Single-cell Force Spectroscopy Research Articles

Probing the Surface Layer Modulation on Archaeal Mechanics and Adhesion at the Single-Cell Level.

Addressing the challenge of understanding how cellular interfaces dictate the mechanical resilience and adhesion of archaeal cells, this study demonstrates the role of the surface layer (S-layer) in methanogenic archaea. Using a combination of atomic force microscopy and single-cell force spectroscopy, we quantified the impact of S-layer disruption on cell morphology, mechanical properties, and adhesion capabilities. We demonstrate that the S-layer is crucial for maintaining cell morphology, where its removal induces significant cellular enlargement and deformation. Mechanical stability of the cell surface is substantially compromised upon S-layer disruption, as evidenced by decreased Young's modulus values. Adhesion experiments revealed that the S-layer primarily facilitates hydrophobic interactions, which are significantly reduced after its removal, affecting both cell-cell and cell-bubble interactions. Our findings illuminate the S-layer's fundamental role in methanogen architecture and provide a chemical understanding of archaeal cell surfaces, with implications for enhancing methane production in biotechnological applications.

Nuclear receptor Nur77 regulates immunomechanics of macrophages

Nuclear receptor Nur77 plays a pivotal role in immune regulation across various tissues, influencing pro-inflammatory signaling pathways and cellular metabolism. While cellular mechanics have been implicated in inflammation, the contribution of Nur77 to these mechanical processes remains elusive. Macrophages exhibit remarkable plasticity in their morphology and mechanics, enabling them to adapt and execute essential inflammatory functions, such as navigating through inflamed tissue and pathogen engulfment. However, the precise regulatory mechanisms governing these dynamic changes in macrophage mechanics during inflammation remain poorly understood. To establish the potential correlation of Nur77 with cellular mechanics, we compared bone marrow-derived macrophages (BMDMs) from wild-type (WT) and Nur77-deficient (Nur77-KO) mice and employed cytoskeletal imaging, single-cell acoustic force spectroscopy (AFS), migration and phagocytosis assays, and RNA-sequencing. Our findings reveal that Nur77-KO BMDMs exhibit changes to their actin networks compared to WT BMDMs, which is associated with a stiffer and more rigid phenotype. Subsequent in vitro experiments validated our observations, showcasing that Nur77 deficiency leads to enhanced migration, reduced adhesion, and increased phagocytic activity. The transcriptomics data confirmed altered mechanics-related pathways in Nur77-deficient macrophage that are accompanied by a robust pro-inflammatory phenotype. Utilizing previously obtained ChIP-data, we revealed that Nur77 directly targets differentially expressed genes associated with cellular mechanics. In conclusion, while Nur77 is recognized for its role in reducing inflammation of macrophages by inhibiting the expression of pro-inflammatory genes, our study identifies a novel regulatory mechanism where Nur77 governs macrophage inflammation through the modulation of expression of genes involved in cellular mechanics. Our findings suggest that immune regulation by Nur77 may be partially mediated through alterations in cellular mechanics, highlighting a potential avenue for therapeutic targeting.

Abstract 2748: Single cell biomechanical profiling of the metastatic potential of MDA-MB-231 cells

