Hierarchical Determination of Nuclear Deformability by Lamin Isoforms During Adult Hematopoiesis: Implications in Blood Cell Trafficking

Abstract 1200

Over the last few decades, it has become clear that different blood lineages have distinct deformability, and that some blood cell types become softer in maturation perhaps to facilitate trafficking from marrow through the endothelial barrier and into the circulation (Lichtman, NEJM, 1970; 283:943–8). However, any common molecular basis of this phenomenon remains unclear. Cellular deformability is determined by elasticity of the cortex and the nucleus, and the latter is dynamically regulated by changes in expression and organization of the lamins (Pajerowski… Discher, PNAS, 2007; 104:15619–24). While gene regulation of hematopoiesis has been extensively studied, roles of lamins in blood lineages are less well understood. To address this, we developed a novel protein isoform expression analysis algorithm, “mass spectrometry calibrated intracellular flow cytometry”, to quantify lamin stoichiometries in human hematopoietic stem cells and progenitors through different mature blood lineages. This approach reveals the hematopoietic lineage map of lamins, showing that lamin A varies by 4-fold, while the normally ‘constitutive’ lamin B varies by 30-fold. During differentiation, lymphoid and myeloid lineages show decreased total lamin intensity and pliable nuclei as measured by micropipette aspiration, consistent with their ability to transmigrate into circulation. In contrast, megakaryocytes (MKs) remain in marrow because their polyploid nuclei are too large and rigid, as indicated by high lamin levels; this nuclear anchorage allows MKs to extend membrane projections into blood, where shear generates circulating platelets. Maturation of MKs is further regulated by serine phosphorylation of lamin A, since overexpression of a phospho-inactive mutant leads to increased polyploidization. Erythroid lineages share the same progenitor with MKs and migrate into blood as enucleated RBCs, because of high lamin A intensity relative to B in the progenitors and stiff chromatin. Consistent with this observation, microarray analysis of primary CD34⁺-derived cells indicates that some key erythroid genes are strongly correlated with lamin isoforms. Functional studies indicate that lamin A overexpression increases MK and erythroid differentiation by 2-fold, while the knockdown increases migration through pores by 2-fold. Surprisingly, increasing the lamin A to B ratio by lamin B1 knockdown decreases nuclear deformability by up to 50%, highlighting the importance of lamin isoform ratios, rather than absolute expression levels, in specifying nuclear rheology and hence traffickability of blood lineages. Finally, lamin A can be transcriptionally downregulated by retinoic acid by 2-fold, consistent with its well-established role in driving myeloid differentiation from progenitors. Together, the study suggests that nuclear deformability is hierarchically programmed by differential expression of the nucleoskeletal lamin A and B isoforms during hematopoietic differentiation, which in turn influence the ability of blood cells to migrate through marrow.