect to the gold surface with nanometer accuracy. Further, we use drugs influencing the cellular motor protein myosin or mechanosensitive ion channels to get deeper insight into focal adhesions at different tension states. We show here that in particular actin is affected by the rationally tuned force balance. Blocking mechanosensitive ion channels has a particularly high influence on the actin and focal adhesion architecture, resulting in larger focal adhesions with elevated paxillin and vinculin and strongly lowered actin stress fibres. Our results can be explained by a balance of adhesion tension with cellular tension together with ion channel-controlled focal adhesion homeostasis, where high cellular tension leads to an elevation of vinculin and actin, while high adhesion tension lowers these proteins.

Hierarchical Composite Self-Sorted Supramolecular Gel Noodles.

Multicomponent supramolecular systems can be used to achieve different properties and new behaviors compared to their corresponding single component systems. Here, a two-component system is used, showing that a non-gelling component modifies the assembly of the gelling component, allowing access to co-assembled structures that cannot be formed from the gelling component alone. The systems are characterized across multiple length scales, from the molecular level by NMR and CD spectroscopy to the microstructure level by SANS and finally to the material level using nanoindentation and rheology. By exploiting the enhanced mechanical properties achieved through addition of the second component, multicomponent noodles are formed with superior mechanical properties to those formed by the single-component system. Furthermore, the non-gelling component can be triggered to crystallize within the multicomponent noodles, allowing the preparation of new types of hierarchical composite noodles.

Photothermal Self-Excitation of a Phase-Controlled Microcantilever for Viscosity or Viscoelasticity Sensing.

This work presents a feedback closed-loop platform to be used for viscosity or viscoelasticity sensing of Newtonian or non-Newtonian fluids. The system consists of a photothermally excited microcantilever working in a digital Phase-Locked Loop, in which the phase between the excitation signal to the cantilever and the reference demodulating signals is chosen and imposed in the loop. General analytical models to describe the frequency and amplitude of oscillation of the cantilever immersed in viscous and viscoelastic fluids are derived and validated against experiments. In particular, the sensitivity of the sensor to variations of viscosity of Newtonian fluids, or to variations of elastic/viscous modulus of non-Newtonian fluids, are studied. Interestingly, it is demonstrated the possibility of controlling the sensitivity of the system to variations of these parameters by choosing the appropriate imposed phase in the loop. A working point with maximum sensitivity can be used for real-time detection of small changes of rheological parameters with low-noise and fast-transient response. Conversely, a working point with zero sensitivity to variations of rheological parameters can be potentially used to decouple the effect of simultaneous external factors acting on the resonator.

Cellular Mechanosensitivity: Validation of an Adaptable 3D-Printed Device for Microindentation.

Mechanotransduction refers to the cellular ability to sense mechanical stimuli from the surrounding environment and convert them into biochemical signals that regulate cellular physiology and homeostasis. Mechanosensitive ion channels (MSCs), especially ones of Piezo family (Piezo1 and Piezo2), play a crucial role in mechanotransduction. These transmembrane proteins directly react to mechanical cues by triggering the onset of an ionic current. The relevance of this mechanism in driving physiology and pathology is emerging, and there is a growing need for the identification of an affordable and reliable assay to measure it. Setting up a mechanosensitivity assay requires exerting a mechanical stimulus on single cells while observing the downstream effects of channels opening. We propose an open-hardware approach to stimulate single adherent cells through controlled microindentation, using a 3D-printed actuation platform. We validated the device by measuring the mechanosensitivity of a neural mice cell line where the expression level and activity of Piezo1 were genetically and pharmacologically manipulated. Moreover, this extremely versatile device could be integrated with different read-out technologies, offering a new tool to improve the understanding of mechanotransduction in living cells.

Relationship between Liver Stiffness and Steatosis in Obesity Conditions: In Vivo and In Vitro Studies.

Obesity is a major risk factor for metabolic dysfunction such as non-alcoholic fatty liver disease (NAFLD). The NAFLD spectrum ranges from simple steatosis, to steatohepatitis, fibrosis, and cirrhosis. The aim of this study is to characterize the grade of steatosis being associated with overnutrition and obesity, both at the level of single hepatocyte and whole liver, and to correlate it with the hepatocyte/liver stiffness and dysfunction. For the in vivo study, 60 subjects were enrolled and grouped based on the stage of liver steatosis/fibrosis according to biochemical analyses, liver ultrasonography (USG) and acoustic radiation force impulse shear wave elastography (ARFI-SWE). For single hepatocyte analyses we employed in vitro models of moderate and severe steatosis on which to assess the single cell biomechanics by Single Cell Force Spectroscopy (SCFS) and Quantitative Phase Microscopy (QPM). Results show that in vivo liver stiffness depends mainly on the extent of fat accumulation and not on fibrosis. These results parallel the in vitro observations showing that hepatocyte stiffness and dysfunction increase with increasing fat accumulation and lipid droplet enlargement. Our findings indicate that the extent of steatosis markedly affects the biomechanical properties of both liver and single hepatocytes thus proving insights about the role of modulation of liver/hepatocyte elasticity as a physical mechanism transducing the obesity-dependent excess of plasmatic lipids towards liver steatosis and dysfunction.

Experimental and Data Analysis Workflow for Soft Matter Nanoindentation.

