Finite-element analysis of the adhesion-cytoskeleton-nucleus mechanotransduction pathway during endothelial cell rounding: axisymmetric model

Endothelial cells possess a mechanical network connecting adhesions on the basal surface, the cytoskeleton, and the nucleus. Transmission of force at adhesions via this pathway can deform the nucleus, ultimately resulting in an alteration of gene expression and other cellular changes (mechanotransduction). Previously, we measured cell adhesion area and apparent nuclear stretch during endothelial cell rounding. Here, we reconstruct the stress map of the nucleus from the observed strains using finite-element modeling. To simulate the disruption of adhesions, we prescribe displacement boundary conditions at the basal surface of the axisymmetric model cell. We consider different scenarios of the cytoskeletal arrangement, and represent the cytoskeleton as either discrete fibers or as an effective homogeneous layer When the nucleus is in the initial (spread) state, cytoskeletal tension holds the nucleus in an elongated, ellipsoidal configuration. Loss of cytoskeletal tension during cell rounding is represented by reactive forces acting on the nucleus in the model. In our simulations of cell rounding, we found that, for both representations of the cytoskeleton, the loss of cytoskeletal tension contributed more to the observed nuclear deformation than passive properties. Since the simulations make no assumption about the heterogeneity of the nucleus, the stress components both within and on the surface of the nucleus were calculated. The nuclear stress map showed that the nucleus experiences stress on the order of magnitude that can be significant for the function of DNA molecules and chromatin fibers. This study of endothelial cell mechanobiology suggests the possibility that mechanotransduction could result, in part, from nuclear deformation, and may be relevant to angiogenesis, wound healing, and endothelial barrier dysfunction.     

Traction Stresses and Translational Distortion of the Nucleus During Fibroblast Migration on a Physiologically Relevant ECM Mimic

Cellular traction forces, resulting in cell-substrate physical interactions, are generated by actin-myosin complexes and transmitted to the extracellular matrix through focal adhesions. These processes are highly dynamic under physiological conditions and modulate cell migration. To better understand the precise dynamics of cell migration, we measured the spatiotemporal redistribution of cellular traction stresses (force per area) during fibroblast migration at a submicron level and correlated it with nuclear translocation, an indicator of cell migration, on a physiologically relevant extracellular matrix mimic. We found that nuclear translocation occurred in pulses whose magnitude was larger on the low ligand density surfaces than on the high ligand density surfaces. Large nuclear translocations only occurred on low ligand density surfaces when the rear traction stresses completely relocated to a posterior nuclear location, whereas such relocation took much longer time on high ligand density surfaces, probably due to the greater magnitude of traction stresses. Nuclear distortion was also observed as the traction stresses redistributed. Our results suggest that the reinforcement of the traction stresses around the nucleus as well as the relaxation of nuclear deformation are critical steps during fibroblast migration, serving as a speed regulator, which must be considered in any dynamic molecular reconstruction model of tissue cell migration. A traction gradient foreshortening model was proposed to explain how the relocation of rear traction stresses leads to pulsed fibroblast migration.     

Actomyosin bundles serve as a tension sensor and a platform for ERK activation

Tensile forces generated by stress fibers drive signal transduction events at focal adhesions. Here, we report that stress fibers per se act as a platform for tension-induced activation of biochemical signals. The MAP kinase, ERK is activated on stress fibers in a myosin II-dependent manner. In myosin II-inhibited cells, uniaxial stretching of cell adhesion substrates restores ERK activation on stress fibers. By quantifying myosin II- or mechanical stretch-mediated tensile forces in individual stress fibers, we show that ERK activation on stress fibers correlates positively with tensile forces acting on the fibers, indicating stress fibers as a tension sensor in ERK activation. Myosin II-dependent ERK activation is also observed on actomyosin bundles connecting E-cadherin clusters, thus suggesting that actomyosin bundles, in general, work as a platform for tension-dependent ERK activation.     

