Micrometric segregation of fluorescent membrane lipids: relevance for endogenous lipids and biogenesis in erythrocytes.

Micrometric membrane lipid segregation is controversial. We addressed this issue in attached erythrocytes and found that fluorescent boron dipyrromethene (BODIPY) analogs of glycosphingolipids (GSLs) [glucosylceramide (BODIPY-GlcCer) and monosialotetrahexosylganglioside (GM1BODIPY)], sphingomyelin (BODIPY-SM), and phosphatidylcholine (BODIPY-PC inserted into the plasma membrane spontaneously gathered into distinct submicrometric domains. GM1BODIPY domains colocalized with endogenous GM1 labeled by cholera toxin. All BODIPY-lipid domains disappeared upon erythrocyte stretching, indicating control by membrane tension. Minor cholesterol depletion suppressed BODIPY-SM and BODIPY-PC but preserved BODIPY-GlcCer domains. Each type of domain exchanged constituents but assumed fixed positions, suggesting self-clustering and anchorage to spectrin. Domains showed differential association with 4.1R versus ankyrin complexes upon antibody patching. BODIPY-lipid domains also responded differentially to uncoupling at 4.1R complexes [protein kinase C (PKC) activation] and ankyrin complexes (in spherocytosis, a membrane fragility disease). These data point to micrometric compartmentation of polar BODIPY-lipids modulated by membrane tension, cholesterol, and differential association to the two nonredundant membrane:spectrin anchorage complexes. Micrometric compartmentation might play a role in erythrocyte membrane deformability and fragility.

analyzing the mechanisms governing their biogenesis. RBCs are the simplest and very robust living cell model, where membrane composition ( 32 ) and cytoskeletal control ( 29,33,34 ) are best characterized and are crucial for cell plasticity and high resistance to shear stress. Analysis of RBC PM is compounded by neither vesicular traffi cking nor metabolic lipid turnover. Pharmacological tools and genetic defi ciencies in patients allow dissection of the reciprocal interactions between membrane lipids and spectrin. Using RBCs attached onto poly-L -lysine, we fi rst observed that fl uorescent GSLs and endogenous GM1, which can be decorated by the fl uorescent pentameric cholera toxin B subunit, segregate into visible domains. We next observed the differential effects on the three classes of fl uorescent lipid micrometric domains (BODIPY-GlcCer, BODIPY-SM, and BODIPY-PC) of various controlled manipulations : i ) stretching, ii ) cholesterol depletion, iii ) membrane:cytoskeleton uncoupling at 4.1R complexes upon acute PKC activation, and iv ) uncoupling at ankyrin complexes in spherocytosis. This was confi rmed by preferential association of micrometric domains with antibody-patched components of the two anchorage com plexes. These observations further support the differential clustering of polar lipids at the RBC PM up to micrometric domains that can be manipulated by membrane tension and oppositely controlled by cholesterol and membrane:cytoskeleton anchorage. A dual level of lateral asymmetry, transient nanometric rafts and stable micrometric domains, most likely play a crucial role in RBC deformability and vulnerability to membrane fragility diseases such as spherocytosis.

