Model for transformations of the clathrin lattice in the coated vesicle pathway

Transport of receptors by the coated vesicle pathway entails assembly of clathrin triskelions into a lattice in conjunction with receptors in a membrane. The processes by which the receptors are concentrated, the lattice is assembled, transformed into a cage during vesiculation, and subsequently removed from pinched off vesicles are not understood in regard to mechanism, energetics or control. Tubulin and actin assembly are looked to for analogies applicable to clathrin. The present model supposes that clathrin assembly is energy linked and can be described by kinetic equations of the same general form as those for treadmilling in linear polymers. The coat lattice assembles in a steady state involving the degradation of a high energy form of the clathrin triskelions. Diffuse endocytosis receptors are assumed to be associated with individual triskelions and to be able to trigger clustering and coated pit formation by influencing the assembly kinetics of the bound triskelions. A generalization of the treadmilling scheme is proposed by which the kinetic parameters associated with clathrin polymerization can shift simultaneously for an entire lattice to favor alternatively net assembly or disassembly. This shift is effected by a coordinated conversion of the lattice bound receptors. The conversion of the receptors in turn depends on some global property of the membrane compartments (arguably pH, calcium concentration or transmembrane voltage) which is likely to change as a consequence of vesiculation. Thereby, lattice disassembly can be coordinated with the topological conversion from coated pit to coated vesicle.     

Structure determination of clathrin coats to subnanometer resolution by single particle cryo-electron microscopy

Clathrin triskelions can assemble into lattices of different shapes, sizes and symmetries. For many years, the structures of clathrin lattices have been studied by single particle cryo-electron microscopy, which probed the architecture of the D6 hexagonal barrel clathrin coat at the molecular level. By introducing additional image processing steps we have recently produced a density map for the D6 barrel clathrin coat at subnanometer resolution, enabling us to generate an atomic model for this lattice [Fotin, A., Cheng, Y., Sliz, P., Grigorieff, N., Harrison, S.C., Kirchhausen, T., Walz, T., 2004. Molecular model for a complete clathrin lattice from electron cryomicroscopy. Nature 432, 573-579]. We describe in detail here the image processing steps that we have added to produce a density map at this high resolution. These procedures should be generally applicable and may thus help determine the structures of other large protein assemblies to higher resolution by single particle cryo-electron microscopy.     

Free clathrin triskelions are required for the stability of clathrin-associated adaptor protein (AP-2) coated pit nucleation sites.

In this study image correlation spectroscopy was used to demonstrate the presence of two populations of clathrin in situ, on intact cells. In the periphery of the cell approximately 35% of the clathrin triskelions are free within the cytosol while approximately 65% are in large aggregates, presumably coated pits. Although endocytosis is inhibited at low temperature, free clathrin triskelions are still present and small AP-2 aggregates (of approximately 20 proteins), or coated pit nucleation sites, are still observed. Following hypertonic treatment, or cytoplasmic acidification, free clathrin triskelions within the cytosol are depleted and all of the clathrin becomes associated with the membrane. Under these conditions coated pit associated AP-2 remains while the smaller AP-2 aggregates, or coated pit nucleation sites, dissociate. This indicates that the stabilization of AP-2 coated pit nucleation sites requires the presence of free clathrin triskelions within the cytosol. Furthermore, this indicates that free clathrin is required for the early stages of coated pit formation and presumably the continuation of the clathrin-mediated endocytic process. We also provide indirect evidence that AP-2 binding to the membrane in coated pit nucleation sites may be regulated in part by binding to internalization-competent membrane receptors.     

Energetics of clathrin basket assembly

A minimal thermodynamic model is used to study the in vitro equilibrium assembly of reconstituted clathrin baskets. The model contains parameters accounting for i) the combined bending and flexing rigidities of triskelion legs and hubs, ii) the intrinsic curvature of an isolated triskelion, and iii) the free energy changes associated with interactions between legs of neighboring triskelions. Analytical expressions for basket size distributions are derived, and published size distribution data (Zaremba S, Keen JH. J Cell Biol 1983;97: 1339–1347) are then used to provide estimates for net total basket assembly energies. Results suggest that energies involved in adding triskelions to partially formed clathrin lattices are small (of the order of kBT), in accord with the notion that lattice remodeling during basket formation occurs as a result of thermodynamic fluctuations. In addition, analysis of data showing the effects of assembly proteins (APs) on basket size indicates that the binding of APs increases the intrinsic curvature of an elemental triskelial subunit, the stabilizing energy of leg interactions, and the effective leg/hub rigidity. Values of effective triskelial rigidity determined in this investigation are similar to those estimated by previous analysis of shape fluctuations of isolated triskelia.     

