Sensing the squeeze: nuclear mechanotransduction in health and disease.

The nucleus not only is a repository for DNA but also a center of cellular and nuclear mechanotransduction. From nuclear deformation to the interplay between mechanosensing components and genetic control, the nucleus is poised at the nexus of mechanical forces and cellular function. Understanding the stresses acting on the nucleus, its mechanical properties, and their effects on gene expression is therefore crucial to appreciate its mechanosensitive function. In this review, we examine many elements of nuclear mechanotransduction, and discuss the repercussions on the health of cells and states of illness. By describing the processes that underlie nuclear mechanosensation and analyzing its effects on gene regulation, the review endeavors to open new avenues for studying nuclear mechanics in physiology and diseases.

Nuclear curvature determines Yes-associated protein localization and differentiation of mesenchymal stem cells.

Controlling mesenchymal stem cell (MSC) differentiation remains a critical challenge in MSCs' therapeutic application. Numerous biophysical and mechanical stimuli influence stem cell fate; however, their relative efficacy and specificity in mechanically directed differentiation remain unclear. Yes-associated protein (YAP) is one key mechanosensitive protein that controls MSC differentiation. Previous studies have related nuclear mechanics with YAP activity, but we still lack an understanding of what nuclear deformation specifically regulates YAP and its relationship with mechanical stimuli. Here, we report that maximum nuclear curvature is the most precise biophysical determinant for YAP mechanotransduction-mediated MSC differentiation and is a relevant parameter for stem cell-based therapies. We employed traction force microscopy and confocal microscopy to characterize the causal relationships between contractility and nuclear deformation in regulating YAP activity in MSCs. We observed that an increase in contractility compresses nuclei anisotropically, whereby the degree of asymmetric compression increased the bending curvature of the nuclear membrane. We then examined membrane curvature and tension using thin micropatterned adhesive substrate lines and an FRET-based tension sensor, revealing the direct role of curvature in YAP activity driven by both active and passive nuclear import. Finally, we employed micropatterned lines to control nuclear curvature and precisely direct MSC differentiation. This work illustrates that nuclear curvature subsumes other biophysical aspects to control YAP-mediated differentiation in MSCs and may provide a deterministic solution to some of the challenges in mesenchymal stem cell therapies.

Tissue tension permits ß-catenin phosphorylation to drive mesoderm specification in human embryonic stem cells.

The role of morphogenetic forces in cell fate specification is an area of intense interest. Our prior studies suggested that the development of high cell-cell tension in human embryonic stem cells (hESC) colonies permits the Src-mediated phosphorylation of junctional ß-catenin that accelerates its release to potentiate Wnt-dependent signaling critical for initiating mesoderm specification. Using an ectopically expressed nonphosphorylatable mutant of ß-catenin (Y654F), we now provide direct evidence that impeding tension-dependent Src-mediated ß-catenin phosphorylation impedes the expression of Brachyury (T) and the epithelial-to-mesenchymal transition (EMT) necessary for mesoderm specification. Addition of exogenous Wnt3a or inhibiting GSK3ß activity rescued mesoderm expression, emphasizing the importance of force dependent Wnt signaling in regulating mechanomorphogenesis. Our work provides a framework for understanding tension-dependent ß-catenin/Wnt signaling in the self-organization of tissues during developmental processes including gastrulation.

Nuclear compression regulates YAP spatiotemporal fluctuations in living cells.

Yes-associated protein (YAP) is a key mechanotransduction protein in diverse physiological and pathological processes; however, a ubiquitous YAP activity regulatory mechanism in living cells has remained elusive. Here, we show that YAP nuclear translocation is highly dynamic during cell movement and is driven by nuclear compression arising from cell contractile work. We resolve the mechanistic role of cytoskeletal contractility in nuclear compression by manipulation of nuclear mechanics. Disrupting the linker of nucleoskeleton and cytoskeleton complex reduces nuclear compression for a given contractility and correspondingly decreases YAP localization. Conversely, decreasing nuclear stiffness via silencing of lamin A/C increases nuclear compression and YAP nuclear localization. Finally, using osmotic pressure, we demonstrated that nuclear compression even without active myosin or filamentous actin regulates YAP localization. The relationship between nuclear compression and YAP localization captures a universal mechanism for YAP regulation with broad implications in health and biology.

