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Envelope glycoproteins from genetically-divergent virus families comprise fusion peptides (FPs) that have been posited to insert and perturb the membranes of target cells upon activation of the virus-cell fusion reaction. Conserved sequences rich in aromatic residues juxtaposed to the external leaflet of the virion-wrapping membranes are also frequently found in viral fusion glycoproteins. These membrane-proximal external regions (MPERs) have been implicated in the promotion of the viral membrane restructuring event required for fusion to proceed, hence, proposed to comprise supplementary FPs. However, it remains unknown whether the structure-function relationships governing canonical FPs also operate in the mirroring MPER sequences. Here, we combine infrared spectroscopy-based approaches with cryo-electron microscopy to analyze the alternating conformations adopted, and perturbations generated in membranes by CpreTM, a peptide derived from the MPER of the HIV-1 Env glycoprotein. Altogether, our structural and morphological data support a cholesterol-dependent conformational plasticity for this HIV-1 sequence, which could assist cell-virus fusion by destabilizing the viral membrane at the initial stages of the process., This study was supported by the Spanish MCIU (Grants RTI2018-095624-B-C21; MCIU/AEI/FEDER, UE to JLN and BA; and PID2019-111096GA-I00; MCIU/AEI/FEDER, UE to AC) and Basque Government (Grant: IT1196-19).

Sphingolipids 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 human 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, 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 glycosylphosphatidylinositol (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.

During mitochondrial fission, key molecular and cellular factors assemble on the outer mitochondrial membrane, where they coordinate to generate constriction. Constriction sites can eventually divide or reverse upon disassembly of the machinery. However, a role for membrane tension in mitochondrial fission, although speculated, has remained undefined. We capture the dynamics of constricting mitochondria in mammalian cells using live-cell structured illumination microscopy (SIM). By analyzing the diameters of tubules that emerge from mitochondria and implementing a fluorescence lifetime-based mitochondrial membrane tension sensor, we discover that mitochondria are indeed under tension. Under perturbations that reduce mitochondrial tension, constrictions initiate at the same rate, but are less likely to divide. We propose a model based on our estimates of mitochondrial membrane tension and bending energy in living cells which accounts for the observed probability distribution for mitochondrial constrictions to divide., This work was supported in part by the National Centre of Competence in Research Chemical Biology (S. Manley, S. Matile, and A.R.). S. Manley also acknowledges SNSF Project Grant 31003A_182429 (to T.K. and D.M). T.K. received funding from the European Molecular Biology Organization (ALTF-739-2016) and the Munich Cluster for Systems Neurology (SyNergy). A.C. received funding from MCIU, MINECOG19/P66, RYC-18/02, and T1270-19.

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.

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.

Membrane shape is a key feature of many cellular processes, including cell differentiation, division, migration, and trafficking. The development of nanostructured surfaces allowing for the in situ manipulation of membranes in living cells is crucial to understand these processes, but this requires complicated and limited-access technologies. Here, we investigate the self-organization of cellular membranes by using a customizable and bench top method allowing to engineer 1D SiO2 nanopillar arrays of defined sizes and shapes on high-performance glass compatible with advanced microscopies. As a result of this original combination, we provide a mapping of the morphology-induced modulation of the cell membrane mechanics, dynamics and steady-state organization of key protein complexes implicated in cellular trafficking and signal transduction.

