A DNA-based optical force sensor for live-cell applications

Mechanical forces are relevant for many biological processes, from wound healing or tumour formation to cell migration and differentiation. Cytoskeletal actin is largely responsible for responding to forces and transmitting them in cells, while also maintaining cell shape and integrity. Here, we describe a novel approach to employ a FRET-based DNA force sensor in vitro and in cellulo for non-invasive optical monitoring of intracellular mechanical forces. We use fluorescence lifetime imaging to determine the FRET efficiency of the sensor, which makes the measurement robust against intensity variations. We demonstrate the applicability of the sensor by monitoring cross-linking activity in in vitro actin networks by bulk rheology and confocal microscopy. We further demonstrate that the sensor readily attaches to stress fibers in living cells which opens up the possibility of live-cell force measurements.

Using Knock-Out Mutants to Investigate the Adhesion of Staphylococcus aureus to Abiotic Surfaces

The adhesion of Staphylococcus aureus to abiotic surfaces is crucial for establishing device-related infections. With a high number of single-cell force spectroscopy measurements with genetically modified S. aureus cells, this study provides insights into the adhesion process of the pathogen to abiotic surfaces of different wettability. Our results show that S. aureus utilizes different cell wall molecules and interaction mechanisms when binding to hydrophobic and hydrophilic surfaces. We found that covalently bound cell wall proteins strongly interact with hydrophobic substrates, while their contribution to the overall adhesion force is smaller on hydrophilic substrates. Teichoic acids promote adhesion to hydrophobic surfaces as well as to hydrophilic surfaces. This, however, is to a lesser extent. An interplay of electrostatic effects of charges and protein composition on bacterial surfaces is predominant on hydrophilic surfaces, while it is overshadowed on hydrophobic surfaces by the influence of the high number of binding proteins. Our results can help to design new models of bacterial adhesion and may be used to interpret the adhesion of other microorganisms with similar surface properties.

IgM regulates airway hyperresponsiveness via modulation of actin associated genes.

Allergic asthma is a disease driven by T helper 2 (Th2) cells, eosinophilia, airway hyperresponsiveness (AHR) and IgE-secreting B cells. Asthma is largely controlled by corticosteroids and β2 adregenic receptor agonists that target and relax airway smooth muscle (ASM). Immunoglobulin M (IgM) isotype secreted by naïve B cells is important for class switching but may have other undefined functions. We investigated the role of IgM in a house dust mite (HDM)-induced Th2 allergic asthma model by sensitising wild type (WT) and IgM-deficient (IgM-/-) mice with HDM. We validated our findings using CRISPR and single cell force cytometry in human ASM. We found IgM to be essential in AHR but not Th2 airway inflammation or eosinophilia. RNA sequencing of lung tissue suggested that IgM regulated AHR through modulating brain-specific angiogenesis inhibitor 1-associated protein 2-like protein 1 (Baiap2l1) and erythroid differentiation regulator 1 (Erdr1). Deletion of BAIAP2L1 and ERDR1 reduced human ASM contraction when stimulated with TNF-α. These are unprecedented findings and have implications in future treatment of asthma beyond current therapies.

Quantifying the Effect of Anti-cancer Compound (Piperlongumine) on Cancer Cells Using Single-Cell Force Spectroscopy

Natural compounds have shown a great potential in anti-cancer research by tumor growth inhibition and anti-metastatic properties. Piperlongumine (PL) is a natural compound derived from pepper species that has been demonstrated to have anti-cancer effect on HeLa cells. Here we focus on understanding the mechanical properties of HeLa cells under PL treatment, using Atomic Force Microscopy (AFM) based single-cell manipulation technique. We used AFM to pull single HeLa cells and acquired the force-distance curves that presented stepwise patterns. We analyzed the step force (SF) and observed that cells treated with PL exhibit higher force compared to control cells. This SF increase was also observed in experiments performed on substrates of different stiffness. Therefore, analyzing SF, it is possible to investigate the effect of PL on the mechanical properties of the HeLa cells. The understanding of the PL action on HeLa cells’ mechanical properties may help in the development of effective therapeutic drugs against cancers.

