Imaging and manipulating the structural machinery of living cells on the micro- and nanoscale

The structure, physiology, and fate of living cells are all highly sensitive to mechanical forces in the cellular microenvironment, including stresses and strains that originate from encounters with the extracellular matrix (ECM), blood and other flowing materials, and neighbouring cells. This relationship between context and physiology bears tremendous implications for the design of cellular micro-or nanotechnologies, since any attempt to control cell behavior in a device must provide the appropriate physical microenvironment for the desired cell behavior. Cells sense, process, and respond to biophysical cues in their environment through a set of integrated, multi-scale structural complexes that span length scales from single molecules to tens of microns, including small clusters of force-sensing molecules at the cell surface, micron-sized cell-ECM focal adhesion complexes, and the cytoskeleton that permeates and defines the entire cell. This review focuses on several key technologies that have recently been developed or adapted for the study of the dynamics of structural micro-and nanosystems in living cells and how these systems contribute to spatially-and temporally-controlled changes in cellular structure and mechanics. We begin by discussing subcellular laser ablation, which permits the precise incision of nanoscale structural elements in living cells in order to discern their mechanical properties and contributions to cell structure. We then discuss fluorescence recovery after photobleaching and fluorescent speckle microscopy, two live-cell fluorescence imaging methods that enable quantitative measurement of the binding and transport properties of specific proteins in the cell. Finally, we discuss methods to manipulate cellular structural networks by engineering the extracellular environment, including microfabrication of ECM distributions of defined geometry and microdevices designed to measure cellular traction forces at micron-scale resolution. Together, these methods form a powerful arsenal that is already adding significantly to our understanding of the nanoscale architecture and mechanics of living cells and may contribute to the rational design of new cellular micro-and nanotechnologies.     

The emergence of ECM mechanics and cytoskeletal tension as important regulators of cell function

The ability to harvest and maintain viable cells from mammalian tissues represented a critical advance in biomedical research, enabling individual cells to be cultured and studied in molecular detail. However, in these traditional cultures, cells are grown on rigid glass or polystyrene substrates, the mechanical properties of which often do not match those of the in vivo tissue from which the cells were originally derived. This mechanical mismatch likely contributes to abrupt changes in cellular phenotype. In fact, it has been proposed that mechanical changes in the cellular microenvironment may alone be responsible for driving specific cellular behaviors. Recent multidisciplinary efforts from basic scientists and engineers have begun to address this hypothesis more explicitly by probing the effects of ECM mechanics on cell and tissue function. Understanding the consequences of such mechanical changes is physiologically relevant in the context of a number of tissues in which altered mechanics may either correlate with or play an important role in the onset of pathology. Examples include changes in the compliance of blood vessels associated with atherosclerosis and intimal hyperplasia, as well as changes in the mechanical properties of developing tumors. Compelling evidence from 2-D in vitro model systems has shown that substrate mechanical properties induce changes in cell shape, migration, proliferation, and differentiation, but it remains to be seen whether or not these same effects translate to 3-D systems or in vivo. Furthermore, the molecular “mechanotransduction” mechanisms by which cells respond to changes in ECM mechanics remain unclear. Here, we provide some historical context for this emerging area of research, and discuss recent evidence that regulation of cytoskeletal tension by changes in ECM mechanics (either directly or indirectly) may provide a critical switch that controls cell function.     

Invited review: engineering approaches to cytoskeletal mechanics.

An outstanding problem in cell biology is how cells sense mechanical forces and how those forces affect cellular functions. Various biophysical and biochemical mechanisms have been invoked to answer this question. A growing body of evidence indicates that the deformable cytoskeleton (CSK), an intracellular network of interconnected filamentous biopolymers, provides a physical basis for transducing mechanical signals into biochemical signals. Therefore, to understand how mechanical forces regulate cellular functions, it is important to know how cells respond to changes in the CSK force balance and to identify the underlying mechanisms that control transmission of mechanical forces throughout the CSK and bring it to equilibrium. Recent developments of new experimental techniques for measuring cell mechanical properties and novel theoretical models of cellular mechanics make it now possible to identify and quantitate the contributions of various CSK structures to the overall balance of mechanical forces in the cell. This review focuses on engineering approaches that have been used in the past two decades in studies of the mechanics of the CSK.     

