Computational modelling of cell motility modes emerging from cell-matrix adhesion dynamics.

Lymphocytes have been described to perform different motility patterns such as Brownian random walks, persistent random walks, and Lévy walks. Depending on the conditions, such as confinement or the distribution of target cells, either Brownian or Lévy walks lead to more efficient interaction with the targets. The diversity of these motility patterns may be explained by an adaptive response to the surrounding extracellular matrix (ECM). Indeed, depending on the ECM composition, lymphocytes either display a floating motility without attaching to the ECM, or sliding and stepping motility with respectively continuous or discontinuous attachment to the ECM, or pivoting behaviour with sustained attachment to the ECM. Moreover, on the long term, lymphocytes either perform a persistent random walk or a Brownian-like movement depending on the ECM composition. How the ECM affects cell motility is still incompletely understood. Here, we integrate essential mechanistic details of the lymphocyte-matrix adhesions and lymphocyte intrinsic cytoskeletal induced cell propulsion into a Cellular Potts model (CPM). We show that the combination of de novo cell-matrix adhesion formation, adhesion growth and shrinkage, adhesion rupture, and feedback of adhesions onto cell propulsion recapitulates multiple lymphocyte behaviours, for different lymphocyte subsets and various substrates. With an increasing attachment area and increased adhesion strength, the cells' speed and persistence decreases. Additionally, the model predicts random walks with short-term persistent but long-term subdiffusive properties resulting in a pivoting type of motility. For small adhesion areas, the spatial distribution of adhesions emerges as a key factor influencing cell motility. Small adhesions at the front allow for more persistent motility than larger clusters at the back, despite a similar total adhesion area. In conclusion, we present an integrated framework to simulate the effects of ECM proteins on cell-matrix adhesion dynamics. The model reveals a sufficient set of principles explaining the plasticity of lymphocyte motility.

Mechanical compartmentalization of the intestinal organoid enables crypt folding and collective cell migration.

Intestinal organoids capture essential features of the intestinal epithelium such as crypt folding, cellular compartmentalization and collective movements. Each of these processes and their coordination require patterned forces that are at present unknown. Here we map three-dimensional cellular forces in mouse intestinal organoids grown on soft hydrogels. We show that these organoids exhibit a non-monotonic stress distribution that defines mechanical and functional compartments. The stem cell compartment pushes the extracellular matrix and folds through apical constriction, whereas the transit amplifying zone pulls the extracellular matrix and elongates through basal constriction. The size of the stem cell compartment depends on the extracellular-matrix stiffness and endogenous cellular forces. Computational modelling reveals that crypt shape and force distribution rely on cell surface tensions following cortical actomyosin density. Finally, cells are pulled out of the crypt along a gradient of increasing tension. Our study unveils how patterned forces enable compartmentalization, folding and collective migration in the intestinal epithelium.

Integrin-linked kinase tunes cell-cell and cell-matrix adhesions to regulate the switch between apoptosis and EMT downstream of TGFß1.

Epithelial-mesenchymal transition (EMT) is a morphogenetic process that endows epithelial cells with migratory and invasive potential. Mechanical and chemical signals from the tumor microenvironment can activate the EMT program, thereby permitting cancer cells to invade the surrounding stroma and disseminate to distant organs. Transforming growth factor ß1 (TGFß1) is a potent inducer of EMT that can also induce apoptosis depending on the microenvironmental context. In particular, stiff microenvironments promote EMT while softer ones promote apoptosis. Here, we investigated the molecular signaling downstream of matrix stiffness that regulates the phenotypic switch in response to TGFß1 and uncovered a critical role for integrin-linked kinase (ILK). Specifically, depleting ILK from mammary epithelial cells precludes their ability to sense the stiffness of their microenvironment. In response to treatment with TGFß1, ILK-depleted cells undergo apoptosis on both soft and stiff substrata. We found that knockdown of ILK decreases focal adhesions and increases cell-cell adhesions, thus shifting the balance from cell-matrix to cell-cell adhesion. High cell-matrix adhesion promotes EMT whereas high cell-cell adhesion promotes apoptosis downstream of TGFß1. These results highlight an important role for ILK in controlling cell phenotype by regulating adhesive connections to the local microenvironment.

Myotendinous junction adaptations to ladder-based resistance training: identification of a new telocyte niche.

