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.

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

Summary 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) [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 phospho-regulation. In modeling the system as a parsimonious gene circuit, tension-dependent stabilization of lamin-A,C and myosin-IIA is shown to suitably couple nuclear and cell morphology downstream of matrix mechanics.

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.

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.rheology | biophysics | hematopoiesis | nucleus | mechanobiology H ematopoietic cells that enter the circulation are seen to squeeze through small pores in the basement membrane and endothelium that partition bone marrow and blood (1). Retention within the marrow niche as well as trafficking into the circulation might therefore be regulated by cell deformability and the structural molecules responsible for it. Indeed, human polymorphonuclear neutrophils (PMNs) were shown decades ago to become more deformable upon differentiation in the marrow (2), with mature PMNs more capable of entering and exiting small capillaries (3). Leukemic cells are more rigid than normal, potentially explaining the interrupted blood flow and marrow hypercellularity in disease (4). Normal hematopoiesis has a well-characterized hierarchy, but it is unclear whether deformability factors into the program (3). Importantly, because of the high nucleus-to-cytoplasm ratio of hematopoietic cells, key processes such as sorting between marrow and blood could be based in part on nuclear deformability (Fig. 1A).Lamins are intermediate filament proteins that assemble into "lamina" networks at the interface between chromatin and the inner nuclear membrane (5), conferring stiffness to the nucleus (6). In addition, the lamina is often proximal to heterochromatin, and, at least with embryonic stem cells, some genes alter their interactions with the lamina during cell-fate determination (7). In nearly all mammalian cells, A-type lamins (splice-forms A and C from LMNA) and B-type lamins (from LMNB1 and LMNB2) are detectable. In blood progenitors versus various mature cells, past studies appear at odds, even for the same cell type in reporting either decreased levels of bo...

Summary In development and differentiation, morphological changes often accompany mechanical changes [1], but it is unclear if 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], but the sensitivity of cells to microenvironment elasticity is debated [3]. Regenerative cardiology provides strong motivation because rigid post-infarct regions limit pumping by the adult heart [4]. Here we focus on embryonic heart and isolated cardiomyocytes, which both beat spontaneously. Tissue elasticity, Et, increases daily for heart to 1-2 kiloPascal 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 heart's 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. Mechanosensitive stem cell cardiogenesis helps generalize tissue results, which demonstrate how myosin-II organization and contractile function is optimally matched to the load presented by matrix elasticity.

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.

Highlights d Fast-moving cells in 3D matrix establish low membrane tension at the rear d Caveolae form in response to low membrane tension and recruit the GEF Ect2 d Ect2 activates RhoA to promote F-actin organization and rear retraction d Positive feedback between membrane tension and contractility reinforces retraction