The Spindle Midzone Microtubule-Associated Proteins Ase1p and Cin8p Affect the Number and Orientation of Astral Microtubules in Saccharomyces cerevisiae

The nucleus of the budding yeast S. cerevisiae has to move to the bud neck during mitosis in order for proper DNA segregation to take place. This movement is mediated by spindle and astral microtubules, and it relies on forces generated by microtubule-associated motor proteins. When budding yeast cells express the non-cleavable cohesin subunit, Scc1-RRDD, sister chromatid separation is blocked, preventing the spindle from elongating. Thus, in the presence of Scc1-RRDD nuclear positioning is mediated solely by forces acting through astral microtubules. We have previously shown that under these conditions cells exit mitosis with the nucleus in the mother cells, and that the position of the nucleus is determined, at least in part, by the FEAR pathway, which regulates various aspects of mitotic exit. When the FEAR pathway is inactivated, cells expressing Scc1-RRDD exit mitosis with the nucleus in the daughter cells (referred to as a “daughterly phenotype”). In order to find additional proteins that participate in nuclear positioning, we screened a series of mutant strains for those that displayed a daughterly phenotype when Scc1-RRDD was expressed. The most prominent defects were seen in ase1Δ and cin8Δ mutant cells. Both Ase1p and Cin8p were previously shown to be nuclear and to be involved in spindle function. We show here that deletion of ASE1 or CIN8 causes a defect in SPB separation and leads to an abnormal number of astral microtubules and a change in their orientation within the cell. Taken together, these results suggest that in budding yeast Ase1p and Cin8p affect nuclear positioning through astral microtubule-dependent mechanisms.     

Calculation of the force field required for nucleus deformation during cell migration through constrictions

During cell migration in confinement, the nucleus has to deform for a cell to pass through small constrictions. Such nuclear deformations require significant forces. A direct experimental measure of the deformation force field is extremely challenging. However, experimental images of nuclear shape are relatively easy to obtain. Therefore, here we present a method to calculate predictions of the deformation force field based purely on analysis of experimental images of nuclei before and after deformation. Such an inverse calculation is technically non-trivial and relies on a mechanical model for the nucleus. Here we compare two simple continuum elastic models of a cell nucleus undergoing deformation. In the first, we treat the nucleus as a homogeneous elastic solid and, in the second, as an elastic shell. For each of these models we calculate the force field required to produce the deformation given by experimental images of nuclei in dendritic cells migrating in microchannels with constrictions of controlled dimensions. These microfabricated channels provide a simplified confined environment mimicking that experienced by cells in tissues. Our calculations predict the forces felt by a deforming nucleus as a migrating cell encounters a constriction. Since a direct experimental measure of the deformation force field is very challenging and has not yet been achieved, our numerical approaches can make important predictions motivating further experiments, even though all the parameters are not yet available. We demonstrate the power of our method by showing how it predicts lateral forces corresponding to actin polymerisation around the nucleus, providing evidence for actin generated forces squeezing the sides of the nucleus as it enters a constriction. In addition, the algorithm we have developed could be adapted to analyse experimental images of deformation in other situations.     

A predictive model of cell traction forces based on cell geometry

Recent work has indicated that the shape and size of a cell can influence how a cell spreads, develops focal adhesions, and exerts forces on the substrate. However, it is unclear how cell shape regulates these events. Here we present a computational model that uses cell shape to predict the magnitude and direction of forces generated by cells. The predicted results are compared to experimentally measured traction forces, and show that the model can predict traction force direction, relative magnitude, and force distribution within the cell using only cell shape as an input. Analysis of the model shows that the magnitude and direction of the traction force at a given point is proportional to the first moment of area about that point in the cell, suggesting that contractile forces within the cell act on the entire cytoskeletal network as a single cohesive unit. Through this model, we demonstrate that intrinsic properties of cell shape can facilitate changes in traction force patterns, independently of heterogeneous mechanical properties or signaling events within the cell.     

