Ludovic D'Auria

Recent progress on lipid lateral heterogeneity in plasma membranes: From rafts to submicrometric domains

The concept of transient nanometric domains known as lipid rafts has brought interest to reassess the validity of the Singer-Nicolson model of a fluid bilayer for cell membranes. However, this new view is still insufficient to explain the cellular control of surface lipid diversity or membrane deformability. During the past decades, the hypothesis that some lipids form large (submicrometric/mesoscale vs nanometric rafts) and stable (>min vs s) membrane domains has emerged, largely based on indirect methods. Morphological evidence for stable submicrometric lipid domains, well-accepted for artificial and highly specialized biological membranes, was further reported for a variety of living cells from prokaryot es to yeast and mammalian cells. However, results remained questioned based on limitations of available fluorescent tools, use of poor lipid fixatives, and imaging artifacts due to non-resolved membrane projections. In this review, we will discuss recent evidence generated using powerful and innovative approaches such as lipid-specific toxin fragments that support the existence of submicrometric domains. We will integrate documented mechanisms involved in the formation and maintenance of these domains, and provide a perspective on their relevance on membrane deformability and regulation of membrane protein distribution.

Amyloid precursor protein controls cholesterol turnover needed for neuronal activity.

Perturbation of lipid metabolism favours progression of Alzheimer disease, in which processing of Amyloid Precursor Protein (APP) has important implications. APP cleavage is tightly regulated by cholesterol and APP fragments regulate lipid homeostasis. Here, we investigated whether up or down regulation of full‐length APP expression affected neuronal lipid metabolism. Expression of APP decreased HMG‐CoA reductase (HMGCR)‐mediated cholesterol biosynthesis and SREBP mRNA levels, while its down regulation had opposite effects. APP and SREBP1 co‐immunoprecipitated and co‐localized in the Golgi. This interaction prevented Site‐2 protease‐mediated processing of SREBP1, leading to inhibition of transcription of its target genes. A GXXXG motif in APP sequence was critical for regulation of HMGCR expression. In astrocytes, APP and SREBP1 did not interact nor did APP affect cholesterol biosynthesis. Neuronal expression of APP decreased both HMGCR and cholesterol 24‐hydroxylase mRNA levels and consequently cholesterol turnover, leading to inhibition of neuronal activity, which was rescued by geranylgeraniol, generated in the mevalonate pathway, in both APP expressing and mevastatin treated neurons. We conclude that APP controls cholesterol turnover needed for neuronal activity.

Three unrelated sphingomyelin analogs spontaneously cluster into plasma membrane micrometric domains

Micrometric lipid compartmentation at the plasma membrane is disputed. Using live confocal imaging, we found that three unrelated fluorescent sphingomyelin (SM) analogs spontaneously clustered at the outer leaflet into micrometric domains, contrasting with homogeneous labelling by DiIC18 and TMA-DPH. In erythrocytes, these domains were round, randomly distributed, and reversibly coalesced under hypotonicity. BODIPY-SM and -glucosylceramide showed distinct temperature-dependence, in the same ranking as Tm for corresponding natural lipids, indicating phase behaviour. Scanning electron microscopy excluded micrometric surface structural features. In CHO cells, similar surface micrometric patches were produced by either direct BODIPY-SM insertion or intracellular processing from BODIPY-ceramide, ruling out aggregation artefacts. BODIPY-SM surface micrometric patches were refractory to endocytosis block or actin depolymerization and clustered upon cholesterol deprivation, indicating self-clustering at the plasma membrane. BODIPY-SM excimers further suggested clustering in ordered domains. Segregation of BODIPY-SM and -lactosylceramide micrometric domains showed coexistence of distinct phases. Consistent with micrometric domain boundaries, fluorescence recovery after photobleaching (FRAP) revealed restriction of BODIPY-SM lateral diffusion over long-range, but not short-range, contrasting with comparable high mobile fraction of BODIPY-lactosylceramide in both ranges. Controlled perturbations of endogenous SM pool similarly affected BODIPY-SM domain size by confocal imaging and its mobile fraction by FRAP. The latter evidence supports the hypothesis that, as shown for BODIPY-SM, endogenous SM spontaneously clusters at the plasmalemma outer leaflet of living cells into ordered micrometric domains, defined in shape by liquid-phase coexistence and in size by membrane tension and cholesterol. This proposal remains speculative and calls for further investigations.

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.

Mechanism of Neuromuscular Dysfunction in Krabbe Disease

The atrophy of skeletal muscles in patients with Krabbe disease is a major debilitating manifestation that worsens their quality of life and limits the clinical efficacy of current therapies. The pathogenic mechanism triggering muscle wasting is unknown. This study examined structural, functional, and metabolic changes conducive to muscle degeneration in Krabbe disease using the murine (twitcher mouse) and canine [globoid cell leukodystrophy (GLD) dog] models. Muscle degeneration, denervation, neuromuscular [neuromuscular junction (NMJ)] abnormalities, and axonal death were investigated using the reporter transgenic twitcher–Thy1.1–yellow fluorescent protein mouse. We found that mutant muscles had significant numbers of smaller-sized muscle fibers, without signs of regeneration. Muscle growth was slow and weak in twitcher mice, with decreased maximum force. The NMJ had significant levels of activated caspase-3 but limited denervation. Mutant NMJ showed reduced surface areas and lower volumes of presynaptic terminals, with depressed nerve control, increased miniature endplate potential (MEPP) amplitude, decreased MEPP frequency, and increased rise and decay rate constants. Twitcher and GLD dog muscles had significant capacity to store psychosine, the neurotoxin that accumulates in Krabbe disease. Mechanistically, muscle defects involved the inactivation of the Akt pathway and activation of the proteasome pathway. Our work indicates that muscular dysfunction in Krabbe disease is compounded by a pathogenic mechanism involving at least the failure of NMJ function, activation of proteosome degradation, and a reduction of the Akt pathway. Akt, which is key for muscle function, may constitute a novel target to complement in therapies for Krabbe disease.

