Cellular Force Microscopy Research Articles

Visualizing and quantifying dynamic cellular forces with photonic crystal hydrogels.

Cellular forces play a crucial role in numerous biological processes, including tissue development, morphogenesis, and disease progression. However, existing methods for detecting cellular forces, such as traction force microscopy and atomic force microscopy, often face limitations in terms of high throughput, real-time monitoring, and applicability to complex biological systems. In this study, we utilized a novel Photonic Crystal Cellular Force Microscopy (PCCFM) system to visualize and quantify dynamic cellular forces. This system consists of a conventional optical microscope and a photonic crystal substrate formed by the periodic arrangement of silica nanoparticles within polyacrylamide hydrogels. Taking MDCK cells and BMSCs as examples, we found that PCCFM can capture dynamic cellular forces with high spatial and temporal resolution during the cell adhesion, spread, proliferation, and osteogenic differentiation. The application of this technique revealed distinct force patterns in different cellular stages, offering insights into the interplay between cellular forces and morphological changes. By investigating the migration of cells from MDCK cyst fragments, we could gain significant insights into tumour cell migration behaviours. The real-time, high-throughput analysis of cellular biomechanics from the PCCFM system offers valuable information on the mechanisms of tumour metastasis, potentially guiding therapeutic development and improving disease treatment strategies.

Imaging cellular forces with photonic crystals

Current techniques for visualizing and quantifying cellular forces have limitations in live cell imaging, throughput, and multi-scale analysis, which impede progress in cell force research and its practical applications. We developed a photonic crystal cellular force microscopy (PCCFM) to image vertical cell forces over a wide field of view (1.3 mm ⨯ 1.0 mm, a 10 ⨯ objective image) at high speed (about 20 frames per second) without references. The photonic crystal hydrogel substrate (PCS) converts micro-nano deformations into perceivable color changes, enabling in situ visualization and quantification of tiny vertical cell forces with high throughput. It enabled long-term, cross-scale monitoring from subcellular focal adhesions to tissue-level cell sheets and aggregates.

Functional α7 nicotinic receptors in human airway smooth muscle increase intracellular calcium concentration and contractility in asthmatics.

Although nicotinic acetylcholine receptors (nAChRs) are commonly associated with neurons in the brain and periphery, recent data indicate that they are also expressed in non-neuronal tissues. We recently found the alpha7 (α7nAChR) subunit is highly expressed in human airway smooth muscle (hASM) with substantial increase in asthmatics, but their functionality remains unknown. We investigated the location and functional role of α7nAChRs in hASM cells from normal versus mild-moderate asthmatic patients. Immunostaining and protein analyses showed α7nAChR in the plasma membrane including in asthmatics. In asthmatic hASM, patch-clamp recordings revealed significantly higher functional homomeric α7nAChR channels. Real-time fluorescence imaging showed nicotine, via α7nAChR, increases intracellular Ca2+ ([Ca2+]i) independent of ACh effects, particularly in asthmatic hASM, while cellular traction force microscopy showed nicotine-induced contractility including in asthmatics. These results indicate functional homomeric and heteromeric nAChRs that are increased in asthmatic hASM, with pharmacology that likely differ owing to different subunit interfaces that form the orthosteric sites. nAChRs may represent a novel target in alleviating airway hyperresponsiveness in asthma.NEW & NOTEWORTHY Cigarette smoking and vaping exacerbate asthma. Understanding the mechanisms of nicotine effects in asthmatic airways is important. This study demonstrates that functional alpha7 nicotinic acetylcholine receptors (α7nAChRs) are expressed in human airway smooth muscle, including from asthmatics, and enhance intracellular calcium and contractility. Although a7nAChRs are associated with neuronal pathways, α7nAChR in smooth muscle suggests inhaled nicotine (e.g., vaping) can directly influence airway contractility. Targeting α7nAChR may represent a novel approach to alleviating airway hyperresponsiveness in asthma.