Novel nuclear translocation of inositol polyphosphate 4-phosphatase is associated with cell cycle,proliferation and survival

Inositol polyphosphate 4 phosphatase type I enzyme (INPP4A) has a well-documented function in the cytoplasm where it terminates the phosphatidylinositol 3-kinase (PI 3-K) pathway by acting as a negative regulator. In this study, we demonstrate for the first time that INPP4A shuttles between the cytoplasm and the nucleus. Nuclear INPP4A is enzymatically active and in dynamic equilibrium between the nucleus and cytoplasm depending on the cell cycle stage, with highest amounts detected in the nucleus during the G₀/G₁ phase. Moreover, nuclear INPP4A is found to have direct proliferation suppressive activity. Cells constitutively overexpressing nuclear INPP4A exhibit massive apoptosis. In human tissues as well as cell lines, lower nuclear localization of INPP4A correlate with cancerous growth. Together, our findings suggest that nuclear compartmentalization of INPP4A may be a mechanism to regulate cell cycle progression, proliferation and apoptosis. Our results imply a role for nuclear-localized INPP4A in tumor suppression in humans.     

The assembly and function of perinuclear actin cap in migrating cells

Stress fibers are actin bundles encompassing actin filaments, actin-crosslinking, and actin-associated proteins that represent the major contractile system in the cell. Different types of stress fibers assemble in adherent cells, and they are central to diverse cellular processes including establishment of the cell shape, morphogenesis, cell polarization, and migration. Stress fibers display specific cellular organization and localization, with ventral fibers present at the basal side, and dorsal fibers and transverse actin arcs rising at the cell front from the ventral to the dorsal side and toward the nucleus. Perinuclear actin cap fibers are a specific subtype of stress fibers that rise from the leading edge above the nucleus and terminate at the cell rear forming a dome-like structure. Perinuclear actin cap fibers are fixed at three points: both ends are anchored in focal adhesions, while the central part is physically attached to the nucleus and nuclear lamina through the linker of nucleoskeleton and cytoskeleton (LINC) complex. Here, we discuss recent work that provides new insights into the mechanism of assembly and the function of these actin stress fibers that directly link extracellular matrix and focal adhesions with the nuclear envelope.     

Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates

Mechanical forces play a major role in the regulation of cell adhesion and cytoskeletal organization. In order to explore the molecular mechanism underlying this regulation, we have investigated the relationship between local force applied by the cell to the substrate and the assembly of focal adhesions. A novel approach was developed for real-time, high-resolution measurements of forces applied by cells at single adhesion sites. This method combines micropatterning of elastomer substrates and fluorescence imaging of focal adhesions in live cells expressing GFP-tagged vinculin. Local forces are correlated with the orientation, total fluorescence intensity and area of the focal adhesions, indicating a constant stress of 5.5 +/- 2 nNmicrom(-2). The dynamics of the force-dependent modulation of focal adhesions were characterized by blocking actomyosin contractility and were found to be on a time scale of seconds. The results put clear constraints on the possible molecular mechanisms for the mechanosensory response of focal adhesions to applied force.     

A Nondimensional Model Reveals Alterations in Nuclear Mechanics upon Hepatitis C Virus Replication

Morphology of the nucleus is an important regulator of gene expression. Nuclear morphology is in turn a function of the forces acting on it and the mechanical properties of the nuclear envelope. Here, we present a two-parameter, nondimensional mechanical model of the nucleus that reveals a relationship among nuclear shape parameters, such as projected area, surface area, and volume. Our model fits the morphology of individual nuclei and predicts the ratio between forces and modulus in each nucleus. We analyzed the changes in nuclear morphology of liver cells due to hepatitis C virus (HCV) infection using this model. The model predicted a decrease in the elastic modulus of the nuclear envelope and an increase in the pre-tension in cortical actin as the causes for the change in nuclear morphology. These predictions were validated biomechanically by showing that liver cells expressing HCV proteins possessed enhanced cellular stiffness and reduced nuclear stiffness. Concomitantly, cells expressing HCV proteins showed downregulation of lamin-A,C and upregulation of β-actin, corroborating the predictions of the model. Our modeling assumptions are broadly applicable to adherent, monolayer cell cultures, making the model amenable to investigate changes in nuclear mechanics due to other stimuli by merely measuring nuclear morphology. Toward this, we present two techniques, graphical and numerical, to use our model for predicting physical changes in the nucleus.     

