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Measuring forces within living cells remains a technical challenge. We developed hydrophobic mechanosensing fluorescent probes called Flippers, whose fluorescence lifetime depends on lipid packing and can report on membrane tension. Here, we describe technical optimization of the probe imaging, and diverse characterizations in various biological and in vitro systems. We provide a guideline to measure biophysical parameters of cellular membranes by FLIM microscopy with Flipper probes, providing evidences that flippers can report long range forces in cells, tissues and organsi.

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.

SUMMARYHow junctional proteins regulate the mechanics of the plasma membrane and how actin and myosin isoforms are selectively localized at epithelial cell-cell junctions is poorly understood. Here we show by atomic force indentation microscopy, immunofluorescence analysis and FLIM membrane tension imaging that the tight junction (TJ) protein cingulin maintains apical surface stiffness and TJ membrane tortuosity and down-regulates apico-lateral membrane tension in MDCK cells. KO of cingulin in MDCK, mCCD and Eph4 cells results in a decrease in the juxta-membrane accumulation of labeling for cytoplasmic myosin-2B (NM2B), γ-actin, phalloidin and ARHGEF18, but no detectable effect on myosin-2A (NM2A) and β-actin. Loss of paracingulin leads to weaker mechanical phenotypes in MDCK cells, correlating with no detectable effect on the junctional accumulation of myosins and actins. Cingulin and paracingulin form biomolecular condensates, bind to the ZU5 domain of ZO-1, and are recruited as clients into ZO-1 condensates in a ZU5-dependent manner. Cingulin binding to ZO-1 promotes the unfolding of ZO-1, as determined by interaction with DbpA in cells lacking ZO-2 and in vitro. Cingulin promotes the accumulation of a pool of ZO-1 at the TJ and is required in a ZU5-dependent manner for the recruitment of phalloidin-labelled actin filaments into ZO-1 condensates, suggesting that ZU5-cingulin interaction promotes ZO-1 interaction with actin filaments. Our results indicate that cingulin tethers the juxta-membrane and apical branched γ-actin-NM2B network to TJ to modulate ZO-1 conformation and the TJ assembly of a pool of ZO-1 and fine-tune the distribution of forces to apical and TJ membranes.

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.

We report the 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 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.

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 its 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 colabeling 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 intra-cellularly targeted fluorescent tension probes to address essential questions that were previously beyond reach.

Dynamin proteins assemble into characteristic helical structures around necks of clathrin-coated membrane buds. Hydrolysis of dynamin-bound GTP results in both fission of the membrane neck and partial disruption of the dynamin oligomer. Imaging by atomic force microscopy reveals that, on GTP hydrolysis, dynamin oligomers undergo a dynamic remodeling and lose their distinctive helical shape. While breakup of the dynamin helix is a critical stage in clathrin-mediated endocytosis, the mechanism for this remodeling of the oligomer has not been resolved. In this paper, we formulate an analytical, elasticity-based model for the reshaping and disassembly of the dynamin scaffold. We predict that the shape of the oligomer is modulated by the orientation of dynamin’s pleckstrin homology (PH) domain relative to the underlying membrane. Our results indicate that tilt of the PH domain drives deformation and fragmentation of the oligomer, in agreement with experimental observations. This model motivated the introduction of the tilted helix: a curve that maintains a fixed angle between its normal and the normal of the embedding surface. Our findings highlight the importance of tilt as a key regulator of size and morphology of membrane-bound oligomers.

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.

Many molecular factors required for mitochondrial division have been identified; however, how they combine to physically trigger division remains unknown. Here, we report that constriction by the division machinery does not ensure mitochondria will divide. Instead, potential division sites accumulate molecular components and can constrict before either dividing, or relaxing back to an unconstricted state. Using time-lapse structured illumination microscopy (SIM), we find that constriction sites with higher local curvatures – reflecting an increased membrane bending energy – are more likely to divide. Furthermore, analyses of mitochondrial motion and shape changes demonstrate that dividing mitochondria are typically under an externally induced membrane tension. This is corroborated by measurements using a newly synthesized fluorescent mitochondrial membrane tension sensor, which reveal that depolymerizing the microtubule network diminishes mitochondrial membrane tension. We find that under reduced tension, the number of constrictions is maintained, but the probability that constrictions will divide is concomitantly reduced. These measurements allow us to establish a physical model, based on in situ estimates of membrane bending energy and tension, which accounts for the observed fates of mitochondrial constriction events.

