Inferring cellular contractile forces and work using deep morphology traction microscopy.

Traction-force microscopy (TFM) has emerged as a widely used standard methodology to measure cell-generated traction forces and determine their role in regulating cell behavior. While TFM platforms have enabled many discoveries, their implementation remains limited due to complex experimental procedures, specialized substrates, and the ill-posed inverse problem whereby low-magnitude high-frequency noise in the displacement field severely contaminates the resulting traction measurements. Here, we introduce deep morphology traction microscopy (DeepMorphoTM), a deep-learning alternative to conventional TFM approaches.

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.

Tissue tension permits β-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 β-catenin that accelerates its release to potentiate Wnt-dependent signaling critical for initiating mesoderm specification. Using an ectopically expressed nonphosphorylatable mutant of β-catenin (Y654F), we now provide direct evidence that impeding tension-dependent Src-mediated β-catenin phosphorylation impedes the expression of Brachyury (T) and the epithelial-to-mesenchymal transition (EMT) necessary for mesoderm specification.

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.

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.

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 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.

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, 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.

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).

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.

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.

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.

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).

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.

Engineering bioprintable alginate/gelatin composite hydrogels with tunable mechanical and cell adhesive properties to modulate tumor spheroid growth kinetics.

Tunable bioprinting materials are capable of creating a broad spectrum of physiological mimicking 3D models enabling in vitro studies that more accurately resemble in vivo conditions. Tailoring the material properties of the bioink such that it achieves both bioprintability and biomimicry remains a key challenge. Here we report the development of engineered composite hydrogels consisting of gelatin and alginate components.

Multiplexed, high-throughput measurements of cell contraction and endothelial barrier function.

Vascular leakage, protein exudation, and edema formation are events commonly triggered by inflammation and facilitated by gaps that form between adjacent endothelial cells (ECs) of the vasculature. In such paracellular gap formation, the role of EC contraction is widely implicated, and even therapeutically targeted. However, related measurement approaches remain slow, tedious, and complex to perform.

Traction Force Screening Enabled by Compliant PDMS Elastomers.

Actomyosin contractility is an essential element of many aspects of cellular biology and manifests as traction forces that cells exert on their surroundings. The central role of these forces makes them a novel principal therapeutic target in diverse diseases. This requires accurate and higher-capacity measurements of traction forces; however, existing methods are largely low throughput, limiting their utility in broader applications.

Robust mechanobiological behavior emerges in heterogeneous myosin systems.

Biological complexity presents challenges for understanding natural phenomenon and engineering new technologies, particularly in systems with molecular heterogeneity. Such complexity is present in myosin motor protein systems, and computational modeling is essential for determining how collective myosin interactions produce emergent system behavior. We develop a computational approach for altering myosin isoform parameters and their collective organization, and support predictions with in vitro experiments of motility assays with α-actinins as molecular force sensors.

Composite alginate gels for tunable cellular microenvironment mechanics.

The mechanics of the cellular microenvironment can be as critical as biochemistry in directing cell behavior. Many commonly utilized materials derived from extra-cellular-matrix create excellent scaffolds for cell growth, however, evaluating the relative mechanical and biochemical effects independently in 3D environments has been difficult in frequently used biopolymer matrices. Here we present 3D sodium alginate hydrogel microenvironments over a physiological range of stiffness (E = 1.

Alpha-actinin binding kinetics modulate cellular dynamics and force generation.

The actin cytoskeleton is a key element of cell structure and movement whose properties are determined by a host of accessory proteins. Actin cross-linking proteins create a connected network from individual actin filaments, and though the mechanical effects of cross-linker binding affinity on actin networks have been investigated in reconstituted systems, their impact on cellular forces is unknown. Here we show that the binding affinity of the actin cross-linker α-actinin 4 (ACTN4) in cells modulates cytoplasmic mobility, cellular movement, and traction forces.

Structurally governed cell mechanotransduction through multiscale modeling.

Mechanotransduction has been divided into mechanotransmission, mechanosensing, and mechanoresponse, although how a cell performs all three functions using the same set of structural components is still highly debated. Here, we bridge the gap between emerging molecular and systems-level understandings of mechanotransduction through a multiscale model linking these three phases. Our model incorporates a discrete network of actin filaments and associated proteins that responds to stretching through geometric relaxation.

Microtubule sliding drives proplatelet elongation and is dependent on cytoplasmic dynein.

Bone marrow megakaryocytes produce platelets by extending long cytoplasmic protrusions, designated proplatelets, into sinusoidal blood vessels. Although microtubules are known to regulate platelet production, the underlying mechanism of proplatelet elongation has yet to be resolved. Here we report that proplatelet formation is a process that can be divided into repetitive phases (extension, pause, and retraction), as revealed by differential interference contrast and fluorescence loss after photoconversion time-lapse microscopy.

Probing the stochastic, motor-driven properties of the cytoplasm using force spectrum microscopy.

Molecular motors in cells typically produce highly directed motion; however, the aggregate, incoherent effect of all active processes also creates randomly fluctuating forces, which drive diffusive-like, nonthermal motion. Here, we introduce force-spectrum-microscopy (FSM) to directly quantify random forces within the cytoplasm of cells and thereby probe stochastic motor activity. This technique combines measurements of the random motion of probe particles with independent micromechanical measurements of the cytoplasm to quantify the spectrum of force fluctuations.