Joe Swift

Nuclear Lamin-A Scales with Tissue Stiffness and Enhances Matrix-Directed Differentiation

Introduction Tissues can be soft like brain, bone marrow, and fat, which bear little mechanical stress, or stiff like muscle, cartilage, and bone, which sustain high levels of stress. Systematic relationships between tissue stiffness, protein abundance, and differential gene expression are unclear. Recent studies of stem cells cultured on matrices of different elasticity, E, have suggested that differentiation is mechanosensitive, but the molecular mechanisms involved in particular tissues remain elusive. Tissue micromechanics correlate with abundance of collagens and nuclear lamins, which influence cell differentiation. (Left) Collagen and lamin-A levels scale with E, consistent with matching tissue stress to nuclear mechanics. (Right) Matrix stiffness in tissue culture increases cell tension and stabilizes lamin-A, regulating its own transcription and that of stress fiber genes, enhancing differentiation. RA, retinoic acid, i.e., vitamin A; RARG, YAP1, and SRF, transcription factors. Methods We developed quantitative mass spectrometry algorithms to measure protein abundance, stoichiometry, conformation, and interactions within tissues and cells in relation to stiffness of tissues and extracellular matrix. Manipulations of lamin-A levels with small interfering RNA, overexpression, and retinoic acid or antagonist were applied to stem cells cultured on different matrices to assess lamin-A’s role in mechanosensitive differentiation. To characterize molecular mechanisms, promoter analyses, transcriptional profiling, and localization of transcription factors were complemented by measurements of nuclear mechanics and by modeling of the core gene circuit. Results Proteomic profiling of multiple adult solid tissues showed that widely varied levels of collagens in extracellular matrix and of lamin-A in nuclei followed power-law scaling versus E. Scaling for mechanoresponsive lamin-A conformed to predictions from polymer physics, whereas lamin-B’s varied weakly. Tumor xenograft studies further demonstrated that matrix determined tissue E, whereas lamin-A levels responded to changes in E. In tissue culture cells, both lamin-A conformation and expression were mechanosensitive, with phosphorylation and turnover of lamin-A correlating inversely with matrix E. Lamin-A knockdown enhanced mesenchymal stem cell differentiation on soft matrix that favored a low-stress, fat phenotype. Lamin-A overexpression or transcriptional induction with a retinoic acid (RA) antagonist enhanced differentiation on stiff matrix toward a high-stress, bone phenotype. Downstream of matrix stiffness, the RA pathway regulated lamin-A transcription, but feedback by lamin-A regulated RA receptor (RARG) translocation into nuclei. High lamin-A levels physically impeded nuclear remodeling under stress but also coregulated other key factors. These factors included both serum response factor (SRF), which promoted expression of stress fiber–associated proteins involved in differentiation, and a Hippo pathway factor (YAP1) involved in growth. Discussion The characteristic stress in normal tissue favors collagen accumulation and a characteristic stiffness that cells transduce through nuclear lamin-A to enhance tissue-specific differentiation. Tension-inhibited turnover of rope-like filaments of lamin-A provides sufficient mechanochemical control of a core gene circuit to explain the steady-state scaling of lamin-A with E. High lamin-A physically stabilizes the nucleus against stress and thereby stabilizes the nuclear lamina and chromatin, with implications for epigenetic stabilization and limiting of DNA breaks. Moreover, lamin-A levels directly or indirectly regulate many proteins involved in tissue-specific gene expression, and, because lamin-A levels can vary by a factor of 10 or more downstream of tissue mechanics, an important fraction of tissue-specific gene expression depends on tissue mechanics, which changes in development, injury, and many diseases. Lamins and Tissue Stiffness Microenvironment can influence cell fate and behavior; for example, extracellular matrix (ECM) stiffness increases cell proliferation, and ECM rigidity induces disorders in tissue morphogenesis by increasing cell tension. Swift et al. (1240104; see the Perspective by Bainer and Weaver) used proteomics to identify molecules that are mechanical sensors for tissue elasticity in the nucleus and discovered that expression of lamin-A levels apparently functions as a “mechanostat.” Tissues that need to remain stiff under stress rely on lamin-A to keep the cell nucleus whole. [Also see Perspective by Bainer and Weaver] 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.