Deep-learning-based 3D cellular force reconstruction directly from volumetric images

The forces exerted by single cells in the three-dimensional (3D) environments play a crucial role in modulating cellular functions and behaviors closely related to physiological and pathological processes. Cellular force microscopy (CFM) provides a feasible solution for quantifying mechanical interactions, which usually regains cellular forces from deformation information of extracellular matrices embedded with fluorescent beads. Owing to computational complexity, traditional 3D-CFM is usually extremely time consuming, which makes it challenging for efficient force recovery and large-scale sample analysis. With the aid of deep neural networks, this study puts forward a novel, data-driven 3D-CFM to reconstruct 3D cellular force fields directly from volumetric images with random fluorescence patterns. The deep-learning-based network is established through stacking deep convolutional neural networks (DCNN) and specific function layers. Some necessary physical information associated with constitutive relation of extracellular matrix material is coupled to the data-driven network. The mini-batch stochastic-gradient-descent and back-propagation algorithms are introduced to ensure its convergence and training efficiency. The networks not only have good generalization ability and robustness but also can recover 3D cellular forces directly from the input fluorescence image pairs. Particularly, the computational efficiency of the deep-learning-based network is at least one to two orders of magnitude higher than that of traditional 3D-CFM. This study provides a novel scheme for developing high-performance 3D-CFM to quantitatively characterize mechanical interactions between single cells and surrounding extracellular matrices, which is of vital importance for quantitative investigations in biomechanics and mechanobiology.

Force microscopy of the Caenorhabditis elegans embryonic eggshell

Assays focusing on emerging biological phenomena in an animal’s life can be performed during embryogenesis. While the embryo of Caenorhabditis elegans has been extensively studied, its biomechanical properties are largely unknown. Here, we demonstrate that cellular force microscopy (CFM), a recently developed technique that combines micro-indentation with high resolution force sensing approaching that of atomic force microscopy, can be successfully applied to C. elegans embryos. We performed, for the first time, a quantitative study of the mechanical properties of the eggshell of living C. elegans embryos and demonstrate the capability of the system to detect alterations of its mechanical parameters and shell defects upon chemical treatments. In addition to investigating natural eggshells, we applied different eggshell treatments, i.e., exposure to sodium hypochlorite and chitinase solutions, respectively, that selectively modified the multilayer eggshell structure, in order to evaluate the impact of the different layers on the mechanical integrity of the embryo. Finite element method simulations based on a simple embryo model were used to extract characteristic eggshell parameters from the experimental micro-indentation force-displacement curves. We found a strong correlation between the severity of the chemical treatment and the rigidity of the shell. Furthermore, our results showed, in contrast to previous assumptions, that short bleach treatments not only selectively remove the outermost vitelline layer of the eggshell, but also significantly degenerate the underlying chitin layer, which is primarily responsible for the mechanical stability of the egg.

Extracellular and intercellular force distribution in circularly shaped epithelia

Stress homeostasis in multicellular organisms is essential to tissue growth, development, and repair. However, how cellular forces are generated and transmitted within a multicellular organism to maintain stress homeostasis remains poorly understood. Here we employ cellular force microscopies to quantify extracellular traction and intercellular tension of circularly shaped cohesive epithelia seeded on hydrogels of various stiffness. Our experimental measurements show that the traction force is both colony size and gel stiffness dependent. A minimal mechanics model is developed and predicts exponential decay of extracellular traction from the periphery to the center of the epithelial monolayers, which agrees with the experimental data. Our modeling analyses further show that the colony size and gel stiffness dependent traction is originated from the line tension effect and stiffness-dependent cell contractility, respectively. Our study lays down a foundation for future modeling and experimental measurements of cellular force generation, transmission, and distribution in multicellular organisms, which is critical to mechanobiological understanding of normal and disease development.

