Biophysical approaches to protein-induced membrane deformations in trafficking

Membrane traffic requires membrane deformation to generate vesicles and tubules. Strong evidence suggests that assembly of curvature-active proteins can drive such membrane shape changes. Well-documented pathways often involve protein scaffolds, in particular coats (clathrin or COP). However, membrane curvature should, in principle, be influenced by any protein binding asymmetrically on a membrane; large membrane morphological changes could result from their aggregation. In the case of Shiga toxin or viral matrix proteins, tubules and buds appear to result from the cargo-driven formation of protein-lipid nanodomains, showing that collective protein behaviour is crucial in the process. We argue here that a combination of in vitro experiments on giant unilamellar vesicles and theoretical modelling based on statistical physics is ideally suited to tackle these collective effects.     

Clathrin senses membrane curvature

The ability of proteins to assemble at sites of high membrane curvature is essential to diverse membrane remodeling processes, including clathrin-mediated endocytosis. Multiple adaptor proteins within the clathrin pathway have been shown to sense regions of high membrane curvature, leading to local recruitment of the clathrin coat. Because clathrin triskelia do not bind to the membrane directly, it has remained unclear whether the clathrin coat plays an active role in sensing membrane curvature or is passively recruited by adaptor proteins. Using a synthetic tag to assemble clathrin directly on membrane surfaces, here we show that clathrin is a strong sensor of membrane curvature, comparable with previously studied adaptor proteins. Interestingly, this sensitivity arises from clathrin assembly rather than from the properties of unassembled triskelia, suggesting that triskelia have preferred angles of interaction, as predicted by earlier structural data. Furthermore, when clathrin is recruited by adaptors, its curvature sensitivity is amplified by 2- to 10-fold, such that the resulting protein complex is up to 100 times more likely to assemble on a highly curved surface compared with a flatter one. This exquisite sensitivity points to a synergistic relationship between the coat and its adaptor proteins, which enables clathrin to pinpoint sites of high membrane curvature, an essential step in ensuring robust membrane traffic. More broadly, these findings suggest that protein networks, rather than individual protein domains, are likely the most potent drivers of membrane curvature sensing.     

Effect of Clathrin Light Chains on the Stiffness of Clathrin Lattices and Membrane Budding

Clathrin‐dependent transport processes require the polymerization of clathrin triskelia into polygonal scaffolds. Together with adapter proteins, clathrin collects cargo and induces membrane bud formation. It is not known to what extent clathrin light chains affect the structural and functional properties of clathrin lattices and the ability of clathrin to deform membranes. To address these issues, we have developed a novel procedure for analyzing clathrin lattice formation on rigid surfaces. We found that lattices can form on adaptor‐coated convex‐, planar‐ and even shallow concave surfaces, but the rate of formation and resistance to thermal dissociation of the lattice are greatly enhanced on convex surfaces. Atomic force microscopy on planar clathrin lattices demonstrates that the stiffness of the clathrin lattice is strictly dependent on light chains. The reduced stiffness of the lattice also compromised the ability of clathrin to generate coated buds on the surface of rigid liposomal membranes.      

Membrane Curvature Sensing by Amphipathic Helices: Insights from Implicit Membrane Modeling

Sensing and generation of lipid membrane curvature, mediated by the binding of specific proteins onto the membrane surface, play crucial roles in cell biology. A number of mechanisms have been proposed, but the molecular understanding of these processes is incomplete. All-atom molecular dynamics simulations have offered valuable insights but are extremely demanding computationally. Implicit membrane simulations could provide a viable alternative, but current models apply only to planar membranes. In this work, the implicit membrane model 1 is extended to spherical and tubular membranes. The geometric change from planar to curved shapes is straightforward but insufficient for capturing the full curvature effect, which includes changes in lipid packing. Here, these packing effects are taken into account via the lateral pressure profile. The extended implicit membrane model 1 is tested on the wild-types and mutants of the antimicrobial peptide magainin, the ALPS motif of arfgap1, α-synuclein, and an ENTH domain. In these systems, the model is in qualitative agreement with experiments. We confirm that favorable electrostatic interactions tend to weaken curvature sensitivity in the presence of strong hydrophobic interactions but may actually have a positive effect when those are weak. We also find that binding to vesicles is more favorable than binding to tubes of the same diameter and that the long helix of α-synuclein tends to orient along the axis of tubes, whereas shorter helices tend to orient perpendicular to it. Adoption of a specific orientation could provide a mechanism for coupling protein oligomerization to tubule formation.     