RBC isolation and immobilization
This study was approved by the Medical Ethics Institutional Committee; each donor gave written informed consent. RBCs were isolated from three healthy volunteers, two with their spleen (named C#2 and C#3) and one splenectomized for traumatic rupture (C#1); and from two splenectomized adult spherocytotic patients (P#1 and P#2). Blood was collected by venopuncture into dry EDTA (K + salt)-coated tubes, diluted 1:10 in medium (DMEM containing 25 mM glucose and 25 mM HEPES) and washed twice by centrifugation at 133 g for 2 min and resuspension. For spreading onto poly-L -lysine-coated coverslips (PLKcoverslips), RBCs were plated at ‫ف‬ 20 · 10 6 cells/ml onto 2 cm 2 coverslips precoated with 0.1 mg/ml 70-150 kDa PLK (Sigma) at 20°C for exactly 4 min after which the suspension was removed and replaced with fresh medium in which RBCs were allowed to spread for another 4 min (unless otherwise stated). This caused a variable level of stretching exploited in Fig. 1A .
We recently compared by vital imaging the lateral organization of fl uorescent boron dipyrromethene (BODIPY) lipid analogs of GlcCer, SM, and PC at the outer PM leafl et of Chinese hamster ovary (CHO) cells and human red blood cells (RBCs) ( 19,20 ). These studies visualized welldefi ned brilliant spots corresponding to submicrometric domains ( ‫ف‬ 0.5-1 m; i.e., well-above the resolution limit of confocal microscopy), with 5-8-fold enrichment over the rest of the labeled cell surface. In CHO cells, the three analogs segregated into distinct domains, exhibiting decreasing packing (GSLsBODIPY > BODIPY-SM > BODIPY-PC without detectable fl ip-fl op from the outer to the inner leafl et, and covering together up to ‫ف‬ 70% of the cell surface. As shown by inhibition of SM biosynthesis and sphingomyelinase surface cleavage, BODIPY-SM domains depended on endogenous SM, supporting the hypothesis that fl uorescent patches could refl ect genuine lasting micrometric compartmentation, complementary to the transient nanometric rafts. The remarkable deformability of RBCs allows squeezing into narrow blood capillaries and the narrower pores of spleen sinusoids, where they undergo >10,000 "quality controls" in their 120-day lifetime. Two properties are essential for RBC stability: a uniquely high cholesterol content ( ‫ف‬ 40 mol% vs. ‫ف‬ 30 mol% in fi broblasts vs. ‫ف‬ 15 mol% in blood platelets) and a strong membrane:cytoskeleton anchorage. Cholesterol itself is a key regulator of two membrane biophysical properties: i ) membrane fl uidity via lipid ordering, and ii ) membrane deformability via modulation of PM protein interactions at the cortical cytoskeleton interface ( 28 ). Interactions between the PM bilayer and the underlying spectrin network dominate elastic properties of RBCs, thanks to two nonredundant 4.1R-and ankyrin-based complexes ( 29,30 ) (see supplementary Fig. I). These provide RBCs with a 20-fold higher adhesive energy than fi broblasts ( 31 ). Linkage via 4.1R complexes is regulated by protein kinase C (PKC)-dependent phosphorylation. Ankyrin-based complexes are frequently defective in spherocytosis, a genetic disease impairing resistance to shear stress and hypotonicity. When spherocytosis leads to severe hemolytic anemia, blood counts can be restored by spleen removal (splenectomy).
The present investigation aimed at testing the relevance of micrometric domains labeled by fl uorescent lipids at the outer PM leafl et of RBCs for endogenous lipids and

Statistical analyses
Values are mean ± SEM. Statistical signifi cance of comparisons was tested by Student's t -test.

Spontaneous clustering of GSLsBODIPY, BODIPY-SM, and BODIPY-PC into submicrometric domains on RBCs partially spread onto PLK-coverslips
Labeling of RBCs partially spread on PLK-coverslips with trace levels of BODIPY analogs of GlcCer, SM, or PC (respectively, 0.7 ± 0.5%, 1.1 ± 0.7%, and 1.0 ± 0.3% of RBC major membrane lipids; mean ± SEM pooled from 3 independent experiments) revealed submicrometric domains readily visible by confocal microscopy ( Fig. 1A ). Increasing BODIPY-SM concentration from 0.5 M to 3 M changed neither the number nor the size of micrometric domains (data not shown). In most spread cells (projected diameter >8 m; arrows in Fig. 1A ), lipid analogs were still inserted into the PM but no longer clustered into visible domains. When compared to CHO cells where membrane tension is much lower, fl uorescent lipid domains of RBCs were overall less abundant, showed a more uniform size, and had a more round shape, but showed comparable enrichment (>5-fold) over the rest of the labeled cell surface. For each class of BODIPY-lipids, relative domain abundance was undistinguishable between conventional donors and a healthy donor splenectomized for traumatic spleen rupture, but domain number consistently varied according to the lipid analog (4.8 ± 0.1 for GlcCer-BODIPY; 3.8 ± 0.2 for BODIPY-SM 3.2 ± 0.3 for BODIPY-PC mean ± SEM of 984-1,323 RBCs pooled from 6 to 10 independent experiments).

