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In native states, animal cells of many types are supported by a fibrous network that forms the main structural component of the ECM. Mechanical interactions between cells and the 3D ECM critically regulate cell function, including growth and migration. However, the physical mechanism that governs the cell interaction with fibrous 3D ECM is still not known. In this article, we present single-cell traction force measurements using breast tumor cells embedded within 3D collagen matrices. We recreate the breast tumor mechanical environment by controlling the microstructure and density of type I collagen matrices. Our results reveal a positive mechanical feedback loop: cells pulling on collagen locally align and stiffen the matrix, and stiffer matrices, in return, promote greater cell force generation and a stiffer cell body. Furthermore, cell force transmission distance increases with the degree of strain-induced fiber alignment and stiffening of the collagen matrices. These findings highlight the importance of the nonlinear elasticity of fibrous matrices in regulating cell–ECM interactions within a 3D context, and the cell force regulation principle that we uncover may contribute to the rapid mechanical tissue stiffening occurring in many diseases, including cancer and fibrosis.

Exposed to mechanical confinement, mammalian cells can establish remarkable unspecific adhesion, which is independent of integrins. How cells facilitate such adhesion remains unclear. Here, it is investigated how mammalian cells exposed to compression initiate unspecific and integrin‐mediated adhesion. It is observed that with increasing compression, cells increase adhesion to collagen I or fibronectin and strengthen adhesion faster. Under low and medium compression, cells minimally increase unspecific adhesion to substrates that lack specific binding sites for cell surface receptors, such as integrins. However, under high compression, mammalian cells switch to a strong unspecific adhesion state, which significantly contributes to cell‐extracellular matrix (ECM) adhesion. Thereby cells use the glycocalyx to directly facilitate strong unspecific adhesion and to enhance early integrin‐mediated adhesion. The mechanistic insight of how cells unspecifically adhere to substrates under confinement opens avenues to better understand cell adhesion in development, homeostasis, disease, and in a wide range of biotechnological and medical applications in which cells are exposed to mechanical confinement. This study quantifies how cell compression and confinement triggers cell adhesion. Under low compression, integrins dominate cell adhesion to extracellular matrix (ECM) proteins, whereas the glycocalyx contributes weak unspecific adhesion. With increasing compression, the glycocalyx increases its unspecific adhesion to ECM and non‐ECM proteins and can contribute considerably to the integrin‐mediated cell adhesion to the ECM.

Controlling cell migration is important in tissue engineering and medicine. Cell motility depends on factors such as nutrient concentration gradients and soluble factor signaling. In particular, cell–cell signaling can depend on cell–cell separation distance and can influence cellular arrangements in bulk cultures. Here, we seek a physicalbased approach, which identifies a potential governed by cell–cell signaling that induces a directed cell–cell motion. A single-cell barcode chip (SCBC) was used to experimentally interrogate secreted proteins in hundreds of isolated glioblastoma brain cancer cell pairs and to monitor their relative motions over time. We used these trajectories to identify a range of cell–cell separation distances where the signaling was most stable. We then used a thermodynamics-motivated analysis of secreted protein levels to characterize free-energy changes for different cell–cell distances. We show that glioblastoma cell–cell movement can be described as Brownian motion biased by cell–cell potential. To demonstrate that the free-energy potential as determined by the signaling is the driver of motion, we inhibited two proteins most involved in maintaining the free-energy gradient. Following inhibition, cell pairs showed an essentially random Brownian motion, similar to the case for untreated, isolated single cells.

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold enormous potential in cardiac disease modeling, drug screening, and regenerative medicine. Furthermore, patient-specific iPSC-CMS can be tested for personalized medicine. To provide a deeper understanding of the contractile force dynamics of iPSC-CMs, we employed Atomic Force Microscopy (AFM) as an advanced detection tool to distinguish the characteristics of force dynamics at a single cell level. We measured normal (vertical) and lateral (axial) force at different pacing frequencies. We found a significant correlation between normal and lateral force. We also observed a significant force–frequency relationship for both types of forces. This work represents the first demonstration of the correlation of normal and lateral force from individual iPSC-CMs. The identification of this correlation is relevant because it validates the comparison across systems and models that can only account for either normal or lateral force. These findings enhance our understanding of iPSC-CM properties, thereby paving the way for the development of therapeutic strategies in cardiovascular medicine.

Cell adhesive force, exerting on the local matrix or neighboring cells, plays a critical role in regulating many cell functions and physiological processes. In the past four decades, significant efforts have been dedicated to cell adhesive force detection, visualization and quantification. A recent important methodological advancement in cell adhesive force visualization is to adopt force-to-fluorescence conversion instead of force-to-substrate strain conversion, thus greatly improving the sensitivity and resolution of force imaging. This review summarizes the recent development of force imaging techniques (collectively termed as cell adhesive force microscopy or CAFM here), with a particular focus on the improvement of CAFM’s spatial resolution and the biomaterial choices for constructing the tension sensors used in force visualization. This review also highlights the importance of DNA-based tension sensors in cell adhesive force imaging and the recent breakthrough in the development of super-resolution CAFM.