The present study shows chronic adjustments in the myotendinous junction (MTJ) in response to different ladder-based resistance training (LRT) protocols. Thirty adult male Wistar rats were divided into groups: sedentary (S), calisthenics (LRT without additional load [C]), and resistance-trained (LRT with extra weight [R]). We demonstrated longer lengths of sarcoplasmatic invaginations in the trained groups; however, evaginations were seen mainly in group R. We showed a greater thickness of sarcoplasmatic invaginations in groups C and R, in addition to greater evaginations in R. We also observed thinner basal lamina in trained groups. The support collagen layer (SCL) adjacent to the MTJ and the diameters of the transverse fibrils were larger in R. We also discovered a niche of telocytes in the MTJ with electron micrographs of the plantar muscle and with immunostaining with CD34+ in the gastrocnemius muscle near the blood vessels and pericytes. We concluded that the continuous adjustments in the MTJ ultrastructure were the result of tissue plasticity induced by LRT, which is causally related to muscle hypertrophy and, consequently, to the remodeling of the contact interface. Also, we reveal the existence of a collagen layer adjacent to MTJ and discover a new micro anatomic location of telocytes.

Coupling to substrate adhesions drives the maturation of muscle stress fibers into myofibrils within cardiomyocytes.

Forces generated by heart muscle contraction must be balanced by adhesion to the extracellular matrix (ECM) and to other cells for proper heart function. Decades of data have suggested that cell-ECM adhesions are important for sarcomere assembly. However, the relationship between cell-ECM adhesions and sarcomeres assembling de novo remains untested. Sarcomeres arise from muscle stress fibers (MSFs) that are translocating on the top (dorsal) surface of cultured cardiomyocytes. Using an array of tools to modulate cell-ECM adhesion, we established a strong positive correlation between the extent of cell-ECM adhesion and sarcomere assembly. On the other hand, we found a strong negative correlation between the extent of cell-ECM adhesion and the rate of MSF translocation, a phenomenon also observed in nonmuscle cells. We further find a conserved network architecture that also exists in nonmuscle cells. Taken together, our results show that cell-ECM adhesions mediate coupling between the substrate and MSFs, allowing their maturation into sarcomere-containing myofibrils.

The Interplay Between Cell-Cell and Cell-Matrix Forces Regulates Cell Migration Dynamics.

Cells in vivo encounter and exert forces as they interact with the extracellular matrix (ECM) and neighboring cells during migration. These mechanical forces play crucial roles in regulating cell migratory behaviors. Although a variety of studies have focused on describing single-cell or the collective cell migration behaviors, a fully mechanistic understanding of how the cell-cell (intercellular) and cell-ECM (extracellular) traction forces individually and cooperatively regulate single-cell migration and coordinate multicellular movement in a cellular monolayer is still lacking. Here, we developed an integrated experimental and analytical system to examine both the intercellular and extracellular traction forces acting on individual cells within an endothelial cell colony as well as their roles in guiding cell migratory behaviors (i.e., cell translation and rotation). Combined with force, multipole, and moment analysis, our results revealed that traction force dominates in regulating cell active translation, whereas intercellular force actively modulates cell rotation. Our findings advance the understanding of the intricacies of cell-cell and cell-ECM forces in regulating cellular migratory behaviors that occur during the monolayer development and may yield deeper insights into the single-cell dynamic behaviors during tissue development, embryogenesis, and wound healing.

Calcium-induced differentiation in normal human colonoid cultures: Cell-cell / cell-matrix adhesion, barrier formation and tissue integrity.

BACKGROUND AND AIMS: The goal of the study was to assess calcium alone and Aquamin, a multi-mineral natural product that contains magnesium and detectable levels of 72 trace elements in addition to calcium, for capacity to affect growth and differentiation in colonoid cultures derived from histologically-normal human colon tissue. METHODS: Colonoid cultures were maintained in a low-calcium (0.25 mM) medium or in medium supplemented with an amount of calcium (1.5-3.0 mM), either from calcium alone or Aquamin for a period of two weeks. This was shown in a previous study to induce differentiation in colonoids derived from large adenomas. Changes in growth, morphological features and protein expression profile were assessed at the end of the incubation period using a combination of phase-contrast and scanning electron microscopy, histology and immunohistology, proteomic assessment and transmission electron microscopy. RESULTS: Unlike the previously-studied tumor-derived colonoids (which remained un-differentiated in the absence of calcium-supplementation), normal tissue colonoids underwent differentiation as indicated by gross and microscopic appearance, a low proliferative index and high-level expression of cytokeratin 20 in the absence of intervention (i.e., in control condition). Only modest additional changes were seen in these parameters with either calcium alone or Aquamin (providing up to 3.0 mM calcium). In spite of this, proteomic analysis and immunohistochemistry revealed that both interventions induced strong up-regulation of proteins that promote cell-cell and cell-matrix adhesive functions, barrier formation and tissue integrity. Transmission electron microscopy revealed an increase in desmosomes in response to intervention. CONCLUSIONS: These findings demonstrate that colonoids derived from histologically normal human tissue can undergo differentiation in the presence of a low ambient calcium concentration. However, higher calcium levels induce elaboration of proteins that promote cell-cell and cell-matrix adhesion. These changes could lead to improved barrier function and improved colon tissue health.

