Benoit Sorre

Curvature-driven lipid sorting needs proximity to a demixing point and is aided by proteins

Sorting of lipids and proteins is a key process allowing eukaryotic cells to execute efficient and accurate intracellular transport and to maintain membrane homeostasis. It occurs during the formation of highly curved transport intermediates that shuttle between cell compartments. Protein sorting is reasonably well described, but lipid sorting is much less understood. Lipid sorting has been proposed to be mediated by a physical mechanism based on the coupling between membrane composition and high curvature of the transport intermediates. To test this hypothesis, we have performed a combination of fluorescence and force measurements on membrane tubes of controlled diameters pulled from giant unilamellar vesicles. A model based on membrane elasticity and nonideal solution theory has also been developed to explain our results. We quantitatively show, using 2 independent approaches, that a difference in lipid composition can build up between a curved and a noncurved membrane. Importantly, and consistent with our theory, lipid sorting occurs only if the system is close to a demixing point. Remarkably, this process is amplified when even a low fraction of lipids is clustered upon cholera toxin binding. This can be explained by the reduction of the entropic penalty of lipid sorting when some lipids are bound together by the toxin. Our results show that curvature-induced lipid sorting results from the collective behavior of lipids and is even amplified in the presence of lipid-clustering proteins. In addition, they suggest a generic mechanism by which proteins can facilitate lipid segregation in vivo.

A method to recapitulate early embryonic spatial patterning in human embryonic stem cells

Embryos allocate cells to the three germ layers in a spatially ordered sequence. Human embryonic stem cells (hESCs) can generate the three germ layers in culture; however, differentiation is typically heterogeneous and spatially disordered. We show that geometric confinement is sufficient to trigger self-organized patterning in hESCs. In response to BMP4, colonies reproducibly differentiated to an outer trophectoderm-like ring, an inner ectodermal circle and a ring of mesendoderm expressing primitive-streak markers in between. Fates were defined relative to the boundary with a fixed length scale: small colonies corresponded to the outer layers of larger ones. Inhibitory signals limited the range of BMP4 signaling to the colony edge and induced a gradient of Activin-Nodal signaling that patterned mesendodermal fates. These results demonstrate that the intrinsic tendency of stem cells to make patterns can be harnessed by controlling colony geometries and provide a quantitative assay for studying paracrine signaling in early development.

Membrane curvature controls dynamin polymerization

The generation of membrane curvature in intracellular traffic involves many proteins that can curve lipid bilayers. Among these, dynamin-like proteins were shown to deform membranes into tubules, and thus far are the only proteins known to mechanically drive membrane fission. Because dynamin forms a helical coat circling a membrane tubule, its polymerization is thought to be responsible for this membrane deformation. Here we show that the force generated by dynamin polymerization, 18 pN, is sufficient to deform membranes yet can still be counteracted by high membrane tension. Importantly, we observe that at low dynamin concentration, polymer nucleation strongly depends on membrane curvature. This suggests that dynamin may be precisely recruited to membrane buds’ necks because of their high curvature. To understand this curvature dependence, we developed a theory based on the competition between dynamin polymerization and membrane mechanical deformation. This curvature control of dynamin polymerization is predicted for a specific range of concentrations (∼0.1–10 μM), which corresponds to our measurements. More generally, we expect that any protein that binds or self-assembles onto membranes in a curvature-coupled way should behave in a qualitatively similar manner, but with its own specific range of concentration.

Nature of curvature coupling of amphiphysin with membranes depends on its bound density

Cells are populated by a vast array of membrane-binding proteins that execute critical functions. Functions, like signaling and intracellular transport, require the abilities to bind to highly curved membranes and to trigger membrane deformation. Among these proteins is amphiphysin 1, implicated in clathrin-mediated endocytosis. It contains a Bin-Amphiphysin-Rvs membrane-binding domain with an N-terminal amphipathic helix that senses and generates membrane curvature. However, an understanding of the parameters distinguishing these two functions is missing. By pulling a highly curved nanotube of controlled radius from a giant vesicle in a solution containing amphiphysin, we observed that the action of the protein depends directly on its density on the membrane. At low densities of protein on the nearly flat vesicle, the distribution of proteins and the mechanical effects induced are described by a model based on spontaneous curvature induction. The tube radius and force are modified by protein binding but still depend on membrane tension. In the dilute limit, when practically no proteins were present on the vesicle, no mechanical effects were detected, but strong protein enrichment proportional to curvature was seen on the tube. At high densities, the radius is independent of tension and vesicle protein density, resulting from the formation of a scaffold around the tube. As a consequence, the scaling of the force with tension is modified. For the entire density range, protein was enriched on the tube as compared to the vesicle. Our approach shows that the strength of curvature sensing and mechanical effects on the tube depends on the protein density.