Labeling of endogenous GM1 by cholera toxin also reveals submicrometric domains that colocalize with GM1BODIPY
In view of the lasting controversy on the very existence of micrometric domains for endogenous lipids in living cells, as opposed to labeling by fl uorescent lipid analogs with a grafted BODIPY acyl tail, we fi rst asked whether endogenous GM1 decorated by the fl uorescent CTxB subunit (Alexa 568-CTxB) also segregates into visible domains. Of note, distribution of this probe was reported to be patchy in erythrocytes at both room temperature and 37°C ( 40 ). As shown in Fig. 1B (second row), CTxB-labeled submicrometric patches that largely colocalized with GM1BODIPY (arrowheads at left) segregated from BODIPY-SM and BODIPY-PC domains ( Fig. 1B b", c"). The latter observation also indicated that patches revealed by CTxB were not induced by exogenous GM1BODIPY insertion. These results supported the interpretation that micrometric BODIPY lipid domains could refl ect endogenous lipid compartmentation, as previously suggested for BODIPY-SM domains in CHO cells based on endogenous SM depletion ( 19,20 ).

Spontaneous formation of BODIPY lipid submicrometric domains depends on membrane tension
The consistent decrease of BODIPY lipid domain abundance upon RBC spreading suggested a role for membrane then DiIC18. For vital imaging of endogenous GM1, RBCs were sequentially labeled in suspension at 37°C for 15 min with BODIPY-lipids then with 4 g/ml Alexa568-cholera toxin B (CTxB) subunit (Invitrogen) in the continued presence of BODIPY-lipids in DMEM/BSA. RBCs were then spread on PLKcoverslips and washed twice with DMEM/BSA. For confocal imaging, coverslips were placed bottom-up into Lab-Tek chambers and examined with a Zeiss LSM510 confocal microscope using a plan-Apochromat 63× NA 1.4 oil immersion objective in a thermostatically controlled cabinet set at 37 ± 1°C (XL/LSM incubator, Zeiss; Tempcontrol 37-2, PeCon) ( 19,20 ). FRAP and scanning electron microscopy were performed as described ( 19,20 ).

Pharmacological treatments
For cholesterol depletion, RBCs were preincubated in suspension with 0-0.25 mM methyl-␤ -cyclodextrin (m ␤ CD) (Sigma) at 37°C for 30 min, pelleted, and resuspended in DMEM. RBCs were then either plated onto PLK-coverslips for imaging or lipids were extracted ( 35 ) and cholesterol was measured using the Amplex Red Cholesterol kit (Invitrogen) with omission of cholesterol esterase ( 19 ). To phosphorylate 4.1R complexes by PKC, RBCs were treated on PLK-coverslips in DMEM containing 20 nM calyculin A (CalA) for 4 min, washed 3 times, reincubated with CalA and 5 M PMA (both from Sigma) for 5 min, then labeled with BODIPY-lipids and imaged in the continued presence of PMA and CalA for <15 min.

RBC immunolabeling
After spreading on PLK-coverslips, RBCs were labeled with BODIPY-lipids and washed as above. After blocking for 30 min with 1% BSA and 0.1% L -lysine (w/v, BSA/lysine), RBCs were incubated for 1 h with rabbit monoclonal antibody to glycophorin C together with mouse monoclonal antibody to CD47 (both from Abcam), washed 6 times in BSA/lysine, incubated for 1 h with the appropriate Alexa-secondary antibodies (5 g/ml of each), washed 6 times with PBS, then transferred for 5 min into ice-cold dimethylsuberimidate fi xative [extemporaneously prepared at 15 mM in 0.135 M borate buffer containing MgCl 2 , pH 8.0 ( 36 )] or 4% formaldehyde, rinsed, and mounted in Mowiol in the dark for 24 h. Preparations were examined with an LSM510 confocal microscope as above.