The appendage domain of the AP-2 subunit is not required for assembly or invagination of clathrin-coated pits

Coated pits contain a resident membrane molecule(s) that binds clathrin AP-2 with high affinity. AP-2 binding to this site is likely to be the first step in coated pit assembly because this subunit functions as a template for the polymerization of clathrin into flat polygonal lattices. Integral membrane proteins involved in receptor mediated endocytosis cluster in the newly assembled pits as they invaginate and bud from the membrane. The AP-2 subunit is a multi-domain, molecular complex that can be separated by proteolysis into a brick-shaped core and ear-like appendage domains. We have used this property to identify the domain involved in the various stages of coated pit assembly and budding. We found that the core of AP-2 is the domain that binds both to membranes and to triskelions during assembly. Triskelions are perfectly capable of forming lattices on the membrane bound cores. Clathrin lattices bound only to core domains were also able to invaginate normally. Limited proteolysis was also useful for further characterizing the AP-2 binding site. Elastase treatment of the inside membrane surface released a peptide fraction that is able to bind AP-2 in solution and prevent it from interacting with membranes. Affinity purification of binding activity yielded a collection of peptides that was dominated by a 45-kD species. This is the candidate peptide for containing the AP-2-binding site. Therefore, the appendage domain does not directly participate in any of the assembly or invagination events required for coated pit function.     

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-coated pits contain an integral membrane protein that binds the AP-2 subunit with high affinity

Coated pits will assemble onto purified plasma membranes that are attached to a poly-L-lysine coated substratum (Moore, M. S., Mahaffey, D. T., Brodsky, F. M., and Anderson, R. G. W. (1987) Science 236, 558-563; Mahaffey, D. T., Moore, M. S., Brodsky, F. M., and Anderson, R. G. W. (1989) J. Cell Biol. 108, 1615-1624). To better understand the assembly reaction, we have purified both clathrin triskelion and AP-2 subunits from bovine brain and assayed for their ability to bind to the cytoplasmic surface of attached membranes. Two types of membranes were analyzed: those washed with a high pH buffer that selectively removes triskelions and those washed with a high salt buffer that removes both the AP-2 and the triskelion subunits. We found that purified AP-2 subunits bind with high affinity (Kd approximately 3 x 10(-8) M) to salt stripped membranes. Binding is saturable and abolished by treating membranes with less than 20 micrograms/ml of elastase. When membranes were treated with elastase before the salt wash and then salt washed and assayed for AP-2 binding, normal binding was seen, which indicates that the presence of clathrin-coated pits protects the binding site from the protease. Membranes that had rebound AP-2 did not bind purified triskelions, even though high pH buffer-washed membranes that bear endogenous AP-2 bound triskelions with high affinity (Kd approximately 3 x 10(-9) M) and supported lattice assembly. We conclude that coated pit assembly is initiated by the binding of AP-2 to an integral membrane protein but that the AP-2 complex must be activated by an unknown process before the coated pit lattice will assemble.     

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.     

Clathrin lattice reorganization: theoretical considerations

We wish to postulate a mechanism by which flat hexagonal lattices of clathrin trimers transform into coated pits. Using an established model for packing trimers into lattices, we explored the assembly process by single addition of trimers to form polygons. Subject to favorable conditions, removal of a single trimer from a hexagon could lead to the formation of a pentagon. Elimination of trimers from polygonal sheets can occur either at the center of the network or at the edges. Removal of a trimer from the center of these adjacent polygons, "hub transformation," is possible in very few instances, whereas removal from the edges of a polygonal sheet, "fringe transformation," is possible in a host of cases. These hypothetical constructs can be used effectively to explain intermediate structures actually observed in flat hexagonal lattices. The geometry of a purely hexagonal lattice seems to dictate that the first step in transformation must be a "fringe transformation," which then will allow subsequent "hub transformation" to take place leading to the introduction of pentagons into the center of the lattice and ultimately to the curvature of the clathrin lattice.     

Functional organization of clathrin in coats: combining electron cryomicroscopy and X-ray crystallography.