Curvature-Driven Lipid Sorting in Biomembranes

It has often been suggested that the high curvature of transport intermediates in cells may be a sufficient means to segregate different lipid populations based on the relative energy costs of forming bent membranes. In this review, we present in vitro experiments that highlight the essential physics of lipid sorting at thermal equilibrium: It is driven by a trade-off between bending energy, mixing entropy, and interactions between species. We collect evidence that lipid sorting depends strongly on lipid-lipid and protein-lipid interactions, and hence on the underlying composition of the membrane and on the presence of bound proteins.

Dynamics of TGF-β signaling reveal adaptive and pulsatile behaviors reflected in the nuclear localization of transcription factor Smad4

The TGF-β pathway plays a vital role in development and disease and regulates transcription through a complex composed of receptor-regulated Smads (R-Smads) and Smad4. Extensive biochemical and genetic studies argue that the pathway is activated through R-Smad phosphorylation; however, the dynamics of signaling remain largely unexplored. We monitored signaling and transcriptional dynamics and found that although R-Smads stably translocate to the nucleus under continuous pathway stimulation, transcription of direct targets is transient. Surprisingly, Smad4 nuclear localization is confined to short pulses that coincide with transcriptional activity. Upon perturbation, the dynamics of transcription correlate with Smad4 nuclear localization rather than with R-Smad activity. In Xenopus embryos, Smad4 shows stereotyped, uncorrelated bursts of nuclear localization, but activated R-Smads are uniform. Thus, R-Smads relay graded information about ligand levels that is integrated with intrinsic temporal control reflected in Smad4 into the active signaling complex.

Lipid cosorting mediated by shiga toxin induced tubulation.

To maintain cell membrane homeostasis, lipids must be dynamically redistributed during the formation of transport intermediates, but the mechanisms driving lipid sorting are not yet fully understood. Lowering sphingolipid concentration can reduce the bending energy of a membrane, and this effect could account for sphingolipid depletion along the retrograde pathway. However, sphingolipids and cholesterol are enriched along the anterograde pathway, implying that other lipid sorting mechanisms, such as protein‐mediated sorting, can dominate. To characterize the influence of protein binding on the lipid composition of highly curved membranes, we studied the interactions of the B‐subunit of Shiga toxin (STxB) with giant unilamellar vesicles containing its glycosphingolipid receptor [globotriaosylceramide (Gb3)]. STxB binding induced the formation of tubular membrane invaginations, and fluorescence microscopy images of these highly curved membranes were consistent with co‐enrichment of Gb3 and sphingolipids. In agreement with theory, sorting was stronger for membrane compositions close to demixing. These results strongly support the hypothesis that proteins can indirectly mediate the sorting of lipids into highly curved transport intermediates via interactions between lipids and the membrane receptor of the protein.

Studying In Vitro Membrane Curvature Recognition by Proteins and its Role in Vesicular Trafficking

In recent years, the interest for proteins that exert key functions in vesicular trafficking through their ability to sense or induce positive membrane curvature has expanded. In this chapter, we first present simple protocols to determine whether a protein targets positively curved membranes with liposomes of well-defined size. Next we describe more sophisticated approaches based on the controlled deformation of giant liposomes. These approaches allow visualization and quantification of protein binding to membrane regions of high curvature by real-time fluorescence microscopy. Last we describe several functional assays to measure how membrane curvature controls the activation state of Arf1 via ArfGAP1 or the asymmetric tethering between flat and curved membranes via the golgin GMAP-210.

Coco is a dual activity modulator of TGFβ signaling

The TGFβ signaling pathway is a crucial regulator of developmental processes and disease. The activity of TGFβ ligands is modulated by various families of soluble inhibitors that interfere with the interactions between ligands and receptors. In an unbiased, genome-wide RNAi screen to identify genes involved in ligand-dependent signaling, we unexpectedly identified the BMP/Activin/Nodal inhibitor Coco as an enhancer of TGFβ1 signaling. Coco synergizes with TGFβ1 in both cell culture and Xenopus explants. Molecularly, Coco binds to TGFβ1 and enhances TGFβ1 binding to its receptor Alk5. Thus, Coco acts as both an inhibitor and an enhancer of signaling depending on the ligand it binds. This finding raises the need for a global reconsideration of the molecular mechanisms regulating TGFβ signaling. Summary: A genome-wide RNAi screen identifies the BMP/Activin/Nodal inhibitor Coco as an enhancer of the binding of TGFβ1 to its receptor Alk5, thus potentiating TGFβ1 signaling in murine cells and Xenopus explants.