Physics of Bacterial Morphogenesis

Summary: Bacterial cells utilize three-dimensional (3D) protein assemblies to perform important cellular functions such as growth, division, chemoreception, and motility. These assemblies are composed of mechanoproteins that can mechanically deform and exert force. Sometimes, small-nucleotide hydrolysis is coupled to mechanical deformations. In this review, we describe the general principle for an understanding of the coupling of mechanics with chemistry in mechanochemical systems. We apply this principle to understand bacterial cell shape and morphogenesis and how mechanical forces can influence peptidoglycan cell wall growth. We review a model that can potentially reconcile the growth dynamics of the cell wall with the role of cytoskeletal proteins such as MreB and crescentin. We also review the application of mechanochemical principles to understand the assembly and constriction of the FtsZ ring. A number of potential mechanisms are proposed, and important questions are discussed.     

Musculoskeletal mechanobiology: Interpretation by external force and engineered substratum

Mechanobiology aims to discover how the mechanical environment affects the biological activity of cells and how cells’ ability to sense these mechanical cues is converted into elicited cellular responses. Musculoskeletal mechanobiology is of particular interest given the high mechanical loads that musculoskeletal tissues experience on a daily basis. How do cells within these mechanically active tissues interpret external loads imposed on their extracellular environment, and, how are cell–substrate interactions converted into biochemical signals? This review outlines many of the main mechanotransduction mechanisms known to date, and describes recent literature examining effects of both external forces and cell–substrate interactions on musculoskeletal cells. Whether via application of external forces and/or cell–substrate interactions, our understanding and regulation of musculoskeletal mechanobiology can benefit by expanding upon traditional models, and shedding new light through novel investigative approaches. Current and future work in this field is focused on identifying specific forces, stresses, and strains at the cellular and tissue level through both experimental and computational approaches, and analyzing the role of specific proteins through fluorescence-based investigations and knockdown models.     

The mechanobiology of NK cells- ‘Forcing NK to Sense’ target cells

Natural killer (NK) cells are innate immune lymphocytes that recognize and kill cancer and infected cells, which makes them unique ‘off-the-shelf’ candidates for a new generation of immunotherapies. Biomechanical forces in homeostasis and pathophysiology accrue additional immune regulation for NK immune responses. Indeed, cellular and tissue biomechanics impact NK receptor clustering, cytoskeleton remodeling, NK transmigration through endothelial cells, nuclear mechanics, and even NK-dendritic cell interaction, offering a plethora of unexplored yet important dynamic regulation for NK immunotherapy. Such events are made more complex by the heterogeneity of human NK cells. A significant question remains on whether and how biochemical and biomechanical cues collaborate for NK cell mechanotransduction, a process whereby mechanical force is sensed, transduced, and translated to downstream mechanical and biochemical signalling. Herein, we review recent advances in understanding how NK cells perceive and mechanotransduce biophysical cues. We focus on how the cellular cytoskeleton crosstalk regulates NK cell function while bearing in mind the heterogeneity of NK cells, the direct and indirect mechanical cues for NK anti-tumor activity, and finally, engineering advances that are of translational relevance to NK cell biology at the systems level.     

Mechanobiology of neural development

As the brain develops, proliferating cells organize into structures, differentiate, migrate, extrude long processes, and connect with other cells. These biological processes produce mechanical forces that further shape cellular dynamics and organ patterning. A major unanswered question in developmental biology is how the mechanical forces produced during development are detected and transduced by cells to impact biochemical and genetic programs of development. This gap in knowledge stems from a lack of understanding of the molecular players of cellular mechanics and an absence of techniques for measuring and manipulating mechanical forces in tissue. In this review article, we examine recent advances that are beginning to clear these bottlenecks and highlight results from new approaches that reveal the role of mechanical forces in neurodevelopmental processes.     