MAP4K4 regulates forces at cell-cell and cell-matrix adhesions to promote collective cell migration.

Collective cell migration is not only important for development and tissue homeostasis but can also promote cancer metastasis. To migrate collectively, cells need to coordinate cellular extensions and retractions, adhesion sites dynamics, and forces generation and transmission. Nevertheless, the regulatory mechanisms coordinating these processes remain elusive. Using A431 carcinoma cells, we identify the kinase MAP4K4 as a central regulator of collective migration. We show that MAP4K4 inactivation blocks the migration of clusters, whereas its overexpression decreases cluster cohesion. MAP4K4 regulates protrusion and retraction dynamics, remodels the actomyosin cytoskeleton, and controls the stability of both cell-cell and cell-substrate adhesion. MAP4K4 promotes focal adhesion disassembly through the phosphorylation of the actin and plasma membrane crosslinker moesin but disassembles adherens junctions through a moesin-independent mechanism. By analyzing traction and intercellular forces, we found that MAP4K4 loss of function leads to a tensional disequilibrium throughout the cell cluster, increasing the traction forces and the tension loading at the cell-cell adhesions. Together, our results indicate that MAP4K4 activity is a key regulator of biomechanical forces at adhesion sites, promoting collective migration.

Prostate cancer cells of increasing metastatic potential exhibit diverse contractile forces, cell stiffness, and motility in a microenvironment stiffness-dependent manner.

During metastasis, all cancer types must migrate through crowded multicellular environments. Simultaneously, cancers appear to change their biophysical properties. Indeed, cell softening and increased contractility are emerging as seemingly ubiquitous biomarkers of metastatic progression which may facilitate metastasis. Cell stiffness and contractility are also influenced by the microenvironment. Stiffer matrices resembling the tumor microenvironment cause metastatic cells to contract more strongly, further promoting contractile tumorigenic phenotypes. Prostate cancer (PCa), however, appears to deviate from these common cancer biophysics trends; aggressive metastatic PCa cells appear stiffer, rather than softer, to their lowly metastatic PCa counterparts. Although metastatic PCa cells have been reported to be more contractile than healthy cells, how cell contractility changes with increasing PCa metastatic potential has remained unknown. Here, we characterize the biophysical changes of PCa cells of various metastatic potential as a function of microenvironment stiffness. Using a panel of progressively increasing metastatic potential cell lines (22RV1, LNCaP, DU145, and PC3), we quantified their contractility using traction force microscopy (TFM), and measured their cortical stiffness using optical magnetic twisting cytometry (OMTC) and their motility using time-lapse microscopy. We found that PCa contractility, cell stiffness, and motility do not universally scale with metastatic potential. Rather, PCa cells of various metastatic efficiencies exhibit unique biophysical responses that are differentially influenced by substrate stiffness. Despite this biophysical diversity, this work concludes that mechanical microenvironment is a key determinant in the biophysical response of PCa with variable metastatic potentials. The mechanics-oriented focus and methodology of the study is unique and complementary to conventional biochemical and genetic strategies typically used to understand this disease, and thus may usher in new perspectives and approaches.

Cell monolayer deformation microscopy reveals mechanical fragility of cell monolayers following EMT.