Thin layer chromatography
BODIPY-lipids were inserted in the RBC membrane at 0.75-1 M. After washing, all lipids (endogenous and inserted BODIPY) were extracted, separated by thin layer chromatography (TLC) in chloroform:methanol:15 mM CaCl 2 (65:35:8; v/v/v) ( 38 ) and revealed by charring densitometry after staining with 10% cupric sulfate in 8% orthophosphoric acid ( 39 ). Band intensity of inserted fl uorescent BODIPY-lipids was quantifi ed and expressed by reference to the sum of major lipids (cholesterol, PC, phosphatidylethanolamine, ceramide, and SM) from the same sample, after correction for band intensity of corresponding endogenous lipid. tension in BODIPY lipid domain formation/biogenesis. To clarify this role, we tested the relation between membrane stretching due to cell spreading and BODIPY-lipids clustering into submicrometric membrane domains, by titration of the adhesion time onto PLK-coverslips and PLK concentration. Both parameters increased spreading and abrogated domains (illustrated for BODIPY-GlcCer in Fig. 2A ). In contrast, the number of domains was greatly increased at very low PLK concentration (0.01 mg/ml; Fig. 2B a), but large variation in size precluded rigorous quantifi cation. Likewise, micrometric BODIPY-lipid domains could be evidenced on loosely attached stomatocytes/discocytes ( Fig. 2B b, c), ruling out artifact due to PLK-coverslip spreading. We concluded that BODIPY-lipid micrometric domains depended on membrane tension.

The high cholesterol level of RBCs promotes BODIPY-SM and BODIPY-PC domains
We next turned our attention to cholesterol because of its particular abundance at the RBC PM where it could regulate membrane tension by modulating both lateral membrane fl uidity and transversal anchorage to the cytoskeleton ( 28,41 ). Cholesterol abundance can be precisely decreased by controlled extraction with empty m ␤ CD. An  ( 20 ). To further test whether cholesterol depletion could instead favor other domains, we looked at the artifi cial dialkylindocarbocyanine DiIC18, a well-established membrane probe that homogenously labels untreated RBCs ( 19,42 ), but which is recognized to preferentially partition into the liquid-disordered phase in giant unilamellar vesicles, segregated from the liquid-ordered phase decorated by fl uor escent cholera toxin that binds ganglioside GM1 ( 8 ). Using a minimal m ␤ CD concentration (0.06 mM) that barely affected total cholesterol (supplementary Fig. IIIAa), DiIC18 already started concentrating into bright submicrometric patches ( Fig. 3n-p ). At low m ␤ CD concentrations that combined preservation of BODIPY-SM domains with induction of DiIC18 patches, the two types of assemblies were fully segregated (supplementary Fig. IIIC). We concluded that the high cholesterol level of RBCs favored BODIPY-SM and BODIPY-PC micrometric domain formation, also implying that local cholesterol variations could fi nely modulate their stability.

BODIPY-lipid domains are immobile clusters of highly mobile constituents
To analyze the stability of BODIPY-lipid domains and their constituents, we next combined time-lapse imaging with FRAP ( 19,20 ). Prolonged imaging revealed that domains were remarkably immobile in the adherent RBCs (examples in Fig. 4A ). Moreover, bleached domains were regenerated exactly at their original position. Domain stability contrasted with the very fast ( t 1/2 ‫ف‬ 10 sec) and high recovery of their constituents ( Fig. 4B ), indicating that domains were stable large-scale assemblies of highly dynamic lipids or small clusters nondetectable by conventional confocal microscopy. We thus looked at a relation between domain stability and membrane anchorage to the underlying spectrin network, by addressing the respective contribution of the two nonredundant 4.1R-and ankyrin-based complexes.

Membrane:spectrin anchorage at 4.1R complexes restricts BODIPY-GlcCer and BODIPY-SM domains
Membrane:spectrin anchorage via 4.1R complexes can be acutely and selectively uncoupled by acute phosphorylation upon PKC activation via phorbol 12-myristate 13-acetate (PMA) combined with CalA as phosphatase inhibitor ( 43 ).  ( 44 ). Scanning electron micro scopy further disclosed circumferential retraction (arrowhead in supplementary Fig. IIC, right) and central bulging (arrow), compatible with decreased spreading and membrane tension upon partial release from the spectrin network, with preservation of a featureless smooth RBC surface. PKC activation increased the abundance of BODIPY-GlcCer domains by ‫ف‬ 50% ( Fig. 5A d ) and BODIPY-SM domains by ‫ف‬ 90% ( Fig. 5A e) with out a detectable effect on BODIPY-PC  Fig. 6B . These results indicated that ankyrin-based complexes preferentially restricted BODIPY-SM and BODIPY-PC clustering into submicrometric domains.

