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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.

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.

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.

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

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.

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