Chronic exposure to elevated levels of glucose and free fatty acids impairs beta-cell function, leading to insulin secretion defects and eventually beta-cell failure. Using a semi-high throughput approach applied to INS-1E beta-cells, we tested multiple conditions of chronic exposure to basal, intermediate and high glucose, combined with saturated versus mono- and polyunsaturated fatty acids in order to assess cell integrity, lipid metabolism, mitochondrial function, glucose-stimulated calcium rise and secretory kinetics. INS-1E beta-cells were cultured for 3 days at different glucose concentrations (5.5, 11.1, 25 mM) without or with BSA-complexed 0.4 mM saturated (C16:0 palmitate), monounsaturated (C18:1 oleate) or polyunsaturated (C18:2 linoleate, C18:3 linolenate) fatty acids, resulting in 0.1–0.5 μM unbound fatty acids. Accumulation of triglycerides in cells exposed to fatty acids was glucose-dependent, oleate inducing the strongest lipid storage and protecting against glucose-induced cytotoxicity. The combined chronic exposure to both high glucose and either palmitate or oleate altered mitochondrial function as well as glucose-induced calcium rise. This pattern did not directly translate at the secretory level since palmitate and oleate exhibited distinct effects on the first and the second phases of glucose-stimulated exocytosis. Both fatty acids changed the activity of kinases, such as the MODY-associated BLK. Additionally, chronic exposure to fatty acids modified membrane physicochemical properties by increasing membrane fluidity, oleate exhibiting larger effects compared to palmitate. Chronic fatty acids differentially and specifically exacerbated some of the glucotoxic effects, without promoting cytotoxicity on their own. Each of the tested fatty acids functionally modified INS-1E beta-cell, oleate inducing the strongest effects.

Giant vesicles (GVs) are widely-used model systems for biological membranes. The formulation of these vesicles, however, can be problematic and artifacts, such as degraded molecules or left-over oil, may be present in the final liposomes. The rapid formulation of a high number of artifact-free vesicles of uniform size using standard laboratory equipment is, therefore, highly desirable. Here, the gentle hydration method of glass bead-supported thin lipid films has been enhanced by adding a vortexing step. This led to the formulation of a uniform population of giant vesicles. Batches of glass beads coated with different lipids can be combined to produce vesicles of hybrid lipid compositions. This method represents a stable approach to rapidly generate giant vesicles.

International audience; Dynamin is a dimeric GTPase that assembles into a helix around the neck of endocytic buds. Upon GTP hydrolysis, dynamin breaks these necks, a reaction called membrane fission. Fission requires dynamin to first constrict the membrane. It is unclear, however, how dynamin helix constriction works. Here we undertake a direct high-speed atomic force microscopy imaging analysis to visualize the constriction of single dynamin-coated membrane tubules. We show GTP-induced dynamic rearrangements of the dynamin helix turns: the average distances between turns reduce with GTP hydrolysis. These distances vary, however, over time because helical turns were observed to transiently pair and dissociate. At fission sites, these cycles of association and dissociation were correlated with relative lateral displacement of the turns and constriction. Our findings show relative longitudinal and lateral displacements of helical turns related to constriction. Our work highlights the potential of high-speed atomic force microscopy for the observation of mechanochemical proteins onto membranes during action at almost molecular resolution.

The endosomal sorting complex required for transport (ESCRT) mediates membrane remodelling in cells. When ESCRT oligomerize, it is able to bud the membrane forming constriction necks that will break resulting in vesicular bodies or the viral envelope, to name a few of its implications. So far, relatively little is known about the molecular fine structure and less about the dynamics of ESCRT assembly, essential for our understanding how it deforms and cleaves the membrane.In this work, we used high-speed atomic force microscopy (HS-AFM) to study the ESCRT machinery, in particular the ESCRT-III complex, Snf7. HS-AFM allows simultaneous observation of structure, dynamics and function of biological assemblies, with nanometer spatial and sub-second temporal resolution. We show HS-AFM movies of the Snf7 complex formation and its dynamics from filament to the maturated circular assembly around the membrane constriction site. We observe interfilament dynamics that provide a basis for a mechanistic explanation how the machinery creates tension for membrane fission by a buckling mechanism.

