Cells and organelles are delimited by lipid bilayers in which high deformability is essential to many cell processes, including motility, endocytosis and cell division. Membrane tension is therefore a major regulator of the cell processes that remodel membranes, albeit one that is very hard to measure in vivo. Here we show that a planarizable push-pull fluorescent probe called FliptR (fluorescent lipid tension reporter) can monitor changes in membrane tension by changing its fluorescence lifetime as a function of the twist between its fluorescent groups. The fluorescence lifetime depends linearly on membrane tension within cells, enabling an easy quantification of membrane tension by fluorescence lifetime imaging microscopy. We further show, using model membranes, that this linear dependency between lifetime of the probe and membrane tension relies on a membrane-tension-dependent lipid phase separation. We also provide calibration curves that enable accurate measurement of membrane tension using fluorescence lifetime imaging microscopy.

SummaryESCRT-III is required for lipid membrane remodeling in many cellular processes, from abscission to viral budding and multi-vesicular body biogenesis. However, how ESCRT-III polymerization generates membrane curvature remains debated. Here, we show that Snf7, the main component of ESCRT-III, polymerizes into spirals at the surface of lipid bilayers. When covering the entire membrane surface, these spirals stopped growing when densely packed: they had a polygonal shape, suggesting that lateral compression could deform them. We reasoned that Snf7 spirals could function as spiral springs. By measuring the polymerization energy and the rigidity of Snf7 filaments, we showed that they were deformed while growing in a confined area. Furthermore, we observed that the elastic expansion of compressed Snf7 spirals generated an area difference between the two sides of the membrane and thus curvature. This spring-like activity underlies the driving force by which ESCRT-III could mediate membrane deformation and fission.

In this report, “fluorescent flippers” are introduced to create planarizable push–pull probes with the mechanosensitivity and fluorescence lifetime needed for practical use in biology. Twisted push–pull scaffolds with large and bright dithienothiophenes and their S,S-dioxides as the first “fluorescent flippers” are shown to report on the lateral organization of lipid bilayers with quantum yields above 80% and lifetimes above 4 ns. Their planarization in liquid-ordered (Lo) and solid-ordered (So) membranes results in red shifts in excitation of up to +80 nm that can be transcribed into red shifts in emission of up to +140 nm by Förster resonance energy transfer (FRET). These unique properties are compatible with multidomain imaging in giant unilamellar vesicles (GUVs) and cells by confocal laser scanning or fluorescence lifetime imaging microscopy. Controls indicate that strong push–pull macrodipoles are important, operational probes do not relocate in response to lateral membrane reorganization, and two flippers are indeed needed to “really swim,” i.e., achieve high mechanosensitivity.

land § These two authors contributed equally ABSTRACT. Measuring forces inside cells is particularly challenging. With the development of quantitative microscopy, fluorophores which allow the measurement of forces became highly desirable. We have previously introduced a mechanosensitive flipper probe, which responds to the change of plasma membrane tension by changing fluorescence lifetime and thus allows tension imaging by FLIM. Herein, we describe the design, synthesis, and evaluation of flipper probes that selectively label intracellular organelles, i.e., lysosomes, mitochondria, and the endoplasmic reticulum. The probes respond uniformly to osmotic shocks applied extracellularly, thus confirming sensitivity toward changes in membrane tension.At rest, different lifetimes found for different organelles relate to known differences in membrane organization rather than membrane tension and allow co-labeling in the same cells. At the organelle scale, lifetime heterogeneity provides unprecedented insights on ER tubules and sheets, and nuclear membranes.Examples on endosomal trafficking or increase of tension at mitochondrial constriction sites outline the potential of intracellularly targeted fluorescent tension probes to address essential questions that were previously beyond reach.The importance of mechanical forces in biological processes is only starting to emerge. 1-3 Plasma membrane tension is a topic of particular current interest because mounting evidence suggests its involvement in regulating various biochemical processes in cells. 2 Although membrane tension should also regulate membranous organelles' functions, standard techniques of force measurements, such as optical tweezers or force microscopes are difficult to apply inside of cells. 3 Therefore, the role of membrane tension in

The target of rapamycin complex 2 (TORC2) plays a key role in maintaining the homeostasis of plasma membrane (PM) tension. TORC2 activation following increased PM tension involves redistribution of the Slm1 and 2 paralogues from PM invaginations known as eisosomes into membrane compartments containing TORC2. How Slm1/2 relocalization is triggered, and if/how this plays a role in TORC2 inactivation with decreased PM tension, is unknown. Using osmotic shocks and palmitoylcarnitine as orthogonal tools to manipulate PM tension, we demonstrate that decreased PM tension triggers spontaneous, energy-independent reorganization of pre-existing phosphatidylinositol-4,5-bisphosphate into discrete invaginated membrane domains, which cluster and inactivate TORC2. These results demonstrate that increased and decreased membrane tension are sensed through different mechanisms, highlighting a role for membrane lipid phase separation in mechanotransduction.

Systematic headgroup engineering yields planarizable push-pull flipper probes that are ready for use in biology - stable, accessible, modifiable -, and affords non-trivial insights into chalcogen-bond mediated mechanophore degradation and fluorescence enhancement.

We report design, synthesis, and evaluation of fluorescent flipper probes for single-molecule super-resolution imaging of membrane tension in living cells. Reversible switching from bright-state ketones to dark-state hydrates, hemiacetals, and hemithioacetals is demonstrated for twisted and planarized mechanophores in solution and membranes. Broadband femtosecond fluorescence up-conversion spectroscopy evinces ultrafast chalcogen-bonding cascade switching in the excited state in solution. According to fluorescence lifetime imaging microscopy, the new flippers image membrane tension in live cells with record red shifts and photostability. Single-molecule localization microscopy with the new tension probes resolves membranes well below the diffraction limit. The imaging of physical forces in living systems is a central challenge in current biology. 1 To contribute solutions, we have introduced small-molecule chemistry tools to image membrane tension in living cells. 2Based on the concept of planarizable push-pull probes, 3,4 the so-called flipper probes allowed us to image the change of plasma 5 and organellar membrane tension in living cells. 6 Flipper probes are twisted dithienothiophene (DTT) push-pull dimers (Figure 1a). Their mechanical planarization in the ground state

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...