Adai Colom

Relaxation of Loaded ESCRT-III Spiral Springs Drives Membrane Deformation

Summary ESCRT-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.

Fluorescent Flippers for Mechanosensitive Membrane Probes

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.

A hybrid high-speed atomic force–optical microscope for visualizing single membrane proteins on eukaryotic cells

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.

High-Speed Atomic Force Microscopy: Cooperative Adhesion and Dynamic Equilibrium of Junctional Microdomain Membrane Proteins

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: Imaging and force spectroscopy

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.

Headgroup engineering in mechanosensitive membrane probes

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.

Nanomechanical Characterization of the Stiffness of Eye Lens Cells: A Pilot Study

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 Youngs 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 Youngs 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 Youngs 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.

Dynamic remodeling of the dynamin helix during membrane constriction

Significance The GTPase dynamin catalyzes membrane fission and is essential in endocytosis and other events such as organelle division. Dynamin is a unique molecular motor with torsional and contractile abilities. Because these abilities involve a conformational change at the whole-polymer level, standard structural biology tools have not been able to fully unravel the mechanism by which it constricts and twists. Here we used high-speed atomic force microscopy to image the constriction and fission of dynamin-coated tubules with subnanometer and subsecond resolution. Our results provide important findings to establish the contribution of the various constriction mechanisms. 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.

Decrease in plasma membrane tension triggers PtdIns(4,5)P 2 phase separation to inactivate TORC2

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.Using a small-molecule modulator of TORC2 signalling and a mechanosensitive probe, Riggi et al. reveal that decreased plasma membrane tension induces distinct PIP2-enriched domains that sequester and inactivate TORC2.

A fluorescent membrane tension probe

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.Lipid membranes—which separate cells and organelles from their environment—experience tension during various cell processes; however, measuring membrane tension is notoriously difficult. Now, a new fluorescent, mechanosensitive membrane probe called FliptR has been developed. FliptR enables simple, direct membrane tension measurements in cellular and artificial membranes.