Three-dimensional visualization of coated vesicle formation in fibroblasts

Fibroblasts apparently ingest low density lipoproteins (LDL) by a selective mechanism of receptor-mediated endocytosis involving the formation of coated vesicles from the plasma membrane. However, it is not known exactly how coated vesicles collect LDL receptors and pinch off from the plasma membrane. In this report, the quick-freeze, deep- etch, rotary-replication method has been applied to fibroblasts; it displays with unusual clarity the coats that appear under the plasma membrane at the start of receptor-mediated endocytosis. These coats appear to be polygonal networks of 7-nm strands or struts arranged into 30-nm polygons, most of which are hexagons but some of which are 5- and 7-sided rings. The proportion of pentagons in each network increases as the coated area of the plasma membrane puckers up from its planar configuration (where the network is mostly hexagons) to its most sharply curved condition as a pinched-off coated vesicle. Coats around the smallest vesicles (which are icosahedrons of hexagons and pentagons) appear only slightly different from "empty coats" purified from homogenized brain, which are less symmetrical baskets containing more pentagons than hexagons. A search for structural intermediates in this coat transformation allows a test of T. Kanaseki and K. Kadota's (1969. J. Cell Biol. 42:202--220.) original idea that an internal rearrangement in this basketwork from hexagons to pentagons could "power" coated vesicle formation. The most noteworthy variations in the typical hexagonal honeycomb are focal juxtapositions of 5- and 7-sided polygons at points of partial contraction and curvature in the basketwork. These appear to precede complete contraction into individual pentagons completely surrounded by hexagons, which is the pattern that characterizes the final spherical baskets around coated vesicles.     

Assembly and packing of clathrin into coats

We present a model for the packing of clathrin molecules into the characteristic hexagons and pentagons covering coated pits and vesicles. The assembly unit is a symmetrical trimer with three extended legs. Polymerization of these units occurs in seconds under suitable conditions, giving empty polyhedral cages resembling the structures around coated vesicles. Images of small, negatively stained fragments of cages, assembled directly on electron microscope grids, reveal details of the structure, which correlate well with the predicted features of the model. There is one clathrin trimer at each polyhedral vertex, and each leg of the trimer extends along two neighboring polyhedral edges. Quasi-equivalent packing in pentagons and hexagons in polyhedra of different sizes requires a variable joint at the vertex of the molecule and a hinge in each leg. The construction of clathrin coats is remarkable for the extended fibrous contacts that each molecule makes with many others. Such contacts may confer mechanical strength combined with flexibility needed when a vesicle is pinched off from the membrane.     

On the structural and functional components of coated vesicles.

Despite the diversity of their known functions, coated vesicles from different tissues contain a rather similar spectrum of proteins, in addition to their major coat protein, clathrin. In particular, each coated vesicle preparation shows a doublet of polypeptide species, on sodium dodecyl sulphate-containing gel electrophoresis, of apparent molecular weight in the region 30,000 to 36,000. Using bullock brain as a source, these molecules are found in association with possible trimers or higher multiples of clathrin, obtained by dissolving coated vesicles in cholate. They may play a structural role relating to the vertices or edges of the lattices of pentagons and hexagons of the polyhedral coats.Purified coated vesicles (e.g. from chicken oocytes) seem to contain relatively small amounts of specific proteins in terms of “contents”. This suggests that the bulk of the isolated particles, especially those in the small size range (500 to 800 Å diam.), may be “empty” of contents, although many still retain a lipid vesicle. These empty structures could represent a pool of recycling coated vesicle components formed after release (possibly from larger vesicles (800 to 1500 Å diam.)) of the specific contents, at their particular destination.     

The preendosomal compartment comprises distinct coated and noncoated endocytic vesicle populations

The transfer of molecules from the cell surface to the early endosomes is mediated by preendosomal vesicles. These vesicles, which have pinched off completely from the plasma membrane but not yet fused with endosomes, form the earliest compartment along the endocytic route. Using a new assay to distinguish between free and cell surface connected vesicle profiles, we have characterized the preedosomal compartment ultrastructurally. Our basic experimental setup was labeling of the entire cell surface at 4 degrees C with Con A-gold, warming of the cells to 37 degrees C to allow endocytosis, followed by replacing incubation medium with fixative, all within either 30 or 60 s. Then the fixed cells were incubated with anti-Con A-HRP to distinguish truly free (gold labeled) endocytic vesicles from surface-connected structures. Finally, analysis of thin (20-30 nm) serial sections and quantification of vesicle diameters were carried out. Based on this approach it is shown that the preendosomal compartment comprises both clathrin-coated and non-coated endocytic vesicles with approximately the same frequency but with distinct diameter distributions, the average noncoated vesicle being smaller (95 nm) than the average coated one (110 nm). In parallel experiments, using an anti-transferrin receptor gold-conjugate as a specific marker for clathrin-dependent endocytosis it is also shown that uncoating of coated vesicles plays only a minor role for the total frequency of noncoated vesicles. Furthermore, after perturbation of clathrin-dependent endocytosis by potassium depletion where uptake of transferrin is blocked, noncoated endocytic vesicles with Con A-gold, but not coated vesicles, exist already after 30 and 60 s. Finally, it is shown that the existence of small, free vesicles in the short-time experiments cannot be ascribed to recycling from the early endosomes.     

