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.

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.

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 where 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. DeepMorphoTM first infers cell-induced substrate displacement solely from a sequence of cell shapes and subsequently computes cellular traction forces, thus avoiding the requirement of a specialized fiducial-marked deformable substrate or force-free reference image. Rather, this technique drastically simplifies the overall experimental methodology, imaging, and analysis needed to conduct cell contractility measurements. We demonstrate that DeepMorphoTM quantitatively matches conventional TFM results, while offering stability against the biological variability in cell contractility for a given cell shape. Without high-frequency noise in the inferred displacement, DeepMorphoTM also resolves the ill-posedness of traction computation, increasing the consistency and accuracy of traction analysis. We demonstrate the accurate extrapolation across several cell types and substrate materials, suggesting robustness of the methodology. Accordingly, we present DeepMorphoTM as a capable yet simpler alternative to conventional TFM for characterizing cellular contractility in 2D.

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.

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.

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.

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.