Abstract Cancer metastasis is a multistep complex process accompanied by changes in the mechanical properties of cancer cells, such as adhesion, viscoelasticity, deformability, and motility. Connexin 43 (Cx43), a gap junction protein with a putative tumor suppressor function, plays a pivotal role in Epithelial to Mesenchymal transition (EMT). It affects cell motility indicators and promotes cytoskeleton reorganization and, ultimately, metastasis when downregulated, corroborating resistance to chemotherapy. The lack of universal markers that can predict the metastatic ability of cancer cells necessitates the evaluation of parameters, such as cell adhesion, viscoelasticity, and stiffness, to serve as unique biophysical metrics that predict cancer cell metastatic potential and elucidate how metastasis and cell mechanics are coupled. Therefore, we utilized fluidic-based single-cell force spectroscopy (SCFS) and Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) to measure the mechanical properties of single cancer cells with different metastatic potential. To explore the relationship between cell mechanics and metastatic cell behavior, we quantitatively demonstrate the progression of cancer cell biomechanics across different metastatic states using MDA-MB-231 breast cancer cells with varying connexin 43 (Cx43) expression as a model of cells with varying metastatic potential. We measured, at a single-cell level and in real-time, the adhesion and viscoelastic properties of MDA-MB-231 cells and their response to chemotherapy. Our data show that cells with higher metastatic potential (down-regulated Cx43 expression) exhibited a softer, less adherent, and more viscoelastic phenotype, consistent with their metastatic state when in clusters compared to cells at the lower spectrum of metastatic potential (up-regulated Cx43 expression). These mechanical biomarkers are significantly absent in single cells outside of a cluster. Of particular interest, we also observed significant cellular stiffening upon treatment with docetaxel (DTX), a chemotherapeutic agent that targets microtubules, with a rapid and profound response of highly aggressive cells when compared to cells with attenuated metastatic potential. Such treatment schemes could render cancer cells less deformable, potentially impeding their ability to invade and metastasize to other tissues. Our study demonstrates a unique quantitative metric that unfolds biomechanical markers for discerning the metastatic state of cancer cells. Our data represents a stepping stone to understanding how mechanical changes in cancer cells may determine how cancer progresses and responds to chemotherapeutic agents and may provide novel modalities to inhibit metastasis. Citation Format: Zeina Habli, Ahmad Zantout, Nadine Al Hajj, Raya Saab, Marwan El-Sabban, Massoud L. Khraiche. Single cell biomechanical profiling of the metastatic potential of MDA-MB-231 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2748.

Regulatory role of interfacial adhesion and mechanical microenvironments in microbe-host interactions

A recent study published in Nature Communications showed that essential modulatory roles of interfacial adhesion and mechanical microenvironments such as geometric constraints and extracellular matrix stiffness, in microbe-host cell interactions. This study utilized single-cell force spectroscopy and RNA sequencing to gain insight into the intrinsic mechanisms by which the mechanical microenvironment regulates bacterial-host interactions and therefore reveal potential interventions against bacterial invasion. Meanwhile, the adhesion forces involved in the bacterial–host interactions were recognized as a new indicator for assessing the extent of bacterial infection. Taken together, these findings demonstrate that interfacial adhesion forces and mechanical microenvironments play a dominant role in modulating functions and behaviors of microorganisms and host cells, which also provide a mechanobiology-inspired idea for the development of subsequent drug-resistant antimicrobials and broad-spectrum antiviral drugs.

Adhesion preference of the sticky bacterium Acinetobacter sp. Tol 5.

Gram-negative bacterium Acinetobacter sp. Tol 5 exhibits high adhesiveness to various surfaces of general materials, from hydrophobic plastics to hydrophilic glass and metals, via AtaA, an Acinetobacter trimeric autotransporter adhesin Although the adhesion of Tol 5 is nonspecific, Tol 5 cells may have prefer materials for adhesion. Here, we examined the adhesion of Tol 5 and other bacteria expressing different TAAs to various materials, including antiadhesive surfaces. The results highlighted the stickiness of Tol 5 through the action of AtaA, which enabled Tol 5 cells to adhere even to antiadhesive materials, including polytetrafluoroethylene with a low surface free energy, a hydrophilic polymer brush with steric hindrance, and mica with an ultrasmooth surface. Single-cell force spectroscopy as an atomic force microscopy technique revealed the strong cell adhesion force of Tol 5 to these antiadhesive materials. Nevertheless, Tol 5 cells showed a weak adhesion force toward a zwitterionic 2-methacryloyloxyethyl-phosphorylcholine (MPC) polymer-coated surface. Dynamic flow chamber experiments revealed that Tol 5 cells, once attached to the MPC polymer-coated surface, were exfoliated by weak shear stress. The underlying adhesive mechanism was presumed to involve exchangeable, weakly bound water molecules. Our results will contribute to the understanding and control of cell adhesion of Tol 5 for immobilized bioprocess applications and other TAA-expressing pathogenic bacteria of medical importance.

