Nuclear lamin-A scales with tissue stiffness and enhances matrix-directed differentiation

Abstract

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.

Fig. 1. Lamin-A and collagen levels scale with tissue stiffness, but collagen determines stiffness while lamin-A responds

(A) Tissue deformation under force is quantified by E and transfers stresses through the extracellular matrix and the cytoskeleton into the nucleus. (B and C) The proteomes of adult mouse tissues were profiled to determine whether scaling of mechanical properties with biopolymer concentration exists across tissues. (D) Quantitative proteomics of multiple human and mouse tissues and cells revealed scaling with E of the absolute ratio or stoichiometry of lamin-A to lamin-B through MS quantification of a pan-lamin peptide. Differences in ratios are significant with brain ≪ liver < fat < heart, lung, and muscle ≪ skull ≪ femur and cartilage, where < indicates P ≤ 0.05 and ≪ indicates P ≤ 0.01. Nuclei with abundant lamin-A are stiff (20). Cultured cells showed the same trend as their primary source tissue. HSCP, human hematopoietic stem cell progenitors from marrow; U251, human glioblastoma cells from brain; A549, human adenocarcinoma epithelial cells from lung; C2C12, mouse myoblast cells from muscle; MSC, osteo-prone human mesenchymal stem cells from marrow. (E) MS trends were validated by immunoblotting (representative blots taken from fig. S5A). (F) Lamin-B1 scales very weakly with E, whereas lamin-B2 is constant on average. (G) Collagen-1 isoforms scale strongly with E. (H) Human glioblastoma cells U251^Luc (expressing luciferase for imaging) were xenografted into mouse brain and flank, and 4-week-old tumors were profiled by MS proteomics. (I) Mouse-derived collagens in U251 grown in mouse brain and flank scale with E as observed for adult mouse tissues. (J) Stiffness of flank tumors made with high (A549) or low (U251) lamin-A:B cells was similar to the stiffness of the subcutaneous site (subQ). Tumors were 50% softer after only a brief treatment with collagenase (col’ase). (K) Lamin composition and stiffness of the tumors fit adult tissue scaling. All points are significantly different where indicated (n ≥ 3 MS measurements).

Fig. 2. Nuclear stability is conferred by lamin-A, which unfolds under stress and couples to phosphorylation

(A) High-resolution images of the nuclear envelope of U251s, A549s, and MSCs show juxtaposed regions of lamins A (green) and B (red), consistent with earlier observations in HeLa cells (24). Triangles highlight domains of lamins A (green), B (red), and overlap (yellow). (B) Higher-order assembly of lamin typical of intermediate filament proteins and the lamin-A dimer solubilized by phosphorylation (38), annotated with MS-detectable phosphorylation and cysteine sites. (C) Shearing of nuclei showed that lamin conformation responds to mechanical stress. A cysteine-reactive label [monobromobimane (mBBr)] was added to nuclei and sheared for 40 min at the indicated stresses in a cone and plate rheometer. All protein was then solubilized and the extent of reaction at each detected cysteine quantified by MS, scaled by the unlabeled protein. (D) A549 nuclei imaged following shear stress. Greater lamin expression confers mechanical robustness to the nuclei, limiting disruption of chromatin. (E) The Ig-like domain of lamin-A has a cryptic cysteine, Cys⁵²², that is buried in the crystal structure (Protein Data Bank accession number 1IFR) but showed 70% more labeling in stressed A549 nuclei. Labeling of Cys⁵⁹¹ in the tail of lamin-A did not change with stress (mean ± SEM from curve fit; P ≤ 0.05, n ≥ 3 MS measurements). (F) A point mutant R453W within the lamin Ig domain that is known to cause muscular dystrophy showed decreased domain stability at 37°C as measured by cysteine labeling rates and tryptophan fluorescence (inset). (G) Images of adherent A549 cells transfected with wild-type or mutant GFP-tagged lamin were labeled with 400 μM mBBr for 10 min. (H) Labeling of wild-type and R453W lamin of mBBr measured by MS after immunoprecipitation of GFP (IP-MS) showed greater in vivo labeling of mutant in adherent cells; the tail domain showed no significant difference. (I) In contrast, phosphorylation at Ser³⁹⁰ was fivefold higher in wildtype lamin-A. All points are significantly different, as indicated (n ≥ 3 MS measurements).

