Circulating primitive erythroblasts establish a functional, protein 4.1R-dependent cytoskeletal network prior to enucleating

Abstract

Hematopoietic ontogeny is characterized by distinct primitive and definitive erythroid lineages. Definitive erythroblasts mature and enucleate extravascularly and form a unique membrane skeleton, composed of spectrin, 4.1R-complex, and ankyrinR-complex components, to survive the vicissitudes of the adult circulation. However, little is known about the formation and composition of the membrane skeleton in primitive erythroblasts, which progressively mature while circulating in the embryonic bloodstream. We found that primary primitive erythroblasts express the major membrane skeleton genes present in similarly staged definitive erythroblasts, suggesting that the composition and formation of this membrane network is conserved in maturing primitive and definitive erythroblasts despite their respective intravascular and extravascular locations. Membrane deformability and stability of primitive erythroblasts, assayed by microfluidic studies and fluorescence imaged microdeformation, respectively, significantly increase prior to enucleation. These functional changes coincide with protein 4.1 R isoform switching and protein 4.1R-null primitive erythroblasts fail to establish normal membrane stability and deformability. We conclude that maturing primitive erythroblasts initially navigate the embryonic vasculature prior to establishing a deformable cytoskeleton, which is ultimately formed prior to enucleation. Formation of an erythroid-specific, protein 4.1R-dependent membrane skeleton is an important feature not only of definitive, but also of primitive, erythropoiesis in mammals.

Comparative genomics provides insights into the aquatic adaptations of mammals

“…The red cell membrane cytoskeletal network consists of spectrin, actin, and protein 4.1R ( 72 ). Protein 4.1R in mature red blood cell is a key component of the erythroid membrane skeleton, regulating red cell morphology and mechanical stability ( 73 – 76 ). The gene EPB41 (encoding the protein 4.1R) shares a convergent site in cetaceans and pinnipeds and is under rapid evolution in the sirenian lineage ( SI Appendix , Fig.…”

Continuous Change in Membrane and Membrane-Skeleton Organization During Development From Proerythroblast to Senescent Red Blood Cell

“…In definitive erythropoiesis, instead, erythroblasts do not enucleate until just before leaving the bone marrow and circulate with a stable membrane-skeleton, and therefore with higher deformability, although with an excess lipid bilayer that must be shed by the circulating retic. In both cases, for stable membrane-skeleton assembly, a switch to the expression of the mature spliceoform of protein 4.1R appears to be fundamental (Huang et al, 2017 ). However, the timing and modalities for the establishment of a stable membrane-skeleton in erythropoiesis before and after enucleation are still poorly understood.…”

Microfluidic assay of the deformability of primitive erythroblasts

“…We have shown recently an increased expression of 4.1 R transcripts as erythroblast cells transit from E10.5 to E12.5 and that an erythroid-specific isoform switch occurs between E10.5 and E11.5. 32 Because 4.1 R can form a ternary complex with actin and spectrin and plays important roles in maintaining the integration of the cytoskeletal network of mature erythrocytes, 35 it is likely that protein 4.1 R in the cytoskeletal composition between E10.5 and E12.5 is, in part, responsible for the observed phenomena in erythroblast cells. To confirm the role of 4.1 R in erythroblast deformability, we tested the shear-induced deformation of primitive erythroblasts from Epb4.1 knockout (KO) mice.…”

“…It attributes to the failure of forming the functional membrane skeleton network. 32 A recent study shows that due to the absence of 4.1 R, E12.5 primitive cells cannot transform from spherical to partially concave in shape, leading to a relatively small surface-to-volume ratio, 32 which consequently results in decreased cell deformability. 37,38 FIG Last, to examine the erythroblast deformability in capillaries where cells have to squeeze through the narrow capillary, we flew erythroblasts through a microfluidic device with a much narrower constriction channel that has dimensions of w c ¼ 5 lm, l c ¼ 170 lm, and h ¼ 7 lm.…”

“…Cells were the resuspended in PB2 and stained on ice using DRAQ5 (eBioscience) for another 5 min, after which the cells were ready for imaging. 32…”