Redistribution of clathrin-coated vesicle adaptor complexes during adipocytic differentiation of 3T3-L1 cells

Mechanisms for intracellular retention of proteins are induced during adipocytic differentiation of 3T3-L1 cells. To investigate the potential role of clathrin lattices in these retention processes, we performed a morphological and biochemical analysis of coated vesicle components in 3T3-L1 cells. Optical sectioning and image restoration revealed a marked increase in the staining of clathrin and beta adaptins in the perinuclear region of cells with differentiation. In addition, predominance of beta (subunit of the AP-2, plasma membrane adaptor) over beta' (subunit of the AP-1, Golgi adaptor) adaptin was observed in immunoblots of clathrin-coated vesicles purified from nondifferentiated fibroblasts, and this ratio was reversed in coated vesicles purified from differentiated adipocytes. These results indicate that the relative abundance of TGN-derived clathrin lattices increases markedly during adipocytic differentiation. Subcellular fractionation indicated that cytosolic AP-1 and AP-2 adaptors comprised approximately 70% of the total cellular adaptor pool. Interestingly, neither the concentration nor the relative ratio of cytosolic AP-1 to AP-2 adaptors increased significantly during differentiation. These data suggest that the increase in TGN-derived lattices results from differentiation-induced mechanisms for enhanced assembly or stabilization of adaptors on Golgi membranes. Interestingly, double- immunofluorescence microscopy also revealed that whereas extensive colocalization between clathrin and beta adaptins occurred both in fibroblasts and adipocytes, structures stained only with anti-adaptin antibody could be detected. Taken together these results suggest that membranes coated with adaptors, but not clathrin, can exist in these cells.     

Clathrin and HA2 adaptors: effects of potassium depletion, hypertonic medium, and cytosol acidification

The effects of methods known to perturb endocytosis from clathrin- coated pits on the localization of clathrin and HA2 adaptors in HEp-2 carcinoma cells have been studied by immunofluorescence and ultrastructural immunogold microscopy, using internalization of transferrin as a functional assay. Potassium depletion, as well as incubation in hypertonic medium, remove membrane-associated clathrin lattices: flat clathrin lattices and coated pits from the plasma membrane, and clathrin-coated vesicles from the cytoplasm, as well as those budding from the TGN. In contrast, immunofluorescence microscopy using antibodies specific for the alpha- and beta-adaptins, respectively, and immunogold labeling of cryosections with anti-alpha- adaptin antibodies shows that under these conditions HA2 adaptors are aggregated at the plasma membrane to the same extent as in control cells. After reconstitution with isotonic K(+)-containing medium, adaptor aggregates and clathrin lattices colocalize at the plasma membrane as normally and internalization of transferrin resumes. Acidification of the cytosol affects neither clathrin nor HA2 adaptors as studied by immunofluorescence microscopy. However, quantitative ultrastructural observations reveal that acidification of the cytosol results in formation of heterogeneously sized and in average smaller clathrin-coated pits at the plasma membrane and buds on the TGN. Collectively, our observations indicate that the methods to perturb formation of clathrin-coated vesicles act by different mechanisms: acidification of the cytosol by affecting clathrin-coated membrane domains in a way that interferes with budding of clathrin-coated vesicles from the plasma membrane as well as from the TGN; potassium depletion and incubation in hypertonic medium by preventing clathrin and adaptors from interacting. Furthermore our observations show that adaptor aggregates can exist at the plasma membrane independent of clathrin lattices and raise the possibility that adaptor aggregates can form nucleation sites for clathrin lattices.     

Capturing the mechanics of clathrin-mediated endocytosis

Clathrin-mediated endocytosis enables selective uptake of molecules into cells in response to changing cellular needs. It occurs through assembly of coat components around the plasma membrane that determine vesicle contents and facilitate membrane bending to form a clathrin-coated transport vesicle. In this review we discuss recent cryo-electron microscopy structures that have captured a series of events in the life cycle of a clathrin-coated vesicle. Both single particle analysis and tomography approaches have revealed details of the clathrin lattice structure itself, how AP2 may interface with clathrin within a coated vesicle and the importance of PIP2 binding for assembly of the yeast adaptors Sla2 and Ent1 on the membrane. Within cells, cryo-electron tomography of clathrin in flat lattices and high-speed AFM studies provided new insights into how clathrin morphology can adapt during CCV formation. Thus, key mechanical processes driving clathrin-mediated endocytosis have been captured through multiple techniques working in partnership.     