Bending membranes

It is widely assumed that peripheral membrane proteins induce intracellular membrane curvature by the asymmetric insertion of a protein segment into the lipid bilayer, or by imposing shape by adhesion of a curved protein domain to the membrane surface. Two papers now provide convincing evidence challenging these views. The first shows that specific assembly of a clathrin protein scaffold, coupled to the membrane, seems to be the most prevalent mechanism for bending a lipid bilayer in a cell. The second reports that membrane crowding, driven by protein-protein interactions, can also drive membrane bending, even in the absence of any protein insertion into the bilayer.     

Effects of cytoplasmic acidification on clathrin lattice morphology

Reducing the internal pH of cultured cells by several different protocols that block endocytosis is found to alter the structure of clathrin lattices on the inside of the plasma membrane. Lattices curve inward until they become almost spherical yet remain stubbornly attached to the membrane. Also, the lattices bloom empty "microcages" of clathrin around their edges. Correspondingly, broken-open cells bathed in acidified media demonstrate similar changes in clathrin lattices. Acidification accentuates the normal tendency of lattices to round up in vitro and also stimulates them to nucleate microcage formation from pure solutions of clathrin. On the other hand, several conditions that also inhibit endocytosis have been found to create, instead of unusually curved clathrin lattices with extraneous microcages, a preponderance of unusually flat lattices. These treatments include pH-"clamping" cells at neutrality with nigericin, swelling cells with hypotonic media, and sticking cells to the surface of a culture dish with soluble polylysine. Again, the unusually flat lattices in such cells display a tendency to round up and to nucleate clathrin microcage formation during subsequent in vitro acidification. This indicates that regardless of the initial curvature of clathrin lattices, they all display an ability to grow and increase their curvature in vitro, and this is enhanced by lowering ambient pH. Possibly, clathrin lattice growth and curvature in vivo may also be stimulated by a local drop in pH around clusters of membrane receptors.     

Clathrin exchange during clathrin-mediated endocytosis

During clathrin-mediated endocytosis, clathrin-coated pits invaginate to form clathrin-coated vesicles (CVs). Since clathrin-coated pits are planar structures, whereas CVs are spherical, there must be a structural rearrangement of clathrin as invagination occurs. This could occur through simple addition of clathrin triskelions to the edges of growing clathrin-coated pits with very little exchange occurring between clathrin in the pits and free clathrin in the cytosol, or it could occur through large scale exchange of free and bound clathrin. In the present study, we investigated this question by studying clathrin exchange both in vitro and in vivo. We found that in vitro clathrin in CVs and clathrin baskets do not exchange with free clathrin even in the presence of Hsc70 and ATP where partial uncoating occurs. However, surprisingly FRAP studies on clathrin-coated pits labeled with green fluorescent protein-clathrin light chains in HeLa cells show that even when endocytosis is blocked by expression of a dynamin mutant or depletion of cholesterol from the membrane, replacement of photobleached clathrin in coated pits on the membrane occurs at almost the same rate and magnitude as when endocytosis is occurring. Furthermore, very little of this replacement is due to dissolution of old pits and reformation of new ones; rather, it is caused by a rapid ATP-dependent exchange of clathrin in the pits with free clathrin in the cytosol. On the other hand, consistent with the in vitro data both potassium depletion and hypertonic sucrose, which have been reported to transform clathrin-coated pits into clathrin cages just below the surface of the plasma membrane, not only block endocytosis but also block exchange of clathrin. Taken together, these data show that ATP-dependent exchange of free and bound clathrin is a fundamental property of clathrin-coated pits, but not clathrin baskets, and may be involved in a structural rearrangement of clathrin as clathrin-coated pits invaginate.     

