Abstract
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.
Highlights
The red blood cell is perhaps the simplest cell in the human body
Cell maturation was gauged by the gestation day at which the cells were harvested, (E10.5 through E14.5), since primitive erythroid cells differentiate as a semi-synchronous cohort, reaching the basophilic erythroblast stage at E10.5 and the orthochromatic erythroblast stage by E12.5-E13.5 (Kingsley et al, 2004, 2013)
This was significant because many theoretical treatments of red blood cell mechanical behavior had previously made the assumption that the density of the membraneassociated cytoskeleton (MAS) was uniform in deformation (Evans and Skalak, 1979)
Summary
The red blood cell is perhaps the simplest cell in the human body. It has no nucleus, its interior consists largely of a concentrated solution of hemoglobin, and its mechanical stability resides entirely in its plasma membrane and a thin, membraneassociated cytoskeleton (MAS). The most advanced studies have combined fluorescence labeling of membrane components and controlled mechanical deformation of the cell (fluorescence imaged microdeformation, FIMD) to gain insights into how specific membrane components are redistributed during deformation (Lee et al, 1999; Picart et al, 2000; Dahl et al, 2003; Huang et al, 2017). We applied this approach to document and quantify the changes in the mechanical stability of the MAS in primary mammalian erythroid cells at different stages of maturation
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