Extracellular vesicles (EVs) contribute to several pathophysiological processes and appear as emerging targets for disease diagnosis and therapy. However, successful translation from bench to bedside requires deeper understanding of EVs, in particular their diversity, composition, biogenesis and shedding mechanisms. In this review, we focus on plasma membrane-derived microvesicles (MVs), far less appreciated than exosomes. We integrate documented mechanisms involved in MV biogenesis and shedding, focusing on the red blood cell as a model. We then provide a perspective for the relevance of plasma membrane lipid composition and biophysical properties in microvesiculation on red blood cells but also platelets, immune and nervous cells as well as tumor cells. Although only a few data are available in this respect, most of them appear to converge to the idea that modulation of plasma membrane lipid content, transversal asymmetry and lateral heterogeneity in lipid domains may play a significant role in the vesiculation process. We suggest that lipid domains may represent platforms for inclusion/exclusion of membrane lipids and proteins into MVs and that MVs could originate from distinct domains during physiological processes and disease evolution.

The shedding of extracellular vesicles (EVs) from the red blood cell (RBC) surface is observed during senescence in vivo and RBC storage in vitro . Two main models for EV shedding, respectively based on calcium rise and oxidative stress, have been proposed in the literature but the role of the plasma membrane lipid composition and properties is not understood. Using blood in K + /EDTA tubes stored for up to 4 weeks at 4°C as a relevant RBC vesiculation model, we showed here that the RBC plasma membrane lipid composition, organization in domains and biophysical properties were progressively modified during storage and contributed to the RBC vesiculation. First, the membrane content in cholesterol and linoleic acid decreased whereas lipid peroxidation and spectrin:membrane occupancy increased, all compatible with higher membrane rigidity. Second, phosphatidylserine surface exposure showed a first rapid rise due to membrane cholesterol decrease, followed by a second calcium-dependent increase. Third, lipid domains mainly enriched in GM1 or sphingomyelin strongly increased from the 1st week while those mainly enriched in cholesterol or ceramide decreased during the 1st and 4th week, respectively. Fourth, the plasmatic acid sphingomyelinase activity considerably increased upon storage following the sphingomyelin-enriched domain rise and potentially inducing the loss of ceramide-enriched domains. Fifth, in support of the shedding of cholesterol- and ceramide-enriched domains from the RBC surface, the number of cholesterol-enriched domains lost and the abundance of EVs released during the 1st week perfectly matched. Moreover, RBC-derived EVs were enriched in ceramide at the 4th week but depleted in sphingomyelin. Then, using K + /EDTA tubes supplemented with glucose to longer preserve the ATP content, we better defined the sequence of events. Altogether, we showed that EV shedding from lipid domains only represents part of the global vesiculation mechanistics, for which we propose four successive events (cholesterol domain decrease, oxidative stress, sphingomyelin/sphingomyelinase/ceramide/calcium alteration and phosphatidylserine exposure).

Background/Aims: Red blood cells (RBC) have been shown to exhibit stable submicrometric lipid domains enriched in cholesterol (chol), sphingomyelin (SM), phosphatidylcholine (PC) or ganglioside GM1, which represent the four main lipid classes of their outer plasma membrane leaflet. However, whether those lipid domains co-exist at the RBC surface or are spatially related and whether and how they are subjected to reorganization upon RBC deformation are not known. Methods: Using fluorescence and/or confocal microscopy and well-validated probes, we compared these four lipid-enriched domains for their abundance, curvature association, lipid order, temperature dependence, spatial dissociation and sensitivity to RBC mechanical stimulation. Results: Our data suggest that three populations of lipid domains with decreasing abundance coexist at the RBC surface: (i) chol-enriched ones, associated with RBC high curvature areas; (ii) GM1/PC/chol-enriched ones, present in low curvature areas; and (iii) SM/PC/chol-enriched ones, also found in low curvature areas. Whereas chol-enriched domains gather in increased curvature areas upon RBC deformation, low curvature-associated lipid domains increase in abundance either upon calcium influx during RBC deformation (GM1/PC/chol-enriched domains) or upon secondary calcium efflux during RBC shape restoration (SM/PC/chol-enriched domains). Hence, abrogation of these two domain populations is accompanied by a strong impairment of the intracellular calcium balance. Conclusion: Lipid domains could contribute to calcium influx and efflux by controlling the membrane distribution and/or the activity of the mechano-activated ion channel Piezo1 and the calcium pump PMCA. Whether this results from lipid domain biophysical properties, the strength of their anchorage to the underlying cytoskeleton and/or their correspondence with inner plasma membrane leaflet lipids remains to be demonstrated.

PIEZO1 ion channels are activated by mechanical stimuli, triggering intracellular chemical signals. Recent structural studies suggest that plasma membrane tension or local curvature changes modulate PIEZO1 channel gating and activation. However, whether PIEZO1 localization is governed by tension gradients or long-range mechanical perturbations across the cells is still unclear. Here, we probe the nanoscale localization of PIEZO1 on red blood cells (RBCs) at high resolution (∼30 nm), and we report for the first time the existence of submicrometric PIEZO1 clusters in native conditions. Upon interaction with Yoda1, an allosteric modulator, PIEZO1 clusters increase in abundance in regions of higher membrane tension and lower curvature. We further show that PIEZO1 ion channels interact with the spectrin cytoskeleton in both resting and activated states. Our results point toward a strong interplay between plasma membrane tension gradients, curvature, and cytoskeleton association of PIEZO1.