Cell migration through solid tissue often involves large contortions of the nucleus, but biological significance is largely unclear. The nucleoskeletal protein lamin-A varies both within and between ...cell types and was shown here to contribute to cell sorting and survival in migration through constraining micropores. Lamin-A proved rate-limiting in 3D migration of diverse human cells that ranged from glioma and adenocarcinoma lines to primary mesenchymal stem cells (MSCs). Stoichiometry of A- to B-type lamins established an activation barrier, with high lamin-A:B producing extruded nuclear shapes after migration. Because the juxtaposed A and B polymer assemblies respectively conferred viscous and elastic stiffness to the nucleus, subpopulations with different A:B levels sorted in 3D migration. However, net migration was also biphasic in lamin-A, as wild-type lamin-A levels protected against stress-induced death, whereas deep knockdown caused broad defects in stress resistance. In vivo xenografts proved consistent with A:B-based cell sorting, and intermediate A:B-enhanced tumor growth. Lamins thus impede 3D migration but also promote survival against migration-induced stresses.
On rigid surfaces, the cytoskeleton of migrating cells is polarized, but tissue matrix is normally soft. We show that nonmuscle MIIB (myosin-IIB) is unpolarized in cells on soft matrix in 2D and also ...within soft 3D collagen, with rearward polarization of MIIB emerging only as cells migrate from soft to stiff matrix. Durotaxis is the tendency of cells to crawl from soft to stiff matrix, and durotaxis of primary mesenchymal stem cells (MSCs) proved more sensitive to MIIB than to the more abundant and persistently unpolarized nonmuscle MIIA (myosin-IIA). However, MIIA has a key upstream role: in cells on soft matrix, MIIA appeared diffuse and mobile, whereas on stiff matrix, MIIA was strongly assembled in oriented stress fibers that MIIB then polarized. The difference was caused in part by elevated phospho-S1943-MIIA in MSCs on soft matrix, with site-specific mutants revealing the importance of phosphomoderated assembly of MIIA. Polarization is thus shown to be a highly regulated compass for mechanosensitive migration.
Scarring is a long-lasting problem in higher animals, and reductionist approaches could aid in developing treatments. Here, we show that copolymerization of collagen I with polyacrylamide produces ...minimal matrix models of scars (MMMS), in which fractal-fibre bundles segregate heterogeneously to the hydrogel subsurface. Matrix stiffens locally-as in scars-while allowing separate control over adhesive-ligand density. The MMMS elicits scar-like phenotypes from mesenchymal stem cells (MSCs): cells spread and polarize quickly, increasing nucleoskeletal lamin-A yet expressing the 'scar marker' smooth muscle actin (SMA) more slowly. Surprisingly, expression responses to MMMS exhibit less cell-to-cell noise than homogeneously stiff gels. Such differences from bulk-average responses arise because a strong SMA repressor, NKX2.5, slowly exits the nucleus on rigid matrices. NKX2.5 overexpression overrides rigid phenotypes, inhibiting SMA and cell spreading, whereas cytoplasm-localized NKX2.5 mutants degrade in well-spread cells. MSCs thus form a 'mechanical memory' of rigidity by progressively suppressing NKX2.5, thereby elevating SMA in a scar-like state.
Tissues can be soft like fat, which bears little stress, or stiff like bone, which sustains high stress, but whether there is a systematic relationship between tissue mechanics and differentiation is ...unknown. Here, proteomics analyses revealed that levels of the nucleoskeletal protein lamin-A scaled with tissue elasticity, E, as did levels of collagens in the extracellular matrix that determine E. Stem cell differentiation into fat on soft matrix was enhanced by low lamin-A levels, whereas differentiation into bone on stiff matrix was enhanced by high lamin-A levels. Matrix stiffness directly influenced lamin-A protein levels, and, although lamin-A transcription was regulated by the vitamin A/retinoic acid (RA) pathway with broad roles in development, nuclear entry of RA receptors was modulated by lamin-A protein. Tissue stiffness and stress thus increase lamin-A levels, which stabilize the nucleus while also contributing to lineage determination.
G-protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors in eukaryotes and detect a wide array of cues in the human body. Here we describe a molecular device ...that couples CRISPR-dCas9 genome regulation to diverse natural and synthetic extracellular signals via GPCRs. We generate alternative architectures for fusing CRISPR to GPCRs utilizing the previously reported design, Tango, and our design, ChaCha. Mathematical modeling suggests that for the CRISPR ChaCha design, multiple dCas9 molecules can be released across the lifetime of a GPCR. The CRISPR ChaCha is dose-dependent, reversible, and can activate multiple endogenous genes simultaneously in response to extracellular ligands. We adopt the design to diverse GPCRs that sense a broad spectrum of ligands, including synthetic compounds, chemokines, mitogens, fatty acids, and hormones. This toolkit of CRISPR-coupled GPCRs provides a modular platform for rewiring diverse ligand sensing to targeted genome regulation for engineering cellular functions.
The broad promise of stem cells for regenerative medicine is founded on decades of clinical success already achieved with bone marrow transplantation of haematopoietic stem cells. Current ...capabilities to induce pluripotency with any persons terminally dierentiated cells could also revolutionize therapies for repairingor replacing many other damaged tissues. Yet direct implantation of such cells causes teratomas that is, tumours with regionsof disorganized fat, bone, epithelium or other tissues.