Nuclear lamin stiffness is a barrier to 3D migration, but softness can limit survival.

Lamins impede 3D migration but also promote survival against migration-induced stresses.

Crawling from soft to stiff matrix polarizes the cytoskeleton and phosphoregulates myosin-II heavy chain

Cytoskeletal polarization occurs in response to mechanosensing of a transition from soft to stiff matrix during migration and promotes dephosphorylation of myosin-IIA, rearward localization of myosin-IIB, and durotaxis.

Matrix elasticity regulates lamin-A,C phosphorylation and turnover with feedback to actomyosin.

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. 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. 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.

Lamins regulate cell trafficking and lineage maturation of adult human hematopoietic cells.

Significance Comparing human blood cell types, nuclear diversity is visually striking but unexplained: quasi-spherical nuclei in stem/progenitor cells and T cells contrast with multilobed nuclei in neutrophils, giant nuclei in megakaryocytes, and anuclear erythrocytes. We hypothesized broad roles for the major nuclear structure proteins—lamins—and developed mass spectrometry-calibrated intracellular flow cytometry to quantify lamin-A:B ratios. This ratio controls both nuclear viscoelasticity and cell trafficking across microporous barriers. High A:B rigidifies erythroblast nuclei to favor marrow retention and also enhances erythropoiesis. Intermediate A:B enhances thrombopoiesis and opposes cell division to favor marrow anchorage of megakaryocytes. Human stem/progenitor cells have moderate lamin levels and reside in marrow whereas white cells are favored by low lamins and predominantly circulate. Hematopoietic stem and progenitor cells, as well as nucleated erythroblasts and megakaryocytes, reside preferentially in adult marrow microenvironments whereas other blood cells readily cross the endothelial barrier into the circulation. Because the nucleus is the largest organelle in blood cells, we hypothesized that (i) cell sorting across microporous barriers is regulated by nuclear deformability as controlled by lamin-A and -B, and (ii) lamin levels directly modulate hematopoietic programs. Mass spectrometry-calibrated intracellular flow cytometry indeed reveals a lamin expression map that partitions human blood lineages between marrow and circulating compartments (P = 0.00006). B-type lamins are highly variable and predominate only in CD34+ cells, but migration through micropores and nuclear flexibility in micropipette aspiration both appear limited by lamin-A:B stoichiometry across hematopoietic lineages. Differentiation is also modulated by overexpression or knockdown of lamins as well as retinoic acid addition, which regulates lamin-A transcription. In particular, erythroid differentiation is promoted by high lamin-A and low lamin-B1 expression whereas megakaryocytes of high ploidy are inhibited by lamin suppression. Lamins thus contribute to both trafficking and differentiation.

Heart-Specific Stiffening in Early Embryos Parallels Matrix and Myosin Expression to Optimize Beating

In development and differentiation, morphological changes often accompany mechanical changes [1], but it is unclear whether or when cells in embryos sense tissue elasticity. The earliest embryo is uniformly pliable, while adult tissues vary widely in mechanics from soft brain and stiff heart to rigid bone [2]. However, cell sensitivity to microenvironment elasticity is debated based in part on results from complex three-dimensional culture models [3]. Regenerative cardiology provides strong motivation to clarify any cell-level sensitivities to tissue elasticity because rigid postinfarct regions limit pumping by the adult heart [4]. Here, we focus on the spontaneously beating embryonic heart and sparsely cultured cardiomyocytes, including cells derived from pluripotent stem cells. Tissue elasticity, Et, increases daily for heart to 1-2 kPa by embryonic day 4 (E4), and although this is ~10-fold softer than adult heart, the beating contractions of E4 cardiomyocytes prove optimal at ~Et,E4 both in vivo and in vitro. Proteomics reveals daily increases in a small subset of proteins, namely collagen plus cardiac-specific excitation-contraction proteins. Rapid softening of the hearts matrix with collagenase or stiffening it with enzymatic crosslinking suppresses beating. Sparsely cultured E4 cardiomyocytes on collagen-coated gels likewise show maximal contraction on matrices with native E4 stiffness, highlighting cell-intrinsic mechanosensitivity. While an optimal elasticity for striation proves consistent with the mathematics of force-driven sarcomere registration, contraction wave speed is linear in Et as theorized for excitation-contraction coupled to matrix elasticity. Pluripotent stem cell-derived cardiomyocytes also prove to be mechanosensitive to matrix and thus generalize the main observation that myosin II organization and contractile function are optimally matched to the load contributed by matrix elasticity.