Fluidic force microscopy (FluidFM), which combines atomic force microscopy (AFM) with microchanneled cantilevers connected to a pressure controller, is a technique allowing the realization of force-sensitive nanopipette under aqueous conditions. FluidFM has unique advantages in simultaneous three-dimensional manipulations and mechanical measurements of biological specimens at the micro-/nanoscale. Over the past decade, FluidFM has shown its potential in biophysical assays particularly in the investigations at single-cell level, offering novel possibilities for discovering the underlying mechanisms guiding life activities. Here, we review the utilization of FluidFM to address biomechanical and biophysical issues in the life sciences. Firstly, the fundamentals of FluidFM are represented. Subsequently, the applications of FluidFM for biophysics at single-cell level are surveyed from several facets, including single-cell manipulations, single-cell force spectroscopy, and single-cell electrophysiology. Finally, the challenges and perspectives for future progressions are provided.

The adhesiveness of cancerous cells to their neighboring cells significantly contributes to tumor progression and metastasis. The single-cell force spectroscopy (SCFS) approach was implemented to survey the cell–cell adhesion force between cancerous cells in three cancerous breast cell lines (MCF-7, T47D, and MDA-MB-231). The gene expression levels of two dominant cell adhesion markers (E-cadherin and N-cadherin) were quantified by real-time PCR. Additionally, the local stiffness of the cell bodies was measured by atomic force microscopy (AFM), and the actin cytoskeletal organization was examined by confocal microscopy. Results indicated that the adhesion force between cells was conversely correlated with their invasion potential. The highest adhesion force was observed in the MCF-7 cells. A reduction in cell–cell adhesion, which is required for the detachment of cells from the main tumor during metastasis, is partly due to the loss of E-cadherin expression and the enhanced expression of N-cadherins. The reduced adhesion was accompanied by the softening of cells, as described by the rearrangement of actin filaments through confocal microscopy observations. The softening of the cell body and the reduced cellular adhesiveness are two adaptive mechanisms through which malignant cells achieve the increased deformability, motility, and strong metastasis potential necessary for passage through endothelial junctions and positioning in host tissue. This study presented application of SCFS to survey cell phenotype transformation during cancer progression. The results can be implemented as a platform for further investigations that target the manipulation of cellular adhesiveness and stiffness as a therapeutic choice.

Tissue formation and healing both require cell proliferation and migration, but also extracellular matrix production and tensioning. In addition to restricting proliferation of damaged cells, increasing evidence suggests that cellular senescence also has distinct modulatory effects during wound healing and fibrosis. Yet, a direct role of senescent cells during tissue formation beyond paracrine signaling remains unknown. We here report how individual modules of the senescence program differentially influence cell mechanics and ECM expression with relevance for tissue formation. We compared DNA damage‐mediated and DNA damage‐independent senescence which was achieved through over‐expression of either p16Ink4a or p21Cip1 cyclin‐dependent kinase inhibitors in primary human skin fibroblasts. Cellular senescence modulated focal adhesion size and composition. All senescent cells exhibited increased single cell forces which led to an increase in tissue stiffness and contraction in an in vitro 3D tissue formation model selectively for p16 and p21‐overexpressing cells. The mechanical component was complemented by an altered expression profile of ECM‐related genes including collagens, lysyl oxidases, and MMPs. We found that particularly the lack of collagen and lysyl oxidase expression in the case of DNA damage‐mediated senescence foiled their intrinsic mechanical potential. These observations highlight the active mechanical role of cellular senescence during tissue formation as well as the need to synthesize a functional ECM network capable of transferring and storing cellular forces. Cellular senescence modulates single cell mechanics and ECM formation with consequences for macroscopic tissue tensioning. Components of the SASP and a reduced collagen expression as a result of DNA damage‐mediated senescence competes with the increased mechanical potential of these cells.

It is an understatement that mating and DNA transfer are key events for living organisms. Among the traits needed to facilitate mating, cell adhesion between gametes is a universal requirement. Thus, there should be specific properties for the adhesion proteins involved in mating. Biochemical and biophysical studies have revealed structural information about mating adhesins, as well as their specificities and affinities, leading to some ideas about these specialized adhesion proteins. Recently, single-cell force spectroscopy (SCFS) has added important findings. In SCFS, mating cells are brought into contact in an atomic force microscope (AFM), and the adhesive forces are monitored through the course of mating. The results have shown some remarkable characteristics of mating adhesins and add knowledge about the design and evolution of mating adhesins.

Epithelial cells form continuous sheets of cells that exist in tensional homeostasis. Homeostasis is maintained through cell-to-cell junctions that distribute tension and balance forces between cells and their underlying matrix. Disruption of tensional homeostasis can lead to epithelial–mesenchymal transition (EMT), a transdifferentiation process in which epithelial cells adopt a mesenchymal phenotype, losing cell–cell adhesion and enhancing cellular motility. This process is critical during embryogenesis and wound healing, but is also dysregulated in many disease states. To further understand the role of intercellular tension in spatial patterning of epithelial cell monolayers, we developed a multicellular computational model of cell–cell and cell–substrate forces. This work builds on a hybrid cellular Potts model (CPM)–finite element model to evaluate cell–matrix mechanical feedback of an adherent multicellular cluster. Cellular movement is governed by thermodynamic constraints from cell volume, cell–cell and cell–matrix contacts, and durotaxis, which arises from cell-generated traction forces on a finite element substrate. Junction forces at cell–cell contacts balance these traction forces, thereby producing a mechanically stable epithelial monolayer. Simulations were compared to in vitro experiments using fluorescence-based junction force sensors in clusters of cells undergoing EMT. Results indicate that the multicellular CPM model can reproduce many aspects of EMT, including epithelial monolayer formation dynamics, changes in cell geometry, and spatial patterning of cell–cell forces in an epithelial tissue.