Abstract 5150: Adipose-derived stem cell disruption of gap junction intercellular communication in obesity-associated endometrial cancer

Abstract Endometrial cancer is the most common gynecologic cancer. It is divided into two major types, a less aggressive endometrioid type and a more aggressive serous type. Recent clinical observations underscore obesity as a major risk factor for endometrial cancer. In this study, we hypothesized that obesity can result in long term epigenetic modifications in the endometrial compartment, leading to the promotion of endometrial cancer. We examined the role of adipose-derived stem cells (ASCs), which are adipocyte progenitors, in the endometrial tumor microenvironment. By staining for ASCs in a tumor tissue microarray, we observed ASC infiltration that was specific to samples from obese patients, correlating with body mass index (R2=0.77, p<0.001). Using a co-culture system to simulate ASC microenvironmental effects and transcriptomic analysis, we showed that exposure of immortalized endometrial epithelial cells (EECs) to ASC secreted factors led to the repression of cell-cell communication pathways, most notably gap junctions and related factors tight junction proteins TJP and PKC genes. This gene repression was associated with induction of DNA Methylation in the promoter of the major GJ gene GJA1 (encoding connexin 43, Cx43) in the ASC-exposed EECs. Importantly, we further demonstrated this DNA hypermethylation in the promoters of the GJA1, TJP2 and PKC genes in primary endometrial tumors from obese patients compared to non-obese patients. We assessed the effects of epigenetic regulation on gap junction intercellular communication (GJIC) and cell-cell interactions using cellular calcein dye transfer assays and atomic force microscopy (for nano-scale assessment of cell-cell adhesion) by treating endometrial cancer cells with a demethylating agent (DAC). DAC treatment resulted in increased level of cell-cell adhesiveness and communication via gap junction coupling. Specific reactivation of GJA1 (Cx43) by expression vector in endometrial cancer cells led to decreased cellular motility. Because we found that PAI-1 is a major adipokine in the ASC secretome, inhibition of PAI-1 in ASC-exposed EECs led to a cell population expression profile with lower GJ expression, based on single-cell PCR studies. Collectively, the data demonstrate multi-scale regulation of cellular communication via paracrine actions and direct cell-cell coupling via gap junctions by epigenetic silencing influenced by ASCs. This leads to disruption of cellular homeostasis and enhanced motility, promoting endometrial cancer in obese patients. Citation Format: Li-Ling Lin, Srikanth Polusani, Guangcun Huang, Chun-Lin Lin, Chiou-Miin Wang, Nicholas Lucio, Mikhail Kolonin, Alexes Daquinag, Pawel Osmulski, Bruce Nicholson, Edward Kost, Tim Huang, Nameer B. Kirma. Adipose-derived stem cell disruption of gap junction intercellular communication in obesity-associated endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5150.

Cellular Force Microscopy to Measure Mechanical Forces in Plant Cells.

Cellular force microscopy (CFM) is a noninvasive microindentation method used to measure plant cell stiffness in vivo. CFM is a scanning probe microscopy technique similar in operation to atomic force microscopy (AFM); however, the scale of movement and range of forces are much larger, making it suitable for stiffness measurements on turgid plant cells in whole organs. CFM experiments can be performed on living samples over extended time periods, facilitating the exploration of the dynamics of processes involving mechanics. Different sensor technologies can be used, along with a variety of probe shapes and sizes that can be tailored to specific applications. Measurements can be made for specific indentation depths, forces and timing, allowing for very precise mechanical stimulation of cells with known forces. High forces with sharp tips can also be used for mechanical ablation of cells with force feedback.

Mechanotargeting: Mechanics-Dependent Cellular Uptake of Nanoparticles.

Targeted delivery of nanoparticle (NP)-based diagnostic and therapeutic agents to malignant cells and tissues has exclusively relied on chemotargeting, wherein NPs are surface-coated with ligands that specifically bind to overexpressed receptors on malignant cells. Here, it is demonstrated that cellular uptake of NPs can also be biased to malignant cells based on the differential mechanical states of cells, enabling mechanotargeting. Owing to mechanotransduction, cell lines (HeLa and HCT-8) cultured on hydrogels of various stiffness are directed into different stress states, measured by cellular force microscopies. In vitro NP delivery reveals that increases in cell stress suppress cellular uptake, counteracting the enhanced uptake that occurs with increases in exposed surface area of spread cells. Upon prolonged culture on stiff hydrogels, cohesive HCT-8 cell colonies undergo metastatic phenotypic change and disperse into individual malignant cells. The metastatic cells are of extremely low stress state and adopt an unspread, 3D morphology, resulting in several-fold higher uptake than the nonmetastatic counterparts. This study opens a new paradigm of harnessing mechanics for the design of future strategies in nanomedicine.