Cell-substratum and cell-cell adhesion forces and single-cell mechanical properties in mono- and multilayer assemblies from robotic fluidic force microscopy

The epithelium covers, protects, and actively regulates various formations and cavities of the human body. During embryonic development the assembly of the epithelium is crucial to the organoid formation, and the invasion of the epithelium is an essential step in cancer metastasis. Live cell mechanical properties and associated forces presumably play an important role in these biological processes. However, the direct measurement of cellular forces in a precise and high-throughput manner is still challenging. We studied the cellular adhesion maturation of epithelial Vero monolayers by measuring single-cell force-spectra with high-throughput fluidic force microscopy (robotic FluidFM). Vero cells were grown on gelatin-covered plates in different seeding concentrations, and cell detachment forces were recorded from the single-cell state, through clustered island formation, to their complete assembly into a sparse and then into a tight monolayer. A methodology was proposed to separate cell-substratum and cell-cell adhesion force and energy (work of adhesion) contributions based on the recorded force-distance curves. For comparison, cancerous HeLa cells were also measured in the same settings. During Vero monolayer formation, a significantly strengthening adhesive tendency was found, showing the development of cell-cell contacts. Interestingly, this type of step-by-step maturation was absent in HeLa cells. The attachment of cancerous HeLa cells to the assembled epithelial monolayers was also measured, proposing a new high-throughput method to investigate the biomechanics of cancer cell invasion. We found that HeLa cells adhere significantly stronger to the tight Vero monolayer than cells of the same origin. Moreover, the mechanical characteristics of Vero monolayers upon cancerous HeLa cell influence were recorded and analyzed. All these results provide insight into the qualitative assessment of cell-substratum and cell-cell mechanical contacts in mono- and multilayered assemblies and demonstrate the robustness and speed of the robotic FluidFM technology to reveal biomechanical properties of live cell assemblies with statistical significances.     

Dynamic and structural signatures of lamellar actomyosin force generation

The regulation of cellular traction forces on the extracellular matrix is critical to cell adhesion, migration, proliferation, and differentiation. Diverse lamellar actin organizations ranging from contractile lamellar networks to stress fibers are observed in adherent cells. Although lamellar organization is thought to reflect the extent of cellular force generation, understanding of the physical behaviors of the lamellar actin cytoskeleton is lacking. To elucidate these properties, we visualized the actomyosin dynamics and organization in U2OS cells over a broad range of forces. At low forces, contractile lamellar networks predominate and force generation is strongly correlated to actomyosin retrograde flow dynamics with nominal change in organization. Lamellar networks build ～60% of cellular tension over rapid time scales. At high forces, reorganization of the lamellar network into stress fibers results in moderate changes in cellular tension over slower time scales. As stress fibers build and tension increases, myosin band spacing decreases and α-actinin bands form. On soft matrices, force generation by lamellar networks is unaffected, whereas tension-dependent stress fiber assembly is abrogated. These data elucidate the dynamic and structural signatures of the actomyosin cytoskeleton at different levels of tension and set a foundation for quantitative models of cell and tissue mechanics.     