The monomeric clathrin assembly protein, AP180, regulates contractile vacuole size in Dictyostelium discoideum

AP180, one of many assembly proteins and adaptors for clathrin, stimulates the assembly of clathrin lattices on membranes, but its unique contribution to clathrin function remains elusive. In this study we identified the Dictyostelium discoideum ortholog of the adaptor protein AP180 and characterized a mutant strain carrying a deletion in this gene. Imaging GFP-labeled AP180 showed that it localized to punctae at the plasma membrane, the contractile vacuole, and the cytoplasm and associated with clathrin. AP180 null cells did not display defects characteristic of clathrin mutants and continued to localize clathrin punctae on their plasma membrane and within the cytoplasm. However, like clathrin mutants, AP180 mutants, were osmosensitive. When immersed in water, AP180 null cells formed abnormally large contractile vacuoles. Furthermore, the cycle of expansion and contraction for contractile vacuoles in AP80 null cells was twice as long as that of wild-type cells. Taken together, our results suggest that AP180 plays a unique role as a regulator of contractile vacuole morphology and activity in Dictyostelium.     

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 Fluctuations Destabilize Clathrin Protein Lattice Order

We develop a theoretical model of a clathrin protein lattice on a flexible cell membrane. The clathrin subunit is modeled as a three-legged pinwheel with elastic deformation modes and intersubunit binding interactions. The pinwheels are constrained to lie on the surface of an elastic sheet that opposes bending deformation and is subjected to tension. Through Monte Carlo simulations, we predict the equilibrium phase behavior of clathrin lattices at various levels of tension. High membrane tensions, which correspond to suppressed membrane fluctuations, tend to stabilize large, flat crystalline structures similar to plaques that have been observed in vivo on cell membranes that are adhered to rigid surfaces. Low tensions, on the other hand, give rise to disordered, defect-ridden lattices that behave in a fluidlike manner. The principles of two-dimensional melting theory are applied to our model system to further clarify how high tensions can stabilize crystalline order on flexible membranes. These results demonstrate the importance of environmental physical cues in dictating the collective behavior of self-assembled protein structures.     

Membrane Fluctuations Destabilize Clathrin Protein Lattice Order

We develop a theoretical model of a clathrin protein lattice on a flexible cell membrane. The clathrin subunit is modeled as a three-legged pinwheel with elastic deformation modes and intersubunit binding interactions. The pinwheels are constrained to lie on the surface of an elastic sheet that opposes bending deformation and is subjected to tension. Through Monte Carlo simulations, we predict the equilibrium phase behavior of clathrin lattices at various levels of tension. High membrane tensions, which correspond to suppressed membrane fluctuations, tend to stabilize large, flat crystalline structures similar to plaques that have been observed in vivo on cell membranes that are adhered to rigid surfaces. Low tensions, on the other hand, give rise to disordered, defect-ridden lattices that behave in a fluidlike manner. The principles of two-dimensional melting theory are applied to our model system to further clarify how high tensions can stabilize crystalline order on flexible membranes. These results demonstrate the importance of environmental physical cues in dictating the collective behavior of self-assembled protein structures.     

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.     

Tyrosine-based endocytic motifs stimulate oligomerization of AP-2 adaptor complexes

The clathrin adaptor complex AP-2 functions in the assembly of clathrin-coated vesicles at the plasma membrane where it serves to couple endocytic vesicle formation to the selection of membrane cargo proteins. Recent evidence suggests that binding of tyrosine-based endocytic sorting motifs may induce a conformational change within the AP-2 adaptor complex that could enhance its interaction with other cargo molecules and with the membrane. We report here that soluble tyrosine-based endocytic sorting motif peptides facilitate clathrin/AP-2 recruitment to liposomal membranes and induce adaptor oligomerization even in the absence of a lipid bilayer. These effects are specific for endocytic motifs of the type Yxxphi whereas peptides corresponding to NPxY- or di-leucine-containing sorting signals are ineffective. Our data may help to explain how the highly cooperative assembly of clathrin and adaptors could be linked to the selection of membrane cargo proteins.     

Two distinct interaction motifs in amphiphysin bind two independent sites on the clathrin terminal domain beta-propeller

During the assembly of clathrin-coated vesicles, many peripheral membrane proteins, including the amphiphysins, use LLDLD-type clathrin-box motifs to interact with the N-terminal beta-propeller domain (TD) of clathrin. The 2.3 A-resolution structure of the clathrin TD in complex with a TLPWDLWTT peptide from amphiphysin 1 delineates a second clathrin-binding motif, PWXXW (the W box), that binds at a site on the TD remote from the clathrin box-binding site. The presence of both sequence motifs within the unstructured region of the amphiphysins allows them to bind more tightly to free TDs than do other endocytic proteins that contain only clathrin-box motifs. This property, along with the propensity of the N-terminal BAR domain to bind curved membranes, will preferentially localize amphiphysin and its partner, dynamin, to the periphery of invaginated clathrin lattices.     