Breast adenocarcinoma cells adhere stronger to brain pericytes than to endothelial cells

Most of the malignancies detected within the brain parenchyma are of metastatic origin. As the brain lacks classical lymphatic circulation, the primary way for metastasis relies on hematogenous routes. Dissemination of metastatic cells to the brain implies attachment to the luminal surface of brain endothelial cells, transmigration through the vessel wall, and adhesion to the brain surface of the vasculature. During this process, tumor cells must interact with brain endothelial cells and later on with pericytes. Physical interaction between tumor cells and brain vascular cells might be crucial in the successful extravasation of metastatic cells through blood vessels and later in their survival within the brain environment. Therefore, we applied single-cell force spectroscopy to investigate the nanoscale adhesive properties of living breast adenocarcinoma cells to brain endothelial cells and pericytes. We found target cell type-dependent adhesion characteristics, i.e. increased adhesion of the tumor cells to pericytes in comparison to endothelial cells, which underlines the existence of metastatic potential-related nanomechanical differences relying partly on membrane tether dynamics. Varying adhesion strength of the tumor cells to different cell types of brain vessels presumably reflects the transitory adhesion to endothelial cells before extravasation and the long-lasting strong interaction with pericytes during survival and proliferation in the brain. Our results highlight the importance of specific mechanical interactions between tumor cells and host cells during metastasis formation.

Factors influencing initial bacterial adhesion to antifouling surfaces studied by single-cell force spectroscopy

Biofilm formation, a major concern for healthcare systems, is initiated when bacteria adhere to surfaces. Escherichia coli adhesion is mediated by appendages, including type-1 fimbriae and curli amyloid fibers. Antifouling surfaces prevent the adhesion of bacteria to combat biofilm formation. Here, we used single-cell force-spectroscopy to study the interaction between E.coli and glass or two antifouling surfaces: the tripeptide DOPA-Phe(4F)-Phe(4F)-OMe and poly(ethylene glycol) polymer-brush. Our results indicate that both antifoulants significantly deter E.coli initial adhesion. By using two mutant strains expressing no type-1 fimbriae or curli amyloids, we studied the adhesion mechanism. Our results suggest that the bacteria adhere to different antifoulants via separate mechanisms. Finally, we show that some bacteria adhere much better than others, illustrating how the variability of bacterial cultures affects biofilm formation. Our results emphasize how additional study at the single-cell level can enhance our understanding of bacterial adhesion, thus leading to novel antifouling technologies.

Targeting KCa3.1 channels to overcome erlotinib resistance in non-small cell lung cancer cells

Almost all non-small cell lung cancer (NSCLC) patients initially responding to EGFR tyrosine kinase inhibitors (TKIs) develop acquired resistance. Since KCa3.1 channels, expressed in mitochondria and plasma membrane, regulate similar behavioral traits of NSCLC cells as EGFR, we hypothesized that their blockade contributes to overcoming EGFR-TKI resistance. Meta-analysis of microarray data revealed that KCa3.1 channel expression in erlotinib-resistant NSCLC cells correlates with that of genes of integrin and apoptosis pathways. Using erlotinib-sensitive and –resistant NSCLC cells we monitored the role of mitochondrial KCa3.1 channels in integrin signaling by studying cell-matrix adhesion with single-cell force spectroscopy. Apoptosis was quantified with fluorescence-based assays. The function of mitochondrial KCa3.1 channels in these processes was assessed by measuring the mitochondrial membrane potential and by quantifying ROS production. Functional assays were supplemented by biochemical analyses. We show that KCa3.1 channel inhibition with senicapoc in erlotinib-resistant NSCLC cells increases cell adhesion by increasing β1-integrin expression, that in turn depends on mitochondrial ROS release. Increased adhesion impairs migration of NSCLC cells in a 3D matrix. At the same time, the senicapoc-dependent ROS production induces cytochrome C release and triggers apoptosis of erlotinib-resistant NSCLC cells. Thus, KCa3.1 channel blockade overcomes EGFR-TKI resistance by inhibiting NSCLC motility and inducing apoptosis.

Single-cell fluid-based force spectroscopy reveals near lipid size nano-topography effects on neural cell adhesion.