Tissue and cell mechanics are crucial factors in maintaining homeostasis and in development, with aberrant mechanics contributing to many diseases. During the epithelial-to-mesenchymal transition (EMT), a highly conserved cellular program in organismal development and cancer metastasis, cells gain the ability to detach from their original location and autonomously migrate. While a great deal of biochemical and biophysical changes at the single-cell level have been revealed, how the physical properties of multicellular assemblies change during EMT, and how this may affect disease progression, is unknown. Here we introduce cell monolayer deformation microscopy (CMDM), a new methodology to measure the planar mechanical properties of cell monolayers by locally applying strain and measuring their resistance to deformation. We employ this new method to characterize epithelial multicellular mechanics at early and late stages of EMT, finding the epithelial monolayers to be relatively compliant, ductile, and mechanically homogeneous. By comparison, the transformed mesenchymal monolayers, while much stiffer, were also more brittle, mechanically heterogeneous, displayed more viscoelastic creep, and showed sharp yield points at significantly lower strains. Here, CMDM measurements identify specific biophysical functional states of EMT and offer insight into how cell aggregates fragment under mechanical stress. This mechanical fingerprinting of multicellular assemblies using new quantitative metrics may also offer new diagnostic applications in healthcare to characterize multicellular mechanical changes in disease.

Advancing the Mechanical Performance of Glasses: Perspectives and Challenges.

Glasses are materials that lack a crystalline microstructure and long-range atomic order. Instead, they feature heterogeneity and disorder on superstructural scales, which have profound consequences for their elastic response, material strength, fracture toughness, and the characteristics of dynamic fracture. These structure-property relations present a rich field of study in fundamental glass physics and are also becoming increasingly important in the design of modern materials with improved mechanical performance. A first step in this direction involves glass-like materials that retain optical transparency and the haptics of classical glass products, while overcoming the limitations of brittleness. Among these, novel types of oxide glasses, hybrid glasses, phase-separated glasses, and bioinspired glass-polymer composites hold significant promise. Such materials are designed from the bottom-up, building on structure-property relations, modeling of stresses and strains at relevant length scales, and machine learning predictions. Their fabrication requires a more scientifically driven approach to materials design and processing, building on the physics of structural disorder and its consequences for structural rearrangements, defect initiation, and dynamic fracture in response to mechanical load. In this article, a perspective is provided on this highly interdisciplinary field of research in terms of its most recent challenges and opportunities.

Substrate stiffening promotes VEGF-A functions via the PI3K/Akt/mTOR pathway.

While it is now well-established that substrate stiffness regulates vascular endothelial growth factor-A (VEGF-A) mediated signaling and functions, causal mechanisms remain poorly understood. Here, we report an underlying role for the PI3K/Akt/mTOR signaling pathway. This pathway is activated on stiffer substrates, is amplified by VEGF-A stimulation, and correlates with enhanced endothelial cell (EC) proliferation, contraction, pro-angiogenic secretion, and capillary-like tube formation. In the settings of advanced age-related macular degeneration, characterized by EC and retinal pigment epithelial (RPE)-mediated angiogenesis, these data implicate substrate stiffness as a novel causative mechanism and Akt/mTOR inhibition as a novel therapeutic pathway.

Endocytic proteins with prion-like domains form viscoelastic condensates that enable membrane remodeling.

Membrane invagination and vesicle formation are key steps in endocytosis and cellular trafficking. Here, we show that endocytic coat proteins with prion-like domains (PLDs) form hemispherical puncta in the budding yeast, Saccharomyces cerevisiae These puncta have the hallmarks of biomolecular condensates and organize proteins at the membrane for actin-dependent endocytosis. They also enable membrane remodeling to drive actin-independent endocytosis. The puncta, which we refer to as endocytic condensates, form and dissolve reversibly in response to changes in temperature and solution conditions. We find that endocytic condensates are organized around dynamic protein-protein interaction networks, which involve interactions among PLDs with high glutamine contents. The endocytic coat protein Sla1 is at the hub of the protein-protein interaction network. Using active rheology, we inferred the material properties of endocytic condensates. These experiments show that endocytic condensates are akin to viscoelastic materials. We use these characterizations to estimate the interfacial tension between endocytic condensates and their surroundings. We then adapt the physics of contact mechanics, specifically modifications of Hertz theory, to develop a quantitative framework for describing how interfacial tensions among condensates, the membrane, and the cytosol can deform the plasma membrane to enable actin-independent endocytosis.