Myosin-II inhibition and soft 2D matrix maximize multinucleation and cellular projections typical of platelet-producing megakaryocytes

Cell division, membrane rigidity, and strong adhesion to a rigid matrix are all promoted by myosin-II, and so multinucleated cells with distended membranes—typical of megakaryocytes (MKs)—seem predictable for low myosin activity in cells on soft matrices. Paradoxically, myosin mutations lead to defects in MKs and platelets. Here, reversible inhibition of myosin-II is sustained over several cell cycles to produce 3- to 10-fold increases in polyploid MK and a number of other cell types. Even brief inhibition generates highly distensible, proplatelet-like projections that fragment readily under shear, as seen in platelet generation from MKs in vivo. The effects are maximized with collagenous matrices that are soft and 2D, like the perivascular niches in marrow rather than 3D or rigid, like bone. Although multinucleation of other primary hematopoietic lineages helps to generalize a failure-to-fission mechanism, lineage-specific signaling with increased polyploidy proves possible and novel with phospho-regulation of myosin-II heavy chain. Label-free mass spectrometry quantitation of the MK proteome uses a unique proportional peak fingerprint (ProPF) analysis to also show upregulation of the cytoskeletal and adhesion machinery critical to platelet function. Myosin-inhibited MKs generate more platelets in vitro and also in vivo from the marrows of xenografted mice, while agonist stimulation activates platelet spreading and integrin αIIbβ3. Myosin-II thus seems a central, matrix-regulated node for MK-poiesis and platelet generation.

The nuclear lamina is mechano-responsive to ECM elasticity in mature tissue.

ABSTRACT How cells respond to physical cues in order to meet and withstand the physical demands of their immediate surroundings has been of great interest for many years, with current research efforts focused on mechanisms that transduce signals into gene expression. Pathways that mechano-regulate the entry of transcription factors into the cell nucleus are emerging, and our most recent studies show that the mechanical properties of the nucleus itself are actively controlled in response to the elasticity of the extracellular matrix (ECM) in both mature and developing tissue. In this Commentary, we review the mechano-responsive properties of nuclei as determined by the intermediate filament lamin proteins that line the inside of the nuclear envelope and that also impact upon transcription factor entry and broader epigenetic mechanisms. We summarize the signaling pathways that regulate lamin levels and cell-fate decisions in response to a combination of ECM mechanics and molecular cues. We will also discuss recent work that highlights the importance of nuclear mechanics in niche anchorage and cell motility during development, hematopoietic differentiation and cancer metastasis, as well as emphasizing a role for nuclear mechanics in protecting chromatin from stress-induced damage.

Directing noble metal ion chemistry within a designed ferritin protein

Human H ferritin (HuHF) assembles from 24 four-helix bundles to form an approximately 500 kDa protein with an 8 nm internal cavity. HuHF provides a useful model for studying the transport of metal ions in solution to buried reaction sites in proteins. In this study, HuHF was redesigned to facilitate noble metal ion (Au(3+), Ag(+)) binding, reduction, and nanoparticle formation within the cavity. Computationally determined amino acid substitutions were targeted at four external and four internal surface sites. A variant with a total of 96 cysteines and histidines removed from the exterior surface and 96 non-native cysteines added to the interior surface retained wild-type stability and structure, as confirmed by X-ray crystallography, and promoted the formation of silver or gold nanoparticles within the protein cavity. Crystallographic studies with HuHF variants provide insight into how ferritins control access of metal ions to interior residues that perform chemistry.

Contractile Forces Sustain and Polarize Hematopoiesis from Stem and Progenitor Cells

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.