Structural basis of membrane invagination by F-BAR domains

BAR superfamily domains shape membranes through poorly understood mechanisms. We solved structures of F-BAR modules bound to flat and curved bilayers using electron (cryo)microscopy. We show that membrane tubules form when F-BARs polymerize into helical coats that are held together by lateral and tip-to-tip interactions. On gel-state membranes or after mutation of residues along the lateral interaction surface, F-BARs adsorb onto bilayers via surfaces other than their concave face. We conclude that membrane binding is separable from membrane bending, and that imposition of the module's concave surface forces fluid-phase bilayers to bend locally. Furthermore, exposure of the domain's lateral interaction surface through a change in orientation serves as the crucial trigger for assembly of the helical coat and propagation of bilayer bending. The geometric constraints and sequential assembly of the helical lattice explain how F-BAR and classical BAR domains segregate into distinct microdomains, and provide insight into the spatial regulation of membrane invagination.     

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.     

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.     

ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type Igamma

Clathrin-mediated endocytosis of synaptic vesicle membranes involves the recruitment of clathrin and AP-2 adaptor complexes to the presynaptic plasma membrane. Phosphoinositides have been implicated in nucleating coat assembly by directly binding to several endocytotic proteins including AP-2 and AP180. Here, we show that the stimulatory effect of ATP and GTPgammaS on clathrin coat recruitment is mediated at least in part by increased levels of PIP2. We also provide evidence for a role of ADP-ribosylation factor 6 (ARF6) via direct stimulation of a synaptically enriched phosphatidylinositol 4-phosphate 5-kinase type Igamma (PIPKIgamma), in this effect. These data suggest a model according to which activation of PIPKIgamma by ARF6-GTP facilitates clathrin-coated pit assembly at the synapse.     

Dynamic Interactions between Clathrin and Locally Structured Elements in a Disordered Protein Mediate Clathrin Lattice Assembly

Assembly of clathrin lattices is mediated by assembly/adaptor proteins that contain domains that bind lipids or membrane-bound cargo proteins and clathrin binding domains (CBDs) that recruit clathrin. Here, we characterize the interaction between clathrin and a large fragment of the CBD of the clathrin assembly protein AP180. Mutational, NMR chemical shift, and analytical ultracentrifugation analyses allowed us to precisely define two clathrin binding sites within this fragment, each of which is found to bind weakly to the N-terminal domain of the clathrin heavy chain (TD). The locations of the two clathrin binding sites are consistent with predictions from sequence alignments of previously identified clathrin binding elements and, by extension, indicate that the complete AP180 CBD contains ∼ 12 degenerate repeats, each containing a single clathrin binding site. Sequence and circular dichroism analyses have indicated that the AP180 CBD is predominantly unstructured and our NMR analyses confirm that this is largely the case for the AP180 fragment characterized here. Unexpectedly, unlike the many proteins that undergo binding-coupled folding upon interaction with their binding partners, the AP180 fragment is similarly unstructured in its bound and free states. Instead, we find that this fragment exhibits localized β-turn-like structures at the two clathrin binding sites both when free and when bound to clathrin. These observations are incorporated into a model in which weak binding by multiple, pre-structured clathrin binding elements regularly dispersed throughout a largely unstructured CBD allows efficient recruitment of clathrin to endocytic sites and dynamic assembly of the clathrin lattice.     

The ～16 kDa C-Terminal Sequence of Clathrin Assembly Protein AP180 Is Essential for Efficient Clathrin Binding

Brain-specific AP180 is present in clathrin coats at equal concentration to the adapter complex, AP2, and assembles clathrin faster than any other protein in vitro. Both AP180 and its ubiquitously expressed homolog clathrin assembly lymphoid myeloid leukemia protein (CALM) control vesicle size and shape in clathrin mediated endocytosis. The clathrin assembly role of AP180 is mediated by a long disordered C-terminal assembly domain. Within this assembly domain, a central acidic clathrin and adapter binding (CLAP) sub-domain contains all of the known short binding motifs for clathrin and AP2. The role of the remaining ∼16 kDa C-terminal sequence has not been clear. We show that this sequence has a separate function in ensuring efficient binding of clathrin, based on in vitro binding and ex vivo transferrin uptake assays. Sequence alignment suggests the C-terminal sub-domain is conserved in CALM.     

