Adherent cells, crawling slugs, peeling paint, sessile liquid drops, bearings and many other living and non-living systems apply forces to solid substrates. Traction force microscopy (TFM) provides spatially-resolved measurements of interfacial forces through the quantification and analysis of the deformation of an elastic substrate. Although originally developed for adherent cells, TFM has no inherent size or force scale, and can be applied to a much broader range of mechanical systems across physics and biology. In this paper, we showcase the wide range of applicability of TFM, describe the theory, and provide experimental details and code so that experimentalists can rapidly adopt this powerful technique.
Cell-cell and cell-matrix adhesions play essential roles in the function of tissues. There is growing evidence for the importance of cross talk between these two adhesion types, yet little is known about the impact of these interactions on the mechanical coupling of cells to the extracellular matrix (ECM). Here, we combine experiment and theory to reveal how intercellular adhesions modulate forces transmitted to the ECM. In the absence of cadherin-based adhesions, primary mouse keratinocytes within a colony appear to act independently, with significant traction forces extending throughout the colony. In contrast, with strong cadherin-based adhesions, keratinocytes in a cohesive colony localize traction forces to the colony periphery. Through genetic or antibody-mediated loss of cadherin expression or function, we show that cadherin-based adhesions are essential for this mechanical cooperativity. A minimal physical model in which cell-cell adhesions modulate the physical cohesion between contractile cells is sufficient to recreate the spatial rearrangement of traction forces observed experimentally with varying strength of cadherin-based adhesions. This work defines the importance of cadherin-based cell-cell adhesions in coordinating mechanical activity of epithelial cells and has implications for the mechanical regulation of epithelial tissues during development, homeostasis, and disease. mechanotransduction | traction force microscopy M echanical interactions of individual cells have a crucial role in the spatial organization of tissues (1, 2) and in embryonic development (3-5). The mechanical cooperation of cells is evident in dynamic processes such as flow-induced alignment of vascular endothelial cells (6) and muscle contraction (7). However, mechanical interactions of cells within a tissue also affect the tissue's static mechanical properties including elastic modulus (8), surface tension (9), and fracture toughness (10). Little is known about how these tissue-scale mechanical phenomena emerge from interactions at the molecular and cellular levels (11).Tissue-scale mechanical phenomena are particularly important in developmental morphogenesis (12), homeostasis (13), and wound healing (14) in epithelial tissues. Cells exert mechanical force on each other at sites of intercellular adhesion, typically through cadherins (15, 16), as well as on the underlying extracellular matrix (ECM) through integrins (17-19). Cadherin-based adhesions can alter physical aspects of cells such as the surface tension of cellular aggregates (20) and the spreading (21) and migration (22) of single cells adherent to cadherin-patterned substrates. Integrity of intercellular adhesions may also contribute to metastatic potential (23). We and others have shown that epithelial cell clusters with strong cell-cell adhesions exhibit coordinated mechanical behavior over length scales much larger than a single cell (24-27). Several studies have implicated cross talk between cell-ECM and cell-cell adhesions (28, 29) that can be modulated by ac...
To understand how the mechanical properties of tissues emerge from interactions of multiple cells, we measure traction stresses of cohesive colonies of 1–27 cells adherent to soft substrates. We find that traction stresses are generally localized at the periphery of the colony and the total traction force scales with the colony radius. For large colony sizes, the scaling appears to approach linear, suggesting the emergence of an apparent surface tension of the order of 10−3 N/m. A simple model of the cell colony as a contractile elastic medium coupled to the substrate captures the spatial distribution of traction forces and the scaling of traction forces with the colony size.