The Target of Rapamycin Complex 2 (TORC2) plays a key role in maintaining the homeostasis of plasma membrane (PM) tension. TORC2 activation upon increased PM tension involves redistribution of the Slm1 and 2 paralogs 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 upon decreased PM tension is unknown. Using osmotic shocks and Palmitoylcarnitine (PalmC) 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 (PtdIns(4,5)P2) into discrete invaginated membrane domains which cluster and inactivate TORC2. These results demonstrate that an increase and a decrease in membrane tension are sensed through different mechanisms and highlight a role for membrane lipid phase separation in mechanotransduction.

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.

Planarizable and polarizable dithieno[3,2-b;2′,3′-d]thiophene (DTT) dimers have been introduced recently as fluorescent probes that report on membrane fluidity with red shifts in excitation, i.e. planarization in the ground state. In this study, we elaborate on the hypothesis that twisted push-pull probes could perform best in the presence of one unorthodox substituent that acts as a weak acceptor with electron-rich and as a strong donor with electron-poor aromatics. According to Hammett constants, we thought that sulfides could provide access to such a conceptually innovative donor-acceptor switch. To elaborate on this hypothesis, we here describe the design, synthesis and evaluation of a comprehensive series of twisted push-pull probes with turn-on sulfide donors. Their planarization is explored in lipid bilayer membranes of different thickness and fluidity from liquid-disordered to liquid-ordered and solid-ordered phases. Results from membranes are compared to the planarization of turn-on mechanophores in crystals, proteins, and cyclodextrin macrocycles of varied diameter.

Three-dimensional (3D) cultures are increasingly used as tissue surrogates to study many physiopathological processes. However, to what extent current 3D culture protocols provide physiologic oxygen tension conditions remains ill defined. To address this limitation, oxygen tension was measured in a panel of acellular or cellularized extracellular matrix (ECM) gels with A549 cells, and analyzed in terms of oxygen diffusion and consumption. Gels included reconstituted basement membrane, fibrin and collagen. Oxygen diffusivity in acellular gels was up to 40% smaller than that of water, and the lower values were observed in the denser gels. In 3D cultures, physiologic oxygen tension was achieved after 2 days in dense (≥3 mg/mL) but not sparse gels, revealing that the latter gels are not suitable tissue surrogates in terms of oxygen distribution. In dense gels, we observed a dominant effect of ECM composition over density in oxygen consumption. All diffusion and consumption data were used in a simple model to estimate ranges for gel thickness, seeding density and time-window that may support physiologic oxygen tension. Thus, we identified critical variables for oxygen tension in ECM gels, and introduced a model to assess initial values of these variables, which may short-cut the optimization step of 3D culture studies. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 2776–2784, 2014.

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.

International audience; 13 ESCRT-III is required for lipid membrane remodeling in many cellular processes, from abscission 14 to viral budding and multi-vesicular body biogenesis. However, how ESCRT-III polymerization 15 generates membrane curvature remains debated. Here we show that Snf7, the main component 16 of ESCRT-III, polymerizes into spirals at the surface of lipid bilayers. When covering the entire 17 membrane surface, these spirals stopped growing when densely packed: they had a polygonal 18 shape, suggesting that lateral compression could deform them. We reasoned that Snf7 spirals 19 could function as spiral springs. By measuring the polymerization energy and the rigidity of Snf7 20 filaments, we showed that they were deformed while growing in a confined area. Furthermore, 21 we observed that the elastic expansion of compressed Snf7 spirals generated an area difference 22 between the two sides of the membrane and thus curvature. This spring-like activity underlies the 23 driving force by which ESCRT-III could mediate membrane deformation and fission. 24 2

There is growing interest in the development of cell culture assays that enable the rigidity of the extracellular matrix to be increased. A promising approach is based on three-dimensional collagen type I matrices that are stiffened by cross-linking through non-enzymatic glycation with reducing sugars.The present study evaluated the biomechanical changes in the non-enzymatically glycated type I collagen matrices, including collagen organization, the advanced glycation end products formation and stiffness achievement. Gels were glycated with ribose at different concentrations (0, 5, 15, 30 and 240 mM). The viability and the phenotypic changes of primary human lung fibroblasts cultured within the non-enzymatically glycated gels were also evaluated along three consecutive weeks. Statistical tests used for data analyze were Mann-Whitney U, Kruskal Wallis, Student's t-test, two-way ANOVA, multivariate ANOVA, linear regression test and mixed linear model.Our findings indicated that the process of collagen glycation increases the stiffness of the matrices and generates advanced glycation end products in a ribose concentration-dependent manner. Furthermore, we identified optimal ribose concentrations and media conditions for cell viability and growth within the glycated matrices. The microenvironment of this collagen based three-dimensional culture induces α-smooth muscle actin and tenascin-C fibroblast protein expression. Finally, a progressive contractile phenotype cell differentiation was associated with the contraction of these gels.The use of non-enzymatic glycation with a low ribose concentration may provide a suitable model with a mechanic and oxidative modified environment with cells embedded in it, which allowed cell proliferation and induced fibroblast phenotypic changes. Such culture model could be appropriate for investigations of the behavior and phenotypic changes in cells that occur during lung fibrosis as well as for testing different antifibrotic therapies in vitro.