Clathrin adaptor GGA1 polymerizes clathrin into tubules

GGAs, a class of monomeric clathrin adaptors, are involved in the sorting of cargo at the trans-Golgi network of eukaryotic cells. They are modular structures consisting of the VHS, the GAT, hinge, and GAE domains, which have been shown to interact directly with cargo, ARF, clathrin, and accessory proteins, respectively. Previous studies have shown that GGAs interact with clathrin both in solution and in the cell, but it has yet been shown whether they assemble clathrin. We find that GGA1 promoted assembly of clathrin with complete assembly achieved when one GGA1 molecule is bound per heavy chain. In the presence of excess GGA1, we obtained the unusual stoichiometry of five GGA1s per heavy chain, and even at this stoichiometry the binding was not saturated. The assembled structures were mostly baskets, but approximately 10% of the structures were tubular with an average length of 180 +/- 40 nm and width of approximately 50 nm. The truncated GGA1 fragment consisting of the hinge+GAE domains bound to clathrin with similar affinity as the full-length molecule and polymerized clathrin into baskets. Unlike the full-length molecule, this fragment saturated the lattices at one molecule per heavy chain and assembled clathrin only into baskets. The separated hinge and GAE domains bound much weaker to clathrin than the intact molecule, and these domains do not significantly polymerize clathrin into baskets. We conclude that clathrin adaptor GGA1 is a clathrin assembly protein, but it is unique in its ability to polymerize clathrin into tubules.     

Multiple roles of auxilin and hsc70 in clathrin-mediated endocytosis

The ATP-dependent dissociation of clathrin from clathrin-coated vesicles (CCVs) by the molecular chaperone Hsc70 requires J-domain cofactor proteins, either auxilin or cyclin-G-associated kinase (GAK). Both the nerve-specific auxilin and the ubiquitous GAK induce CCVs to bind to Hsc70. The removal of auxilin or GAK from various organisms and cells has provided definitive evidence that Hsc70 uncoats CCVs in vivo. In addition, evidence from various studies has suggested that Hsc70 and auxilin are involved in several other key processes that occur during clathrin-mediated endocytosis. First, Hsc70 and auxilin are required for the clathrin exchange that occurs during coated-pit invagination and constriction; this clathrin exchange may catalyze any rearrangement of the clathrin-coated pit (CCP) structure that is required during invagination and constriction. Second, Hsc70 and auxilin may chaperone clathrin after it dissociates from CCPs so that it does not aggregate in the cytosol. Third, auxilin and Hsc70 may be involved in the rebinding of clathrin to the plasma membrane to form new CCPs and independently appear to chaperone adaptor proteins so that they can also rebind to membranes to nucleate the formation of new CCPs. Finally, if formation of the curved clathrin coat induces membrane curvature, then Hsc70 and auxilin provide the energy for this curvature by inducing ATP-dependent clathrin exchange and rearrangement during endocytosis and ATP-dependent dissociation of clathrin at the end of the cycle so that it is energetically primed to rebind to the plasma membrane.     

Distinct Dynamics of Endocytic Clathrin-Coated Pits and Coated Plaques

Clathrin is the scaffold of a conserved molecular machinery that has evolved to capture membrane patches, which then pinch off to become traffic carriers. These carriers are the principal vehicles of receptor-mediated endocytosis and are the major route of traffic from plasma membrane to endosomes. We report here the use of in vivo imaging data, obtained from spinning disk confocal and total internal reflection fluorescence microscopy, to distinguish between two modes of endocytic clathrin coat formation, which we designate as “coated pits” and “coated plaques.” Coated pits are small, rapidly forming structures that deform the underlying membrane by progressive recruitment of clathrin, adaptors, and other regulatory proteins. They ultimately close off and bud inward to form coated vesicles. Coated plaques are longer-lived structures with larger and less sharply curved coats; their clathrin lattices do not close off, but instead move inward from the cell surface shortly before membrane fission. Local remodeling of actin filaments is essential for the formation, inward movement, and dissolution of plaques, but it is not required for normal formation and budding of coated pits in the cells we have studied. We conclude that there are at least two distinct modes of clathrin coat formation at the plasma membrane—classical coated pits and coated plaques—and that these two assemblies interact quite differently with other intracellular structures.     

Coat proteins isolated from clathrin coated vesicles can assemble into coated pits

Isolated human fibroblast plasma membranes that were attached by their extracellular surface to a solid substratum contained numerous clathrin coated pits that could be removed with a high pH buffer (Moore, M.S., D.T. Mahaffey, F.M. Brodsky, and R.G.W. Anderson. 1987. Science [Wash. DC]. 236:558-563). When these membranes were incubated with coat proteins extracted from purified bovine coated vesicles, new coated pits formed that were indistinguishable from native coated pits. Assembly was dependent on the concentration of coat protein with half maximal assembly occurring at 7 micrograms/ml. Assembly was only slightly affected by the presence of divalent cations. Whereas normal appearing lattices formed in a low ionic strength buffer, when assembly was carried out in a low pH buffer, few coated pits were evident but numerous small clathrin cages decorated the membrane. Coated pits did not form randomly on the surface; instead, they assembled at differentiated regions of membrane that could be distinguished in carbon/platinum replicas of frozen and etched membranes by the presence of numerous particles clustered into patches the size and shape of a coated pit.