In the middle of it all: Mutual mechanical regulation between the nucleus and the cytoskeleton

The nucleus is typically treated as the large phase-dense or easy-to-label structure at the center of the cell which is manipulated by the governing mechanical machinery inside the cytoplasm. However, recent evidence has suggested that the mechanical properties of the nucleus are important to cell fate. We will discuss many aspects of the structural and functional interconnections between nuclear mechanics and cellular mechanics in this review. There are numerous implications for the progression of many disease states associated with both nuclear structural proteins and cancers. The nucleus itself is a large organelle taking up significant volume within the cell, and most studies agree that nuclei are significantly stiffer than the surrounding cytoplasm. Thus when a cell is exposed to force, the nucleus is exposed to and helps resist that force. The nucleus and nucleoskeleton are interconnected with the cellular cytoskeleton, and these connections may aid in helping disperse forces within tissues and/or with mechanotransduction. During translocation and transmigration the nucleus can act as a resistive element. Understanding the role of mechanical regulation of the nucleus may aid in understanding cellular motility and crawling through confined geometries. Thus the nucleus plays a role in developing mechanical territories and niches, affecting rates of wound healing and allowing cells to transmigrate through tissues for developmental, repair or pathological means.     

Mechanical stimuli on C2C12 myoblasts affect myoblast differentiation, focal adhesion kinase phosphorylation and galectin-1 expression: a proteomic approach

Mechanical forces are crucial in the regulation of cell morphology and function. At the cellular level, these forces influence myoblast differentiation and fusion. In this study, we applied mechanical stimuli to embryonic muscle cells using magnetic microbeads, a method shown to apply stress to specific receptors on the cell surface. We showed that mechanical stimuli promote an increase in FAK (focal adhesion kinase) phosphorylation. In order to further shed light in the process of myoblast-induced differentiation by mechanical stimuli, we performed a proteomic analysis. Thirteen proteins were found to be affected by mechanical stimulation including galectin-1, annexin III and RhoGDI (Rho guanine-nucleotide-dissociation inhibitor). In this study, we demonstrate how the combination of this method of mechanical stimuli and proteomic analysis can be a powerful tool to detect proteins that are potentially interacting in biochemical pathways or complex cellular mechanisms during the process of myoblast differentiation. We determined an increase in expression and changes in cellular localization of galectin-1 in mechanically stimulated myoblasts. A potential involvement of galectin-1 in myoblast differentiation is presented.     

Contributions of turgor pressure, the contractile ring, and septum assembly to forces in cytokinesis in fission yeast

A paradigm of cytokinesis in animal cells is that the actomyosin contractile ring provides the primary force to divide the cell [1]. In the fission yeast Schizosaccharomyces pombe, cytokinesis also involves a conserved cytokinetic ring, which has been generally assumed to provide the force for cleavage [2-4] (see also [5]). However, in contrast to animal cells, cytokinesis in yeast cells also requires the assembly of a cell wall septum [6], which grows centripetally inward as the ring closes. Fission yeast, like other walled cells, also possess high (MPa) turgor pressure [7-9]. Here, we show that turgor pressure is an important factor in the mechanics of cytokinesis. Decreasing effective turgor pressure leads to an increase in cleavage rate, suggesting that the inward force generated by the division apparatus opposes turgor pressure. The contractile ring, which is predicted to provide only a tiny fraction of the mechanical stress required to overcome turgor, is largely dispensable for ingression; once septation has started, cleavage can continue in the absence of the contractile ring. Scaling arguments and modeling suggest that the large forces for cytokinesis are not produced by the contractile ring but are driven by the assembly of cell wall polymers in the growing septum.     

Shear Force at the Cell-Matrix Interface: Enhanced Analysis for Microfabricated Post Array Detectors

The interplay of mechanical forces between the extracellular environment and the cytoskeleton drives development, repair, and senescence in many tissues. Quantitative definition of these forces is a vital step in understanding cellular mechanosensing. Microfabricated post array detectors (mPADs) provide direct measurements of cell-generated forces during cell adhesion to extracellular matrix. A new approach to mPAD post labeling, volumetric imaging, and an analysis of post bending mechanics determined that cells apply shear forces and not point moments at the matrix interface. In addition, these forces could be accurately resolved from post deflections by using images of post tops and bases. Image analysis tools were then developed to increase the precision and throughput of post centroid location. These studies resulted in an improved method of force measurement with broad applicability and concise execution using a fully automated force analysis system. The new method measures cell-generated forces with less than 5% error and less than 90 seconds of computational time. Using this approach, we demonstrated direct and distinct relationships between cellular traction force and spread cell surface area for fibroblasts, endothelial cells, epithelial cells and smooth muscle cells.     