Fig. 3. Cell and nuclei spread on stiff matrix, suppressing lamin-A phosphorylation and increasing lamin-A and cell tension

Response of MSCs to substrate stiffness was characterized. (A) Cells are more rounded on soft (0.3 kPa) matrix, whereas on stiff (40 kPa) matrix they spread with more pronounced stress fibers, consistent with higher cell tension. (B and C) Levels of α-smooth muscle actin were higher on stiff matrix. (D) Confocal microscopy showed wrinkled nuclei on soft matrix, and smoothed-out and flattened nuclei on stiff matrix. Images are of the middle z-section of different nuclei. (E and F) Cell and nuclei rapidly label with mBBr, but quantitation of lamin-A labeling by IP-MS showed no significant difference in labeling of either the Ig domain or tail sites on soft versus stiff substrate. (G and H) Phosphorylation at Ser³⁹⁰ is ~30% higher on soft substrate, predictive of solubilization. (I and J) Quantitative immunofluorescence and immunoblot show lamin-A increased with substrate stiffness. This tends to reduce the mechanical stress per molecule and maintain the Ig fold. Blots were taken from the same membrane. All points are significantly different (P ≤ 0.05; n ≥ 3 MS and IF measurements).

Fig. 4. Matrix elasticity directs stem cell differentiation, which is enhanced by laminA as it regulates SRF and YAP1

(A) Partial knockdown (KD) of laminA in MSCs with si-LMNA in combination with soft matrix (0.3 kPa) and an adipo-inducing media-maximized adipogenesis (P ≤ 0.02 knockdown versus control). Stiff matrix (40 kPa) suppressed adipogenesis in parallel cultures, with no significant effect of knockdown. Knockdown of lamin-A was to 35% of wild-type or scrambled-siRNA. (B) Adipogenesis in MSCs on plastic showed that cells with oil droplets (phase contrast microscopy; nucleus indicated by blue arrow with asterisk) had minimal stress fibers (myosin-IIa immunofluorescence) compared with cells without oil droplets (nucleus indicated by blue arrow without asterisk). (C) Overexpression (OE) of lamin-A in MSCs in combination with stiff matrix and an osteo-inducing media-maximized osteogenesis (P ≤ 0.0001). Soft matrix suppressed osteogenesis in parallel cultures, with no significant effect of overexpression. (D) Alkaline phosphatase (ALP) staining was done after 1 week as a measure of osteogenic signal, together with the fraction of cells with staining. (E) Correlation between nuclear area and lamin-A level with treatments on soft and stiff matrix (normalized to Hoechst stain). NT, non-treated control. Inset cartoons highlight the relationship between cell and nuclear spread area as well as cell tension. (F) Pathway analyses after knockdown of LMNA in three different MSCs. Gene symbols are colored according to mRNA abundance in MSCs (green, low; red, high) from microarray data for 11 soft tissues in human and 10 soft tissues in adult mouse (of 14,985 gene annotations common to mouse and human), and genes are ranked based on Pearson correlations with lamin-A. SRF and related transcription factors and target genes all show reduced levels with lamin-A knockdown, whereas neither YAP1 nor its target genes were affected. TEAD1 has been implicated in both YAP1 and SRF pathways, but lamin-A knockdown suppresses TEAD1 similar to SRF, suggesting that it is in the SRF pathway. A transcription factor predicted to regulate lamin-A (RARG) was not affected by lamin-A knockdown, and few RA pathway transcripts changed with LMNA knockdown except CRABP2 (89), which decreased. CRABP2 is up-regulated in osteoarthritis models where COL1A1 increases in osteogenic-like processes (90). The SREBF1-regulated gene, FABP5, increases to give an average ratio for message of CRABP2/FABP5 ~ 0.3 relative to untreated cells; both CRABP2 and FABP5 are known to bind RA, and the change in the RA signaling ratio (CRABP2/FABP5) was consistent with switching of differentiation pathways (91). (G) The decrease in ACTA2, downstream of SRF, was confirmed at the protein level in MSCs by immunofluorescence. (H) High-resolution confocal microscopy of YAP1 in MSCs cultured on substrates of increasing stiffness show increasing nuclear localization, as reported previously (11). Insets highlight observation of enrichment at the nuclear envelope, which was especially evident with lamin-A overexpression. (I) Plot shows a fourfold increase in nuclear to cytoplasmic ratio of YAP1 with increasing matrix stiffness in MSCs, except that lamin-A overexpression decreases nuclear YAP1. (J) YAP1 was also bimodally distributed on substrates of intermediate stiffness (10 kPa). (K) YAP1 protein and mRNA levels in tissues of increasing stiffness showed nonmonotonic trends, with the mRNA data averaged from human and mouse microarrays. All points are significantly different, as indicated (n ≥ 3 imaging, IF, and immunoblot experiments).