Decellularized Extracellular Matrix Composite Hydrogel Bioinks for the Development of 3D Bioprinted Head and Neck in Vitro Tumor Models.

Reinforced extracellular matrix (ECM)-based hydrogels recapitulate several mechanical and biochemical features found in the tumor microenvironment (TME) in vivo. While these gels retain several critical structural and bioactive molecules that promote cell-matrix interactivity, their mechanical properties tend toward the viscous regime limiting their ability to retain ordered structural characteristics when considered as architectured scaffolds. To overcome this limitation characteristic of pure ECM hydrogels, we present a composite material containing alginate, a seaweed-derived polysaccharide, and gelatin, denatured collagen, as rheological modifiers which impart mechanical integrity to the biologically active decellularized ECM (dECM). After an optimization process, the reinforced gel proposed is mechanically stable and bioprintable and has a stiffness within the expected physiological values. Our hydrogel's elastic modulus has no significant difference when compared to tumors induced in preclinical xenograft head and neck squamous cell carcinoma (HNSCC) mouse models. The bioprinted cell-laden model is highly reproducible and allows proliferation and reorganization of HNSCC cells while maintaining cell viability above 90% for periods of nearly 3 weeks. Cells encapsulated in our bioink produce spheroids of at least 3000 µm2 of cross-sectional area by day 15 of culture and are positive for cytokeratin in immunofluorescence quantification, a common marker of HNSCC model validation in 2D and 3D models. We use this in vitro model system to evaluate the standard-of-care small molecule therapeutics used to treat HNSCC clinically and report a 4-fold increase in the IC50 of cisplatin and an 80-fold increase for 5-fluorouracil compared to monolayer cultures. Our work suggests that fabricating in vitro models using reinforced dECM provides a physiologically relevant system to evaluate malignant neoplastic phenomena in vitro due to the physical and biological features replicated from the source tissue microenvironment.

Centrifugation and index matching yield a strong and transparent bioinspired nacreous composite.

Glasses have numerous applications because of their exceptional transparency and stiffness; however, poor fracture, impact resistance, and mechanical reliability limit the range of their applications. Recent bioinspired glasses have shown superior mechanical performance, but they still suffer from reduced optical quality. Here, we present a nacreous glass composite that offers a combination of strength, toughness, and transparency. Micrometer-sized glass tablets and poly(methyl methacrylate) (PMMA) were mixed and structured by centrifugation, creating dense PMMA-glass layers. A transparent composite was created by tuning the refractive index of PMMA to that of glass and using chemical functionalization to create continuous interfaces. The fabrication method is robust and scalable, and the composite may prove to be a glass alternative in diverse applications.

Toward embryo cryopreservation-on-a-chip: A standalone microfluidic platform for gradual loading of cryoprotectants to minimize cryoinjuries.

Embryo vitrification is a fundamental practice in assisted reproduction and fertility preservation. A key step of this process is replacing the internal water with cryoprotectants (CPAs) by transferring embryos from an isotonic to a hypertonic solution of CPAs. However, this applies an abrupt osmotic shock to embryos, resulting in molecular damages that have long been a source of concern. In this study, we introduce a standalone microfluidic system to automate the manual process and minimize the osmotic shock applied to embryos. This device provides the same final CPA concentrations as the manual method but with a gradual increase over time instead of sudden increases. Our system allows the introduction of the dehydrating non-permeating CPA, sucrose, from the onset of CPA-water exchange, which in turn reduced the required time of CPA loading for successful vitrification without compromising its outcomes. We compared the efficacy of our device and the conventional manual procedure by studying vitrified-warmed mouse blastocysts based on their re-expansion and hatching rates and transcription pattern of selected genes involved in endoplasmic reticulum stress, oxidative stress, heat shock, and apoptosis. While both groups of embryos showed comparable re-expansion and hatching rates, on-chip loading reduced the detrimental gene expression of cryopreservation. The device developed here allowed us to automate the CPA loading process and push the boundaries of cryopreservation by minimizing its osmotic stress, shortening the overall process, and reducing its molecular footprint.