In vivo topology converts competition for cell-matrix adhesion into directional migration.

When migrating in vivo, cells are exposed to numerous conflicting signals: chemokines, repellents, extracellular matrix, growth factors. The roles of several of these molecules have been studied individually in vitro or in vivo, but we have yet to understand how cells integrate them. To start addressing this question, we used the cephalic neural crest as a model system and looked at the roles of its best examples of positive and negative signals: stromal-cell derived factor 1 (Sdf1/Cxcl12) and class3-Semaphorins. Here we show that Sdf1 and Sema3A antagonistically control cell-matrix adhesion via opposite effects on Rac1 activity at the single cell level. Directional migration at the population level emerges as a result of global Semaphorin-dependent confinement and broad activation of adhesion by Sdf1 in the context of a biased Fibronectin distribution. These results indicate that uneven in vivo topology renders the need for precise distribution of secreted signals mostly dispensable.

Cell-matrix adhesion controls Golgi organization and function through Arf1 activation in anchorage-dependent cells.

Cell-matrix adhesion regulates membrane trafficking controlling anchorage-dependent signaling. While a dynamic Golgi complex can contribute to this pathway, its regulation by adhesion remains unclear. Here we report that loss of adhesion dramatically disorganized the Golgi in mouse and human fibroblast cells. Golgi integrity is restored rapidly upon integrin-mediated re-adhesion to FN and is disrupted by integrin blocking antibody. In suspended cells, the cis, cis-medial and trans-Golgi networks differentially disorganize along the microtubule network but show no overlap with the ER, making this disorganization distinct from known Golgi fragmentation. This pathway is regulated by an adhesion-dependent reduction and recovery of Arf1 activation. Constitutively active Arf1 disrupts this regulation and prevents Golgi disorganization due to loss of adhesion. Adhesion-dependent Arf1 activation regulates its binding to the microtubule minus-end motor protein dynein to control Golgi reorganization, which is blocked by ciliobrevin. Adhesion-dependent Golgi organization controls its function, regulating cell surface glycosylation due to loss of adhesion, which is blocked by constitutively active Arf1. This study, hence, identified integrin-dependent cell-matrix adhesion to be a novel regulator of Arf1 activation, controlling Golgi organization and function in anchorage-dependent cells. This article has an associated First Person interview with the first author of the paper.

Regulation of cell cycle progression by cell-cell and cell-matrix forces.

It has long been proposed that the cell cycle is regulated by physical forces at the cell-cell and cell-extracellular matrix (ECM) interfaces1-12. However, the evolution of these forces during the cycle has never been measured in a tissue, and whether this evolution affects cell cycle progression is unknown. Here, we quantified cell-cell tension and cell-ECM traction throughout the complete cycle of a large cell population in a growing epithelium. These measurements unveil temporal mechanical patterns that span the entire cell cycle and regulate its duration, the G1-S transition and mitotic rounding. Cells subjected to higher intercellular tension exhibit a higher probability to transition from G1 to S, as well as shorter G1 and S-G2-M phases. Moreover, we show that tension and mechanical energy are better predictors of the duration of G1 than measured geometric properties. Tension increases during the cell cycle but decreases 3 hours before mitosis. Using optogenetic control of contractility, we show that this tension drop favours mitotic rounding. Our results establish that cell cycle progression is regulated cooperatively by forces between the dividing cell and its neighbours.

Force Generation via ß-Cardiac Myosin, Titin, and α-Actinin Drives Cardiac Sarcomere Assembly from Cell-Matrix Adhesions.