Nano-roughness has shown great potential in enhancing high-fidelity electrogenic cell interfaces, owing to its characteristic topography comparable to proteins and lipids, which influences a wide range of cellular mechanical responses. Gaining a comprehensive understanding of how cells respond to nano-roughness at the single-cell level is not only imperative for implanted devices but also essential for tissue regeneration and interaction with complex biomaterial surfaces. In this study, we quantify cell adhesion and biomechanics of single cells to nano-roughened surfaces by measuring neural cell adhesion and biomechanics via fluidic-based single-cell force spectroscopy (SCFS). For this, we introduce nanoscale topographical features on polyimide (PI) surfaces achieving roughness up to 25 nm without chemical modifications. Initial adhesion experiments show cell-specific response to nano-roughness for neuroblastoma cells (SH-SY5Y) compared to human astrocytes (NHA) around 15 and 20 nm surface roughness. In addition, our SCFS measurements revealed a remarkable 2.5-fold increase in adhesion forces (150-164 nN) for SH-SY5Y cells cultured on roughened PI (rPI) surfaces compared to smooth surfaces (60-107 nN). Our data also shows that cells can distinguish changes in nano-roughness as small 2 nm (close to the diameter of a single lipid) and show roughness dependence adhesion while favoring 15 nm. Notably, this enhanced adhesion is accompanied by increased cell elongation upon cell detachment without any significant differences in cell area spreading. The study provides valuable insights into the interplay between nano-topography and cellular responses and offers practical implications for designing biomaterial surfaces with enhanced cellular interactions.

Single-cell force spectroscopy of fluid flow-tuned cell adhesion for dissecting hemodynamics in tumor metastasis.

Cell adhesion plays an important role in regulating the metastasis of cancer cells, and atomic force microscopy (AFM)-based single-cell force spectroscopy (SCFS) has become an important method to directly measure the adhesion forces of individual cells. Particularly, bodily fluid flow environments strongly affect the functions and behaviors of metastatic cells for successful dissemination. Nevertheless, the interactions between fluidic flow medium environment and cell adhesion remain poorly understood. In this work, AFM-based SCFS was exploited to examine the effects of fluidic flow environment on cellular adhesion. A fluidic cell culture medium device was used to simulate the fluidic flow environment experienced by cancer cells during metastasis, which was combined with AFM-based SCFS assay. A single living cancer cell was attached to the AFM tipless cantilever to prepare the single-cell probe for performing SCFS experiments on the mesothelial cells grown under the fluidic flow medium conditions, and the effects of experimental parameters (retraction speed, contact time, loading force) on the measured cellular adhesion forces were analyzed. Experimental results of SCFS assay show that cellular adhesion forces significantly decrease after growth in fluidic flow medium, whereas cellular adhesion forces increase after growth in static culture medium. Experiments performed with the use of spherical probes coated with cell adhesion-associated biomolecules also show the weakening of cell adhesion after growth in fluidic flow cell culture medium, which was subsequently confirmed by the confocal fluorescence microscopy experiments of cell adhesion molecules, vividly illustrating the remarkable effects of fluidic flow environment on cellular adhesion. The study provides a new approach to detect adhesion force dynamics involved in the interactions between cells and the fluidic flow environment at the single-cell level, which will facilitate dissecting the role of hemodynamics in tumor metastasis.

Characterization of a unique attachment organelle: Single-cell force spectroscopy of Giardia duodenalis trophozoites.

