Aberrant Membrane Composition and Biophysical Properties Impair Erythrocyte Morphology and Functionality in Elliptocytosis

Abstract

Red blood cell (RBC) deformability is altered in inherited RBC disorders but the mechanism behind this is poorly understood. Here, we explored the molecular, biophysical, morphological, and functional consequences of α-spectrin mutations in a patient with hereditary elliptocytosis (pEl) almost exclusively expressing the Pro260 variant of SPTA1 and her mother (pElm), heterozygous for this mutation. At the molecular level, the pEI RBC proteome was globally preserved but spectrin density at cell edges was increased. Decreased phosphatidylserine vs. increased lysophosphatidylserine species, and enhanced lipid peroxidation, methemoglobin, and plasma acid sphingomyelinase (aSMase) activity were observed. At the biophysical level, although membrane transversal asymmetry was preserved, curvature at RBC edges and rigidity were increased. Lipid domains were altered for membrane:cytoskeleton anchorage, cholesterol content and response to Ca2+ exchange stimulation. At the morphological and functional levels, pEl RBCs exhibited reduced size and circularity, increased fragility and impaired membrane Ca2+ exchanges. The contribution of increased membrane curvature to the pEl phenotype was shown by mechanistic experiments in healthy RBCs upon lysophosphatidylserine membrane insertion. The role of lipid domain defects was proved by cholesterol depletion and aSMase inhibition in pEl. The data indicate that aberrant membrane content and biophysical properties alter pEl RBC morphology and functionality.

Impaired Cytoskeletal and Membrane Biophysical Properties of Acanthocytes in Hypobetalipoproteinemia – A Case Study

“…First, although generalization of observations based on only one patient is difficult, our findings are in agreement with literature data on diseases associated with acanthocytosis. In fact, altered membrane composition and biophysical properties have also been demonstrated in a patient suffering from hereditary elliptocytosis and a series of patients suffering from hereditary spherocytosis, two diseases associated with increased membrane fragility due to cytoskeleton defects ( Pollet et al, 2020 ; unpublished data), thereby supporting the importance of membrane:cytoskeleton interplay in RBC morphology and functionality. Second, although fluorescent lipid analogs were extensively used and validated in previous researches, their validity as bona fide surrogates of endogenous lipid counterparts is debated in view of the bulk fluorophore which can deeply modify biophysical properties.…”

“…RBCs were placed into IBIDI chambers as described above for RBC morphology in suspension. Microscopy images were then analyzed with the last version of Shape Analysis by Fourier Descriptors computation plugging for ImageJ as in Leonard et al (2017b) and Pollet et al (2020) .…”

“…Immunolabeling of spectrin was performed as in Cloos et al (2020) and Pollet et al (2020) . All coverslips were mounted with Dako and examined with a Zeiss Cell Observer Spinning Disk (COSD) confocal microscope using a plan-Apochromat 100× NA 1.4 oil immersion objective and the same settings for illumination.…”

“…The experimental approaches were chosen based on our previous work ( Carquin et al, 2014 , 2015 ; Leonard et al, 2017a ; Pollet et al, 2018 , 2020 ; Cloos et al, 2020 ): (i) for RBC morphology: blood smears, optical microscopy of living RBCs and scanning electron microscopy (SEM) of fixed RBCs; (ii) for RBC deformability: osmotic fragility through Hb release and deformability upon shear stress by ektacytometry; (iii) for cytoskeleton integrity: spectrin confocal imaging; (iv) for intracellular ATP content: a biochemical assay; (v) for the antioxidant defense: extent of oxidative stress through measurement of free intracellular reactive oxygen species (ROS) content, lipid peroxidation and Hb oxidation; (vi) for the ion balance: intracellular calcium content evaluated by flow cytometry; (vii) for membrane lipid composition: determination of fatty acids (FA), cholesterol, sphingolipids and phospholipids content; and (viii) for membrane biophysical properties: evaluation of elastic modulus by atomic force microscopy (AFM), curvature by optical microscopy of living RBCs in suspension, transversal asymmetry by phosphatidylserine (PS) surface exposure and lateral asymmetry by vital confocal imaging using validated fluorescent lipid analogs or toxin fragments specific to endogenous lipids. We finally explored whether impairments resulted from defects during RBC maturation or from acceleration of aging.…”

The Redox Balance and Membrane Shedding in RBC Production, Maturation, and Senescence

“…The disease is characterized by RBCs with sphere shape (instead of biconcave disks), with reduced surface-to-volume ratio and increased osmotic fragility—properties that make them inclined to hemolysis ( Perrotta et al, 2008 ; Chagula et al, 2020 ). Elliptocytosis is another RBC membranopathy with spectrin tetramer self-association, leading to RBCs with an elliptical or elongated shape ( Pollet et al, 2020 ).…”