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

Abstract

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.

Keywords: biophysics; hematopoiesis; mechanobiology; nucleus; rheology.

Fig. 1.

Lamin map of human hematopoiesis. (A) Hematopoietic cells are mostly in marrow or blood, and only a fraction of cells transmigrate through the endothelium with or without their nucleus. EryP, erythroid progenitors; GM, granulocytes and monocytes; HSCP, hematopoietic stem cell and progenitors; Lym, lymphocytes; MK, megakaryocytes; RBC, red blood cells; WBC, white blood cells. (B) Validation of flow cytometry by Western blot with cell numbers adjusted to give similar intensities of B-type lamins. Lamin-A,C splice-forms and B-type lamins are shown for fresh CD34⁺, early and late erythroid cells; densitometry shows rough agreement with MS-IF results. (C) Representative intracellular flow cytometry scatterplots show lamin-A (Alexa-488) and B-type lamin (Alexa-647) expression for indicated cells. (D) Confocal images of MKs immunostained for lamin-A (green) and myosin-II (red). (E) Confocal image of T cell immunostained for B-type lamins (cyan) and myosin-II (red) (Scale bar: 5 μm.) (F) Lamin-A relative to B-type lamins, transformed to a measurable A:B ratio versus calibrated sum intensity A+B (a.u.); on log scales, these are the respective difference and sum of chemical potentials for A and B (Fig. S2A). Mean fluorescent intensity of lamin for each subpopulation from flow cytometry was calibrated to an absolute ratio from MS analyses of a standard A549 cell line (lamin-A:B = 2.3) (Fig. S1). The dashed line schematically illustrates the semipermeable barrier between bone marrow (BM) and peripheral blood (PB), and the net probability of partitioning based on lamin expression can be estimated from the upper half and lower half probabilities: P_TOT = P_UH P_LH = 0.00006. Measurements are mean ± SEM of n ≥ 3, with error bars omitted if <5% of mean. BM G, BM granulocytes (CD33^mid); BM M, BM monocytes (CD33^hi); CD34⁺CD38⁻, early progenitors; CD34⁺CD38⁺, common progenitors; LateEry, late erythroblasts (CD44⁻GPA⁺); MK, polyploid MKs (average 16N); MKP, MK progenitors (CD34⁻CD41⁺); MSC, mesenchymal stromal cells; PB G/M, PB granulocytes/monocytes; Plt, platelets; ProEry, proerythroblasts (CD44⁺GPA⁻); RBC, red blood cells; T, B, lymphoids. Representative MSC results from one donor are shown because the variation in A:B ratios between donors and cultured cells was minimal.

Fig. 2.

Lamin-A:B regulates transmigration of nucleated blood cells. (A) Schematic illustration of hypothesis that cell migration and nuclear deformation through sufficiently small micropores will be facilitated by lamin knockdown (KD). (B) Lamin-A confers nuclear stiffness, based on aspiration of G/M cells with or without knockdown with lamin-A siRNA (siLMNA). (Left) Quantitation of aspirated nuclear length (n = 3 donors, *P < 0.05). (Right) Representative image is for ΔP < 1 kPa at 60 s. (Scale bars: 5 μm.) (C) Migration of fresh CD34⁺ and MK cells. Cells were incubated on top of transwell with different pore sizes (3, 5, or 8 μm) with an SDF-1 (100 ng/mL) gradient for 4 h before analysis by flow cytometry to evaluate % migrated cells. Data were fit to y = a/[1 + b(x-x_c)^-m], where y = %migrated, x = pore area (μm²), a = 100 (% maximal migration when pore size is infinite), b relates to the half-maximum migration area (Fig. S3C), x_c = critical pore area, m = Hill coefficient. Values for b, x_c, and m for each cell type are as follows: CD34⁺CD38⁻ (0.024, 1.44, and 5.4, respectively), CD34⁺CD38⁺ (0.022, 1.44, and 3.2, respectively), MK (0.0006, 0, and 0.9, respectively). (D) Lamin ratios predict migration sensitivity to pore size at small pores: the first derivative of y(x) was estimated for 3-μm pores and then fit to a power law yielding Y(at 3 μm) = 0.90 (A:B – 0.28)^1.55 (R² > 0.95). (E) Lamin-A limits 3D migration. Before transwell migration, cells were transfected with siLMNA for 3 d to give ∼50% knockdown (Fig. S3A). Results from granulocyte/macrophage cells derived from CD34⁺ culture are shown. Data fit per C. All results are mean ± SEM of n ≥ 3. *P < 0.05 in paired t test.

Fig. 3.