LRX Proteins Play a Crucial Role in Pollen Grain and Pollen Tube Cell Wall Development.

Leu-rich repeat extensins (LRXs) are chimeric proteins containing an N-terminal Leu-rich repeat (LRR) and a C-terminal extensin domain. LRXs are involved in cell wall formation in vegetative tissues and required for plant growth. However, the nature of their role in these cellular processes remains to be elucidated. Here, we used a combination of molecular techniques, light microscopy, and transmission electron microscopy to characterize mutants of pollen-expressed LRXs in Arabidopsis (Arabidopsisthaliana). Mutations in multiple pollen-expressed lrx genes cause severe defects in pollen germination and pollen tube growth, resulting in a reduced seed set. Physiological experiments demonstrate that manipulating Ca2+ availability partially suppresses the pollen tube growth defects, suggesting that LRX proteins influence Ca2+-related processes. Furthermore, we show that LRX protein localizes to the cell wall, and its LRR-domain (which likely mediates protein-protein interactions) is associated with the plasma membrane. Mechanical analyses by cellular force microscopy and finite element method-based modeling revealed significant changes in the material properties of the cell wall and the fine-tuning of cellular biophysical parameters in the mutants compared to the wild type. The results indicate that LRX proteins might play a role in cell wall-plasma membrane communication, influencing cell wall formation and cellular mechanics.

On the micro-indentation of plant cells in a tissue context

The effect of geometry on cell stiffness measured with micro-indentation techniques has been explored in single cells, however it is unclear if results on single cells can be readily transferred to indentation experiments performed on a tissue in vivo. Here we explored this question by using simulation models of osmotic treatments and micro-indentation experiments on 3D multicellular tissues with the finite element method. We found that the cellular context does affect measured cell stiffness, and that several cells of context in each direction are required for optimal results. We applied the model to micro-indentation data obtained with cellular force microscopy on the sepal of A. thaliana, and found that differences in measured stiffness could be explained by cellular geometry, and do not necessarily indicate differences in cell wall material properties or turgor pressure.

Two-Layer Elastographic 3-D Traction Force Microscopy

Cellular traction force microscopy (TFM) requires knowledge of the mechanical properties of the substratum where the cells adhere to calculate cell-generated forces from measurements of substratum deformation. Polymer-based hydrogels are broadly used for TFM due to their linearly elastic behavior in the range of measured deformations. However, the calculated stresses, particularly their spatial patterns, can be highly sensitive to the substratum’s Poisson’s ratio. We present two-layer elastographic TFM (2LETFM), a method that allows for simultaneously measuring the Poisson’s ratio of the substratum while also determining the cell-generated forces. The new method exploits the analytical solution of the elastostatic equation and deformation measurements from two layers of the substratum. We perform an in silico analysis of 2LETFM concluding that this technique is robust with respect to TFM experimental parameters, and remains accurate even for noisy measurement data. We also provide experimental proof of principle of 2LETFM by simultaneously measuring the stresses exerted by migrating Physarum amoeboae on the surface of polyacrylamide substrata, and the Poisson’s ratio of the substrata. The 2LETFM method could be generalized to concurrently determine the mechanical properties and cell-generated forces in more physiologically relevant extracellular environments, opening new possibilities to study cell-matrix interactions.

Measuring the Mechanical Properties of Plant Cell Walls

The size, shape and stability of a plant depend on the flexibility and integrity of its cell walls, which, at the same time, need to allow cell expansion for growth, while maintaining mechanical stability. Biomechanical studies largely vanished from the focus of plant science with the rapid progress of genetics and molecular biology since the mid-twentieth century. However, the development of more sensitive measurement tools renewed the interest in plant biomechanics in recent years, not only to understand the fundamental concepts of growth and morphogenesis, but also with regard to economically important areas in agriculture, forestry and the paper industry. Recent advances have clearly demonstrated that mechanical forces play a crucial role in cell and organ morphogenesis, which ultimately define plant morphology. In this article, we will briefly review the available methods to determine the mechanical properties of cell walls, such as atomic force microscopy (AFM) and microindentation assays, and discuss their advantages and disadvantages. But we will focus on a novel methodological approach, called cellular force microscopy (CFM), and its automated successor, real-time CFM (RT-CFM).