The unicellular parasite Giardia duodenalis is the causative agent of giardiasis, a gastrointestinal disease with global spread. In its trophozoite form, G. duodenalis can adhere to the human intestinal epithelium and a variety of other, artificial surfaces. Its attachment is facilitated by a unique microtubule-based attachment organelle, the so-called ventral disc. The mechanical function of the ventral disc, however, is still debated. Earlier studies postulated that a dynamic negative pressure under the ventral disc, generated by persistently beating flagella, mediates the attachment. Later studies suggested a suction model based on structural changes of the ventral discs, substrate clutching or grasping, or unspecific contact forces. In this study, we aim to contribute to the understanding of G. duodenalis attachment by investigating detachment characteristics and determining adhesion forces of single trophozoites on a smooth glass surface (RMS = 1.1 ± 0.2 nm) by fluidic force microscopy (FluidFM)-based single-cell force spectroscopy (SCFS). Briefly, viable adherent trophozoites were approached with a FluidFM micropipette, immobilized to the micropipette aperture by negative pressure, and detached from the surface by micropipette retraction while retract force curves were recorded. These force curves displayed novel and so far undescribed characteristics for a microorganism, namely, gradual force increase on the pulled trophozoite, with localization of adhesion force shortly before cell detachment length. Respective adhesion forces reached 7.7 ± 4.2 nN at 1 μm s-1 pulling speed. Importantly, this unique force pattern was different from that of other eukaryotic cells such as Candida albicans or oral keratinocytes, considered for comparison in this study. The latter both displayed a force pattern with force peaks of different values or force plateaus (for keratinocytes) indicative of breakage of molecular bonds of cell-anchored classes of adhesion molecules or membrane components. Furthermore, the attachment mode of G. duodenalis trophozoites was mechanically resilient to tensile forces, when the pulling speeds were raised up to 10 μm s-1 and adhesion forces increased to 28.7 ± 10.5 nN. Taken together, comparative SCSF revealed novel and unique retract force curve characteristics for attached G. duodenalis, suggesting a ligand-independent suction mechanism, that differ from those of other well described eukaryotes.

The adhesion capability of Staphylococcus aureus cells is heterogeneously distributed over the cell envelope.

Understanding and controlling microbial adhesion is a critical challenge in biomedical research, given the profound impact of bacterial infections on global health. Many facets of bacterial adhesion, including the distribution of adhesion forces across the cell wall, remain poorly understood. While a recent 'patchy colloid' model has shed light on adhesion in Gram-negative Escherichia coli cells, a corresponding model for Gram-positive cells has been elusive. In this study, we employ single cell force spectroscopy to investigate the adhesion force of Staphylococcus aureus. Normally, only one contact point of the entire bacterial surface is measured. However, by using a sine-shaped surface and recording force-distance curves along a path perpendicular to the rippled structures, we can characterize almost a hemisphere of one and the same bacterium. This unique approach allows us to study a greater number of contact points between the bacterium and the surface compared to conventional flat substrata. Distributed over the bacterial surface, we identify sites of higher and lower adhesion, which we call 'patchy adhesion', reminiscent of the patchy colloid model. The experimental results show that only some cells exhibit particularly strong adhesion at certain locations. To gain a better understanding of these locations, a geometric model of the bacterial cell surface was created. The experimental results were best reproduced by a model that features a few (5-6) particularly strong adhesion sites (diameter about 250 nm) that are widely distributed over the cell surface. Within the simulated patches, the number of molecules or their individual adhesive strength is increased. A more detailed comparison shows that simple geometric considerations for interacting molecules are not sufficient, but rather strong angle-dependent molecule-substratum interactions are required. We discuss the implications of our results for the development of new materials and the design and analysis of future studies.

Atomic force microscopy analysis of Pel polysaccharide- and type IV pili-mediated adhesion of Pseudomonas aeruginosa PA14 to an abiotic surface.

Type IV pili (TFP) contribute to the ability of microbes such as Pseudomonas aeruginosa to engage with and move across surfaces. We reported previously that P. aeruginosa TFP generate retractive forces of ∼30 pN and provided indirect evidence that TFP-mediated surface attachment was enhanced in the presence of the Pel polysaccharide. Here, we use different mutants defective in flagellar, Pel production or TFP production - alone or in combination - to decipher the relative contribution of these biofilm-promoting factors for P. aeruginosa adhesion. By means of atomic force microscopy (AFM), we show that mutating the flagellum (ΔflgK mutant) results in an increase in Pel polysaccharide production, but this increase in Pel does not result in an increase in surface adhesive properties compared to those previously described for the WT strain. By blocking Pel production in the ΔflgK mutant (ΔflgKΔpel), we directly show that TFP play a major role in the adhesion of the bacteria to hydrophobic AFM tips, but that the adhesion force is only slightly impaired by the absence of Pel. Inversely, performing single-cell force spectroscopy measurements with the mutant lacking TFP (ΔflgKΔpilA) reveals that the Pel can modulate the attachment of the bacteria to a hydrophobic substrate in a time-dependent manner. Finally, little adhesion was detected for the ΔflgKΔpilAΔpelA triple mutant, suggesting that both TFP and Pel polysaccharide make a substantial contribution to bacteria-substratum interaction events. Altogether, our data allow us to decipher the relative contribution of Pel and TFP in the early attachment by P. aeruginosa.

