Abstract
During their passage through the circulation, red blood cells (RBCs) encounter severe physiological conditions consisting of mechanical stress, oxidative damage and fast changes in ionic and osmotic conditions. In order to survive for 120 days, RBCs adapt to their surroundings by subtle regulation of membrane organization and metabolism. RBC homeostasis depends on interactions between the integral membrane protein band 3 with other membrane and cytoskeletal proteins, and with key enzymes of various metabolic pathways. These interactions are regulated by the binding of deoxyhemoglobin to band 3, and by a signaling network revolving around Lyn kinase and Src family kinase-mediated phosphorylation of band 3. Here we show that manipulation of the interaction between the lipid bilayer and the cytoskeleton, using various pharmacological agents that interfere with protein-protein interactions and membrane lipid organization, has various effects on: (1) morphology, as shown by high resolution microscopy and quantitative image analysis; (2) organization of membrane proteins, as indicated by immunofluorescence confocal microscopy and quantitative as well as qualitative analysis of vesicle generation; (3) membrane lipid organization, as indicated by flow cytometric analysis of phosphatidylserine exposure; (4) deformability, as assessed in capillary-mimicking circumstances using a microfluidics system; (5) deformability as determined using a spleen-mimicking device; (6) metabolic activity as indicated by metabolomics. Our data show that there is a complex relationship between red cell morphology, membrane organization and deformability. Also, our data show that red blood cells have a relatively high resistance to disturbance of membrane organization in vitro, which may reflect their capacity to withstand mechanical, oxidative and osmotic stress in vivo.
Highlights
Despite constant exposure to oxidative and mechanical stress and fast changes in ionic and osmotic conditions, erythrocytes survive for approximately 120 days in the circulation
NEM, that affects the conformation of the cytoplasmic domain did not have a distinct effect on morphology (Figure 1A), which is in agreement with previous observations (Blanc et al, 2010)
Inhibition of Src-family kinase activity by PP2 did not show an effect on cell morphology (Figure 1A)
Summary
Despite constant exposure to oxidative and mechanical stress and fast changes in ionic and osmotic conditions, erythrocytes survive for approximately 120 days in the circulation. Physiological wear and tear leads to the appearance of immune recognition and removal signals, resulting in phagocytosis. This physiological aging process is disturbed by RBC Homeostasis, Morphology and Deformability mutations in hemoglobin, membrane proteins or metabolic enzymes (Bosman, 2004; Da Costa et al, 2013; Koralkova et al, 2014), and by various multifactorial, systemic pathological conditions such as inflammation (Dinkla et al, 2012). RBC homeostasis is regulated by interactions between proteins in and associated with the plasma membrane A signaling network revolving around Lyn and Syk kinase-mediated phosphorylation of band 3 is involved in the regulation of many, if not all these processes. Lyn is activated and translocates to the membrane where it phosphorylates its target residues on band 3 (Campanella et al, 2005; Ferru et al, 2011)
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