Electric fields regulate cellular elasticity through intracellular Ca2+ concentrations.

Cellular elasticity is a key factor related to a broad range of physiological and pathological processes. The elasticity of a single cell has thus emerged as a potential biomarker to characterize the cellular state. Both internal and external stimuli affect cellular elasticity, and changes in elasticity can cause alterations in cellular characteristics or function. The application of electric fields (EFs) is a promising method that can be used to change cellular elasticity; however, the mechanisms underlying its effect remain unknown. Here, we demonstrate EFs-induced elasticity changes in human dermal fibroblasts and discuss the underlying mechanism related to actin polymerization. Cellular elasticity increases after EF (50 mV/mm) stimulation, reaching a maximum at 30 min before decreasing between 30 and 120 min. The cellular elasticity under EF stimulation, regardless of stimulation time, is higher than that of the control. F-actin regulates the elasticity of cells through gelsolin activation. We show changes in intracellular Ca2+ caused by EFs, which induced gelsolin activation and F-actin content changes. This result demonstrates a series of processes in which external electrical stimulation conditions regulate cellular elasticity.

Qualitative analysis of contribution of intracellular skeletal changes to cellular elasticity.

Cells are dynamic structures that continually generate and sustain mechanical forces within their environments. Cells respond to mechanical forces by changing their shape, moving, and differentiating. These reactions are caused by intracellular skeletal changes, which induce changes in cellular mechanical properties such as stiffness, elasticity, viscoelasticity, and adhesiveness. Interdisciplinary research combining molecular biology with physics and mechanical engineering has been conducted to characterize cellular mechanical properties and understand the fundamental mechanisms of mechanotransduction. In this review, we focus on the role of cytoskeletal proteins in cellular mechanics. The specific role of each cytoskeletal protein, including actin, intermediate filaments, and microtubules, on cellular elasticity is summarized along with the effects of interactions between the fibers.

Biomarkers to quantify cell migration characteristics.

BACKGROUND: Because cell movement is primarily driven by the connection between F-actin and integrin through a physical linkage, cellular elasticity and adhesion strength have been considered as biomarkers of cell motility. However, a consistent set of biomarkers that indicate the potential for cell motility is still lacking. METHODS: In this work, we characterize a phenotype of cell migration in terms of cellular elasticity and adhesion strength, which reveals the interdependence of subcellular systems that mediate optimal cell migration. RESULTS: Stiff cells weakly adhered to the substrate revealed superior motility, while soft cell migration with strong adhesion was relatively inhibited. The spatial distribution and amount of F-actin and integrin were highly variable depending on cell type, but their density exhibited linear correlations with cellular elasticity and adhesion strength, respectively. CONCLUSIONS: The densities of F-actin and integrin exhibited linear correlations with cellular elasticity and adhesion strength, respectively, therefore, they can be considered as biomarkers to quantify cell migration characteristics.

Electric field-induced changes in biomechanical properties in human dermal fibroblasts and a human skin equivalent.

PURPOSE: An electric field (EF) can be used to change the mechanical properties of cells and skin tissues. We demonstrate EF-induced elasticity changes in human dermal fibroblasts (HDFs) and a human skin equivalent and identify the underlying principles related to the changes. METHODS: HDFs and human skin equivalent were stimulated with electric fields of 1.0 V/cm. Change in cellular elasticity was determined by using atomic force microscopy. Effects of EF on the biomechanical and chemical properties of a human skin equivalent were analyzed. In cells and tissues, the effects of EF on biomarkers of cellular elasticity were investigated at the gene and protein levels. RESULTS: In HDFs, the cellular elasticity was increased and the expression of biomarkers of cellular elasticity was regulated by the EF. Expression of the collagen protein in the human skin equivalent was changed by EF stimulation; however, changes in density and microstructure of the collagen fibrils were not significant. The viscoelasticity of the human skin equivalent increased in response to EF stimulation, but molecular changes were not observed in collagen. CONCLUSIONS: Elasticity of cells and human skin equivalent can be regulated by electrical stimulation. Especially, the change in cellular elasticity was dependent on cell age.

The apelin receptor influences biomechanical and morphological properties of endothelial cells.

The adaption of endothelial cells to local flow conditions is a multifunctional process which leads to distinct alterations in cell shape, the subcellular distribution of structural proteins, and cellular function. G-protein-coupled receptors (GPCRs) have been identified to be fundamentally involved in such processes. Recently, we and others have shown that the expression of the endothelial GPCR apelin receptor (APJ) is regulated by fluid flow and that activation of APJ participates in signaling pathways which are related to processes of mechanotransduction. The present study aims to illuminate these findings by further visualization of APJ function. We show that APJ is located to the cellular junctions and might thus be associated with platelet endothelial cell adhesion molecule-1 (PECAM-1) in human umbilical vein endothelial cells (HUVEC). Furthermore, siRNA-mediated silencing of APJ expression influences the shear-induced adaption of HUVEC in terms of cytoskeletal remodeling, cellular elasticity, cellular motility, attachment, and distribution of adhesion complexes. Taken together, our results demonstrate that APJ is crucial for complemented endothelial adaption to local flow conditions.

Compressive Force Spectroscopy: From Living Cells to Single Proteins.