High cholesterol level promotes BODIPY-SM and BODIPY-PC domains via 4.1R complexes and BODIPY-GlcCer domains via ankyrin complexes
Cholesterol not only affects lipid cohesion but also impacts on transversal connectivity with the cytoskeleton ( 28 ). Because BODIPY-SM and BODIPY-PC domains vanished upon cholesterol depletion but were conversely promoted by membrane:spectrin uncoupling, we examined domains ( Fig. 5A f). General views are provided in supplementary Fig. IVb, e, h and quantifi cation is shown at Fig. 5B . Thus, because uncoupling of membrane:spectrin anchorage at 4.1R complexes selectively increased the number of BODIPY-GlcCer and BODIPY-SM domains, we concluded that, in untreated cells, 4.1R normally restricts the spontaneous propensity for BODIPY-GlcCer and BODIPY-SM clustering.

Membrane:spectrin anchorage at ankyrin complexes restricts BODIPY-SM and BODIPY-PC domains
In parallel, to evaluate the role of ankyrin-based complexes, we examined the organization of BODIPY-lipids in RBCs from two splenectomized spherocytotic patients (P#1 and P#2) versus two control adults of comparable age, one splenectomized for traumatic spleen rupture (C#1) and one not (C#2). P#2 suffered from a previously described single point mutation (c.3157C>T) at exon 28 of the ankyrin-1 gene ( 45 ). This mutation generates a stop codon within the minimal ZU5-ANK binding domain for ␤ -spectrin ( 46 ) (supplementary Fig. IIF). Although no mutation was found in any of the 42 exons of the ankyrin-1 gene of P#1, RBCs from both patients showed a comparable ‫ف‬ 2-fold lower ankyrin level without obvious spectrin or band 3 defi ciency (supplementary Fig. IID, E), and exhibited a similar osmotic fragility (supplementary Fig. IIG).
RBCs from the two spherocytotic patients showed normal abundance of BODIPY-GlcCer domains (illustrated for  interplay could promote BODIPY-GlcCer domains, suggesting preferential association of BODIPY-GlcCer domains with ankyrin complexes.

Glycophorin C (4.1R complex) overlaps with BODIPY-SM domains and circumscribes BODIPY-PC domains
To look more directly for preferential protein:lipid interactions at the outer PM leafl et, we tested the association of BODIPY-lipid domains with antibody-induced patches of the two anchorage complexes by targeting glycophorin C (GPC) for the 4.1R-based and CD47 for ankyrin-based complexes (see supplementary Fig. I). Patches were previously observed for CD47 ( 47,48 ). GPC and CD47 patches were clearly segregated from one another (compare red and blue labeling in Fig. 8 ) and differentially associated with BODIPY-lipid domains. BODIPY-SM domains largely colocalized with GPC patches (4.1R complex; red in Fig. 8b ), whereas BODIPY-PC domains were instead closely circumscribed by GPC patches (red in Fig. 8c ). In contrast, CD47 patches were never as tightly linked to BODIPY-lipid domains, but frequently seemed in closer proximity to BODIPY-GlcCer and BODIPY-SM (ankyrin complex; blue in Fig. 8a, b ) than to BODIPY-PC domains ( Fig. 8c ). We concluded that differential association of BODIPY-lipid domains with patched proteins of the 4.1R and ankyrin complexes supported the concept whether uncoupling could overcome cholesterol depletion. PKC activation not only abrogated suppression by m ␤ CD of BODIPY-SM and BODIPY-PC domains, but further increased their abundance over untreated controls ( Fig. 7A e , f; quantifi cation in Fig. 7B ). In contrast, m ␤ CD affected BODIPY-GlcCer domains neither in control cells nor upon PKC activation ( Fig. 7A d; quantifi cation in Fig. 7B ), confi rming a differential control of domain biogenesis. Altogether, these data indicated that cholesterol specifi cally promoted BODIPY-SM and BODIPY-PC domains by reducing membrane:spectrin anchorage at 4.1R, suggesting preferential association of BODIPY-SM and BODIPY-PC domains with 4.1R complexes.
Conversely, to test whether BODIPY-GlcCer domains would instead be modulated by the cholesterol:ankyrin interplay, a similar experiment was performed on P#1 spherocytotic RBCs. Whereas BODIPY-GlcCer domains of normal RBCs largely resisted m ␤ CD (see above, Fig. 3a-d ), they were sensitized by spherocytosis to cholesterol depletion ( Fig. 7A g; quantifi cation in Fig. 7B ). In contrast, m ␤ CD equally decreased BODIPY-SM and BODIPY-PC domains both in normal and spherocytotic RBCs ( Fig. 7A b, c, h, i; quantifi cation at Fig. 7B ). Thus, cholesterol:ankyrin  edge remains scarce on: i ) the actual level and stability of lateral membrane lipid organization in living cells (i.e., transient nanometric rafts vs. more stable submicrometric/mesoscale domains); ii ) integration of various assembly/stabilization/restriction mechanisms; and iii ) potential roles in disease, due to limitations of available tools and methods, and to the diversity of cells studied so far. Here we addressed the biological relevance, mechanisms , and relation to a membrane fragility disease of micrometric domains resulting from spontaneous partitioning of fl uorescent (BODIPY ) analogs of three major classes of polar lipids, GSLs (GlcCer and GM1), SM, and PC, when inserted at trace levels into the outer membrane of partially spread RBCs as a simple, robust, and powerful experimental system. Our observations yield new insights on the role of tension, cholesterol enrichment, and preference for proteins of two nonredundant anchorage complexes in micrometric lipid organization.

