Parametric modeling of mechanical effects on circadian oscillators.

Circadian rhythms are archetypal examples of nonlinear oscillations. While these oscillations are usually attributed to circuits of biochemical interactions among clock genes and proteins, recent experimental studies reveal that they are also affected by the cell's mechanical environment. Here, we extend a standard biochemical model of circadian rhythmicity to include mechanical effects in a parametric manner. Using experimental observations to constrain the model, we suggest specific ways in which the mechanical signal might affect the clock. Additionally, a bifurcation analysis of the system predicts that these mechanical signals need to be within an optimal range for circadian oscillations to occur.

Membrane to cortex attachment determines different mechanical phenotypes in LGR5+ and LGR5- colorectal cancer cells.

Colorectal cancer (CRC) tumors are composed of heterogeneous and plastic cell populations, including a pool of cancer stem cells that express LGR5. Whether these distinct cell populations display different mechanical properties, and how these properties might contribute to metastasis is poorly understood. Using CRC patient derived organoids (PDOs), we find that compared to LGR5- cells, LGR5+ cancer stem cells are stiffer, adhere better to the extracellular matrix (ECM), move slower both as single cells and clusters, display higher nuclear YAP, show a higher survival rate in response to mechanical confinement, and form larger transendothelial gaps. These differences are largely explained by the downregulation of the membrane to cortex attachment proteins Ezrin/Radixin/Moesin (ERMs) in the LGR5+ cells. By analyzing single cell RNA-sequencing (scRNA-seq) expression patterns from a patient cohort, we show that this downregulation is a robust signature of colorectal tumors. Our results show that LGR5- cells display a mechanically dynamic phenotype suitable for dissemination from the primary tumor whereas LGR5+ cells display a mechanically stable and resilient phenotype suitable for extravasation and metastatic growth.

Mechanical control of the mammalian circadian clock via YAP/TAZ and TEAD.

Autonomous circadian clocks exist in nearly every mammalian cell type. These cellular clocks are subjected to a multilayered regulation sensitive to the mechanochemical cell microenvironment. Whereas the biochemical signaling that controls the cellular circadian clock is increasingly well understood, mechanisms underlying regulation by mechanical cues are largely unknown. Here we show that the fibroblast circadian clock is mechanically regulated through YAP/TAZ nuclear levels. We use high-throughput analysis of single-cell circadian rhythms and apply controlled mechanical, biochemical, and genetic perturbations to study the expression of the clock gene Rev-erbα. We observe that Rev-erbα circadian oscillations are disrupted with YAP/TAZ nuclear translocation. By targeted mutations and overexpression of YAP/TAZ, we show that this mechanobiological regulation, which also impacts core components of the clock such as Bmal1 and Cry1, depends on the binding of YAP/TAZ to the transcriptional effector TEAD. This mechanism could explain the impairment of circadian rhythms observed when YAP/TAZ activity is upregulated, as in cancer and aging.

Mechanical force application to the nucleus regulates nucleocytoplasmic transport.

Mechanical force controls fundamental cellular processes in health and disease, and increasing evidence shows that the nucleus both experiences and senses applied forces. Such forces can lead to the nuclear translocation of proteins, but whether force controls nucleocytoplasmic transport, and how, remains unknown. Here we show that nuclear forces differentially control passive and facilitated nucleocytoplasmic transport, setting the rules for the mechanosensitivity of shuttling proteins. We demonstrate that nuclear force increases permeability across nuclear pore complexes, with a dependence on molecular weight that is stronger for passive than for facilitated diffusion. Owing to this differential effect, force leads to the translocation of cargoes into or out of the nucleus within a given range of molecular weight and affinity for nuclear transport receptors. Further, we show that the mechanosensitivity of several transcriptional regulators can be both explained by this mechanism and engineered exogenously by introducing appropriate nuclear localization signals. Our work unveils a mechanism of mechanically induced signalling, probably operating in parallel with others, with potential applicability across signalling pathways.

ERK-Mediated Mechanochemical Waves Direct Collective Cell Polarization.