Traction forces exerted through N-cadherin contacts

BACKGROUND INFORMATION: Mechanical forces play an important role in the organization, growth and function of living tissues. The ability of cells to transduce mechanical signals is governed by two types of microscale structures: focal adhesions, which link cells to the extracellular matrix, and adherens junctions, which link adjacent cells through cadherins. Although many studies have examined forces induced by focal adhesions, there is little known about the role of adherens junctions in force-regulation processes. The present study focuses on the determination of force transduction through cadherins at a single cell level. RESULTS: We characterized for the first time the distribution of forces developed by the cell through cadherin contacts. A N-cadherin (neural cadherin)-Fc chimaera, which mimicks the cell adhesion molecule N-cadherin, was immobilized on a muFSA (micro-force sensor array), comprising a dense array of vertical elastomer pillars, which were used both as a cell culture support for N-cadherin-expressing C2 myogenic cells and as detectors for force mapping. We coated the top of the pillars on which cells adhere and recruit adhesion complexes and F-actin. Individual pillar bending allowed the measurement of forces that mainly developed at the cell edge and directed toward their centre. Similar force distribution and amplitude were detected with an unrelated cell line of neuronal origin. Further comparison with forces applied by cells on pillars coated with fibronectin indicates that mechanical stresses transduced through both types of adhesions were comparable in distribution, orientation and amplitude. CONCLUSIONS: These results present a versatile method to measure and map forces exerted by cell-cell adhesion complexes. They show that cells transduce mechanical stress through cadherin contacts which are of the same order as magnitude of those previously characterized for focal adhesions. Altogether, they emphasize the mechanotransduction role of cytoskeleton-linked adhesion receptors of the cadherin family in tissue cohesion and reshaping.     

Image Analysis for the Quantitative Comparison of Stress Fibers and Focal Adhesions

Actin stress fibers (SFs) detect and transmit forces to the extracellular matrix through focal adhesions (FAs), and molecules in this pathway determine cellular behavior. Here, we designed two different computational tools to quantify actin SFs and the distribution of actin cytoskeletal proteins within a normalized cellular morphology. Moreover, a systematic cell response comparison between the control cells and those with impaired actin cytoskeleton polymerization was performed to demonstrate the reliability of the tools. Indeed, a variety of proteins that were present within the string beginning at the focal adhesions (vinculin) up to the actin SFs contraction (non-muscle myosin II (NMMII)) were analyzed. Finally, the software used allows for the quantification of the SFs based on the relative positions of FAs. Therefore, it provides a better insight into the cell mechanics and broadens the knowledge of the nature of SFs.     

Force-Induced Changes in Subnuclear Movement and Rheology

Extracellular mechanical forces result in changes in gene expression, but it is unclear how cells are able to permanently adapt to new mechanical environments because chemical signaling pathways are short-lived. We visualize force-induced changes in nuclear rheology to examine short- and long-time genome organization and movements. Punctate labels in the nuclear interior of HeLa, human umbilical vein endothelial, and osteosarcoma (Saos-2) cells allow tracking of nuclear movements in cells under varying levels of shear and compressive force. Under adequate shear stress two distinct regimes develop in cells under mechanical stimulation: an initial event of increased intranuclear movement followed by a regime of intranuclear movements that reflect the dose of applied force. At early times there is a nondirectionally oriented response with a small increase in nuclear translocations. After 30 min, there is a significant increase in nuclear movements, which scales with the amount of shear or compressive stress. The similarities in the nuclear response to shear and compressive stress suggest that the nucleus is a mechanosensitive element within the cell. Thus, applied extracellular forces stimulate intranuclear movements, resulting in repositioning of nuclear bodies and the associated chromatin within the nucleus.     

Nuclear deformations,from signaling to perturbation and damage

During cell growth and motility in crowded tissues or interstitial spaces, cells must integrate multiple physical and biochemical environmental inputs. After a number of recent studies, the view of the nucleus as a passive object that cells have to drag along has become obsolete, placing the nucleus as a central player in sensing some of these inputs. In the present review, we will focus on changes in nuclear shape caused by external and internal forces. Depending on their magnitude, nuclear deformations can generate signaling events that modulate cell behavior and fate, or be a source of perturbations or even damage, having detrimental effects on cellular functions. On very large deformations, nuclear envelope rupture events become frequent, leading to uncontrolled nucleocytoplasmic mixing and DNA damage. We will also discuss the consequences of repeated compromised nuclear integrity, which can trigger DNA surveillance mechanisms, with critical consequences to cell fate and tissue homeostasis.