International audience; Keywords: High-speed atomic force microscopy High-speed force spectroscopy Membrane protein Membrane structure Titin Actin cortex a b s t r a c t Atomic force microscopy (AFM) is the type of scanning probe microscopy that is probably best adapted for imaging biological samples in physiological conditions with submolecular lateral and vertical resolution. In addition, AFM is a method of choice to study the mechanical unfolding of proteins or for cellular force spectroscopy. In spite of 28 years of successful use in biological sciences, AFM is far from enjoying the same popularity as electron and fluorescence microscopy. The advent of high-speed atomic force microscopy (HS-AFM), about 10 years ago, has provided unprecedented insights into the dynamics of membrane proteins and molecular machines from the single-molecule to the cellular level. HS-AFM imaging at nanometer-resolution and sub-second frame rate may open novel research fields depicting dynamic events at the single bio-molecule level. As such, HS-AFM is complementary to other structural and cellular biology techniques, and hopefully will gain acceptance from researchers from various fields. In this review we describe some of the most recent reports of dynamic bio-molecular imaging by HS-AFM, as well as the advent of high-speed force spectroscopy (HS-FS) for single protein unfolding.

International audience; High-speed atomic force microscopy is a powerful tool for studying structure and dynamics of proteins. So far, however, high-speed atomic force microscopy was restricted to well-controlled molecular systems of purified proteins. Here we integrate an optical microscopy path into high-speed atomic force microscopy, allowing bright field and fluorescence microscopy, without loss of high-speed atomic force microscopy performance. This hybrid high-speed atomic force microscopy/optical microscopy setup allows positioning of the high-speed atomic force microscopy tip with high spatial precision on an optically identified zone of interest on cells. We present movies at 960 ms per frame displaying aquaporin-0 array and single molecule dynamics in the plasma membrane of intact eye lens cells. This hybrid setup allows high-speed atomic force microscopy imaging on cells about 1,000 times faster than conventional atomic force microscopy/optical microscopy setups, and allows first time visualization of unlabelled membrane proteins on a eukaryotic cell under physiological conditions. This development advances high-speed atomic force microscopy from molecular to cell biology to analyse cellular processes at the membrane such as signalling, infection, transport and diffusion. H igh-speed atomic force microscopy (HS-AFM) 1 has been proven to be a unique and powerful tool for the concomitant analysis of the structure and dynamics of single biomolecules 2. HS-AFM was used to visualize myosin-V walking 3 , cellulase cellulose degradation 4 , F 1-ATPase rotary catalysis 5 , bacteriorhodopsin photocycle 6 , and OmpF diffusion and interaction 7. However, HS-AFM has been restricted so far to well-controlled molecular systems of purified proteins under well-controlled conditions. These systems were characterized by a limited number of pure molecular species with small corrugation, mainly because the fast z-piezo that follows the topography profile has a small extension range (typically 400 nm; ref. 8). Recently, the development of a wide-range HS-AFM scanner allowed the first nanoscale observation of molecular movements at two frames per second on living bacteria 9. Historically, AFM debuted as early as 1990 for cell imaging applications 10 , prompted by the need to perform cell structural analysis at a resolution superior to light microscopy. However, AFM images of cells revealed rather the cell interior, like actin filaments, than the membrane structure of the cells. Later, AFM and optical microscopy (OM) were combined in order to take advantage of OM's large-scale overview imaging capacities and its power to analyse fluorescence signal targets of proteins of interest 11. For this purpose, AFMs were built in a table-top configuration and mounted on inverted optical microscopes 12. In order to avoid modifications of the conventional inverted OM, two types of table-top AFM configurations were built. In one of them, the AFM tip and the scanner is combined into a single moving component, and in the other a large optical microscope sample stage on which the AFM sample is mounted needs to be moved. In both cases, there is a loss of AFM performance. In the tip-scanner configuration, the laser detection must be coupled to the moving cantilever, while for the second case, the sample stage that needs to be moved is complex and heavy. Both types of structures are prone to capture environmental noise and feature innate resonance frequencies. Furthermore, massive objects do not allow sub-millisecond mechanical response, thus precluding individual protein imaging at high resolution and high speed. In this work, we integrate an OM path into our HS-AFM, maintaining the structure of the HS-AFM setup 1 , and hence not compromising HS-AFM performance in terms of speed and resolution. To achieve this, we choose a completely different approach compared with most (if not all) AFM/OM integration developments: instead of building a table-top AFM mountable on an inverted optical microscope, accepting loss of AFM performance, we build an optical path into our HS-AFM setup, accepting minimal trade-offs in OM performance. We show that our setup can acquire bright field and fluorescence OM images of biological samples, and that it allows HS-AFM tip positioning with high precision guided by OM. Finally, we show that it achieves HS-AFM imaging of individual membrane proteins on eukaryotic cells and record their dynamics at an imaging rate of 960 ms per frame. This accomplishment opens the door to a wide range of real-time studies of molecular dynamics in membrane processes on living cells. Results Development of hybrid HS-AFM and fluorescence microscope. In order to integrate an OM path into the HS-AFM (Fig. 1 and Fig. 2a,b), we added and exchanged several elements to and from the HS-AFM setup, previously designed by Ando et al 1. The original laser diode of the HS-AFM system that reads the cantilever position was changed to a far-red superluminescent diode (SLD) with 750 nm wavelength and low coherence (Fig. 1, label 1). The use of the SLD 750 nm allowed the optical separation