Self-renewal and differentiation of stem cells depend on asymmetric division and polarized motility processes that in other cell types are modulated by nonmuscle myosin-II (MII) forces and matrix ...mechanics. Here, mass spectrometry-calibrated intracellular flow cytometry of human hematopoiesis reveals MIIB to be a major isoform that is strongly polarized in hematopoietic stem cells and progenitors (HSC/Ps) and thereby downregulated in differentiated cells via asymmetric division. MIIA is constitutive and activated by dephosphorylation during cytokine-triggered differentiation of cells grown on stiff, endosteum-like matrix, but not soft, marrow-like matrix. In vivo, MIIB is required for generation of blood, while MIIA is required for sustained HSC/P engraftment. Reversible inhibition of both isoforms in culture with blebbistatin enriches for long-term hematopoietic multilineage reconstituting cells by 5-fold or more as assessed in vivo. Megakaryocytes also become more polyploid, producing 4-fold more platelets. MII is thus a multifunctional node in polarized division and niche sensing.
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•A and B isoforms of myosin-II switch in hematopoietic differentiation•Polarizable myosin-IIB contributes to asymmetric division•Niche-sensitive, essential myosin-IIA is increasingly activated in differentiation•Inhibiting myosin-II enriches for long-term hematopoietic stem and progenitors
Contractile forces in hematopoietic stem cells mediated by myosin-II isoforms are responsive to the niche environment and regulate self-renewal and differentiation.
Tissue microenvironments are characterized not only in terms of chemical composition but also by collective properties such as stiffness, which influences the contractility of a cell, its adherent ...morphology, and even differentiation 1–8. The nucleoskeletal protein lamin-A,C increases with matrix stiffness, confers nuclear mechanical properties, and influences differentiation of mesenchymal stem cells (MSCs), whereas B-type lamins remain relatively constant 9. Here we show in single-cell analyses that matrix stiffness couples to myosin-II activity to promote lamin-A,C dephosphorylation at Ser22, which regulates turnover, lamina physical properties, and actomyosin expression. Lamin-A,C phosphorylation is low in interphase versus dividing cells, and its levels rise with states of nuclear rounding in which myosin-II generates little to no tension. Phosphorylated lamin-A,C localizes to nucleoplasm, and phosphorylation is enriched on lamin-A,C fragments and is suppressed by a cyclin-dependent kinase (CDK) inhibitor. Lamin-A,C knockdown in primary MSCs suppresses transcripts predominantly among actomyosin genes, especially in the serum response factor (SRF) pathway. Levels of myosin-IIA thus parallel levels of lamin-A,C, with phosphosite mutants revealing a key role for phosphoregulation. In modeling the system as a parsimonious gene circuit, we show that tension-dependent stabilization of lamin-A,C and myosin-IIA can suitably couple nuclear and cell morphology downstream of matrix mechanics.
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•MSCs on soft matrix exhibit a less spread nucleus with low lamin-A,C levels•Lamin-A,C is rapidly phosphorylated in response to reduced cytoskeletal tension•Phosphorylation leads to nuclear softening and lamin-A,C turnover•Levels of lamin-A,C and myosin-IIA are coregulated in response to matrix elasticity
Buxboim et al. show that the nucleoskeletal protein lamin-A,C is phosphorylated upon relaxation of actomyosin tension on the nucleus. Mobility of lamin-A,C increases, as does protein turnover and nuclear flexibility. Downstream of lamin-A,C, the serum response factor (SRF) pathway feeds back into actomyosin expression and a contractile phenotype.
Early in embryogenesis, the heart begins its rhythmic contractions as a tube that helps perfuse the nascent vasculature, but the embryonic heart soon changes shape and mechanical properties, like ...many other developing organs. A key question in the field is whether stresses in development impact the underlying gene circuits and, if so, how? Here, we attempt to address this question as we review the mechanical maturation of heart — and, to a limited extent, lung and blood — with a focus on a few key abundant structural proteins whose expression dynamics have been suggested to be directly sensitive to mechanical stress. In heart maturation, proliferating fibroblasts deposit increasing amounts of collagenous matrix in parallel with cardiomyocytes expressing more sarcomeric proteins that increase the contractile stress and strength of the tissue, which in turn pumps more blood at higher stress throughout the developing vasculature. Feedback of beating cardiomyocytes on the expression of matrix by fibroblasts seems a reasonable model, with both synthesis and turnover of matrix and contractile elements achieving a suitable balance. Based on emerging evidence for coiled-coil biopolymers that are tension-stabilized against degradation, a minimal network model of a dynamic cell–matrix interaction is proposed. This same concept is extended to nuclear mechanics as regulated by stress on the nuclear structural proteins called lamins, which are examined in part because of the prominence of mutations in these coiled-coil proteins in diseases of the heart, amongst other organs/tissues. Variations in lamin levels during development and across adult tissues are to some extent known and appear to correlate with extracellular matrix mechanics, which we illustrate across heart, lung, and blood development. The formal perspective here on the mechanochemistry of tissue development and homeostasis could provide a useful framework for ‘big data’ quantitative biology, particularly of stress-sensitive differentiation, maturation, and disease processes.
Majkut et al. describe the balance between contractile components and the stiffening matrix in cardiac development, and examine how these stresses impact the underlying gene circuits.
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