The biochemical composition of the actomyosin network sets the magnitude of cellular traction forces

The endogenous content of proteins associated with force production and the resultant traction forces were quantified in the same cells using a new traction force-microscopy assay. Focal adhesion size correlated with force in stationary cells. Relative numbers of motors and cross-linkers per actin required an optimum to maximize cell force production.

Mechanical intercellular communication via matrix-borne cell force transmission during vascular network formation

Intercellular communication is critical to the development and homeostatic function of all tissues. Previous work has shown that cells can communicate mechanically via transmission of cell-generated forces through their surrounding extracellular matrix, but this process is not well understood. Here, we utilized synthetic, electrospun fibrous matrices in conjunction with a microfabrication-based cell patterning approach to examine mechanical intercellular communication (MIC) between endothelial cells (ECs) during the assembly of microvascular networks. We found that cell force-mediated matrix displacements in deformable fibrous matrices underly directional migration of neighboring ECs towards each other prior to the formation of stable cell-cell connections. We also identified a critical role for intracellular calcium signaling mediated by focal adhesion kinase and TRPV4 during MIC that extends to multicellular assembly of vessel-like networks in 3D fibrin hydrogels. The results presented here are critical to the design of biomaterials that support cellular self-assembly for tissue engineering applications.

A New Function for Amyloid-Like Interactions: Cross-Beta Aggregates of Adhesins form Cell-to-Cell Bonds

Amyloid structures assemble through a repeating type of bonding called “cross-β”, in which identical sequences in many protein molecules form β-sheets that interdigitate through side chain interactions. We review the structural characteristics of such bonds. Single cell force microscopy (SCFM) shows that yeast expressing Als5 adhesin from Candida albicans demonstrate the empirical characteristics of cross-β interactions. These properties include affinity for amyloid-binding dyes, birefringence, critical concentration dependence, repeating structure, and inhibition by anti-amyloid agents. We present a model for how cross-β bonds form in trans between two adhering cells. These characteristics also apply to other fungal adhesins, so the mechanism appears to be an example of a new type of cell–cell adhesion.

From the first touch to biofilm establishment by the human pathogen Candida glabrata: a genome-wide to nanoscale view

Candida glabrata is an opportunistic pathogen that adheres to human epithelial mucosa and forms biofilm to cause persistent infections. In this work, Single-cell Force Spectroscopy (SCFS) was used to glimpse at the adhesive properties of C. glabrata as it interacts with clinically relevant surfaces, the first step towards biofilm formation. Following a genetic screening, RNA-sequencing revealed that half of the entire transcriptome of C. glabrata is remodeled upon biofilm formation, around 40% of which under the control of the transcription factors CgEfg1 and CgTec1. Using SCFS, it was possible to observe that CgEfg1, but not CgTec1, is necessary for the initial interaction of C. glabrata cells with both abiotic surfaces and epithelial cells, while both transcription factors orchestrate biofilm maturation. Overall, this study characterizes the network of transcription factors controlling massive transcriptional remodelling occurring from the initial cell-surface interaction to mature biofilm formation.

Spatiotemporal model of cellular mechanotransduction via Rho and YAP

How cells sense and respond to mechanical stimuli remains an open question. Recent advances have identified the translocation of Yes-associated protein (YAP) between nucleus and cytoplasm as a central mechanism for sensing mechanical forces and regulating mechanotransduction. We formulate a spatiotemporal model of the mechanotransduction signalling pathway that includes coupling of YAP with the cell force-generation machinery through the Rho family of GTPases. Considering the active and inactive forms of a single Rho protein (GTP/GDP-bound) and of YAP (non-phosphorylated/phosphorylated), we study the cross-talk between cell polarization due to active Rho and YAP activation through its nuclear localization. For fixed mechanical stimuli, our model predicts stationary nuclear-to-cytoplasmic YAP ratios consistent with experimental data at varying adhesive cell area. We further predict damped and even sustained oscillations in the YAP nuclear-to-cytoplasmic ratio by accounting for recently reported positive and negative YAP-Rho feedback. Extending the framework to time-varying mechanical stimuli that simulate cyclic stretching and compression, we show that the YAP nuclear-to-cytoplasmic ratio’s time dependence follows that of the cyclic mechanical stimulus. The model presents one of the first frameworks for understanding spatiotemporal YAP mechanotransduction, providing several predictions of possible YAP localization dynamics, and suggesting new directions for experimental and theoretical studies.