International audience; PURPOSE. The purpose of this study is to probe the mechanical properties of individual eye lens cells isolated from nucleus and cortex of adult sheep eye lens, and to characterize the effect of cytoskeletal drugs. METHODS. We used atomic force microscopy (AFM), featuring a spherical tip at the end of a soft cantilever, to indent single lens cells, and measure the Young's modulus of isolated nuclear and cortical lens cells. Measurements were performed under basal conditions, and after addition of drugs that disrupt actin filaments and microtubules. RESULTS. We found that single lens cells were able to maintain their shape and mechanical properties after being isolated from the lens tissue. The median Young's modulus value for nuclear lens cells (4.83 kPa) was ~ 20-fold higher than for cortical lens cells (0.22 kPa). Surprisingly, disruption of actin filaments and microtubules did not affect the measured Young's moduli. CONCLUSIONS. We found that single cells from the lens nucleus and cortex can be distinguished unambiguously using the elastic modulus as a criterion. The uncommon maintenance of shape and elastic properties after cell isolation together with the null effect of actin filaments and microtubules targeting drugs suggest that the mechanical stability of fiber cells is provided by cellular elements other than the usual cytoskeletal proteins. (Invest Ophthalmol Vis Sci.

International audience; Junctional microdomains, paradigm for membrane protein segregation in functional assemblies, in eye lens fiber cell membranes are constituted of lens-specific aquaporin-0 tetramers (AQP0(4)) and connexin (Cx) hexamers, termed connexons. Both proteins have double function to assure nutrition and mediate adhesion of lens cells. Here we use high-speed atomic force microscopy to examine microdomain protein dynamics at the single-molecule level. We found that the adhesion function of head-to-head associated AQP0(4) and Cx is cooperative. This finding provides first experimental evidence for the mechanistic importance for junctional microdomain formation. From the observation of lateral association-dissociation events of AQP0(4), we determine that the enthalpic energy gain of a single AQP0(4)-AQP0(4) interaction in the membrane plane is -2.7 k(B)T, sufficient to drive formation of microdomains. Connexon association is stronger as dynamics are rarely observed, explaining their rim localization in junctional microdomains.

International audience; Beyond structure, the mechanics of plasma membrane components is of key importance to biological function. Nanoscale mechanics is however poorly described due to the lack of suitable experimental tools. Here, we combined atomic force microscopy and nanomechanical mapping to analyze the structure and mechanical properties of native eye lens cell membranes. Lens membranes mainly comprise two proteins; aquaporin 0 and connexin, forming respectively thin and gap intercellular junctions that sustain mechanical stress during accommodation. Our results reveal the mechanical heterogeneity of the plasma membrane, allowing examination of the mechanical nanoenvironment of individual proteins and the flexibility of supramolecular assemblies. The remarkable rigidity of gap junctions suggests their role as stable intercellular adhesion complexes ensuring maintenance of thin junctions, which form more flexible supramolecular complexes capable of sustaining pressure differences between cells. Our work proposes the mechanical properties of individual proteins and protein domains directly related to biological function as a novel molecular signature.