Background There is growing interest in the development of cell culture assays that enable the rigidity of the extracellular matrix to be increased. A promising approach is based on three-dimensional collagen type I matrices that are stiffened by cross-linking through non-enzymatic glycation with reducing sugars. Methods The present study evaluated the biomechanical changes in the non-enzymatically glycated type I collagen matrices, including collagen organization, the advanced glycation end products formation and stiffness achievement. Gels were glycated with ribose at different concentrations (0, 5, 15, 30 and 240 mM). The viability and the phenotypic changes of primary human lung fibroblasts cultured within the non-enzymatically glycated gels were also evaluated along three consecutive weeks. Statistical tests used for data analyze were Mann–Whitney U, Kruskal Wallis, Student’s t-test, two-way ANOVA, multivariate ANOVA, linear regression test and mixed linear model. Results Our findings indicated that the process of collagen glycation increases the stiffness of the matrices and generates advanced glycation end products in a ribose concentration-dependent manner. Furthermore, we identified optimal ribose concentrations and media conditions for cell viability and growth within the glycated matrices. The microenvironment of this collagen based three-dimensional culture induces α-smooth muscle actin and tenascin-C fibroblast protein expression. Finally, a progressive contractile phenotype cell differentiation was associated with the contraction of these gels. Conclusions The use of non-enzymatic glycation with a low ribose concentration may provide a suitable model with a mechanic and oxidative modified environment with cells embedded in it, which allowed cell proliferation and induced fibroblast phenotypic changes. Such culture model could be appropriate for investigations of the behavior and phenotypic changes in cells that occur during lung fibrosis as well as for testing different antifibrotic therapies in vitro. Electronic supplementary material The online version of this article (doi:10.1186/s12931-015-0237-z) contains supplementary material, which is available to authorized users.

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.

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.

To measure the chemical-mechanic states of lipid membranes, once needs various tools, many of which being incompatible with cell biology protocols. Applying lessons from nature, we developed a mechanosensitive fluorescent probe, the twisted dithienothiophene. This push-pull probe, change planarization state in function of his environment, and we have taken full advantage of this mechano-probe potential and we calibrated based on membrane tension, fluidity and different lipid composition by measuring the push-pull fluorescence lifetime. Likewise, we are able to use this fluorescent probe on life cells, for visualize differences between organelles, as well as to distinguish lipids properties among cells cultured on classic plates or in extracellular matrix.

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.

The design of 3D culture studies remains challenging due to the limited understanding of extracellular matrix (ECM)-dependent hindered diffusion and the lack of simple diffusivity assays. To address these limitations, we set up a cost-effective diffusivity assay based on a Transwell plate and the spectrophotometer of a Microplate Reader, which are readily accessible to cell biology groups. The spectrophotometer-based assay was used to assess the apparent diffusivity D of FITC-dextrans with molecular weight (4-70kDa) spanning the physiological range of signaling factors in a panel of acellular ECM gels including Matrigel, fibrin and type I collagen. Despite their technical differences, D data exhibited ∼15% relative difference with respect to FRAP measurements. Our results revealed that diffusion hindrance of small particles is controlled by the enhanced viscosity of the ECM gel in conformance with the Stokes-Einstein equation rather than by geometrical factors. Moreover, we provided a strong rationale that the enhanced ECM viscosity is largely contributed to by unassembled ECM macromolecules. We also reported that gels with the lowest D exhibited diffusion hindrance closest to the large physiologic hindrance of brain tissue, which has a typical pore size much smaller than ECM gels. Conversely, sparse gels (≤1mg/ml), which are extensively used in 3D cultures, failed to reproduce the hindered diffusion of tissues, thereby supporting that dense (but not sparse) ECM gels are suitable tissue surrogates in terms of macromolecular transport. Finally, the consequences of reduced diffusivity in terms of optimizing the design of 3D culture experiments were addressed in detail.