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft biomaterials and cells. This approach has gained a central role in the fields of mechanobiology, biomaterials design and tissue engineering, to obtain a proper mechanical characterization of soft materials with a resolution comparable to the size of single cells (µm). The most popular strategy to acquire such experimental data is to employ an atomic force microscope (AFM); while this instrument offers an unprecedented resolution in force (down to pN) and space (sub-nm), its usability is often limited by its complexity that prevents routine measurements of integral indicators of mechanical properties, such as Young's Modulus (E). A new generation of nanoindenters, such as those based on optical fiber sensing technology, has recently gained popularity for its ease of integration while allowing to apply sub-nN forces with µm spatial resolution, therefore being suitable to probe local mechanical properties of hydrogels and cells. In this protocol, a step-by-step guide detailing the experimental procedure to acquire nanoindentation data on hydrogels and cells using a commercially available ferrule-top optical fiber sensing nanoindenter is presented. Whereas some steps are specific to the instrument used herein, the proposed protocol can be taken as a guide for other nanoindentation devices, granted some steps are adapted according to the manufacturer's guidelines. Further, a new open-source Python software equipped with a user-friendly graphical user interface for the analysis of nanoindentation data is presented, which allows for screening of incorrectly acquired curves, data filtering, computation of the contact point through different numerical procedures, the conventional computation of E, as well as a more advanced analysis particularly suited for single-cell nanoindentation data.

Non-contact elastography methods in mechanobiology: a point of view.

In recent decades, mechanobiology has emerged as a novel perspective in the context of basic biomedical research. It is now widely recognized that living cells respond not only to chemical stimuli (for example drugs), but they are also able to decipher mechanical cues, such as the rigidity of the underlying matrix or the presence of shear forces. Probing the viscoelastic properties of cells and their local microenvironment with sub-micrometer resolution is required to study this complex interplay and dig deeper into the mechanobiology of single cells. Current approaches to measure mechanical properties of adherent cells mainly rely on the exploitation of miniaturized indenters, to poke single cells while measuring the corresponding deformation. This method provides a neat implementation of the everyday approach to measure mechanical properties of a material, but it typically results in a very low throughput and invasive experimental protocol, poorly translatable towards three-dimensional living tissues and biological constructs. To overcome the main limitations of nanoindentation experiments, a radical paradigm change is foreseen, adopting next generation contact-less methods to measure mechanical properties of biological samples with sub-cell resolution. Here we briefly introduce the field of single cell mechanical characterization, and we concentrate on a promising high resolution optical elastography technique, Brillouin spectroscopy. This non-contact technique is rapidly emerging as a potential breakthrough innovation in biomechanics, but the application to single cells is still in its infancy.

Piezo1 controls cell volume and migration by modulating swelling-activated chloride current through Ca2+ influx.

Regulatory volume decrease (RVD), a homeostatic process responsible for the re-establishment of the original cell volume upon swelling, is critical in controlling several functions, including migration. RVD is mainly sustained by the swelling-activated Cl- current (ICl,swell ), which can be modulated by cytoplasmic Ca2+ . Cell swelling also activates mechanosensitive channels, including the ubiquitously expressed Ca2+ -permeable channel Piezo1. We hypothesized that, by controlling cytoplasmic Ca2+ and in turn ICl,swell , Piezo1 is involved in the fine regulation of RVD and cell migration. We compared RVD and ICl,swell in wild-type (WT) HEK293T cells, which express endogenous levels of Piezo1, and in cells overexpressing (OVER) or knockout (KO) for Piezo1. Compared to WT, RVD was markedly increased in OVER, while virtually absent in KO cells. Consistently, ICl,swell amplitude was highest in OVER and lowest in KO cells, with WT cells displaying an intermediate level, suggesting a Ca2+ -dependent modulation of the current by Piezo1 channels. Indeed, in the absence of external Ca2+ , ICl,swell in both WT and OVER cells, as well as the RVD probed in OVER cells, were significantly lower than in the presence of Ca2+ and no longer different compared to KO cells. However, the Piezo-mediated Ca2+ influx was ineffective in enhancing ICl,swell in the absence of releasable Ca2+ from intracellular stores. The different expression levels of Piezo1 affected also cell migration which was strongly enhanced in OVER, while reduced in KO cells, as compared to WT. Taken together, our data indicate that Piezo1 controls RVD and migration in HEK293T cells by modulating ICl,swell through Ca2+ influx.

Surface-controlled spatially heterogeneous physical properties of a supramolecular gel with homogeneous chemical composition.

Controlling supramolecular self-assembly across multiple length scales to prepare gels with localised properties is challenging. Most strategies concentrate on fabricating gels with heterogeneous components, where localised properties are generated by the stimuli-responsive component. Here, as an alternative approach, we use a spiropyran-modified surface that can be patterned with light. We show that light-induced differences in surface chemistry can direct the bulk assembly of a low molecular weight gelator, 2-NapAV, meaning that mechanical gel properties can be controlled by the surface on which the gel is grown. Using grazing incidence X-ray diffraction and grazing incidence small angle X-ray scattering, we demonstrate that the origin of the different gel properties relates to differences in the architectures of the gels. This provides a new method to prepare a single domain (i.e., chemically homogeneous) hydrogel with locally controlled (i.e., mechanically heterogeneous) properties.

Controlling the formation and alignment of low molecular weight gel 'noodles'.

We show how to control the formation and alignment of gel 'noodles'. Nanostructure alignment can be achieved reproducibly by extensional deformation as the filaments form. Using a spinning technique, very long and highly aligned filaments can be made. The Young's moduli of the gel noodles are similar to that of a bulk gel. By using two syringe pumps in a concentric flow setup, we show that a filament-in-filament morphology can be created.