Spatial Control of Epsin-induced Clathrin Assembly by Membrane Curvature

Epsins belong to the family of highly conserved clathrin-associated sorting proteins that are indispensable for clathrin-mediated endocytosis, but their precise functions remain unclear. We have developed an assay system of budded supported membrane tubes displaying planar and highly curved membrane surfaces to analyze intrinsic membrane curvature preference shown by clathrin-associated sorting proteins. Using real-time fluorescence microscopy, we find that epsin preferentially partitions to and assembles clathrin on highly curved membrane surfaces. Sorting of epsin to regions of high curvature strictly depends on binding to phosphatidylinositol 4,5-bisphosphate. Fluorescently labeled clathrins rapidly assemble as foci, which in turn cluster epsin, while maintaining tube integrity. Clathrin foci grow in intensity with a typical time constant of ∼75 s, similar to the time scales for coated pit formation seen in cells. Epsin therefore effectively senses membrane curvature to spatially control clathrin assembly. Our results highlight the potential role of membrane curvature in orchestrating the myriad molecular interactions necessary for the success of clathrin-mediated membrane budding.     

Minimal Mesoscale Model for Protein-Mediated Vesiculation in Clathrin-Dependent Endocytosis

In eukaryotic cells, the internalization of extracellular cargo via the endocytic machinery is an important regulatory process required for many essential cellular functions. The role of cooperative protein-protein and protein-membrane interactions in the ubiquitous endocytic pathway in mammalian cells, namely the clathrin-dependent endocytosis, remains unresolved. We employ the Helfrich membrane Hamiltonian together with surface evolution methodology to address how the shapes and energetics of vesicular-bud formation in a planar membrane are stabilized by presence of the clathrin-coat assembly. Our results identify a unique dual role for the tubulating protein epsin: multiple epsins localized spatially and orientationally collectively play the role of a curvature inducing capsid; in addition, epsin serves the role of an adapter in binding the clathrin coat to the membrane. Our results also suggest an important role for the clathrin lattice, namely in the spatial- and orientational-templating of epsins. We suggest that there exists a critical size of the coat above which a vesicular bud with a constricted neck resembling a mature vesicle is stabilized. Based on the observed strong dependence of the vesicle diameter on the bending rigidity, we suggest that the variability in bending stiffness due to variations in membrane composition with cell type can explain the experimentally observed variability on the size of clathrin-coated vesicles, which typically range 50–100 nm. Our model also provides estimates for the number of epsins involved in stabilizing a coated vesicle, and without any direct fitting reproduces the experimentally observed shapes of vesicular intermediates as well as their probability distributions quantitatively, in wildtype as well as CLAP IgG injected neuronal cell experiments. We have presented a minimal mesoscale model which quantitatively explains several experimental observations on the process of vesicle nucleation induced by the clathrin-coated assembly prior to vesicle scission in clathrin dependent endocytosis.     

Membrane-Mediated Interaction between Strongly Anisotropic Protein Scaffolds

Specialized proteins serve as scaffolds sculpting strongly curved membranes of intracellular organelles. Effective membrane shaping requires segregation of these proteins into domains and is, therefore, critically dependent on the protein-protein interaction. Interactions mediated by membrane elastic deformations have been extensively analyzed within approximations of large inter-protein distances, small extents of the protein-mediated membrane bending and small deviations of the protein shapes from isotropic spherical segments. At the same time, important classes of the realistic membrane-shaping proteins have strongly elongated shapes with large and highly anisotropic curvature. Here we investigated, computationally, the membrane mediated interaction between proteins or protein oligomers representing membrane scaffolds with strongly anisotropic curvature, and addressed, quantitatively, a specific case of the scaffold geometrical parameters characterizing BAR domains, which are crucial for membrane shaping in endocytosis. In addition to the previously analyzed contributions to the interaction, we considered a repulsive force stemming from the entropy of the scaffold orientation. We computed this interaction to be of the same order of magnitude as the well-known attractive force related to the entropy of membrane undulations. We demonstrated the scaffold shape anisotropy to cause a mutual aligning of the scaffolds and to generate a strong attractive interaction bringing the scaffolds close to each other to equilibrium distances much smaller than the scaffold size. We computed the energy of interaction between scaffolds of a realistic geometry to constitute tens of k_BT, which guarantees a robust segregation of the scaffolds into domains.     