Spatial distribution of lamin A/C determines nuclear stiffness and stress-mediated deformation.

While diverse cellular components have been identified as mechanotransduction elements, the deformation of the nucleus itself is a critical mechanosensory mechanism, implying that nuclear stiffness is essential in determining responses to intracellular and extracellular stresses. Although the nuclear membrane protein lamin A/C is known to contribute to nuclear stiffness, bulk moduli of nuclei have not been reported for various levels of lamin A/C. Here, we measure the nuclear bulk moduli as a function of lamin A/C expression and applied osmotic stress, revealing a linear dependence within the range of 2-4 MPa. We also find that the nuclear compression is anisotropic, with the vertical axis of the nucleus being more compliant than the minor and major axes in the substrate plane. We then related the spatial distribution of lamin A/C with submicron 3D nuclear envelope deformation, revealing that local areas of the nuclear envelope with higher density of lamin A/C have correspondingly lower local deformations. These findings describe the complex dispersion of nuclear deformations as a function of lamin A/C expression and distribution, implicating a lamin A/C role in mechanotransduction. This article has an associated First Person interview with the first author of the paper.

Bioprintable, Stiffness-Tunable Collagen-Alginate Microgels for Increased Throughput 3D Cell Culture Studies.

3D culture platforms with tunable stiffness have the potential to improve many applications, such as drug discovery, organoid studies, and stem cell differentiation. Both dimensionality and stiffness regulate crucial and relevant cellular processes. However, 3D culture models are often limited in throughput and difficult to adopt for widespread use. Here, we demonstrate an accessible 3D, stiffness-tunable tissue culture platform, based on an interpenetrating network of collagen-1 and alginate. When blended with polymers that induce phase separation, these networks can be bioprinted at microliter volumes, using standard liquid handling infrastructure. We demonstrate robust reproducibility in printing these microgels, consistent tunability of mechanical properties, and maintained viability of multiple printed cell types. To highlight the utility and importance of this system, we demonstrate distinct morphological changes to cells in culture, use the system to probe the role of matrix mechanics and soluble factors in a collagen contraction assay, and perform a prototype viability screen against a candidate chemotherapeutic, demonstrating stiffness-dependent responses.

Pattern-Based Contractility Screening, a Reference-Free Alternative to Traction Force Microscopy Methodology.

The sensing and generation of cellular forces are essential aspects of life. Traction force microscopy (TFM) has emerged as a standard broadly applicable methodology to measure cell contractility and its role in cell behavior. While TFM platforms have enabled diverse discoveries, their implementation remains limited in part due to various constraints, such as time-consuming substrate fabrication techniques, the need to detach cells to measure null force images, followed by complex imaging and analysis, and the unavailability of cells for postprocessing. Here we introduce a reference-free technique to measure cell contractile work in real time, with commonly available substrate fabrication methodologies, simple imaging, and analysis with the availability of the cells for postprocessing. In this technique, we confine the cells on fluorescent adhesive protein micropatterns of a known area on compliant silicone substrates and use the cell deformed pattern area to calculate cell contractile work. We validated this approach by comparing this pattern-based contractility screening (PaCS) with conventional bead-displacement TFM and show quantitative agreement between the methodologies. Using this platform, we measure the contractile work of highly metastatic MDA-MB-231 breast cancer cells that is significantly higher than the contractile work of noninvasive MCF-7 cells. PaCS enables the broader implementation of contractile work measurements in diverse quantitative biology and biomedical applications.

A nondestructive contactless technique to assess the viscoelasticity of blood clots in real-time.