Association of the fusion protein NSF with clathrin-coated vesicle membranes.

N-ethylmaleimide-sensitive fusion protein (NSF) is a component of intracellular transport reactions. In order to understand the role of NSF during the fusion of endocytic transport vesicles with the endosome, we have investigated the binding of NSF to purified clathrin-coated vesicle components. First, we have examined whether detergent-solubilized coated vesicle membranes will support formation of NSF-containing 'fusion complexes'. Our results show that these membranes are substantially enriched in components capable of driving formation of these complexes, when compared with membranes from other sources. Secondly, we have analysed coated vesicle preparations for their NSF content. Coated vesicle preparations contain significant amounts of NSF. This was shown to be associated with coated vesicles rather than contaminating membranes by a number of criteria, and was found to be bound in an ATP-independent manner. These findings are discussed in the light of current models for vesicle fusion.     

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.     

The subpopulation of brain coated vesicles that carries synaptic vesicle proteins contains two unique polypeptides

Coated vesicles have been purified in the past on the basis of their remarkably homogeneous structure, not their function. We have succeeded in isolating two subpopulations of bovine brain coated vesicles that carry specific "cargoes," in this case two synaptic vesicle membrane polypeptides (Mr = 95,000 and 65,000). Monoclonal antibodies that recognize cytoplasmic domains of these polypeptides can penetrate the clathrin coat and recognize them on the outer surface of the coated vesicle membrane. An immunoadsorption technique could therefore be used to fractionate coated vesicles on the basis of their membrane composition. The subpopulations have the normal complement of conventional coated vesicle proteins. Exclusive, however, to the subpopulations that carry synaptic vesicle polypeptides are two new coated vesicle polypeptides (Mr = 38,000 and 29,000).     

苜蓿丫纹夜蛾核型多角体病毒囊膜内核衣壳排列图式的电镜观察

核型多角体病毒有单核衣壳包埋型和多核衣壳包埋型之分,单核衣壳包埋型是在一个病毒囊膜内只包含一个核衣壳,而多核衣壳包埋型的特点是在一个病毒囊膜内包含有２个以上的核衣壳,由于多个核衣壳成束地被包装在同一个病毒囊膜内,又称病毒束［１,２］。Ｈｕｎｔｅｒ等表明在干果斑螟核型多角体病毒中,病毒囊膜内包含２～２３个核衣壳［３］。Ｆｒａｓｅｒ将苜蓿丫纹夜蛾核型多角体病毒接种于秋粘虫细胞系,超薄切片电镜观察,病毒囊膜内包含的核衣壳数变动于２～１７粒,但未研究其核衣壳在病毒囊膜内的排列结构［４］。本研究用苜蓿丫纹…     

Statistical mechanical model of the energetics of coated vesicle formation.

Adsorptive endocytosis is mediated by a structural transformation of a two-dimensional hexagonal lattice to a polyhedron or coated vesicle. Clathrin is the structural protein involved in this process. A theoretical model that focuses on the energetics of clathrin in coated vesicle formation is presented. Fisher's cluster model of phase transitions is applied to this problem. The equilibrium constant for the process of converting a large two-dimensional patch to a coated vesicle and a smaller patch is calculated. There are three energetic contributions to be considered. They are the surface energy, the interior or lattice energy, and a loop entropy. Features of the Ising model are introduced into this model by scaling the critical exponents. In determining the configurational partition functions required for equilibrium constant calculations, the polyhedron is represented as a planar graph with no surfaces. The equilibrium constants are extremely sensitive to changes in the lattice and surface energies. The loop entropy contributions favor bimodal distributions in vesicle size under certain conditions. Conditions can also be established where the energy required to form the vesicle is small.     

On the structure of coated vesicles

Electron micrographs of tilted specimens of coated vesicles show that their coats are based on polyhedral lattices constructed from 12 pentagons plus a variable number of hexagons. We have identified three such structures among the smaller particles, two containing 108 molecules of clathrin and a third containing 84. The coats of larger particles are believed to be constructed on similar principles. This polymorphism enables a variety of vesicles to be accommodated in an economical manner.     