To what extent do BODIPY-lipid domains refl ect endogenous lateral compartmentation?
The validity of exogenous fl uorescent polar lipid analogs based on partial replacement of an acyl chain by a BODIPY fl uorophore of comparable length as bona fi de reporters of endogenous membrane lipids has long been questioned ( 50 ). However, polar heads are also important for lateral lipid segregation so that their substitution for of preferential protein:lipid interaction, as predicted from differential inactivation of the two anchorage complexes upon cholesterol depletion.

Overview
The classical model of the organization of lipids in biological membranes as a homogenous bilayer has been abandoned due to clear evidence of lateral heterogeneity and clustering mechanisms ( 2,7,49 ). However, knowl- Fig. 8. Differential association of micrometric BODIPY-lipid domains with patched glycophorin C and CD47. RBCs from a normal donor were attached onto PLK-coverslips, labeled with BODIPYlipid (green), then GPC (4.1R complex; red) and CD47 (ankyrin complex; blue) were patched by antibodies . Notice that GPC patches selectively overlapped BODIPY-SM domains (b), while circumscribing BODIPY-PC domains (c). In contrast, CD47 patches were never as tightly linked to lipid-domains, but frequently seemed in closer proximity to BODIPY-GlcCer and BODIPY-SM (a, b) than to BODIPY-PC domains (c). Scale bars, 2 m; at insets, 1 m. Dashed circles at insets indicate lipid* domain boundaries. *, BODIPY.

BODIPY-lipid domains are differentially promoted by cholesterol
Upon moderate cholesterol depletion, BODIPY-SM and BODIPY-PC domains disappeared, but BODIPY-GlcCer domains were largely preserved. This indicates that cholesterol can fi nely tune the lateral heterogeneity of membrane lipids in a living cell, not only to stabilize nanometric rafts but also up to a micrometric scale. It remains to be clarifi ed to what extent the very stable micrometric GSLBODIPY and BODIPY-SM domains of RBCs result from the coalescence of nanometric rafts, due to the particularly high cholesterol level and strong membrane:cytoskeleton anchorage.
We suggest that the higher vulnerability of BODIPY-SM and BODIPY-PC domains to cholesterol depletion may refl ect a looser intrinsic packing, as already indicated by their weaker propensity than BODIPY-GlcCer domains to generate excimers in CHO cells ( 20 ). Conversely, the relative resistance of BODIPY-GlcCer domains to m ␤ CD likely refl ects their higher intrinsic packing. Refractoriness of BODIPY-GlcCer domains to m ␤ CD cannot be explained by selective inaccessibility (e.g., by cholesterol shielding under extended polar heads of complex GSLs), because we found that BODIPY-GlcCer domains were sensitive to m ␤ CD in spherocytotic RBCs where lipid composition was unaltered ( 54 ). Taking together our comparative data on excimers ( 20 ) and sensitivity to m ␤ CD (this paper), we suggest that the lower the packing of BODIPY lipid domains, the higher their dependence on cholesterol.

