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The combined use of fluorescence labeling and micro-manipulation of red blood cells has proven to be a powerful tool for understanding and characterizing fundamental mechanisms underlying the mechanical behavior of cells. Here we used this approach to study the development of the membrane-associated cytoskeleton (MAS) in primary embryonic erythroid cells. Erythropoiesis comes in two forms in the mammalian embryo, primitive and definitive, characterized by intra- and extra-vascular maturation, respectively. Primitive erythroid precursors in the murine embryo first begin to circulate at embryonic day (E) 8.25 and mature as a semi-synchronous cohort before enucleating between E12.5 and E16.5. Previously, we determined that the major components of the MAS become localized to the membrane between E10.5 and E12.5, and that this localization is associated with an increase in membrane mechanical stability over this same period. The change in mechanical stability was reflected in the creation of MAS-free regions of the membrane at the tips of the projections formed when cells were aspirated into micropipettes. The tendency to form MAS-free regions decreases as primitive erythroid cells continue to mature through E14.5, at least 2 days after all detectable cytoskeletal components are localized to the membrane, indicating continued strengthening of membrane cohesion after membrane localization of cytoskeletal components. Here we demonstrate that the formation of MAS-free regions is the result of a mechanical failure within the MAS, and not the detachment of membrane bilayer from the MAS. Once a “hole” is formed in the MAS, the skeletal network contracts laterally along the aspirated projection to form the MAS-free region. In protein 4.1-null primitive erythroid cells, the tendency to form MAS-free regions is markedly enhanced. Of note, similar MAS-free regions were observed in maturing erythroid cells from human marrow, indicating that similar processes occur in definitive erythroid cells. We conclude that localization of cytoskeletal components to the cell membrane of mammalian erythroid cells during maturation is insufficient by itself to produce a mature MAS, but that subsequent processes are additionally required to strengthen intraskeletal interactions.

Development of Mechanical Stability in Late-Stage Embryonic Erythroid Cells: Insights From Fluorescence Imaged Micro-Deformation Studies