During collective migration of epithelial cells, the migration direction is aligned over a tissue-scale expanse. Although the collective cell migration is known to be directed by mechanical forces transmitted via cell-cell junctions, it remains elusive how the intercellular force transmission is coordinated with intracellular biochemical signaling to achieve collective movements. Here, we show that intercellular coupling of extracellular signal-regulated kinase (ERK)-mediated mechanochemical feedback yields long-distance transmission of guidance cues. Mechanical stretch activates ERK through epidermal growth factor receptor (EGFR) activation, and ERK activation triggers cell contraction. The contraction of the activated cell pulls neighboring cells, evoking another round of ERK activation and contraction in the neighbors. Furthermore, anisotropic contraction based on front-rear polarization guarantees unidirectional propagation of ERK activation, and in turn, the ERK activation waves direct multicellular alignment of the polarity, leading to long-range ordered migration. Our findings reveal that mechanical forces mediate intercellular signaling underlying sustained transmission of guidance cues for collective cell migration.

Biomarkers for tissue engineering of the tendon-bone interface.

The tendon-bone interface (enthesis) is a highly sophisticated biomaterial junction that allows stress transfer between mechanically dissimilar materials. The enthesis encounters very high mechanical demands and the regenerative capacity is very low resulting in high rupture recurrence rates after surgery. Tissue engineering offers the potential to recover the functional integrity of entheses. However, recent enthesis tissue engineering approaches have been limited by the lack of knowledge about the cells present at this interface. Here we investigated the cellular differentiation of enthesis cells and compared the cellular pattern of enthesis cells to tendon and cartilage cells in a next generation sequencing transcriptome study. We integrated the transcriptome data with proteome data of a previous study to identify biomarkers of enthesis cell differentiation. Transcriptomics detected 34468 transcripts in total in enthesis, tendon, and cartilage. Transcriptome comparisons revealed 3980 differentially regulated candidates for enthesis and tendon, 395 for enthesis and cartilage, and 946 for cartilage and tendon. An asymmetric distribution of enriched genes was observed in enthesis and cartilage transcriptome comparison suggesting that enthesis cells are more chondrocyte-like than tenocyte-like. Integrative analysis of transcriptome and proteome data identified ten enthesis biomarkers and six tendon biomarkers. The observed gene expression characteristics and differentiation markers shed light into the nature of the cells present at the enthesis. The presented markers will foster enthesis tissue engineering approaches by setting a bench-mark for differentiation of seeded cells towards a physiologically relevant phenotype.

Mechanics of soft epithelial keratin networks depend on modular filament assembly kinetics.

UNLABELLED: Structural adaptability is a pivotal requirement of cytoskeletal structures, enabling their reorganization to meet the cellular needs. Shear stress, for instance, results in large morphological network changes of the human soft epithelial keratin pair K8:K18, and is accompanied by an increase in keratin phosphorylation levels. Yet the mechanisms responsible for the disruption of the network structure in vivo remain poorly understood. To understand the effect of the stress-related site-specific phosphorylation of the K8:K18 pair, we created phosphomimicry mutants - K8(S431E), K8(S73E), K18(S52E) - in vitro, and investigated the various steps of keratin assembly from monomer to network structure using fluorescence and electron microscopy, and using rheology characterized their network mechanical properties. We find that the addition of a charged group produces networks with depleted intra-connectivity, which translates to a mechanically weaker and more deformable network. This large variation in network structure is achieved by the formation of shorter mutant filaments, which exhibit differing assembly kinetics and a manifestly reduced capacity to form the extended structures characteristic of the wild-type system. The similarity in outcome for all the phosphomimicry mutants explored points to a more general mechanism of structural modulation of intermediate filaments via phosphorylation. Understanding the role of kinetic effects in the construction of these cytoskeletal biopolymer networks is critical to elucidating their structure-function properties, providing new insight for the design of keratin-inspired biomaterials. STATEMENT OF SIGNIFICANCE: Structural remodeling of cytoskeletal networks accompanies many cellular processes. Interestingly, levels of phosphorylation of the human soft epithelial keratin pair K8:K18 increase during their stress-related structural remodeling. Our multi-scale study sheds light on the poorly understood mechanism with which site-specific phosphorylation induces disruption of the keratin network structure in vivo. We show how phosphorylation reduces keratin filament length, an effect that propagates through to the mesoscopic structure, resulting in the formation of connectivity-depleted and mechanically weaker networks. We determine that the intrinsically-set filament-to-filament attractions that drive bundle assembly give rise to the structural variability by enabling the formation of kinetically-arrested structures. Overall, our results shed light on how self-assembled intermediate filament structures can be tailored to exhibit different structural functionalities.