The sorting of specific proteins into clathrin-coated pits and the mechanics of membrane invagination are determined by assembly of the clathrin lattice. Recent structures of a six-fold barrel clathrin coat at 21 A resolution by electron cryomicroscopy and of the clathrin terminal domain and linker at 2.6 A by X-ray crystallography together show how domains of clathrin interact and orient within the coat and reveal the strongly puckered shape and conformational variability of individual triskelions. The beta propeller of the terminal domain faces the membrane so that recognition segments from adaptor proteins can extend along its lateral grooves. Clathrin legs adapt to different coat environments in the barrel by flexing along a segment at the knee that is free of contacts with other molecules.     

The association of clathrin fragments with coated vesicle membranes

The association between clathrin triskelions and the clathrin-stripped membranes of coated vesicles has been investigated using a filter assay to separate bound from unbound clathrin. The filter assay is more sensitive and less cumbersome than a sedimentation assay used previously (1). While confirming the high affinity interaction between clathrin and the vesicle membrane, our results yield Scatchard plots that are curvilinear and consistent with a positively cooperative interaction between clathrin and the vesicle membranes. Controlled digestion with trypsin removes the distal portions of the triskelion legs leaving the proximal 31 nm portions that form the hub of the triskelions. These hubs are trimers of large 112,000- and 124,000-dalton fragments of clathrin heavy chains. They competitively inhibit the binding of 125I-labeled intact triskelions to stripped vesicles with a KI identical to the KD for the association of 125I-labeled intact triskelions to stripped vesicles. Furthermore, these large fragment trimers bind to stripped vesicles with approximately the same high affinity as do intact triskelions and also show evidence of a positively cooperative interaction. It is concluded that clathrin binds to coated vesicles by an interaction that is mediated by the proximal 112,000-dalton fragment of the clathrin heavy chains.     

Mathematical modelling of the formation of coated vesicles. A theoretical geometrical approach.

The mechanism by which flat hexagonal lattices of clathrin trimers transform into pentagonal/hexagonal spheres remains a mystery. In light of the geometrical nature of this process we have pursued a mathematical approach to the question. Through the geometrical analysis of flat hexagonal lattices we have discovered three possible forms of transformation to introduce curvature into the centre of the lattice: hub-centre transformation; hub-edge transformation; fringe transformation. Hub-edge and fringe transformations are used first to close the lattice while introducing localized curvature at the edges of the lattice. Hub-centre transformation is used after closure to relax the severely localized curvature generated during closure. This scheme not only maximizes the size of the coated vesicle generated, but also minimizes the number of transformations, thus minimizing the energy expended.     

A monoclonal antibody to the heavy chain of clathrin.

Monoclonal antibodies have been raised to pig brain triskelions and one clone, DC41, was found to recognize the clathrin heavy chain by immunoblotting. However, both by immunofluorescence and immunoelectron microscopy, and in complete contrast to polyclonal anti-clathrin antibodies, monoclonal DC41 did not label either coated pits or coated vesicles anywhere in the cell. Instead it appeared to label the cell cytoplasm. These data suggest that DC41 recognizes a cytoplasmic form of clathrin, perhaps that form produced by uncoating of coated vesicles which is then ready to re-build another coated pit.     

Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation

The clathrin-coated pit lattice is held onto the plasma membrane by an integral membrane protein that binds the clathrin AP-2 subunit with high affinity. In vitro studies have suggested that this protein controls the assembly of the pit because membrane bound AP-2 is required for lattice assembly. If so, the AP-2 binding site must be a resident protein of the coated pit and recycle with other receptors that enter cells through this pathway. Proper recycling, however, would require the switching off of AP-2 binding to allow the binding site to travel through the endocytic pathway unencumbered. Evidence for this hypothesis has been revealed by the cationic amphiphilic class of drugs (CAD), which have previously been found to inhibit receptor recycling. Incubation of human fibroblasts in the presence of these drugs caused clathrin lattices to assemble on endosomal membranes and at the same time prevented coated pit assembly at the cell surface. These effects suggest that CADs reverse an on/off switch that controls AP-2 binding to membranes. We conclude that cells have a mechanism for switching on and off AP-2 binding during the endocytic cycle.     