Fig. 5. Matrix stiffness is upstream of RA regulation of lamin-A transcription

(A) LMNA message correlates with protein (R² = 0.95) across tissue. (B) Promoter-reporter construct for LMNA is annotated with six predicted binding sites of transcription factors in RA pathway and a deletion construct (Δ-LMNA) lacking four RA factor binding sites. (C) A549 cells transfected with GFP reporter constructs. (D) Antagonist (AGN) and agonist (RA) increase and decrease, respectively, expression from the LMNA promoter-reporter and not Δ-LMNA; lamin-A protein shows the same response. (E) LMNA reporter activity increased significantly in MSCs grown on stiff (40 kPa) versus soft (0.3 kPa) matrix, but Δ-LMNA showed no significant difference. (F) RA and AGN regulate lamin-A in MSCs only on stiff matrix. Gels were coated with collagen-1 for comparison to cultures on untreated plastic, and immunoblotting (G) was performed after 36 hours culture (mean ± SEM from titration; all blots from same membrane; P ≤ 0.05; n ≥ 3 immunoblots). (H) AGN coupled with stiff matrix to increase osteogenesis, determined by ALP staining (I). (J) Increased osteogenic potential was coincident with increased lamin-A levels, measured by immunofluorescence. (K) Lamin-A was necessary for the increased osteogenic potential of MSCs treated with AGN as coincident treatment with siRNA against LMNA-abrogated osteogenesis.

Fig. 6. Lamin-A protein regulates nuclear translocation of RA receptor

(A) RARG protein and message (mouse and human average) increased in tissues of increasing stiffness. (B) High-resolution confocal microscope images of RARG in MSCs on matrices of various stiffnesses and with knockdown or overexpression of lamin-A. Nuclear midsections showed cytoplasmic RARG on soft matrix and increasing localization of RARG to the nuclear periphery with increasing lamin level. (C) Nuclear-to-cytoplasmic ratio of RARG scales with matrix elasticity and was directly affected by lamin-A knockdown or overexpression. All points are significantly different, as indicated (n ≥ 3 IF experiments). (D) Proteins coimmunoprecipitated with lamin-A, common to both A549s and MSCs but not found in a nonspecific control (against GFP, which was not present in the sample), were analyzed by MS. (E) Proteins associated with immunoprecipitated RARG or YAP1, but not with control samples (a combined list of proteins precipitating with antibody to GFP, not present in the sample, and proteins binding to antibody-free beads). Protein lists were compiled by combining hits from duplicate experiments. The nuclear membrane protein SUN2 was common to both lamin-A and RARG immunoprecipitation experiments. N.D., not detected.

Fig. 7. Lamin-A confers a viscous stiffness to nuclei that impedes nuclear remodeling by stress

(A) Micropipette aspiration of an A549 cell nucleus expressing GFP-lamin-A shows extension of the lamina with time. (B) Schematic showing how nuclear compliance is calculated from image analysis as a function of time and aspiration pressure. (C) Modeling compliance over the first 12 s of deformation, with contributions from elasticity (G) and viscosity (η) in nuclei with different lamina compositions. (D and E) Relationship between the characteristic lamin-A:B ratio and (D) the elastic modulus or (E) the viscosity. The outlier points, A549 OE and MSC, indicated by open symbols, were omitted from the linear fits. (F) The response of the lamina can be considered as a combination of elastic and viscous components, with an elongation response time, τ (see Box 1). τ was calculated for nuclei extended to ~5 μm by micropipette aspiration over seconds-to-minutes time scales in cells with different lamin-A:B ratios (G) and in A549 cells overexpressing GFP-lamin-A (H). Lamin ratios were calculated from a combination of immuno-blotting and MS methods. The scaling of τ with changes in the lamin-A:B ratio, β, was found to be the same in both experiments. (I) A potential biological consequence of nuclear distension is the remodeling of chromosome territories and chromatin-envelope interactions. All points are significantly different where indicated (n ≥ 3 nuclei).

Fig. 8. A feedback-based gene circuit for lamin-A exhibits polymer physics scaling if cell tension suppresses protein turnover

(A) Gene circuit connecting matrix stiffness to osteogenesis; not shown is an overlapping circuit for adipogenesis on soft matrix that includes the positive regulator SREBP1. LMNA protein level is regulated by a stress-sensitive phosphorylation mechanism and feeds back into LMNA transcript through interaction with RARG, possibly through an intermediary, α, and can be perturbed with antagonist (AGN) or agonist (RA). LMNA protein also influences location of YAP1 (through a possible intermediary, Ψ) to drive cell fate (11), and LMNA regulates SRF through interaction with nuclear actin (49). A simple model was generated based on this circuit: Time evolution of LMNA mRNA (M) level is dependent on the LMNA protein level (P), whereas the protein level itself is regulated by a tension-dependent degradation term, h. The model shows that tension-regulated protein turnover can produce steady-state (SS) protein levels that scale with cell tension. (B) Trajectories of lamin-A message and protein as the model converges from a range of initial conditions to a single steady-state solution appropriate to the tension. (C) Setting the kinase/protease binding coefficient, K_m, to be proportional to (Tension)^0.3 allows the model to generate steady-state lamin scaling with tension consistent with experiment (Fig. 1D).