Complete reconstitution of clathrin basket formation with recombinant protein fragments: adaptor control of clathrin self-assembly

Clathrin polymerization into a polyhedral basket, surrounding budding membrane vesicles, mediates protein sorting during endocytosis and organelle biogenesis. Adaptor proteins target clathrin assembly to specific membrane sites and sequester receptors into the clathrin coat. We have reconstituted complete clathrin basket formation from recombinantly expressed fragments of clathrin and adaptors. This reconstitution reveals a hierarchy of clathrin self-assembly interactions and demonstrates that adaptors control basket formation by alignment of the distal domains of the clathrin triskelion leg through their binding to the terminal domain.     

An AP-1/clathrin coat plays a novel and essential role in forming the Weibel-Palade bodies of endothelial cells

Clathrin provides an external scaffold to form small 50-100-nm transport vesicles. In contrast, formation of much larger dense-cored secretory granules is driven by selective aggregation of internal cargo at the trans-Golgi network; the only known role of clathrin in dense-cored secretory granules formation is to remove missorted proteins by small, coated vesicles during maturation of these spherical organelles. The formation of Weibel-Palade bodies (WPBs) is also cargo driven, but these are cigar-shaped organelles up to 5 mum long. We hypothesized that a cytoplasmic coat might be required to make these very different structures, and we found that new and forming WPBs are extensively, sometimes completely, coated. Overexpression of an AP-180 truncation mutant that prevents clathrin coat formation or reduced AP-1 expression by small interfering RNA both block WPB formation. We propose that, in contrast to other secretory granules, cargo aggregation alone is not sufficient to form immature WPBs and that an external scaffold that contains AP-1 and clathrin is essential.     

Chirality-induced budding: a raft-mediated mechanism for endocytosis and morphology of caveolae?

The formation of transport carriers (spherical vesicles and tubules) involves membrane budding, growth, and ultimately fission. We propose a mechanism of membrane budding, wherein the tilt and chirality of constituent molecules, confined to a patch of area A, induces buds of approximately 50-100 nm that are comparable to vesicles involved in endocytosis. Because such chiral and tilted lipid molecules are likely to exist in "rafts", we suggest the involvement of this mechanism in generating membrane buds in the clathrin and dynamin-independent, raft-component mediated endocytosis of glycosylphosphatidylinositol-anchored proteins. We argue that caveolae, permanent cell surface structures with characteristic morphology and enriched in raft constituents, are also likely to be formed by this mechanism. Thus, molecular chirality and tilt, and its expression over large spatial scales may be a common organizing principle in membrane budding of transport carriers.     

A new type of coated vesicular carrier that appears not to contain clathrin: its possible role in protein transport within the Golgi stack

Isolated Golgi membranes incubated in the presence of ATP and a cytosolic protein fraction form a population of coated buds or vesicles from the Golgi cisternae. The coats do not have the characteristic hexagonal-pentagonal basketwork of clathrin, and do not react with anti-clathrin polyclonal antibody. The conditions that produce these apparently nonclathrin-coated buds also reconstitute protein transport between compartments of the Golgi stack. The membrane of the buds contains the glycoprotein in transit through these Golgi stacks (VSV-encoded G protein). This suggests that protein transport through the Golgi stack is mediated by a new type of coated vesicle that does not contain clathrin. The concentration of G protein in the coated buds reflects the local concentration of G protein in the cisternae, raising the possibility that the Golgi coated vesicles may be "bulk" membrane carriers.     

Spontaneous-curvature theory of clathrin-coated membranes.

Clathrin-coated membranes are precursors to coated vesicles in the receptor-mediated endocytic pathway. In this paper we present a physical model for the first steps of the transformation of a clathrin-coated membrane into a coated vesicle. The theory is based on in vitro cytoplasmic acidification experiments of Heuser (J. Cell Biol. 108:401-411) that suggest the transformation proceeds by changes in the chemical environment of the clathrin lattice, wherein the chemical environment determines the amount of intrinsic, or spontaneous, curvature of the network. We show that a necessary step of the transformation, formation of free pentagons in the clathrin network, can proceed via dislocation unbinding, driven by changes in the spontaneous curvature. Dislocation unbinding is shown to favor formation of coated vesicles that are quite small compared to those predicted by the current continuum theories, which do not include the topology of the clathrin lattice.     