Conformational changes of coat proteins during vesicle formation

In coated vesicle formation, coat protein recruitment needs to be spatially and temporally controlled. The coating process involves conformational changes of the coat protein complexes that activate them for interaction with cargo or machinery components and coat polymerization. Here we discuss mechanisms that have emerged recently from studies of the clathrin adaptor and the COPI systems.     

Surface potential and surface charge density of the cerebral-cortex synaptic vesicle and stability of vesicle suspension

Using a microelectrophoresis instrument employing the Lazer-Zee system, the electrophoretic mobility of synaptic vesicles isolated from Guinea-pig brain cortex was measured under several conditions. The mobility was found to depend on both pH and ionic concentration of the solution. The surface of the synaptic vesicle was shown to be negatively charged under physiological conditions. The isoelectric point was observed at pH 4.0 in 0.01 M NaCl solution. Effects of divalent cations were examined and reversal of surface charge was observed in 0.1 M CaCl₂ solution. Interaction of vesicles was also considered on the basis of the DLVO theory of colloid stability by using calculated values of surface charge density and surface potential of the synaptic vesicle.     

Structure and properties of the coated vesicle (H+)-ATPase

Clathrin-coated vesicles play an important role in both receptor-mediated endocytosis and intracellular membrane traffic in eukaryotic cells. The coated vesicle (H⁺)-ATPase functions to provide the acidic environment within endosomes and other intracellular compartments necessary for receptor recycling and intracellular membrane traffic. The coated vesicle (H⁺)-ATPase is composed of nine different subunits which are divided into two distinct domains. The peripheral V₁ domain, which has the structure 73₃:58₃:40₁:34₁:33₁, possesses the nucleotide binding sites of the (H⁺)-ATPase. The integral V₀ domain, which has the composition 100₁:38₁:19₁:17₆, contains the pathway for proton conduction across the membrane. Topographical analysis indicates a structure for the coated vesicle (H⁺)-ATPase very similar to that of the F-type ATPases. Reassembly studies have allowed us to probe the function of particular subunits in this complex and the activity properties of the separate domains. These studies have led to insights into possible mechanisms of regulating vacuolar acidification.     

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.     

Involvement of vesicle coat material in casein secretion and surface regeneration

The ultrastructure of the apical zone of lactating rat mammary epithelial cells was studied with emphasis on vesicle coat structures. Typical 40-60 nm ID "coated vesicles" were abundant, frequently associated with the internal filamentous plasma membrane coat or in direct continuity with secretory vesicles (SV) or plasma membrane proper. Bristle coats partially or totally covered membranes of secretory vesicles identified by their casein micelle content. This coat survived SV isolation. Exocytotic fusion of SV membranes and release of the casein micelles was observed. Frequently, regularly arranged bristle coat structures were identified in those regions of the plasma membrane that were involved in exocytotic processes. Both coated and uncoated surfaces of the casein-containing vesicles, as well as typical "coated vesicles", were frequently associated with microtubules and/or microfilaments. We suggest that coat materials of vesicles are related or identical to components of the internal coat of the surface membrane and that new plasma membrane and associated internal coat is produced concomitantly by fusion and integration of bristle coat moieties. Postexocytotic association of secreted casein micelles with the cell surface, mediated by finely filamentous extensions, provided a marker for the integrated vesicle membrane. An arrangement of SV with the inner surface of the plasma membrane is described which is characterized by regularly spaced, heabily stained membrane to membrane cross-bridges (pre-exocytotic attachment plaques). Such membrane-interconnecting elements may represent a form of coat structure important to recognition and interaction of membrane surfaces.     

Dynamin GTPase domain mutants block endocytic vesicle formation at morphologically distinct stages

Abundant evidence has shown that the GTPase dynamin is required for receptor-mediated endocytosis, but its exact role in endocytic clathrin-coated vesicle formation remains to be established. Whereas dynamin GTPase domain mutants that are defective in GTP binding and hydrolysis are potent dominant-negative inhibitors of receptor-mediated endocytosis, overexpression of dynamin GTPase effector domain (GED) mutants that are selectively defective in assembly-stimulated GTPase-activating protein activity can stimulate the formation of constricted coated pits and receptor-mediated endocytosis. These apparently conflicting results suggest that a complex relationship exists between dynamin's GTPase cycle of binding and hydrolysis and its role in endocytic coated vesicle formation. We sought to explore this complex relationship by generating dynamin GTPase mutants predicted to be defective at distinct stages of its GTPase cycle and examining the structural intermediates that accumulate in cells overexpressing these mutants. We report that the effects of nucleotide-binding domain mutants on dynamin's GTPase cycle in vitro are not as predicted by comparison to other GTPase superfamily members. Specifically, GTP and GDP association was destabilized for each of the GTPase domain mutants we analyzed. Nonetheless, we find that overexpression of dynamin mutants with subtle differences in their GTPase properties can lead to the accumulation of distinct intermediates in endocytic coated vesicle formation.     