BODIPY-lipid domains are differentially restricted by the two nonredundant anchorage complexes to spectrin
Fixed positions lasting for at least a half hour and reappearance after bleaching at the exact same place indicated that BODIPY-lipid domains at the outer membrane leafl et were strongly linked to the underlying spectrin network, suggesting dependence on transmembrane components of the two anchorage complexes. Acute uncoupling at 4.1R complexes selectively increased the abundance of GlcCerBODIPY and BODIPY-SM but not BODIPY-PC domains, indicating that the network of 4.1R-based anchors in untreated RBCs preferentially restricts BODIPY-GlcCer and BODIPY-SM coalescence. Alternatively, ankyrin defi ciency in two unrelated spherocytotic patients selectively increased the abundance of BODIPY-SM and BODIPY-PC but did not enhance BODIPY-GlcCer domains, indicating that ankyrin preferentially restricts BODIPY-SM and BODIPY-PC coalescence.

BODIPY-lipid domains show preferential protein association and are fi nely tuned by cholesterol and anchorage interplay
The large colocalization of patched glycophorin C within BODIPY-SM domains and its close association with the borders of BODIPY-PC domains suggested that assembly of BODIPY-SM and BODIPY-PC domains, which are both restricted by ankyrin complexes (see above), could instead be promoted by 4.1R complexes in two different ways, "within" the domains or "around" them . Combination of alternative fl uorophore labeling is not without potential pitfalls. The impact of sphingolipid headgroups on lateral lipid organization has been demonstrated in artifi cial membranes ( 51 ). Moreover, we recently reported that BODIPY-GlcCer/GM1BODIPY and BODIPY-SM, built on the very same BODIPY-ceramide backbone and differing only by their respective polar head, segregated into distinct domains exhibiting different properties ( 20 ). We further reported that BODIPY-SM micrometric domains spontaneously formed in CHO cells, irrespective of direct BODIPY-SM surface insertion versus secondary exocytosis after Golgi synthesis from BODIPY-ceramide and selectively vanished upon depletion of endogenous SM upon biosynthetic inhibition or surface cleavage, indirectly suggesting that BODIPY-SM domains somehow refl ected some endogenous SM compartmentation in a living cell ( 19 ). Now, we show that direct labeling of endogenous GM1 at the RBC PM by fl uorescent CTxB yielded submicrometric domains similar to those seen after insertion of exogenous GM1BODIPY, and that both are largely colocalized . In addition, differential modulation of the three BODIPY-lipid domains in RBCs by temperature ( 19,20 ), stretching, cholesterol, and membrane:cytoskeleton anchorage not only points to a key role of lipid phases and membrane tension in domains biogenesis/maintenance, but also rules out that domains are simply fl uorescent lipid aggregates. We and others further noticed that the size and stability of BODIPY-lipid domains evidenced in RBCs strongly resembled those recorded at the surface of yeast expressing appropriate green fl uorescent protein -tagged membrane proteins ( 21,52,53 ). Thus, while available data do not allow us to conclude that micrometric BODIPY-lipid domains faithfully refl ect the level of the organization of corresponding endogenous lipids, convergent evidence indicates that both must be linked. The level of fi delity of BODIPY-lipids deserves to be further evaluated, e.g., by comparison with the labeling of endogenous lipids using monovalent toxin-derived probes combined with superresolution microscopy ( 6 ), even though this approach is not suited for dynamic vital imaging. We will now discuss mechanistic information gained from the study of micrometric BODIPY-lipid domains .