There is a need for reliable and quantitative real-time assessment of blood properties to study and treat a broad spectrum of disorders and cardiovascular diseases as well as to test the efficacy of hemostatic agents. In this study, the real-time changes in viscoelastic/rheological properties of bovine whole blood during coagulation induced by different concentrations of calcium chloride (CaCl2; 15, 25, 35 and 45 mM) was investigated. For this purpose, a novel, contactless technique was used to accurately measure the clotting characteristics under controlled and sterile conditions. It was demonstrated that, increasing the calcium concentration from low values (i.e., 15 and 25 mM), led to shorter reaction time; however, a further increase in calcium concentration (i.e., 35 and 45 mM) favored longer reaction times. Additionally, increasing the CaCl2 concentration resulted in higher shear storage modulus (i.e., stiffer clots). These results were also comparable to those generated by thromboelastrograph, a clinically established technique, as well as a conventional rheometer, which quantitatively verified the high correlation of the shear storage modulus data. In sum, the non-destructive testing technique used in this study is reproducible and sensitive in measuring clot formation kinetics, which could be applied to assess the efficacy of hemostatic agents, and may also contribute to better diagnosing relevant circulatory system diseases and conditions.

Dynamic actin cross-linking governs the cytoplasm's transition to fluid-like behavior.

Cells precisely control their mechanical properties to organize and differentiate into tissues. The architecture and connectivity of cytoskeletal filaments change in response to mechanical and biochemical cues, allowing the cell to rapidly tune its mechanics from highly cross-linked, elastic networks to weakly cross-linked viscous networks. While the role of actin cross-linking in controlling actin network mechanics is well-characterized in purified actin networks, its mechanical role in the cytoplasm of living cells remains unknown. Here, we probe the frequency-dependent intracellular viscoelastic properties of living cells using multifrequency excitation and in situ optical trap calibration. At long timescales in the intracellular environment, we observe that the cytoskeleton becomes fluid-like. The mechanics are well-captured by a model in which actin filaments are dynamically connected by a single dominant cross-linker. A disease-causing point mutation (K255E) of the actin cross-linker α-actinin 4 (ACTN4) causes its binding kinetics to be insensitive to tension. Under normal conditions, the viscoelastic properties of wild-type (WT) and K255E+/- cells are similar. However, when tension is reduced through myosin II inhibition, WT cells relax 3× faster to the fluid-like regime while K255E+/- cells are not affected. These results indicate that dynamic actin cross-linking enables the cytoplasm to flow at long timescales.

Tissue traction microscopy to quantify muscle contraction within precision-cut lung slices.

In asthma, acute bronchospasm is driven by contractile forces of airway smooth muscle (ASM). These forces can be imaged in the cultured ASM cell or assessed in the muscle strip and the tracheal/bronchial ring, but in each case, the ASM is studied in isolation from the native airway milieu. Here, we introduce a novel platform called tissue traction microscopy (TTM) to measure ASM contractile force within porcine and human precision-cut lung slices (PCLS). Compared with the conventional measurements of lumen area changes in PCLS, TTM measurements of ASM force changes are 1) more sensitive to bronchoconstrictor stimuli, 2) less variable across airways, and 3) provide spatial information. Notably, within every human airway, TTM measurements revealed local regions of high ASM contraction that we call "stress hotspots". As an acute response to cyclic stretch, these hotspots promptly decreased but eventually recovered in magnitude, spatial location, and orientation, consistent with local ASM fluidization and resolidification. By enabling direct and precise measurements of ASM force, TTM should accelerate preclinical studies of airway reactivity.

Nuclei deformation reveals pressure distributions in 3D cell clusters.

Measuring pressures within complex multi-cellular environments is critical for studying mechanobiology as these forces trigger diverse biological responses, however, these studies are difficult as a deeply embedded yet well-calibrated probe is required. In this manuscript, we use endogenous cell nuclei as pressure sensors by introducing a fluorescent protein localized to the nucleus and confocal microscopy to measure the individual nuclear volumes in 3D multi-cellular aggregates. We calibrate this measurement of nuclear volume to pressure by quantifying the nuclear volume change as a function of osmotic pressure in isolated 2D culture. Using this technique, we find that in multicellular structures, the nuclear compressive mechanical stresses are on the order of MPa, increase with cell number in the cluster, and that the distribution of stresses is homogenous in spherical cell clusters, but highly asymmetric in oblong clusters. This approach may facilitate quantitative mechanical measurements in complex and extended biological structures both in vitro and in vivo.