One of the most successful applications of atomic force microscopy (AFM) in biology involves monitoring the effect of force on single biological molecules, often referred to as force spectroscopy. Such studies generally entail the application of pulling forces of different magnitudes and velocities upon individual molecules to resolve individualistic unfolding/separation pathways and the quantification of the force-dependent rate constants. However, a less recognized variation of this method, the application of compressive force, actually pre-dates many of these "tensile" force spectroscopic studies. Further, beyond being limited to the study of single molecules, these compressive force spectroscopic investigations have spanned samples as large as living cells to smaller, multi-molecular complexes such as viruses down to single protein molecules. Correspondingly, these studies have enabled the detailed characterization of individual cell states, subtle differences between seemingly identical viral structures, as well as the quantification of rate constants of functionally important, structural transitions in single proteins. Here, we briefly review some of the recent achievements that have been obtained with compressive force spectroscopy using AFM and highlight exciting areas of its future development.

The roles of cellular nanomechanics in cancer.

The biomechanical properties of cells and tissues may be instrumental in increasing our understanding of cellular behavior and cellular manifestations of diseases such as cancer. Nanomechanical properties can offer clinical translation of therapies beyond what are currently employed. Nanomechanical properties, often measured by nanoindentation methods using atomic force microscopy, may identify morphological variations, cellular binding forces, and surface adhesion behaviors that efficiently differentiate normal cells and cancer cells. The aim of this review is to examine current research involving the general use of atomic force microscopy/nanoindentation in measuring cellular nanomechanics; various factors and instrumental conditions that influence the nanomechanical properties of cells; and implementation of nanoindentation methods to distinguish cancer cells from normal cells or tissues. Applying these fundamental nanomechanical properties to current discoveries in clinical treatment may result in greater efficiency in diagnosis, treatment, and prevention of cancer, which ultimately can change the lives of patients.

Krüppel-like factor 6 (KLF6) requires its amino terminal domain to promote villous trophoblast cell fusion.

INTRODUCTION: Villous cytotrophoblast (vCTB) cells fuse to generate and maintain the syncytiotrophoblast layer required for placental development and function. Krüppel-like factor 6 (KLF6) is a ubiquitous transcription factor with an N-terminal acidic transactivation domain and a C-terminal zinc finger DNA-binding domain. KLF6 is highly expressed in placenta, and it is required for proper placental development. We have demonstrated that KLF6 is necessary for cell fusion in human primary vCTBs, and in the BeWo cell line. MATERIALS AND METHODS: Full length KLF6 or a mutant lacking its N-terminal domain were expressed in BeWo cells or in primary vCTB cells isolated from human term placentas. Cell fusion, gene and protein expression, and cell proliferation were analyzed. Moreover, Raman spectroscopy and atomic force microscopy (AFM) were used to identify biochemical, topography, and elasticity cellular modifications. RESULTS: The increase in KLF6, but not the expression of its deleted mutant, is sufficient to trigger cell fusion and to raise the expression of ß-hCG, syncytin-1, the chaperone protein 78 regulated by glucose (GRP78), the ATP Binding Cassette Subfamily G Member 2 (ABCG2), and Galectin-1 (Gal-1), all molecules involved in vCTB differentiation. Raman and AFM analysis revealed that KLF6 reduces NADH level and increases cell Young's modulus. KLF6-induced differentiation correlates with p21 upregulation and decreased cell proliferation. Remarkable, p21 silencing reduces cell fusion triggered by KLF6 and the KLF6 mutant impairs syncytialization and decreases syncytin-1 and ß-hCG expression. DISCUSSION: KLF6 induces syncytialization through a mechanism that involves its regulatory transcriptional domain in a p21-dependent manner.

Spatial micro-variation of 3D hydrogel stiffness regulates the biomechanical properties of hMSCs.

Human mesenchymal stem cells (hMSCs) are one of the most promising candidates for cell-based therapeutic products. Nonetheless, their biomechanical phenotype afterin vitroexpansion is still unsatisfactory, for example, restricting the efficiency of microcirculation of delivered hMSCs for further cell therapies. Here, we propose a scheme using maleimide-dextran hydrogel with locally varied stiffness in microscale to modify the biomechanical properties of hMSCs in three-dimensional (3D) niches. We show that spatial micro-variation of stiffness can be controllably generated in the hydrogel with heterogeneously cross-linking via atomic force microscopy measurements. The result of 3D cell culture experiment demonstrates the hydrogels trigger the formation of multicellular spheroids, and the derived hMSCs could be rationally softened via adjustment of the stiffness variation (SV) degree. Importantly,in vitro, the hMSCs modified with the higher SV degree can pass easier through capillary-shaped micro-channels. Further, we discuss the underlying mechanics of the increased cellular elasticity by focusing on the effect of rearranged actin networks, via the proposed microscopic model of biomechanically modified cells. Overall, this work highlights the effectiveness of SV-hydrogels in reprogramming and manufacturing hMSCs with designed biomechanical properties for improved therapeutic potential.

Comparison of Cancer Cell Elasticity by Cell Type.

Lower cellular elasticity is a distinguishing feature of cancer cells compared with normal cells. To determine whether cellular elasticity differs based on cancer cell type, cells were selected from three different cancer types including breast, cervix, and lung. For each cancer type, one counterpart normal cell and three types of cancer cells were selected, and their elasticity was measured using atomic force microscopy (AFM). The elasticity of normal cells was in the order of MCF10A > WI-38 ≥ Ect1/E6E7 which corresponds to the counterpart normal breast, lung, and cervical cancer cells, respectively. All cancer cells exhibited lower elasticity than their counterpart normal cells. Compared with the counterpart normal cells, the difference in cellular elasticity was the greatest in cervical cancer cells, followed by lung and breast cancer cells. This result indicates lower elasticity is a unique property of cancer cells; however, the reduction in elasticity may depend on the histological origin of the cells. The F-actin cytoskeleton of cancer cells was different in structure and content from normal cells. The F-actin is mainly distributed at the periphery of cancer cells and its content was mostly lower than that seen in normal cells.