Roles for Lo microdomains and ESCRT in ER stress-induced lipid droplet microautophagy in budding yeast

Microlipophagy (µLP), degradation of lipid droplets (LDs) by microautophagy, occurs by autophagosome-independent direct uptake of LDs at lysosomes/vacuoles in response to nutrient limitations and ER stressors in Saccharomyces cerevisiae. In nutrient-limited yeast, liquid-ordered (Lo) microdomains, sterol-rich raft-like regions in vacuolar membranes, are sites of membrane invagination during LD uptake. The endosome sorting complex required for transport (ESCRT) is required for sterol transport during Lo formation under these conditions. However, ESCRT has been implicated in mediating membrane invagination during µLP induced by ER stressors or the diauxic shift from glycolysis- to respiration-driven growth. Here, we report that ER stress induced by lipid imbalance and other stressors induces Lo microdomain formation. This process is ESCRT-independent and dependent upon Niemann-Pick type C sterol transfer proteins. Inhibition of ESCRT or Lo microdomain formation partially inhibits lipid imbalance-induced µLP, while inhibition of both blocks this µLP. Finally, although the ER stressors dithiothreitol or tunicamycin induce Lo microdomains, µLP in response to these stressors is ESCRT-dependent and Lo microdomain-independent. Our findings reveal that Lo microdomain formation is a yeast stress response, and stress-induced Lo microdomain formation occurs by stressor-specific mechanisms. Moreover, ESCRT and Lo microdomains play functionally distinct roles in LD uptake during stress-induced µLP.

Structural Diversity of Photoswitchable Sphingolipids for Optodynamic Control of Lipid Raft Microdomains

Sphingolipids are a structurally diverse class of lipids predominantly found in the plasma membrane of eukaryotic cells. These lipids can laterally segregate with other saturated lipids and cholesterol into lipid rafts; liquid-ordered (Lo) microdomains that act as organizing centers within biomembranes. Owing the vital role of sphingolipids for lipid segregation, controlling their lateral localization is of utmost significance. Hence, we made use of the light-induced trans-cis isomerization of azobenzene-modified acyl chains, to develop a set of photoswitchable sphingolipids, with different headgroups (hydroxyl, galactosyl, phosphocholine) and backbones (sphingosine, phytosphingosine, tetrahydropyran (THP)-blocked sphingosine), able to shuttle between liquid-ordered (Lo) and liquid-disordered (Ld) regions of model membranes upon irradiation with UV-A (λ = 365 nm) and blue (λ = 470 nm) light, respectively. Using combined high-speed atomic force microscopy, fluorescence microscopy, and force spectroscopy, we investigated how these active sphingolipids laterally remodel supported bilayers upon photo-isomerization, notably in terms of domain area changes, height mismatch, line tension, and membrane piercing. Hereby, we show that all sphingosine-(Azo-β-Gal-Cer, Azo-SM, Azo-Cer) and phytosphingosine-based (Azo-α-Gal-PhCer, Azo-PhCer) photolipids behave similarly, promoting a reduction in Lo domain area when in the UV-adapted cis-isoform. In contrast, azo-sphingolipids having THP groups that block H-bonding at the sphingosine backbone (Azo-THP-SM, Azo-THP-Cer) induce an increase in the Lo domain area when in cis, accompanied by a major rise in height mismatch and line tension. These changes were fully reversible upon blue light-triggered isomerization of the various lipids back to trans, pinpointing the role of interfacial interactions for the formation of stable Lo lipid raft domains.

The Two Faces of the Liquid Ordered Phase

The coexistence of liquid ordered Lo and liquid disordered Ld phases in synthetic and plasma membrane-derived vesicles serves as a model for biomembrane heterogeneity. However, the connection between the structures of microscopic phases present in vesicles at low temperatures and the tiny ordered "raft" domains of biomembranes at body temperature is unclear. To study the Lo phase structure across temperatures, we performed atomistic molecular dynamics simulations, differential scanning calorimetry, and fluorescence spectroscopy on the Lo phase in binary and ternary lipid mixtures. Our results reveal an Lo phase with highly ordered and hexagonally packed clusters of saturated lipid chains at low temperatures. These clusters melt upon heating, and numerous membrane properties reflect this transition as two regimes with different temperature dependence. Still, the transition between the regimes is continuous, and they both match the description of the Lo phase with high order and relatively high mobility. Our findings question the use of vesicles displaying Lo–Ld coexistence as models for heterogeneity in cellular membranes, as they likely correspond to different molecular organizations.

