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.

Myoblast migration is crucial for myogenesis and muscular tissue homeostasis. However, its spatiotemporal control remains elusive. Here, we explored the involvement of plasma membrane cholesterol and sphingolipids in this process. In resting C2C12 mouse myoblasts, those lipids clustered in sphingomyelin/cholesterol/GM1 ganglioside (SM/chol/GM1)- and cholesterol (chol)-enriched domains, which presented a lower stiffness than the bulk membrane. Upon migration, cholesterol and sphingomyelin polarized at the front, forming cholesterol (chol)- and sphingomyelin/cholesterol (SM/chol)-enriched domains, while GM1-enriched domains polarized at the rear. A comparison of domain proportion suggested that SM/chol- and GM1-enriched domains originated from the SM/chol/GM1-coenriched domains found at resting state. Modulation of domain proportion (through cholesterol depletion, combined or not with actin polymerization inhibition, or sphingolipid synthesis inhibition) revealed that the higher the chol- and SM/chol-enriched domains, the higher the myoblast migration. At the front, chol- and SM/chol-enriched domains were found in proximity with F-actin fibers and the lateral mobility of sphingomyelin in domains was specifically restricted in a cholesterol- and cytoskeleton-dependent manner while domain abrogation impaired F-actin and focal adhesion polarization. Altogether, we showed the polarization of cholesterol and sphingomyelin and their clustering in chol- and SM/chol-enriched domains with differential properties and roles, providing a mechanism for the spatial and functional control of myoblast migration.

A specific plasma membrane distribution of the mechanosensitive ion channel Piezo1 is required for cell migration, but the mechanism remains elusive. Here, we addressed this question using WT and Piezo1-silenced C2C12 mouse myoblasts and WT and Piezo1-KO human kidney HEK293T cells. We showed that cell migration in a cell-free area and through a porous membrane decreased upon Piezo1 silencing or deletion, but increased upon Piezo1 activation by Yoda1, whereas migration towards a chemoattractant gradient was reduced by Yoda1. Piezo1 organized into clusters, which were preferentially enriched at the front. This polarization was stimulated by Yoda1, accompanied by Ca²⁺ polarization, and abrogated by partial cholesterol depletion. Piezo1 clusters partially colocalized with cholesterol- and GM1 ganglioside-enriched domains, the proportion of which was increased by Yoda1. Mechanistically, Piezo1 activation induced a differential mobile fraction of GM1 associated with domains and the bulk membrane. Conversely, cholesterol depletion abrogated the differential mobile fraction of Piezo1 associated with clusters and the bulk membrane. In conclusion, we revealed, for the first time, the differential implication of Piezo1 depending on the migration mode and the interplay between GM1/cholesterol-enriched domains at the front during migration in a cell-free area. These domains could provide the optimal biophysical properties for Piezo1 activity and/or spatial dissociation from the PMCA calcium efflux pump.