Protein-protein interactions are of major importance for biological function. Many proteins are oligomers and interact temporaly with other proteins. This is of particular importance in the membrane, where multiprotein assemblies act in important processes like signaling, respiration, photosynthesis, etc. High-speed atomic force microscopy (HS-AFM,[1]) offers unique possibilities to study protein-protein interactions in the membrane. HS-AFM allows not only visualization and tracking of single proteins but also their environment, hence describing the interaction energy between proteins (Fig.1A,[2]), their dynamic supramolecular assemblies (Fig.1B,[3]), and membrane crowding and interaction specificity (Fig.1C, [4]). The interaction profiles can be, though at different time scales, compared to molecular simulations [4]. The perspective of short-cantilever HS-AFM in force spectroscopy will be discussed: the high speed of short cantilevers allows measuring interaction forces at unprecedented loading rates and temporal resolution (Fig.1D).References[1] Ando, PNAS 98 (22):12468-12472 (2001).[2] Casuso, BiophysJ 99 (7):47-49 (2010).[3] Colom, JMB 423 (2):249-256 (2012)[4] Casuso, Nat Nanotechnol 7 (8):525-529 (2012).Fig. 1) Membrane protein interactions by HS-AFM. (a) Dimer interaction of ATP-synthase c-rings, (b) AQP0 array association/dissociation in eye lens membranes, (c) OmpF diffusion and interactions, (d) HS-AFM based force spectrocopy at 1MHz temporal resolution.View Large Image | View Hi-Res Image | Download PowerPoint Slide

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.

High-speed atomic force microscopy (HS-AFM, [1]) offers unique novel possibilities to study single molecule dynamics [2,3]. Here we present two HS-AFM developments, first the integration of optical microscopy into HS-AFM for imaging membrane proteins on cells, and second high-speed force spectroscopy (HS-FS) for fast protein unfolding studies:HS-AFM is a powerful tool for studying structure and dynamics of proteins. So far, however, HS-AFM was restricted to well-controlled molecular systems. Here, we integrate optical microscopy (OM) into HS-AFM, allowing bright field and fluorescence microscopy, without loss of HS-AFM performance. This hybrid HS-AFM/OM setup [4] allows positioning the HS-AFM tip on an optically identified zone of interest on cells. We present movies at 960ms per frame displaying aquaporin-0 array and single molecule dynamics on intact eye lens cells. This hybrid setup allows HS-AFM imaging on cells ∼1000 times faster than conventional AFM/OM setups, and first time visualization of unlabeled membrane proteins on a eukaryotic cell under physiological conditions.The mechanical unfolding of muscle protein titin by AFM is a landmark experiment in single molecule biophysics. Molecular dynamics simulations offered an atomic level mechanistic description of the process. However, experiment and simulation differ in pulling velocity by orders of magnitude. We have developed HS-FS [5] to unfold titin at velocities reached by simulation (∼4 mm/s). Our results show that an intermediate state in a small βeta-strand pair dynamically unfolds and refolds, buffering pulling forces up to ∼100pN. Furthermore, our data indicates that the distance to the transition state of domain unfolding is much larger than previously estimated, but in better agreement with atomistic predictions.[1] Ando, et al., PNAS,2001,98(22):12468-12472.[2] Kodera, et al., Nature,2010,468(7320):72-76.[3] Casuso, et al., Nature Nanotechnology,2012,7(8):525-529.[4] Colom, et al., Nature Communications,2013,4:DOI:10.1038/ncomms3155.[5] Rico, et al., submitted,2013.

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.

Junctional microdomains are specialized membrane protein assemblies in eye lens fiber cell membranes. They are constituted of lens-specific aquaporin-0 (AQP0) and connexins (Cx) that assure nutrition and adhesion of lens cells, mutation or absence of these proteins lead to cataract. In a more general term, junctional microdomains are paradigm for membrane protein segregation in functional assemblies in a membrane with non-random superstructure. Here we use high-speed atomic force microscopy (HS-AFM) and Monte Carlo simulation to analyze the dynamics and assembly of membrane proteins in junctional microdomains. We report cooperative adhesion of head-to-head attached AQP0 and Cx in junctional microdomains. Furthermore we evidence that enthalpy energy gain of protein association dominates entropy leading to square shaped AQP0 arrays of finite size segregated from and edged by connexins. The power of HS-AFM sample manipulation and imaging structure and dynamics at single-molecule resolution is highlighted opening a new avenue of membrane research watching the behavior of unlabelled molecules while also seeing their molecular environment.HS-AFM movie frames showing native AQP0 arrays assembling (A-E) and disassembling (E-H).View Large Image | View Hi-Res Image | Download PowerPoint Slide