Plant Sterol Clustering Correlates with Membrane Microdomains as Revealed by Optical and Computational Microscopy

Local inhomogeneities in lipid composition play a crucial role in the regulation of signal transduction and membrane traffic. This is particularly the case for plant plasma membrane, which is enriched in specific lipids, such as free and conjugated forms of phytosterols and typical phytosphingolipids. Nevertheless, most evidence for microdomains in cells remains indirect, and the nature of membrane inhomogeneities has been difficult to characterize. We used a new push–pull pyrene probe and fluorescence lifetime imaging microscopy (FLIM) combined with all-atom multiscale molecular dynamics simulations to provide a detailed view on the interaction between phospholipids and phytosterol and the effect of modulating cellular phytosterols on membrane-associated microdomains and phase separation formation. Our understanding of the organization principles of biomembranes is limited mainly by the challenge to measure distributions and interactions of lipids and proteins within the complex environment of living cells. Comparing phospholipids/phytosterol compositions typical of liquid-disordered (Ld) and liquid-ordered (Lo) domains, we furthermore show that phytosterols play crucial roles in membrane homeostasis. The simulation work highlights how state-of-the-art modeling alleviates some of the prior concerns and how unrefuted discoveries can be made through a computational microscope. Altogether, our results support the role of phytosterols in the lateral structuring of the PM of plant cells and suggest that they are key compounds for the formation of plant PM microdomains and the lipid-ordered phase.

A palmitoylation code controls PI4KIIIα complex formation and PI(4,5)P2 homeostasis at the plasma membrane

PI4KIIIα is the major enzyme responsible for generating the phosphoinositide PI(4)P at the plasma membrane. This lipid kinase forms two multicomponent complexes, both including a palmitoylated anchor, EFR3. Whereas both PI4KIIIα complexes support production of PI(4)P, the distinct functions of each complex and mechanisms underlying the interplay between them remain unknown. Here, we present roles for differential palmitoylation patterns within a tri-Cys motif in EFR3B (Cys5/Cys7/Cys8) in controlling the distribution of PI4KIIIα between these two complexes at the plasma membrane and corresponding functions in phosphoinositide homeostasis. Spacing of palmitoyl groups within three doubly palmitoylated EFR3B “lipoforms” affects both its interactions with TMEM150A, a transmembrane protein governing formation of a PI4KIIIα complex functioning in rapid PI(4,5)P2 resynthesis following PLC signaling, and its partitioning within liquid-ordered and -disordered regions of the plasma membrane. This work identifies a palmitoylation code in controlling protein-protein and protein-lipid interactions affecting a plasma membrane-resident lipid biosynthetic pathway.

Lipid-based and protein-based interactions synergize transmembrane signaling stimulated by antigen clustering of IgE receptors

Antigen (Ag) crosslinking of immunoglobulin E–receptor (IgE-FcεRI) complexes in mast cells stimulates transmembrane (TM) signaling, requiring phosphorylation of the clustered FcεRI by lipid-anchored Lyn tyrosine kinase. Previous studies showed that this stimulated coupling between Lyn and FcεRI occurs in liquid ordered (Lo)-like nanodomains of the plasma membrane and that Lyn binds directly to cytosolic segments of FcεRI that it initially phosphorylates for amplified activity. Net phosphorylation above a nonfunctional threshold is achieved in the stimulated state but not in the resting state, and current evidence supports the hypothesis that this relies on Ag crosslinking to disrupt a balance between Lyn and tyrosine phosphatase activities. However, the structural interactions that underlie the stimulation process remain poorly defined. This study evaluates the relative contributions and functional importance of different types of interactions leading to suprathreshold phosphorylation of Ag-crosslinked IgE-FcεRI in live rat basophilic leukemia mast cells. Our high-precision diffusion measurements by imaging fluorescence correlation spectroscopy on multiple structural variants of Lyn and other lipid-anchored probes confirm subtle, stimulated stabilization of the Lo-like nanodomains in the membrane inner leaflet and concomitant sharpening of segregation from liquid disordered (Ld)-like regions. With other structural variants, we determine that lipid-based interactions are essential for access by Lyn, leading to phosphorylation of and protein-based binding to clustered FcεRI. By contrast, TM tyrosine phosphatase, PTPα, is excluded from these regions due to its Ld-preference and steric exclusion of TM segments. Overall, we establish a synergy of lipid-based, protein-based, and steric interactions underlying functional TM signaling in mast cells.

Membrane thickness, lipid phase and sterol type are determining factors in the permeability of membranes to small solutes

Cell membranes provide a selective semi-permeable barrier to the passive transport of molecules. This property differs greatly between organisms. While the cytoplasmic membrane of bacterial cells is highly permeable for weak acids and glycerol, yeasts can maintain large concentration gradients. Here we show that such differences can arise from the physical state of the plasma membrane. By combining stopped-flow kinetic measurements with molecular dynamics simulations, we performed a systematic analysis of the permeability through synthetic lipid membranes to obtain detailed molecular insight into the permeation mechanisms. While membrane thickness is an important parameter for the permeability through fluid membranes, the largest differences occur when the membranes transit from the liquid-disordered to liquid-ordered and/or to gel state. By comparing our results with in vivo measurements from yeast, we conclude that the yeast membrane exists in a highly ordered and rigid state, which is comparable to synthetic saturated DPPC-sterol membranes.