Microtubule Motors Power Plasma Membrane Tubulation in Clathrin‐Independent Endocytosis

How the plasma membrane is bent to accommodate clathrin‐independent endocytosis remains uncertain. Recent studies suggest Shiga and cholera toxin induce membrane curvature required for their uptake into clathrin‐independent carriers by binding and cross‐linking multiple copies of their glycosphingolipid receptors on the plasma membrane. But it remains unclear if toxin‐induced sphingolipid crosslinking provides sufficient mechanical force for deforming the plasma membrane, or if host cell factors also contribute to this process. To test this, we imaged the uptake of cholera toxin B‐subunit into surface‐derived tubular invaginations. We found that cholera toxin mutants that bind to only one glycosphingolipid receptor accumulated in tubules, and that toxin binding was entirely dispensable for membrane tubulations to form. Unexpectedly, the driving force for tubule extension was supplied by the combination of microtubules, dynein and dynactin, thus defining a novel mechanism for generating membrane curvature during clathrin‐independent endocytosis.      

Potassium-dependent assembly of coated pits: new coated pits form as planar clathrin lattices

Previous studies have shown that when human fibroblasts are depleted of intracellular K+, coated pits disappear from the cell surface and the receptor-mediated endocytosis of low density lipoprotein (LDL) is inhibited. We have now used the K+ depletion protocol to study several aspects of coated pit function. First, since coated pits rapidly form when K+-depleted fibroblasts are incubated in the presence of 10 mM KCl, we studied the sequence of assembly of coated pits as visualized in carbon-platinum replicas of inner membrane surfaces from cells that had been incubated in the presence of K+ for various times. New coated pits initially appeared as planar clathrin lattices that increased in size by the formation of polygons at the margin of the lattice. Once the lattice reached a critical size it invaginated to form coated vesicles. Second, we determined that LDL-ferritin can induce clustering of LDL receptors over noncoated membrane on the surface of K+-depleted fibroblasts; however, when these cells are subsequently incubated in the presence of K+, these clusters become associated with newly formed coated pits and are internalized. Finally, we determined that K+ depletion inhibits the assembly of coated pits, but that existing coated pits in K+-depleted cells are able to internalize LDL. These results suggest that the clathrin lattice of coated pits is actively involved in membrane shape change during endocytosis and that the structural proteins of the lattice are cyclically assembled and disassembled in the process.     

Molecular dynamics simulations of discoidal bilayers assembled from truncated human lipoproteins

Human apolipoprotein A-1 (apo A-1) is the major protein component of high-density lipoproteins. The apo A-1 lipid-binding domain was used as a template for the synthesis of amphipathic helical proteins termed membrane scaffold proteins, employed to self-assemble soluble monodisperse discoidal particles called Nanodiscs. In these particles, membrane scaffold proteins surround a lipid bilayer in a belt-like fashion forming bilayer disks of discrete size and composition. Here we investigate the structure of Nanodiscs through molecular dynamics simulations in which Nanodiscs were built from scaffold proteins of various lengths. The simulations showed planar or deformed Nanodiscs depending on optimal length and alignment of the scaffold proteins. Based on mean surface area per lipid calculations, comparison of small-angle x-ray scattering curves, and the relatively planar shape of Nanodiscs made from truncated scaffold proteins, one can conclude that the first 17 to 18 residues of the 200-residue apo A-1 lipid-binding domain are not involved in formation of the protein "belts" surrounding the lipid bilayer. To determine whether the addition of an integral membrane protein has an effect on the overall structure of a Nanodisc, bacteriorhodopsin was embedded into a Nanodisc and simulated using molecular dynamics, revealing a planar disk with a slightly rectangular shape.     

Membrane deformation by protein coats

Protein coats deform lipid membranes into spherical buds, which undergo fission at the neck to become vesicles. To induce membrane curvature, protein coats use basic tools including amphipathic helices and concave protein surfaces, and take advantage of the bulk properties of cellular membranes, such as loose lipid packing in the endoplasmic reticulum and cis-Golgi and the abundance of anionic lipids in the cytosolic leaflet of the plasma membrane. Protein scaffolds, sensors of membrane curvature and finely tuned reactions such as GTP hydrolysis permit the spatial and temporal organization of these tools, making protein coats self-organized molecular machines. Because biological membranes generally adhere to a cytoskeleton, the functioning of protein coats is coupled to other large remodeling events at the membrane interface.