Decoding mechanical cues by molecular mechanotransduction

Cells are exposed to a variety of mechanical cues, including forces from their local environment and physical properties of the tissue. These mechanical cues regulate a vast number of cellular processes, relying on a repertoire of mechanosensors that transduce forces into biochemical pathways through mechanotransduction. Forces can act on different parts of the cell, carry information regarding magnitude and direction, and have distinct temporal profiles. Thus, the specific cellular response to mechanical forces is dependent on the ability of cells to sense and transduce these physical parameters. In this review, we will highlight recent findings that provide insights into the mechanisms by which different mechanosensors decode mechanical cues and how their coordinated response determines the cellular outcomes.     

Tunable cell-surface mimetics as engineered cell substrates

Most recent breakthroughs in understanding cell adhesion, cell migration, and cellular mechanosensitivity have been made possible by the development of engineered cell substrates of well-defined surface properties. Traditionally, these substrates mimic the extracellular matrix (ECM) environment by the use of ligand-functionalized polymeric gels of adjustable stiffness. However, such ECM mimetics are limited in their ability to replicate the rich dynamics found at cell-cell contacts. This review focuses on the application of cell surface mimetics, which are better suited for the analysis of cell adhesion, cell migration, and cellular mechanosensitivity across cell-cell interfaces. Functionalized supported lipid bilayer systems were first introduced as biomembrane-mimicking substrates to study processes of adhesion maturation during adhesion of functionalized vesicles (cell-free assay) and plated cells. However, while able to capture adhesion processes, the fluid lipid bilayer of such a relatively simple planar model membrane prevents adhering cells from transducing contractile forces to the underlying solid, making studies of cell migration and cellular mechanosensitivity largely impractical. Therefore, the main focus of this review is on polymer-tethered lipid bilayer architectures as biomembrane-mimicking cell substrate. Unlike supported lipid bilayers, these polymer-lipid composite materials enable the free assembly of linkers into linker clusters at cellular contacts without hindering cell spreading and migration and allow the controlled regulation of mechanical properties, enabling studies of cellular mechanosensitivity. The various polymer-tethered lipid bilayer architectures and their complementary properties as cell substrates are discussed.     

Dissecting the influence of cellular senescence on cell mechanics and extracellular matrix formation in vitro

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 p16^Ink4a or p21^Cip1 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.     

The Mechanical Role of Microtubules in Tissue Remodeling

During morphogenesis, tissues undergo extensive remodeling to get their final shape. Such precise sculpting requires the application of forces generated within cells by the cytoskeleton and transmission of these forces through adhesion molecules within and between neighboring cells. Within individual cells, microtubules together with actomyosin filaments and intermediate filaments form the composite cytoskeleton that controls cell mechanics during tissue rearrangements. While studies have established the importance of actin-based mechanical forces that are coupled via intercellular junctions, relatively little is known about the contribution of other cytoskeletal components such as microtubules to cell mechanics during morphogenesis. In this review the focus is on recent findings, highlighting the direct mechanical role of microtubules beyond its well-established role in trafficking and signaling during tissue formation.     

