Tools to image membrane tension in response to mechanical stimuli are badly needed in mechanobiology. We have recently introduced mechanosensitive flipper probes to report quantitatively global membrane tension changes in fluorescence lifetime imaging microscopy (FLIM) images of living cells. However, to address specific questions on physical forces in biology, the probes need to be localized precisely in the membrane of interest (MOI). Herein we present a general strategy to image the tension of the MOI by tagging our newly introduced HaloFlippers to self-labeling HaloTags fused to proteins in this membrane. The critical challenge in the construction of operational HaloFlippers is the tether linking the flipper and the HaloTag: It must be neither too taut nor too loose, be hydrophilic but lipophilic enough to passively diffuse across membranes to reach the HaloTags, and allow partitioning of flippers into the MOI after the reaction. HaloFlippers with the best tether show localized and selective fluorescence after reacting with HaloTags that are close enough to the MOI but remain nonemissive if the MOI cannot be reached. Their fluorescence lifetime in FLIM images varies depending on the nature of the MOI and responds to myriocin-mediated sphingomyelin depletion as well as to osmotic stress. The response to changes in such precisely localized membrane tension follows the validated principles, thus confirming intact mechanosensitivity. Examples covered include HaloTags in the Golgi apparatus, peroxisomes, endolysosomes, and the ER, all thus becoming accessible to the selective fluorescence imaging of membrane tension.

Sphingolipids have been shown to play important roles in physiology and cell biology, but a systematic examination of their functions is lacking. We performed a genome-wide CRISPRi screen in sphingolipid-depleted cells and identified hypersensitive mutants in genes of membrane trafficking and lipid biosynthesis, including ether lipid synthesis. Systematic lipidomic analysis showed a coordinate regulation of ether lipids with sphingolipids, where depletion of one of these lipid types resulted in increases in the other, suggesting an adaptation and functional compensation. Biophysical experiments on model membranes show common properties of these structurally diverse lipids that also share a known function as GPI anchors in different kingdoms of life. Molecular dynamics simulations show a selective enrichment of ether phosphatidylcholine around p24 proteins, which are receptors for the export of GPI-anchored proteins and have been shown to bind a specific sphingomyelin species. Our results support a model of convergent evolution of proteins and lipids, based on their physico-chemical properties, to regulate GPI-anchored protein transport and maintain homeostasis in the early secretory pathway. INTRODUCTIONThe maintenance of membrane lipid homeostasis is an energetically expensive yet necessary process in cells. Lipid diversity has evolved together with cell complexity to give rise to thousands of different lipids species with specific functions, many of which are still unexplored 1, 2 . Moreover, different lipid metabolic pathways are interconnected, and cells show a high phenotypic plasticity when adapting to changes in membrane lipid composition, which makes it difficult to disentangle the function of individual lipid species 3 . A systematic analysis of the cellular responses to perturbation of specific synthetic pathways is thus needed to reveal co-regulated lipid networks and uncover new lipid functions. Sphingolipids (SL) are a class of lipids that contain a sphingoid-base backbone, in contrast to the more commonly found glycerol backbone in glycerophospholipids (GPL). These bioactive lipids have been extensively studied in the last decades, revealing distinctive physico-chemical properties and connections to diseases 4,5 . SL have been implicated in diabetes 6 , cancer 7 and inflammation 8 , and mutations in SL synthetic or metabolic enzymes are associated with severe genetic disorders [9][10][11] . SL species sphingosine (So) and ceramide (Cer) can permeabilize membranes 12,13 ; Cer induces the flip-flop of neighbouring lipids 14 and can phase-separate to form membrane platforms important for signalling 15 . The most abundant SL species, sphingomyelin (SM), has been shown to modulate membrane properties and regulate signalling pathways 16,17 . Besides direct phosphorylation of ceramide synthases [18][19][20] , the only direct regulators of sphingolipid synthesis identified are Orm proteins (ORMDL in mammalian cells), that associate with serine palmitoyl transferase (SPT), the first enzyme of the sphingolipi...

Ether lipids are glycerolipids further classified into alkyl‐ether and alkenyl‐ether (also termed plasmalogens) lipids. The two ether lipid subclasses share the first steps of their synthesis. However, alkyl‐ether and alkenyl‐ether lipids differ in their structure and physico‐chemical properties (featuring different head groups) and, thus, probably in their functions. Ether lipids have intermittent distribution across the evolutionary tree and defects in their synthesis have been shown to perturb cellular homeostasis and lead to disease in humans. Here, we review their structure, their interactions with other lipids, and their potential roles in cellular functions, such as membrane homeostasis and membrane trafficking. Moreover, we discuss still unclear aspects of these lipids such as their subcellular distribution, and the need to unravel their molecular functions as well as how novel tools to study lipid biology will help clarify these aspects.

This article describes four fluorescent membrane tension probes that have been designed, synthesized, evaluated, commercialized and applied to current biology challenges in the context of the NCCR Chemical Biology. Their names are Flipper-TR®, ER Flipper-TR®, Lyso Flipper-TR®, and Mito Flipper-TR®. They are available from Spirochrome.