Truncating mutations in the sarcomere protein titin cause dilated cardiomyopathy due to sarcomere insufficiency. However, it remains mechanistically unclear how these mutations decrease sarcomere content in cardiomyocytes. Utilizing human induced pluripotent stem cell-derived cardiomyocytes, CRISPR/Cas9, and live microscopy, we characterize the fundamental mechanisms of human cardiac sarcomere formation. We observe that sarcomerogenesis initiates at protocostameres, sites of cell-extracellular matrix adhesion, where nucleation and centripetal assembly of α-actinin-2-containing fibers provide a template for the fusion of Z-disk precursors, Z bodies, and subsequent striation. We identify that ß-cardiac myosin-titin-protocostamere form an essential mechanical connection that transmits forces required to direct α-actinin-2 centripetal fiber assembly and sarcomere formation. Titin propagates diastolic traction stresses from ß-cardiac myosin, but not α-cardiac myosin or non-muscle myosin II, to protocostameres during sarcomerogenesis. Ablating protocostameres or decoupling titin from protocostameres abolishes sarcomere assembly. Together these results identify the mechanical and molecular components critical for human cardiac sarcomerogenesis.

Patterns of Cell-Matrix Interactions during Formation the Distraction Bone Regenerates.

Multiple-day distraction regenerate contains a number of foci or zones merging into one another with different structural organization of the fibrocellular basis that reflects successive stages of reparative regeneration. At the moment of the last traction, the central part of the regenerate represents a zone of fibrogenesis (growth zone), which is common for the proximal and distal parts of the distraction regenerate. The other zones are paired and symmetrical: zones of angiogenesis, fibrous osteogenesis (primary), and osteogenesis (secondary). The latter include subzones of resorption, secondary osteogenesis, and functional remodeling.

A phenomenological cohesive model for the macroscopic simulation of cell-matrix adhesions.

Cell adhesion is crucial for cells to not only physically interact with each other but also sense their microenvironment and respond accordingly. In fact, adherent cells can generate physical forces that are transmitted to the surrounding matrix, regulating the formation of cell-matrix adhesions. The main purpose of this work is to develop a computational model to simulate the dynamics of cell-matrix adhesions through a cohesive formulation within the framework of the finite element method and based on the principles of continuum damage mechanics. This model enables the simulation of the mechanical adhesion between cell and extracellular matrix (ECM) as regulated by local multidirectional forces and thus predicts the onset and growth of the adhesion. In addition, this numerical approach allows the simulation of the cell as a whole, as it models the complete mechanical interaction between cell and ECM. As a result, we can investigate and quantify how different mechanical conditions in the cell (e.g., contractile forces, actin cytoskeletal properties) or in the ECM (e.g., stiffness, external forces) can regulate the dynamics of cell-matrix adhesions.

A Combined In Vitro Imaging and Multi-Scale Modeling System for Studying the Role of Cell Matrix Interactions in Cutaneous Wound Healing.

Many cell types remodel the extracellular matrix of the tissues they inhabit in response to a wide range of environmental stimuli, including mechanical cues. Such is the case in dermal wound healing, where fibroblast migrate into and remodel the provisional fibrin matrix in a complex manner that depends in part on the local mechanical environment and the evolving multi-scale mechanical interactions of the system. In this study, we report on the development of an image-based multi-scale mechanical model that predicts the short-term (24 hours), structural reorganization of a fibrin gel by fibroblasts. These predictive models are based on an in vitro experimental system where clusters of fibroblasts (i.e., explants) were spatially arranged into a triangular geometry onto the surface of fibrin gels that were subjected to either Fixed or Free in-plane mechanical constraints. Experimentally, regional differences in short-term structural remodeling and cell migration were observed for the two gel boundary conditions. A pilot experiment indicated that these small differences in the short-term remodeling of the fibrin gel translate into substantial differences in long-term (4 weeks) remodeling, particularly in terms of collagen production. The multi-scale models were able to predict some regional differences in remodeling and qualitatively similar reorganization patterns for the two boundary conditions. However, other aspects of the model, such as the magnitudes and rates of deformation of gel, did not match the experiments. These discrepancies between model and experiment provide fertile ground for challenging model assumptions and devising new experiments to enhance our understanding of how this multi-scale system functions. These efforts will ultimately improve the predictions of the remodeling process, particularly as it relates to dermal wound healing and the reduction of patient scarring. Such models could be used to recommend patient-specific mechanical-based treatment dependent on parameters such as wound geometry, location, age, and health.

Mechanosensing via cell-matrix adhesions in 3D microenvironments.

The extracellular matrix (ECM) microenvironment plays a central role in cell migration by providing physiochemical information that influences overall cell behavior. Much of this external information is accessed by direct interaction of the cell with ECM ligands and structures via integrin-based adhesions that are hypothesized to act as mechanosensors for testing the surrounding microenvironment. Our current understanding of these mechanical complexes is derived primarily from studies of cellular adhesions formed on two-dimensional (2D) substrates in vitro. Yet the rules of cell/ECM engagement and mechanosensing in three-dimensional (3D) microenvironments are invariably more complex under both in vitro and in vivo conditions. Here we review the current understanding of how cellular mechanosensing occurs through adhesion complexes within 3D microenvironments and discuss how these mechanisms can vary and differ from interactions on 2D substrates.