Transmission of Mechanical Stresses within the Cytoskeleton of Adherent Cells: A Theoretical Analysis Based on a Multi-Component Cell Model

How environmental mechanical forces affect cellular functions is a central problem in cell biology. Theoretical models of cellular biomechanics provide relevant tools for understanding how the contributions of deformable intracellular components and specific adhesion conditions at the cell interface are integrated for determining the overall balance of mechanical forces within the cell. We investigate here the spatial distributions of intracellular stresses when adherent cells are probed by magnetic twisting cytometry. The influence of the cell nucleus stiffness on the simulated nonlinear torque-bead rotation response is analyzed by considering a finite element multi-component cell model in which the cell and its nucleus are considered as different hyperelastic materials. We additionally take into account the mechanical properties of the basal cell cortex, which can be affected by the interaction of the basal cell membrane with the extracellular substrate. In agreement with data obtained on epithelial cells, the simulated behaviour of the cell model relates the hyperelastic response observed at the entire cell scale to the distribution of stresses and strains within the nucleus and the cytoskeleton, up to cell adhesion areas. These results, which indicate how mechanical forces are transmitted at distant points through the cytoskeleton, are compared to recent data imaging the highly localized distribution of intracellular stresses.     

Probing cytoskeletal pre-stress and nuclear mechanics in endothelial cells with spatiotemporally controlled (de-)adhesion kinetics on micropatterned substrates

The mechanical properties of living cells reflect their propensity to migrate and respond to external forces. Both cellular and nuclear stiffnesses are strongly influenced by the rigidity of the extracellular matrix (ECM) through reorganization of the cyto- and nucleoskeletal protein connections. Changes in this architectural continuum affect cell mechanics and underlie many pathological conditions. In this context, an accurate and combined quantification of the mechanical properties of both cells and nuclei can contribute to a better understanding of cellular (dys-)function. To address this challenge, we have established a robust method for probing cellular and nuclear deformation during spreading and detachment from micropatterned substrates. We show that (de-)adhesion kinetics of endothelial cells are modulated by substrate stiffness and rely on the actomyosin network. We combined this approach with measurements of cell stiffness by magnetic tweezers to show that relaxation dynamics can be considered as a reliable parameter of cellular pre-stress in adherent cells. During the adhesion stage, large cellular and nuclear deformations occur over a long time span (>60 min). Conversely, nuclear deformation and condensed chromatin are relaxed in a few seconds after detachment. Finally, our results show that accumulation of farnesylated prelamin leads to modifications of the nuclear viscoelastic properties, as reflected by increased nuclear relaxation times. Our method offers an original and non-intrusive way of simultaneously gauging cellular and nuclear mechanics, which can be extended to high-throughput screens of pathological conditions and potential countermeasures.     

Shape-dependent control of cell growth, differentiation, and apoptosis: switching between attractors in cell regulatory networks

Development of characteristic tissue patterns requires that individual cells be switched locally between different phenotypes or "fates;" while one cell may proliferate, its neighbors may differentiate or die. Recent studies have revealed that local switching between these different gene programs is controlled through interplay between soluble growth factors, insoluble extracellular matrix molecules, and mechanical forces which produce cell shape distortion. Although the precise molecular basis remains unknown, shape-dependent control of cell growth and function appears to be mediated by tension-dependent changes in the actin cytoskeleton. However, the question remains: how can a generalized physical stimulus, such as cell distortion, activate the same set of genes and signaling proteins that are triggered by molecules which bind to specific cell surface receptors. In this article, we use computer simulations based on dynamic Boolean networks to show that the different cell fates that a particular cell can exhibit may represent a preprogrammed set of common end programs or "attractors" which self-organize within the cell's regulatory networks. In this type of dynamic network model of information processing, generalized stimuli (e.g., mechanical forces) and specific molecular cues elicit signals which follow different trajectories, but eventually converge onto one of a small set of common end programs (growth, quiescence, differentiation, apoptosis, etc.). In other words, if cells use this type of information processing system, then control of cell function would involve selection of preexisting (latent) behavioral modes of the cell, rather than instruction by specific binding molecules. Importantly, the results of the computer simulation closely mimic experimental data obtained with living endothelial cells. The major implication of this finding is that current methods used for analysis of cell function that rely on characterization of linear signaling pathways or clusters of genes with common activity profiles may overlook the most critical features of cellular information processing which normally determine how signal specificity is established and maintained in living cells.