Ceramides and dihydroceramides are N-acyl derivatives of sphingosine and sphinganine, respectively, which are the major sphingoid-base backbones of mammals. Recent studies have found that mammals, like certain other organisms, also produce 1-deoxy-(dihydro)ceramides (1-deoxyDHCers) that contain sphingoid bases lacking the 1-hydroxyl- or 1-hydroxymethyl- groups. The amounts of these compounds can be substantial-indeed, we have found comparable levels of 1-deoxyDHCers and ceramides in RAW 264.7 cells maintained in culture. The biophysical properties of 1-deoxyDHCers have not yet been reported, although these lipids might play important roles in normal cell regulation and in the pathology of diseases in which they are elevated, such as hereditary sensory autonomic neuropathies or diabetes. This study uses several approaches, including surface-pressure measurements, differential scanning calorimetry, and confocal microscopy, to study the behavior of 1-deoxyDHCers of different N-acyl-chain lengths and their interaction with sphingomyelin (SM). The thermotropic behaviors of 1-deoxyDHCers alone and in mixtures with SM are described, together with their interactions in monolayers and giant unilamellar vesicles. The gel-fluid transition temperatures of the pure compounds increase in the order 1-deoxyceramide < ceramide ≈ 1-deoxyDHCer < 1-(deoxymethyl)DHCer. In general, canonical ceramides are more miscible with SM in bilayers than are 1-deoxyceramides, and 1-(deoxymethyl)DHCers are the most hydrophobic among them, not even capable of forming monolayers at the air-water interface. Thus, these properties suggest that 1-deoxyDHCer can influence the properties of cellular membranes in ways that might affect biological function/malfunction.

This study was aimed at preparing and characterizing plasma membranes (PM) from Chinese Hamster Ovary (CHO) cells. Two methods of PM preparation were applied, one based on adhering cells to a poly-lysine-coated surface, followed by hypotonic lysis and removal of intracellular components, so that PM patches remain adhered to each other, and a second one consisting of bleb induction in cells, followed by separation of giant plasma membrane vesicles (GPMV). Both methods gave rise to PM in sufficient amounts to allow biophysical and biochemical characterization. Laurdan generalized polarization was used to measure molecular order in membranes, PM preparations were clearly more ordered than the average cell membranes (GP ≈0.450 vs. ≈0.20 respectively). Atomic force microscopy was used in the force spectroscopy mode to measure breakthrough forces of PM, both PM preparations provided values in the 4–6 nN range, while the corresponding value for whole cell lipid extracts was ≈2 nN. Lipidomic analysis of the PM preparations revealed that, as compared to the average cell membranes, PM were enriched in phospholipids containing 30–32 C atoms in their acyl chains but were relatively poor in those containing 34–40 C atoms. PM contained more saturated and less polyunsaturated fatty acids than the average cell membranes. Blebs (GPMV) and patches were very similar in their lipid composition, except that blebs contained four-fold the amount of cholesterol of patches (≈23 vs. ≈6 mol% total membrane lipids) while the average cell lipids contained 3 mol%. The differences in lipid composition are in agreement with the observed variations in physical properties between PM and whole cell membranes.

The thermotropic properties of aqueous dispersions of sphingomyelins (SM) and ceramides (Cer) with N-acyl chains varying from C6:0 to C24:1, either pure or in binary mixtures, have been examined by differential scanning calorimetry. Even in the pure state, Cer and particularly SM exhibited complex endotherms, and their thermal properties did not vary in a predictable way with changes in structure. In some cases, e.g. C18:0 SM, atomic force microscopy revealed coexisting lamellar domains made of a single lipid. Partial chain interdigitation and metastable crystalline states were deemed responsible for the complex behavior. SM:Cer mixtures (90:10mol ratio) gave rise to bilayers containing separate SM-rich and Cer-rich domains. In vesicles made of more complex mixtures (SM:PE:Chol, 2:1:1), it is known that sphingomyelinase degradation of SM to Cer is accompanied by vesicle aggregation and release of aqueous contents. These vesicles did not reveal observable domain separation by confocal microscopy. Vesicle aggregation occurred at a faster rate for those bilayers that appeared to be more fluid according to differential scanning calorimetry. Content efflux rates measured by fluorescence spectroscopy were highest with C18:0 and C18:1 SM, and in general those rates did not vary regularly with other physical properties of SM or Cer. In general the individual SM and Cer appear to have particular thermotropic properties, often unrelated to the changes in N-acyl chain.

To image membrane tension in selected membranes of interest (MOI) inside living systems, the field of mechanobiology requires increasingly elaborated small-molecule chemical tools. We have recently introduced HaloFlipper, i.e., a mechanosensitive flipper probe that can localize in the MOI using HaloTag technology to report local membrane tension changes using fluorescence lifetime imaging microscopy. However, the linker tethering the probe to HaloTag hampers the lateral diffusion of the probe in all the lipid domains of the MOI. For a more global membrane tension measurement in any MOI, we present here a supramolecular chemistry strategy for selective localization and controlled release of flipper into the MOI, using a genetically encoded supramolecular tag. SupraFlippers, functionalized with a desthiobiotin ligand, can selectively accumulate in the organelle having expressed streptavidin. The addition of biotin as a biocompatible external stimulus with a higher affinity for Sav triggers the release of the probe, which spontaneously partitions into the MOI. Freed in the lumen of endoplasmic reticulum (ER), SupraFlippers report the membrane orders along the secretory pathway from the ER over the Golgi apparatus to the plasma membrane. Kinetics of the process are governed by both the probe release and the transport through lipid domains. The concentration of biotin can control the former, while the expression level of a transmembrane protein (Sec12) involved in the stimulation of the vesicular transport from ER to Golgi influences the latter. Finally, the generation of a cell-penetrating and fully functional Sav-flipper complex using cyclic oligochalcogenide (COC) transporters allows us to combine the SupraFlipper strategy and HaloTag technology.