Deep Learning Image Recognition-Assisted Atomic Force Microscopy for Single-Cell Efficient Mechanics in Co-culture Environments.

Atomic force microscopy (AFM)-based force spectroscopy assay has become an important method for characterizing the mechanical properties of single living cells under aqueous conditions, but a disadvantage is its reliance on manual operation and experience as well as the resulting low throughput. Particularly, providing a capacity to accurately identify the type of the cell grown in co-culture environments without the need of fluorescent labeling will further facilitate the applications of AFM in life sciences. Here, we present a study of deep learning image recognition-assisted AFM, which not only enables fluorescence-independent recognition of the identity of single co-cultured cells but also allows efficient downstream AFM force measurements of the identified cells. With the use of the deep learning-based image recognition model, the viability and type of individual cells grown in co-culture environments were identified directly from the optical bright-field images, which were confirmed by the following cell growth and fluorescent labeling results. Based on the image recognition results, the positional relationship between the AFM probe and the targeted cell was automatically determined, allowing the precise movement of the AFM probe to the target cell to perform force measurements. The experimental results show that the presented method was applicable not only to the conventional (microsphere-modified) AFM probe used in AFM indentation assay for measuring the Young's modulus of single co-cultured cells but also to the single-cell probe used in AFM-based single-cell force spectroscopy (SCFS) assay for measuring the adhesion forces of single co-cultured cells. The study illustrates deep learning imaging recognition-assisted AFM as a promising approach for label-free and high-throughput detection of single-cell mechanics under co-culture conditions, which will facilitate unraveling the mechanical cues involved in cell-cell interactions in their native states at the single-cell level and will benefit the field of mechanobiology.

(Invited) Advanced Scanning Probe Microscopy for Studying Biofilm Formation and Single Cell Entities at Electrified Interfaces

Advanced scanning probe microscopy techniques such as atomic force – scanning electrochemical microscopy (AFM-SECM) und scanning electrochemical microscopy (SECM) are highly attractive hybrid techniques for studying biomedically relevant samples down to the single entity level. Our group introduced a new type of AFM-SECM probe having a conductive colloid instead of a sharp AFM tip. As this spherical microelectrode, which is located at the end of an electrically insulated AFM cantilever can be easily modified by electrodeposition with e.g., conductive polymers and electrocatalytic layers, single cell force spectroscopy under potential control allows studying adhesion properties [1,2]. Cell adhesion is a crucial parameter not only for developing new materials serving as substrates for neural scaffolds, electrodes, and biomedical devices but also in biofilm formation. Biofilms are well-organized aggregates of bacteria able to attach to and proliferate at almost every type of solid surface resulting in increased resistance to conventional antimicrobial and antibiofouling agents [3].The first attachment of bacterials is a process highly influenced by the nature of the surface, such as hydrophobicity, chemical structure, surface charge, and presence of antimicrobials [4].In this contribution, we present the potential of various scanning probe microscopy techniques, as suitable tools to locally investigate the early stages of biofilm formation and the effects of various antibiofouling and antimicrobial systems against Escherichia coli and Pseudomonas fluorescens. For example, the properties of polydopamine, a bio-compatible polymer, are studied in respect to early stages of bacterial adhesion in dependence of surface charge density and applied potential [5]. Colloidal conductive AFM probes can also be modified electrocatalytic layers, suitable for detecting signaling molecules and at the same time allow electrochemical force spectroscopy at soft samples like biomedically relevant single entities.

Cell Adhesion, Elasticity, and Rupture Forces Guide Microbial Cell Death on Nanostructured Antimicrobial Titanium Surfaces.