OS18-7 New Cellular Traction Force Microscopy Reveals the Effect of Substrate Curvature on Focal Adhesion Dynamics(Cell and Tissue mechanics 3,OS18 Cell and tissue mechanics,BIOMECHANICS)

OS18-7 New Cellular Traction Force Microscopy Reveals the Effect of Substrate Curvature on Focal Adhesion Dynamics(Cell and Tissue mechanics 3,OS18 Cell and tissue mechanics,BIOMECHANICS)

M307 逆解析による細胞牽引力の推定 : 数理的アプローチによる最適条件の検討(GS2,3 材料力学(4),修士研究発表セッション)

M307 逆解析による細胞牽引力の推定 : 数理的アプローチによる最適条件の検討(GS2,3 材料力学(4),修士研究発表セッション)

Forces in T Cell Antigen Recognition

There are strong indications that mechanical forces are particularly relevant in immune recognition. For the immune system, the enormous variety of antigenic ligands imposes a fundamental challenge to the discriminative power; the mechanism for discriminating between activating and non-activating ligands has remained enigmatic.In a recent theoretical study we showed how forces alters the potency for receptor ligand discrimination by orders of magnitudes(1). For the T cell receptor, which specifically binds to peptides presented by MHC on an antigen-presenting cell, discrimination can be realized with kinetic proofreading, which fails when ligands have only marginal differences in their off-rates. We showed, however, that the specificity of antigen-recognition can be massively improved by putting the TCR-pMHC bond under load: while under no force the bond rupture probability decays exponentially with time, force-induced bond rupture leads to much narrower distributions.Here, we present cellular traction force microscopy data to measure forces involved during T cell activation. Hydrogels were prepared with variable stiffness. Fluorescent beads carrying CD3 antibodies were immobilized onto the top layer of the hydrogel. Forces are read out by measuring the fluorescent bead movement throughout T cell attachment and activation. The bead movement was directly correlated to forces applied to the antibodies immobilized on the beads. Moreover, discrimination between lateral and transversal applied forces was possible by tracking the beads' positions in 3D.1.Klotzsch, E., and G.J. Schutz. 2013. Improved Ligand Discrimination by Force-Induced Unbinding of the T Cell Receptor from Peptide-MHC. Biophysj. 104: 1670-1675.

3 Dimensional Cellular Force Microscopy in Fibrin Gels

The mechanical forces exerted and detected by living cells play integral roles in diverse biological phenomena, including growth and development, wound healing, and cancer metastasis. In the past decade, techniques such as traction force microscopy and micropost arrays have proven to be powerful tools for measuring the forces generated by cells. In particular, traction force microscopy has recently been extended to three-dimensional cell culture environments by embedding tracer beads in either a synthetic polyethylene glycol hydrogel (PEG; Legant et al., Nat. Meth. 2010) or in collagen gels (Koch et al., PLoS ONE 2012). The embedded beads move in response to cell-generated distortions of the matrix, allowing cell-generated forces to be calculated. We sought to develop an experimental system that would exhibit the excellent mechanical properties of the PEG hydrogel while using a naturally occurring biological matrix. Fibrin gels fulfill both of these requirements: fibrin is elastic up to ∼50% strain (Brown et al., Science 2009) and is also widely used for 3D cell culture. Here we describe the use of fluorescently labeled fibrin gels to measure the forces generated by cells in 3D culture. We observe dramatic but elastic deformations of the fibrin matrix surrounding cells as they grow, divide, and migrate. Further, we find that the dynamic forces generated by the cell can be measured using the deformations of the matrix itself, providing a direct observation of how the cell modifies its surroundings. We discuss the use of this new technique in studying matrix remodeling and cell migration.