Lamin ratios predict nuclear stiffness in hematopoietic lineages. (A) Nuclear compliance change versus time at constant pressure ΔP = 0.3–6 kPa. (Upper) A power law fit, J(t) (kPa⁻¹) = a·t^b (t = sec) for each blood cell type, where (b = 1 for fluids), (b = 0 for solids). (Lower) Values for (a, b) in each cell type are as follows: (T cell: 15.2, 0.2), (CD33⁺: 16.4, 0.1), (MkP ≥ 30s: 0.16, 0.5), (MK: 3.4, 0.1), (ProEry: 1.1, 0.2), (LateEry: 0.3, 0). (B) High lamin-A:B correlates with stiff nuclei. (Upper) Images at 2 min of nuclear aspiration at ΔP = 1–2 kPa. (Scale bar: 5 μm.) (Lower) Correlation between nuclear stiffness (at 2 min) and lamin-A:B fits J (at 2 min) = 0.23(A:B – 0.68)^0.91 (R² = 0.96). All results are mean ± SEM of n ≥ 5 for each cell type. (C) Lamin-B1 knockdown stiffens nuclei in proerythroblasts, with no change in viable cell numbers (n = 10, P < 0.05). Nuclear compliance change with ΔP = 1–2 kPa where values for (a, b) in each sample are as follows: (scrambled: 12.8, 0.1), (shLMNB1: 9.4, 0.03). (Scale bars: 5 μm.)

Fig. 4.

Lamins regulate erythroid and MK differentiation. (A) Scheme for in vitro differentiation. MEP, myeloid and erythroid progenitor; SCF, stem cell factor. Epo is erythropoietin and induces erythropoiesis; Tpo is thrombopoietin and induces MK-poiesis. (B and C) Colony-forming assays in methylcellulose medium show a shift to erythroid progenitors after (B) overexpression of GFP-lamin-A (∼40% transfection efficiency) or (C) knockdown of lamin-B1 (shLMNB1, ∼50% efficiency) in the presence of Epo for 3 d. *P < 0.05, GFP vs. GFP-lamin-A or scrambled vs. shLMNB1 (n = 3). (D and E) MK progenitor population enumerated by flow cytometry with CD41 and CD42b. (D) MK progenitors increase with lamin-A overexpression. CD41⁺CD42b⁻, early progenitor; CD41⁺CD42b⁺, late progenitor. *P < 0.05, GFP⁺ vs. GFP-lamin-A⁺ for each progenitor (n = 4). (E) Lamin-B1 knockdown decreases average MK ploidy. *P < 0.01, scrambled vs. shLMNB1 (n = 3). (F) Lamin-A is required for erythroid differentiation and restricts myeloid progenitor number. Cells were transduced with either scrambled or lamin-A shRNA (shLMNA) and cultured in the presence of Epo, IL-3 and Tpo. Functional progenitors (CFU-GM, BFU-E, CFU-GEMM) were quantified by colony-forming assay (Left) whereas differentiated subpopulations were quantified by flow cytometry as per ref. (Right), normalized by 10⁴ initial cell number. CFU-E, CD34⁻CD36⁺IL-3R⁻; EarlyEry, CD44⁺GPA⁺; LateEry, CD44⁻GPA⁺; MkP, CD41⁺; Myeloid, CD33⁺. *P < 0.05, scrambled vs. shLMNA.

Fig. 5.

Lamin-A levels change with physiological agonists. (A) Retinoic Acid (RA) modulates lamin-A in myeloid (Mye) differentiation. CD34⁺-derived cells treated with either DMSO or RA (1 μM) for 3 d with G-CSF (10 ng/mL) before assays. CFU-GM colonies are more abundant and larger with RA (Fig. S6 D and E). (B) Effect of RA on transwell migration of CD34⁺ cells. *P < 0.05 (n = 3) for DMSO vs. RA in paired t tests (unless noted). (C) RA down-regulates lamin-A protein expression for both CD34⁻ and CD34⁺ cells. (Inset) A representative flow cytometry plot (2°, secondary alone). *P < 0.01, DMSO vs. RA (n = 4). (D) RA represses lamin-A transcription. Cells were transiently transfected with a GFP reporter construct driven by a human lamin-A promoter, consisting of the 1,132-base pair upstream region and the first 385-base pair mRNA transcript region, followed by drug treatment. *P < 0.05 (n = 4). (E) RA modulates lamin-A in erythroid (Ery) differentiation. Absolute values of erythroid colony numbers were normalized to initial cell input and fit to dose–response curves. (IC₅₀ or EC₅₀, Hill coefficient) for: RA (20 nM), AGN in the presence of 0.2 μM RA (250 nM). R² > 0.9. (n = 3). (Inset) Lamin A:B ratios with drug treatment (AGN: 5 μM, RA: 0.2 μM). *P < 0.05, one-way ANOVA (n = 3). All results are mean ± SEM.