Naturally occurring and synthetic nanostructured surfaces have been widely reported to resist microbial colonization. The majority of these studies have shown that both bacterial and fungal cells are killed upon contact and subsequent surface adhesion to such surfaces. This occurs because the presence of high-aspect-ratio structures can initiate a self-driven mechanical rupture of microbial cells during the surface adsorption process. While this technology has received a large amount of scientific and medical interest, one important question still remains: what factors drive microbial death on the surface? In this work, the interplay between microbial-surface adhesion, cell elasticity, cell membrane rupture forces, and cell lysis at the microbial-nanostructure biointerface during adsorptive processes was assessed using a combination of live confocal laser scanning microscopy, scanning electron microscopy, in situ amplitude atomic force microscopy, and single-cell force spectroscopy. Specifically, the adsorptive behavior and nanomechanical properties of live Gram-negative (Pseudomonas aeruginosa) and Gram-positive (methicillin-resistant Staphylococcus aureus) bacterial cells, as well as the fungal species Candida albicans and Cryptococcus neoformans, were assessed on unmodified and nanostructured titanium surfaces. Unmodified titanium and titanium surfaces with nanostructures were used as model substrates for investigation. For all microbial species, cell elasticity, rupture force, maximum cell-surface adhesion force, the work of adhesion, and the cell-surface tether behavior were compared to the relative cell death observed for each surface examined. For cells with a lower elastic modulus, lower force to rupture through the cell, and higher work of adhesion, the surfaces had a higher antimicrobial activity, supporting the proposed biocidal mode of action for nanostructured surfaces. This study provides direct quantification of the differences observed in the efficacy of nanostructured antimicrobial surface as a function of microbial species indicating that a universal, antimicrobial surface architecture may be hard to achieve.

Papain-Based Solubilization of Decellularized Extracellular Matrix for the Preparation of Bioactive, Thermosensitive Pregels.

Solubilized, gel-forming decellularized extracellular matrix (dECM) is used in a wide range of basic and translational research and due to its inherent bioactivity can promote structural and functional tissue remodeling. The animal-derived protease pepsin has become the standard proteolytic enzyme for the solubilization of almost all types of collagen-based dECM. In this study, pepsin was compared with papain, α-amylase, and collagenase for their potential to solubilize porcine liver dECM. Maximum preservation of bioactive components and native dECM properties was used as a decisive criterion for further application of the enzymes, with emphasis on minimal destruction of the protein structure and maintained capacity for physical thermogelation at neutral pH. The solubilized dECM digests, and/or their physically gelled hydrogels were characterized for their rheological properties, gelation kinetics, GAG content, proteomic composition, and growth factor profile. This study highlights papain as a plant-derived enzyme that can serve as a cost-effective alternative to animal-derived pepsin for the efficient solubilization of dECM. The resulting homogeneous papain-digested dECM preserved its thermally triggered gelation properties similar to pepsin digests, and the corresponding dECM hydrogels demonstrated their enhanced bioadhesiveness in single-cell force spectroscopy experiments with fibroblasts. The viability and proliferation of human HepaRG cells on dECM gels were similar to those on pure rat tail collagen type I gels. Papain is not only highly effective and economically attractive for dECM solubilization but also particularly interesting when digesting human-tissue-derived dECM for regenerative applications, where animal-derived materials are to be avoided.