Cellular Traction Force Reconstruction Based on a Self-adaptive Filtering Scheme

As an emerging measurement technique in cell mechanics, cellular traction force microscopy has begun to be applied to monitor spatiotemporal dynamics of cell-substrate interactions, in which Fourier transform traction cytometry (FTTC) can be utilized to reconstruct cellular traction fields from substrate displacement knowledge in a computationally cheap way. Owing to the intrinsic ill-posed property, cellular traction recovery founded on FTTC is extremely susceptible to measurement noise so that it is very likely to produce unreliable traction results. This paper investigates the nature of noise amplification during the process of deconvolution and accordingly proposes a set of filtering algorithms to evaluate cellular tractions. By self-adaptively filtering out the noise components in the two-dimensional Fourier space, high-resolution traction fields can thus be recovered in a relatively efficient manner. The feasibility and effectiveness of the set of algorithms are verified by both systematic simulation analyses and actual cellular traction reconstructions.

Cellular Force Microscopy for in Vivo Measurements of Plant Tissue Mechanics

Although growth and morphogenesis are controlled by genetics, physical shape change in plant tissue results from a balance between cell wall loosening and intracellular pressure. Despite recent work demonstrating a role for mechanical signals in morphogenesis, precise measurement of mechanical properties at the individual cell level remains a technical challenge. To address this challenge, we have developed cellular force microscopy (CFM), which combines the versatility of classical microindentation techniques with the high automation and resolution approaching that of atomic force microscopy. CFM's large range of forces provides the possibility to map the apparent stiffness of both plasmolyzed and turgid tissue as well as to perform micropuncture of cells using very high stresses. CFM experiments reveal that, within a tissue, local stiffness measurements can vary with the level of turgor pressure in an unexpected way. Altogether, our results highlight the importance of detailed physically based simulations for the interpretation of microindentation results. CFM's ability to be used both to assess and manipulate tissue mechanics makes it a method of choice to unravel the feedbacks between mechanics, genetics, and morphogenesis.

Chaperonin Containing T-Complex Polypeptide Subunit Eta (CCT-eta) Is a Specific Regulator of Fibroblast Motility and Contractility

Integumentary wounds in mammalian fetuses heal without scar; this scarless wound healing is intrinsic to fetal tissues and is notable for absence of the contraction seen in postnatal (adult) wounds. The precise molecular signals determining the scarless phenotype remain unclear. We have previously reported that the eta subunit of the chaperonin containing T-complex polypeptide (CCT-eta) is specifically reduced in healing fetal wounds in a rabbit model. In this study, we examine the role of CCT-eta in fibroblast motility and contractility, properties essential to wound healing and scar formation. We demonstrate that CCT-eta (but not CCT-beta) is underexpressed in fetal fibroblasts compared to adult fibroblasts. An in vitro wound healing assay demonstrated that adult fibroblasts showed increased cell migration in response to epidermal growth factor (EGF) and platelet derived growth factor (PDGF) stimulation, whereas fetal fibroblasts were unresponsive. Downregulation of CCT-eta in adult fibroblasts with short inhibitory RNA (siRNA) reduced cellular motility, both basal and growth factor-induced; in contrast, siRNA against CCT-beta had no such effect. Adult fibroblasts were more inherently contractile than fetal fibroblasts by cellular traction force microscopy; this contractility was increased by treatment with EGF and PDGF. CCT-eta siRNA inhibited the PDGF-induction of adult fibroblast contractility, whereas CCT-beta siRNA had no such effect. In each of these instances, the effect of downregulating CCT-eta was to modulate the behavior of adult fibroblasts so as to more closely approximate the characteristics of fetal fibroblasts. We next examined the effect of CCT-eta modulation on alpha-smooth muscle actin (α-SMA) expression, a gene product well known to play a critical role in adult wound healing. Fetal fibroblasts were found to constitutively express less α-SMA than adult cells. Reduction of CCT-eta with siRNA had minimal effect on cellular beta-actin but markedly decreased α-SMA; in contrast, reduction of CCT-beta had minimal effect on either actin isoform. Direct inhibition of α-SMA with siRNA reduced both basal and growth factor-induced fibroblast motility. These results indicate that CCT-eta is a specific regulator of fibroblast motility and contractility and may be a key determinant of the scarless wound healing phenotype by means of its specific regulation of α-SMA expression.