The first five seconds in the life of a clathrin-coated pit

Coated pits assemble by growth of a clathrin lattice, which is linked by adaptors to the underlying membrane. How does this process start? We used live-cell TIRF imaging with single-molecule EGFP sensitivity and high temporal resolution to detect arrival of the clathrin triskelions and AP2 adaptors that initiate coat assembly. Unbiased object identification and trajectory tracking, together with?a statistical model, yield the arrival times and numbers of individual proteins, as well as experimentally confirmed estimates of the extent of substitution of endogenous by expressed, fluorescently tagged proteins. Pits initiate by coordinated arrival of clathrin and AP2, which is usually detected as two sequential steps, each of one triskelion with two adaptors. PI-4,5-P(2) is essential for initiation. The accessory proteins FCHo1/2 are not; instead, they are required for sustained growth. This objective picture of coated pit initiation also shows that methods outlined here will be broadly useful for studies of dynamic assemblies in living cells.     

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.      

ATP catalyzes the sequestration of clathrin during enzymatic uncoating

ATP facilitates the sequestration of displaced triskelions by uncoating protein. In so doing, ATP is not hydrolyzed; nor does the concentration of ATP affect the equilibrium of this binding. However, the rates of both the binding of uncoating protein to clathrin and of their dissociation are greatly accelerated by ATP. These properties suggest that ATP acts catalytically to speed the capture of displaced triskelions by uncoating protein, as well as stoichiometrically in its hydrolysis to drive the displacement of triskelions from cages. The nucleotide specificity of this "catalytic" site for ATP on the uncoating protein is much less strict than that of the distinct "hydrolytic" site that drives the ATP-dependent displacement of triskelions from cages.     

Enzymatic dissociation of clathrin cages in a two-stage process

Uncoating ATPase catalyzes the ATP-dependent dissociation of clathrin from coated vesicles and empty cages. Following an uncoating reaction, clathrin triskelions are released intact, in a stoichiometric complex with bound uncoating protein. This overall uncoating process was dissected into two partial reactions. In the first, ATP hydrolysis drives the transient displacement of a portion of a triskelion from a cage. Uncoating protein then captures the displaced triskelion, in the second stage, by binding to a newly exposed site on clathrin that had previously been buried in the cage lattice. Triskelion-uncoating protein complexes are released when all points of attachment of the triskelion to the cage have been severed. The uncoating protein interacts with a distinct site on clathrin for each of these reactions.     

Purification of Clathrin Heavy and Light Chain fromDictyostelium discoideum

Clathrin, a protein important for endocytosis, is a hexamer composed of three heavy chains and three light chains. We report here the purification scheme used to isolate the clathrin protein from the simple eukaryote,Dictyostelium discoideum.Using a combination of differential centrifugation and column chromatography, we isolated ∼2 mg of clathrin triskelions from 150–200 g ofDictyosteliumcells. One additional step purified the 30-kDa clathrin light chain to homogeneity. Glycerol gradient centrifugation was used to determine anSvalue of 7.9 for purified clathrin. Rotary shadowed images ofDictyosteliumclathrin revealed trimeric molecules with extended legs measuring 48 ± 5 nm, similar in length to the legs of mammalian and yeast clathrin triskelions. The single clathrin light chain proved resistant to heat treatment, a property also similar to light chains from other species. The conservation of these physical properties inDictyosteliumclathrin demonstrates the potential of this model organism for the study of clathrin structure and function.     

Compromise of clathrin function and membrane association by clathrin light chain deletion

While clathrin heavy chains from different species are highly conserved in amino acid sequence, clathrin light chains are much more divergent. Thus clathrin light chain may have different functions in different organisms. To investigate clathrin light chain function, we cloned the clathrin light chain, clcA, from Dictyostelium and examined clathrin function in clcA– mutants. Phenotypic deficiencies in development, cytokinesis, and osmoregulation showed that light chain was critical for clathrin function in Dictyostelium . In contrast with budding yeast, we found the light chain did not influence steady-state levels of clathrin, triskelion formation, or contribute to clathrin over-assembly on intracellular membranes. Imaging GFP-CHC in clcA– mutants showed that the heavy chain formed dynamic punctate structures that were remarkably similar to those found in wild-type cells. However, clathrin light chain knockouts showed a decreased association of clathrin with intracellular membranes. Unlike wild-type cells, half of the clathrin in clcA– mutants was cytosolic, suggesting that the absence of light chain compromised the assembly of triskelions onto intracellular membranes. Taken together, these results suggest a role for the Dictyostelium clathrin light chain in regulating the self-assembly of triskelions onto intracellular membranes, and demonstrate a crucial contribution of the light chain to clathrin function in vivo .