Hair follicle stem cells (HFSCs) and their transit amplifying cell (TAC) progeny sense BMPs at defined stages of the hair cycle to control their proliferation and differentiation. Here, we exploit the distinct spatial and temporal localizations of these cells to selectively ablate BMP signaling in each compartment and examine its functional role. We find that BMP signaling is required for HFSC quiescence and to promote TAC differentiation along different lineages as the hair cycle progresses. We also combine in vivo genome-wide chromatin immunoprecipitation and deep-sequencing, transcriptional profiling, and loss-of-function genetics to define BMP-regulated genes. We show that some pSMAD1/5 targets, like Gata3, function specifically in TAC lineage-progression. Others, like Id1 and Id3, function in both HFSCs and TACs, but in distinct ways. Our study therefore illustrates the complex differential roles that a key signaling pathway can play in regulation of closely-related stem/progenitor cells within the context of their overall niche.
Adult stem cell (SC) maintenance and differentiation are known to depend on signals received from the niche. Here however, we demonstrate a mechanism for SC specification and regulation that is niche-independent. Using immunofluorescence, live imaging, genetics, cell-cycle analyses, in utero lentiviral transduction, and lineage-tracing, we show that in developing hair buds, SCs are born from asymmetric divisions that differentially display WNT and SHH signaling. Displaced WNTlo suprabasal daughters become SCs that respond to paracrine SHH and symmetrically expand. By contrast, basal daughters remain WNThi. They express but do not respond to SHH, and hence maintain slow-cycling, asymmetric divisions. Over time, they become short-lived progenitors, generating differentiating daughters rather than SCs. Thus, in contrast to an established niche which harbors a fixed SC pool whose expelled progeny differentiate, asymmetric divisions first specify and displace early SCs into an environment conducive to expansion, and later restrict their numbers by switching asymmetric fates.
Generation of a barrier in multi-layered epithelia like the epidermis requires restricted positioning of functional tight junctions (TJ) to the most suprabasal viable layer. This positioning necessitates tissue-level polarization of junctions and the cytoskeleton through unknown mechanisms. Using quantitative whole-mount imaging, genetic ablation, and traction force microscopy and atomic force microscopy, we find that ubiquitously localized E-cadherin coordinates tissue polarization of tension-bearing adherens junction (AJ) and F-actin organization to allow formation of an apical TJ network only in the uppermost viable layer. Molecularly, E-cadherin localizes and tunes EGFR activity and junctional tension to inhibit premature TJ complex formation in lower layers while promoting increased tension and TJ stability in the granular layer 2. In conclusion, our data identify an E-cadherin-dependent mechanical circuit that integrates adhesion, contractile forces and biochemical signaling to drive the polarized organization of junctional tension necessary to build an in vivo epithelial barrier.
In order to understand the mechanisms that guide cell fate decisions during early human development, we closely examined the differentiation process in adherent colonies of human embryonic stem cells (hESCs). Live imaging of the differentiation process reveals that cells on the outer edge of the undifferentiated colony begin to differentiate first and remain on the perimeter of the colony to eventually form a band of differentiation. Strikingly, this band is of constant width in all colonies, independent of their size. Cells at the edge of undifferentiated colonies show distinct actin organization, greater myosin activity and stronger traction forces compared to cells in the interior of the colony. Increasing the number of cells at the edge of colonies by plating small colonies can increase differentiation efficiency. Our results suggest that human developmental decisions are influenced by cellular environments and can be dictated by colony geometry of hESCs.
Cell competition (CC)—the sensing and elimination of less fit “loser” cells by neighbouring “winner” cells—was first described in Drosophila. Although proposed as a selection mechanism to optimize tissue and organ development, its evolutionary generality remains unclear. Here, by employing live-imaging, lineage-tracing, single cell transcriptomics and genetics, we unearth two intriguing CC mechanisms that sequentially shape and maintain stratified tissue architecture during mouse skin development. In early embryonic epidermis, winner progenitors within the single-layered epithelium kill and clear neighbouring losers by engulfment. Upon stratification and skin barrier formation, the basal layer instead expels losers through a homeostatic upward flux of differentiating progeny. This CC switch is physiologically relevant: when perturbed, so too is barrier formation. Our findings establish CC as a selective force to optimize vertebrate tissue function, and illuminate how a tissue dynamically adjusts CC strategies to preserve fitness as it encounters increased architectural complexity during morphogenesis.
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