BODIPY-lipid domains are regulated by membrane tension
We fi rst found that membrane tension strongly impacted on the number and size of visible BODIPY domains formed by the three main classes of polar BODIPY-lipids. Domains were numerous and polydisperse when RBCs were barely attached, declined to a stable low number with monodisperse size at partial spreading, and vanished upon maximal stretching. Conversely, when membrane tension was relaxed by uncoupling either of the two spectrin anchorage complexes, the number of BODIPY domains increased, albeit to a different extent, according to the BODIPY-lipid class as will be discussed below. Biophysical studies should address the mechanical parameters governing the relation between membrane tension and BODIPY domains cohesion and size in RBCs .
The signifi cance of micrometric BODIPY-lipid domains for RBC deformability and membrane fragility diseases may be viewed by two opposite, yet not mutually exclusive models. In the fi rst one, packed domains would favor membrane resilience by providing a reservoir when lipid spacing is needed for membrane deformation, e.g., for crossing spleen sinusoid pores, somehow analogous to the recruitment of caveolae upon endothelial cell stretching ( 61,62 ). In the second model, domain formation would refl ect a propensity to local rupture, favoring release of microvesicles ( 63 ) known as microspherocytes in spherocytosis. Based on this well-known fact, we recently found that controlled perturbations can trigger differential budding from the three types of BODIPY-lipid domains in RBCs, which may pave the way to defi nition of their respective biochemical composition and biophysical properties . cholesterol depletion with differential anchorage uncoupling provided additional information. The reappearance (and even doubling, as compared with untreated cells) of BODIPY-SM and BODIPY-PC domains in cholesteroldepleted RBCs upon acute uncoupling of 4.1R complexes, but not in ankyrin-defi cient cells, suggested that cholesterol selectively modulates membrane lipid:4.1R interactions, thereby favoring BODIPY-SM and BODIPY-PC domains, in agreement with previous reports demonstrating that cholesterol not only regulates lateral membrane fl uidity, but also regulates transversal mem brane: cytoskeleton anchorage ( 28,41 ). Cholesterol may regulate membrane:cytoskeleton coupling via its strong direct interaction with band 3, as shown in mixed phos pholipid:cholesterol monolayers ( 55,56 ). Cholesterol has also been shown to indirectly affect spectrin:phospholipid interactions in a similar system ( 57 ). In addition, because cholesterol depletion was shown to disrupt the lateral organization of phosphatidylinositol-4,5-biphosphate (PIP 2 ) at the PM ( 58,59 ), and because 4.1R contains a PIP 2 -binding motif ( 60 ), it was tempting to speculate that cholesterol could impact on 4.1R:lipid interaction. By complementarity, the selective decrease of BODIPY-GlcCer domains by m ␤ CD in spherocytosis, but not in normal RBCs, would suggest that BODIPY-GlcCer domains are promoted by cholesterol at ankyrin-based complexes. Taken together, our observations revealed that the three types of BODIPYlipid domains showed differential preference for the two types of nonredundant anchorage complexes, as shown by copatching experiments and functional destabilization studies based on cholesterol depletion and selective anchorage uncoupling. It should be possible to clarify the issue of BODIPY lipid:protein preference based on biochemical studies after differential domain budding into microvesicles and fractionation (see below).

Current models for micrometric BODIPY-lipid domain biogenesis, functions, and role in disease
Biological membrane fl uidity depends on several levels of heterogeneity: i ) complex composition covering a broad Tm range; ii ) differential clustering at the nanometric versus the micrometric scale; and iii ) transversal anchorage to a dynamic cortical cytoskeleton that can be modulated . For RBCs, we here showed that a combinatorial interplay of moderate variations in (local?) cholesterol concentrations with differential preference to, or restriction by, the two anchorage complexes and their regulated linkage to the spectrin network can fi nely tune three types of micrometric assemblies of polar membrane BODIPY-lipids. One can thus interpret BODIPY-lipid domain assembly in erythrocytes as essentially governed by two complementary mechanisms that were recently proposed to generate dynamic lateral heterogeneity ( 7,49 ): i ) differential intrinsic lipid cohesion, and ii ) regulated linkage to the spectrin network thanks to preferential interaction with two nonredundant anchorage complexes and providing either internal stabilization (e.g., BODIPY-SM or peripheral retention (e.g., BODIPY-PC Finally, domain expansion would be limited by fences and membrane tension.