Nuclear receptor peroxisome proliferator activated receptor (PPAR) ß/δ in skin wound healing and cancer.

We review the functions of peroxisome proliferator activated receptor (PPAR) ß/δ in skin wound healing and cancer. In particular, we highlight the roles of PPARß/δ in inhibiting keratinocyte apoptosis at wound edges via activation of the PI3K/PKBα/Akt1 pathway and its role during re-epithelialization in regulating keratinocyte adhesion and migration. In fibroblasts, PPARß/δ controls IL-1 signalling and thereby contributes to the homeostatic control of keratinocyte proliferation. We discuss its therapeutic potential for treating diabetic wounds and inflammatory skin diseases such as psoriasis and acne vulgaris. PPARß/δ is classified as a tumour growth modifier; it is activated by chronic low-grade inflammation, which promotes the production of lipids that, in turn, enhance PPARß/δ transcription activity. Our earlier work unveiled a cascade of events triggered by PPARß/δ that involve the oncogene Src, which promotes ultraviolet-induced skin cancer in mice via enhanced EGFR/Erk1/2 signalling and the expression of epithelial-to-mesenchymal transition (EMT) markers. Interestingly, PPARß/δ expression is correlated with the expression of SRC and EMT markers in human skin squamous cell carcinoma. Furthermore, there is a positive interaction between PPARß/δ, SRC, and TGFß1 at the transcriptional level in various human epithelial cancers. Taken together, these observations suggest the need for evaluating PPARß/δ modulators that attenuate or increase its activity, depending on the therapeutic target.

Modeling the formation of cell-matrix adhesions on a single 3D matrix fiber.

Cell-matrix adhesions are crucial in different biological processes like tissue morphogenesis, cell motility, and extracellular matrix remodeling. These interactions that link cell cytoskeleton and matrix fibers are built through protein clutches, generally known as adhesion complexes. The adhesion formation process has been deeply studied in two-dimensional (2D) cases; however, the knowledge is limited for three-dimensional (3D) cases. In this work, we simulate different local extracellular matrix properties in order to unravel the fundamental mechanisms that regulate the formation of cell-matrix adhesions in 3D. We aim to study the mechanical interaction of these biological structures through a three dimensional discrete approach, reproducing the transmission pattern force between the cytoskeleton and a single extracellular matrix fiber. This numerical model provides a discrete analysis of the proteins involved including spatial distribution, interaction between them, and study of the different phenomena, such as protein clutches unbinding or protein unfolding.

Spatial distribution of cell-cell and cell-ECM adhesions regulates force balance while main-taining E-cadherin molecular tension in cell pairs.

Mechanical linkage between cell-cell and cell-extracellular matrix (ECM) adhesions regulates cell shape changes during embryonic development and tissue homoeostasis. We examined how the force balance between cell-cell and cell-ECM adhesions changes with cell spread area and aspect ratio in pairs of MDCK cells. We used ECM micropatterning to drive different cytoskeleton strain energy states and cell-generated traction forces and used a Förster resonance energy transfer tension biosensor to ask whether changes in forces across cell-cell junctions correlated with E-cadherin molecular tension. We found that continuous peripheral ECM adhesions resulted in increased cell-cell and cell-ECM forces with increasing spread area. In contrast, confining ECM adhesions to the distal ends of cell-cell pairs resulted in shorter junction lengths and constant cell-cell forces. Of interest, each cell within a cell pair generated higher strain energies than isolated single cells of the same spread area. Surprisingly, E-cadherin molecular tension remained constant regardless of changes in cell-cell forces and was evenly distributed along cell-cell junctions independent of cell spread area and total traction forces. Taken together, our results showed that cell pairs maintained constant E-cadherin molecular tension and regulated total forces relative to cell spread area and shape but independently of total focal adhesion area.

Force transmission at cell-cell and cell-matrix adhesions.

All cells are subjected to mechanical forces throughout their lifetimes. These forces are sensed by cell surface adhesion receptors and trigger robust actin cytoskeletal rearrangements and growth of the associated adhesion complex to counter the applied force. In this review, we discuss how integrins and cadherins sense force and transmit these forces into the cell interior. We focus on the complement of proteins each adhesion complex recruits to bear the force and the signal transduction pathways activated to allow the cell to tune its contractility. A discussion of the similarities, differences, and crosstalk between cadherin- and integrin-mediated force transmission is also presented.