Mechanomics study of cardiomyocytes with filamin c truncations

Abstract Background Filamin C (FLNC) is expressed in cardiomyocytes, where it binds to actin and localizes to Z-discs, sarcolemma, and intercalated discs. Although FLNC truncation variants (FLNCtv) are an established cause of arrhythmias and heart failure (arrhythmogenic cardiomyopathy), the underlying mechanisms, in particular changes in mechanical properties of cardiomyocytes, are mostly unknown. Purpose In this study, we applied a biophysical approach to investigate the interaction between altered mechanical properties and biological response in human induced pluripotent stem cells–derived cardiomyocytes (hiPSC-CMs) carrying FLNCtv. Methods CRISPR/Cas9 genome-edited FLNC-/- hiPSC-CMs, a homozygous condition that is lethal in animal models, and FLNC+/- hiPSC-CMs, recapitulating the heterozygous human disease, were analyzed. Wild-type FLNC hiPSC-CMs were used as controls. Atomic Force microscopy (AFM) was used to perform single-cell force spectroscopy (SCFS) microindentation, to evaluate passive and dynamic mechanical properties. From the loading curves, two cell Young’s Moduli were calculated: E1, related to the compression of the plasma membrane and actin cortex, and E2, including the rest of the inner cell layers (cytoskeleton and nucleus). Cell adhesion was assessed using the retraction curve of the SCFS. Data were processed in terms of adhesion force (the highest value of the force exerted by the cell-surface contact to the cantilever during retraction) and work of adhesion (integrating the area under the retraction curve). Finally, FLNC-/- hiPSC-CMs were incubated with 1 µM Crenolanib (a PDGFRA signaling inhibitor) for 3 days, and the microindentation was performed again using DMSO-only-treated FLNC-/- cells as controls. Results The AFM indentation curve for the cells analyzed showed significant differences. Both elastic contributions (E1 and E2) show that wild-type FLNC iPSC-CMs are stiffer than both mutant FLNC+/- and FLNC-/- iPSC-CMs (see Figure upper panel). The adhesion showed the same trend as the elasticity: mutant FLNC iPSC-CMs showed a progressive decrease of work of adhesion as well as max adhesion force from the FLNC+/- to the FLNC-/- models. Rescue of the latter with Crenolanib showed to moderately increase their stiffness. Moreover, a qualitative analysis of the beating traces showed that FLNC+/- but mostly FLNC-/- display "irregular" small peaks, resembling those of delayed after depolarizations (see Figure lower panel). Conclusions We found that FLNCtv causes a decrease in cell stiffness which declines progressively from the heterozygous status, which is associated with FLNC haploinsufficiency, to the homozygous status, which lacks FLNC. This indicates a progressive softening of the cardiomyocytes, suggesting a damaged cytoskeleton. Crenolanib rescue suggests that the inhibition of PDGFRA signaling could partially restore such damage (see Figure upper panel - right corner).

Plasma Treatment of PDMS for Microcontact Printing (μCP) of Lectins Decreases Silicone Transfer and Increases the Adhesion of Bladder Cancer Cells.

The present study investigates silicone transfer occurring during microcontact printing (μCP) of lectins with polydimethylsiloxane (PDMS) stamps and its impact on the adhesion of cells. Static adhesion assays and single-cell force spectroscopy (SCFS) are used to compare adhesion of nonmalignant (HCV29) and cancer (HT1376) bladder cells, respectively, to high-affinity lectin layers (PHA-L and WGA, respectively) prepared by physical adsorption and μCP. The chemical composition of the μCP lectin patterns was monitored by time-of-flight secondary ion mass spectrometry (ToF-SIMS). We show that the amount of transferred silicone in the μCP process depends on the preprocessing of the PDMS stamps. It is revealed that silicone contamination within the patterned lectin layers inhibits the adhesion of bladder cells, and the work of adhesion is lower for μCP lectins than for drop-cast lectins. The binding capacity of microcontact printed lectins was larger when the PDMS stamps were treated with UV ozone plasma as compared to sonication in ethanol and deionized water. ToF-SIMS data show that ozone-based treatment of PDMS stamps used for μCP of lectin reduces the silicone contamination in the imprinting protocol regardless of stamp geometry (flat vs microstructured). The role of other possible contributors, such as the lectin conformation and organization of lectin layers, is also discussed.

Host Cell Geometry and Cytoskeletal Organization Governs Candida-Host Cell Interactions at the Nanoscale.

Candida is one of the most common opportunistic fungal pathogens in humans. Its adhesion to the host cell is required in parasitic states and is important for pathogenesis. Many studies have shown that there is an increased risk of developing candidiasis when normal tissue barriers are weakened or when immune defenses are compromised, for example, during cancer treatment that induces immunosuppression. The mechanical properties of malignant cells, such as adhesiveness and viscoelasticity, which contribute to cellular invasion and migration are different from those of noncancerous cells. To understand host invasion and its relationship with host cell health, we probed the interaction of Candida spp. with cancerous and noncancerous human cell lines using atomic force microscopy in the single-cell force spectroscopy mode. There was significant adhesion between Candida and human cells, with more adhesion to cancerous versus noncancerous cell lines. This increase in adhesion is related to the mechanobiological properties of cancer cells, which have a disorganized cytoskeleton and lower rigidity. Altered geometry and cytoskeletal disruption of the human cells impacted adhesion parameters, underscoring the role of cytoskeletal organization in Candida-human cell adhesion and implicating the manipulation of cell properties as a potential future therapeutic strategy.