Physical aspects of COPI vesicle formation

Abstract

Coat proteins orchestrate membrane budding and molecular sorting during the formation of transport intermediates. Coat protein complex I (COPI) vesicles shuttle between the Golgi apparatus and the endoplasmic reticulum and between Golgi stacks. The formation of a COPI vesicle proceeds in four steps: coat self-assembly, membrane deformation into a bud, fission of the coated vesicle and final disassembly of the coat to ensure recycling of coat components. Although some issues are still actively debated, the molecular mechanisms of COPI vesicle formation are now fairly well understood. In this review, we argue that physical parameters are critical regulators of COPI vesicle formation. We focus on recent real-time in vitro assays highlighting the role of membrane tension, membrane composition, membrane curvature and lipid packing in membrane remodelling and fission by the COPI coat.     

Conical tomography II: A method for the study of cellular organelles in thin sections

We have used conical electron tomography in order to reconstruct neuronal organelles in thin sections of plastic embedded rat somato-sensory cortical tissue. The conical tilt series were collected at a 55 degrees tilt and at 5 degrees rotations, aligned using gold particles as fiduciary markers, and reconstructed using the weighted back projection algorithm. After a refinement process based on projection matching, the 3D maps showed the "unit membrane pattern" along the entire reconstructed volume. This pattern is indicative of the bilayer arrangement of phospholipids in biological membranes. Based on Fourier correlation methods as well as the visualization of the "unit membrane" pattern, we estimated resolutions of approximately 4 nm. To illustrate the prospective advantages of conical tomography, we segmented "coated" vesicles in the reconstructed volumes. These vesicles were comprised of a central core enclosing a small lumen, and a protein "coating" extending into the cytoplasm. The "coated" vesicle was attached to the plasma membrane through a complex structure shaped as an arch where the ends are attached to the membrane and the crook is connected to the vesicle. We concluded that conical electron tomography of thin-sectioned specimens provides a powerful experimental approach for studying thin-sectioned neuronal organelles at resolution levels of approximately 4 nm.     

Enzymatic activities of coated vesicle fractions from bovine and rat cerebrums

The changes in the enzymatic properties of coated vesicle fractions obtained from bovine cerebrums and rat forebrains were investigated as the preparation procedures were modified. Because Mg²⁺-dependent ATPase activity in the coated vesicle fractions that were prepared by conventional centrifugation methods appeared to derive from contaminating particulates, the activity was examined after further purification by column chromatography and confirmed by histochemical technique. Both the coat proteins and the vesicles enclosed in the coat networks failed to show the activity. Since plain synaptic vesicles are known to have ATPase activities, the results may indicate that the membrane structure of synaptic vesicles is modified between the coated vesicle stage and the plain vesicle stage of vesicle recycling as Heuser and Reese proposed (J. Cell Biol.57, 315–344, 1973). The comparison of the specific activity change in ATPase with those of acetylcholinesterase and NADPH-cytochrome c reductase suggested that there were two types of microsomes contaminating coated vesicle fractions that were prepared conventionally.     

Phosphatidylinositol-4,5-bisphosphate is required for endocytic coated vesicle formation

Receptor-mediated endocytosis via clathrin-coated vesicles has been extensively studied and, while many of the protein players have been identified, much remains unknown about the regulation of coat assembly and the mechanisms that drive vesicle formation [1]. Some components of the endocytic machinery interact with inositol polyphosphates and inositol lipids in vitro, implying a role for phosphatidylinositols in vivo [2] and [3]. Specifically, the adaptor protein complex AP2 binds phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P₂), PtdIns(3)P, PtdIns(3,4,5)P₃ and inositol phosphates. Phosphatidylinositol binding regulates AP2 self-assembly and the interactions of AP2 complexes with clathrin and with peptides containing endocytic motifs [4] and [5]. The GTPase dynamin contains a pleckstrin homology (PH) domain that binds PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃ to regulate GTPase activity in vitro [6] and [7]. However, no direct evidence for the involvement of phosphatidylinositols in clathrin-mediated endocytosis exists to date. Using well-characterized PH domains as high affinity and high specificity probes in combination with a perforated cell assay that reconstitutes coated vesicle formation, we provide the first direct evidence that PtdIns(4,5)P₂ is required for both early and late events in endocytic coated vesicle formation.