Live-cell imaging with water-soluble aminophenoxazinone dyes synthesised through laccase biocatalysis

Aminophenoxazinone dyes with variable water solubilities were assayed for the first time in a live‐cell imaging application. Among a library of ten sulfonylated chromophores, one compound gave excellent results as an endocytic marker, showing a precise subcellular distribution. The compound was compared to four commercial vital tracers, including Lucifer Yellow. The first laccase‐mediated regioselective synthesis of a diphosphorylated 2‐aminophenoxazinone dye was also described. This compound, water‐soluble at 10−2 M, displayed modest fluorescence properties and the ability to complex Mg2+ and Ca2+ cations, therefore giving fluorescence quenching.

Surfactins modulate the lateral organization of fluorescent membrane polar lipids: A new tool to study drug:membrane interaction and assessment of the role of cholesterol and drug acyl chain length

The lipopeptide surfactin exhibits promising antimicrobial activities which are hampered by haemolytic toxicity. Rational design of new surfactin molecules, based on a better understanding of membrane:surfactin interaction, is thus crucial. We here performed bioimaging of lateral membrane lipid heterogeneity in adherent living human red blood cells (RBCs), as a new relevant bioassay, and explored its potential to better understand membrane:surfactin interactions. RBCs show (sub)micrometric membrane domains upon insertion of BODIPY analogs of glucosylceramide (GlcCer), sphingomyelin (SM) and phosphatidylcholine (PC). These domains exhibit increasing sensitivity to cholesterol depletion by methyl-β-cyclodextrin. At concentrations well below critical micellar concentration, natural cyclic surfactin increased the formation of PC and SM, but not GlcCer, domains, suggesting preferential interaction with lipid assemblies with the highest vulnerability to methyl-β-cyclodextrin. Surfactin not only reversed disappearance of SM domains upon cholesterol depletion but further increased PC domain abundance over control RBCs, indicating that surfactin can substitute cholesterol to promote micrometric domains. Surfactin sensitized excimer formation from PC and SM domains, suggesting increased lipid recruitment and/or diffusion within domains. Comparison of surfactin congeners differing by geometry, charge and acyl chain length indicated a strong dependence on acyl chain length. Thus, bioimaging of micrometric lipid domains is a visual powerful tool, revealing that intrinsic lipid domain organization, cholesterol abundance and drug acyl chain length are key parameters for membrane:surfactin interaction. Implications for surfactin preferential location in domains or at their boundaries are discussed and may be useful for rational design of better surfactin molecules.

Fluid levity of the cell: Role of membrane lipid architecture in genetic sphingolipidoses

Sphingolipidoses arise from inherited loss of function of key enzymes regulating the sphingolipid (SL) metabolism and the accumulation of large quantities of these lipids in affected cells. Most frequently, toxicity is manifested in the nervous system, where survival and function of neurons and glial cells are most affected. Although detailed information is available on neuroglial alterations during terminal stages of the disease, the initial pathogenic mechanisms triggering neuropathology are largely unclear. Because they are key components of biological membranes, changes in the local concentration of SLs are likely to impact the organization of membrane domains and functions. This Commentary proposes that SL toxicity involves initial defects in the integrity of lipid domains, membrane fluidity, and membrane bending, leading to membrane deformation and deregulation of cell signaling and function. Understanding how SLs alter membrane architecture may provide breakthroughs for more efficient treatment of sphingolipidoses.

Prophylactic treatment against GM-CSF, but not IL-17, abolishes relapses in a chronic murine model of multiple sclerosis

The pathogenic role of IL‐17 and GM‐CSF has been unravelled in experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). However, in most models, EAE is characterised by a monophasic attack which is not representative of the relapsing nature nor the chronicity displayed in MS. Here, we used proteolipid protein peptide (PLP139‐151) to trigger EAE‐relapses (EAE‐II) in SJL mice that had recovered from a primary‐EAE episode (EAE‐I). This procedure resulted in severe and irreversible disease that, unlike EAE‐I, was not abolished by anti‐IL‐17‐mAb. In contrast, prophylactic anti‐GM‐CSF‐mAb treatment prevented EAE‐I and ‐II. Strikingly, the expression of T‐cell transcription factors and cytokines/chemokines in mice treated with anti‐GM‐CSF during both EAE episodes was silenced. Anti‐GM‐CSF‐mAb treatment administered only during EAE‐II did not completely prevent relapses but mice ultimately reached full recovery. Anti‐GM‐CSF treatment also strongly impaired and ultimately resolved monophasic MOG35‐55‐induced EAE in C57Bl/6 mice. In such protected mice, anti‐GM‐CSF treatment also prevented a further relapse induced by MOG‐revaccination. These results underscore the critical role of GM‐CSF on pro‐inflammatory mediator production. Furthermore, we observed a strong preventive and curative effect of anti‐GM‐CSF neutralisation in two EAE models, relapsing and chronic. Altogether these findings are relevant for further MS research.