Endocytosis by random initiation and stabilization of clathrin-coated pits

Clathrin-coated vesicles carry traffic from the plasma membrane to endosomes. We report here the real-time visualization of cargo sorting and endocytosis by clathrin-coated pits in living cells. We have detected the formation of coats by monitoring incorporation of fluorescently tagged clathrin or its adaptor AP-2; we have also followed clathrin-mediated uptake of transferrin and of single LDL or reovirus particles. The intensity of a cargo-loaded clathrin cluster grows steadily during its lifetime, and the time required to complete assembly is proportional to the size of the cargo particle. These results are consistent with a nucleation-growth mechanism and an approximately constant growth rate. There are no strongly preferred nucleation sites. A proportion of the nucleation events are weak and short lived. Cargo incorporation occurs primarily or exclusively in a newly formed coated pit. Our data lead to a model in which coated pits initiate randomly but collapse unless stabilized, perhaps by cargo capture.     

The role of dynamin and its binding partners in coated pit invagination and scission

Plasma membrane clathrin-coated vesicles form after the directed assembly of clathrin and the adaptor complex, AP2, from the cytosol onto the membrane. In addition to these structural components, several other proteins have been implicated in clathrin-coated vesicle formation. These include the large molecular weight GTPase, dynamin, and several Src homology 3 (SH3) domain-containing proteins which bind to dynamin via interactions with its COOH-terminal proline/arginine-rich domain (PRD). To understand the mechanism of coated vesicle formation, it is essential to determine the hierarchy by which individual components are targeted to and act in coated pit assembly, invagination, and scission.To address the role of dynamin and its binding partners in the early stages of endocytosis, we have used well-established in vitro assays for the late stages of coated pit invagination and coated vesicle scission. Dynamin has previously been shown to have a role in scission of coated vesicles. We show that dynamin is also required for the late stages of invagination of clathrin-coated pits. Furthermore, dynamin must bind and hydrolyze GTP for its role in sequestering ligand into deeply invaginated coated pits.We also demonstrate that the SH3 domain of endophilin, which binds both synaptojanin and dynamin, inhibits both late stages of invagination and also scission in vitro. This inhibition results from a reduction in phosphoinositide 4,5-bisphosphate levels which causes dissociation of AP2, clathrin, and dynamin from the plasma membrane. The dramatic effects of the SH3 domain of endophilin led us to propose a model for the temporal order of addition of endophilin and its binding partner synaptojanin in the coated vesicle cycle.     

Disabled-2 exhibits the properties of a cargo-selective endocytic clathrin adaptor

Clathrin-coated pits at the cell surface select material for transportation into the cell interior. A major mode of cargo selection at the bud site is via the micro 2 subunit of the AP-2 adaptor complex, which recognizes tyrosine-based internalization signals. Other internalization motifs and signals, including phosphorylation and ubiquitylation, also tag certain proteins for incorporation into a coated vesicle, but the mechanism of selection is unclear. Disabled-2 (Dab2) recognizes the FXNPXY internalization motif in LDL-receptor family members via an N-terminal phosphotyrosine-binding (PTB) domain. Here, we show that in addition to binding AP-2, Dab2 also binds directly to phosphoinositides and to clathrin, assembling triskelia into regular polyhedral coats. The FXNPXY motif and phosphoinositides contact different regions of the PTB domain, but can stably anchor Dab2 to the membrane surface, while the distal AP-2 and clathrin-binding determinants regulate clathrin lattice assembly. We propose that Dab2 is a typical member of a growing family of cargo-specific adaptor proteins, including beta-arrestin, AP180, epsin, HIP1 and numb, which regulate clathrin-coat assembly at the plasma membrane by synchronizing cargo selection and lattice polymerization events.     

Role of the AP2 beta-appendage hub in recruiting partners for clathrin-coated vesicle assembly

Adaptor protein complex 2 alpha and beta-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of beta-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the beta-appendage (the "top" and "side" sites) that bind motifs distinct from those previously identified on the alpha-appendage. We solved the structure of the beta-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor beta-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the beta-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability ("matricity"). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as beta-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.     

The clathrin adaptor Dab2 recruits EH domain scaffold proteins to regulate integrin β1 endocytosis

Endocytic adaptor proteins facilitate cargo recruitment and clathrin-coated pit nucleation. The prototypical clathrin adaptor AP2 mediates cargo recruitment, maturation, and scission of the pit by binding cargo, clathrin, and accessory proteins, including the Eps-homology (EH) domain proteins Eps15 and intersectin. However, clathrin-mediated endocytosis of some cargoes proceeds efficiently in AP2-depleted cells. We found that Dab2, another endocytic adaptor, also binds to Eps15 and intersectin. Depletion of EH domain proteins altered the number and size of clathrin structures and impaired the endocytosis of the Dab2- and AP2-dependent cargoes, integrin β1 and transferrin receptor, respectively. To test the importance of Dab2 binding to EH domain proteins for endocytosis, we mutated the EH domain-binding sites. This mutant localized to clathrin structures with integrin β1, AP2, and reduced amounts of Eps15. Of interest, although integrin β1 endocytosis was impaired, transferrin receptor internalization was unaffected. Surprisingly, whereas clathrin structures contain both Dab2 and AP2, integrin β1 and transferrin localize in separate pits. These data suggest that Dab2-mediated recruitment of EH domain proteins selectively drives the internalization of the Dab2 cargo, integrin β1. We propose that adaptors may need to be bound to their cargo to regulate EH domain proteins and internalize efficiently.     

Cargo recognition during clathrin-mediated endocytosis: a team effort

Transmembrane proteins destined to endosomes are selectively accumulated in clathrin-coated pits at the plasma membrane and rapidly internalized in clathrin-coated vesicles. The recognition of specific sequence motifs in transmembrane cargo by coated-pit proteins confers specificity on the endocytic process. Interaction of membrane cargo with the clathrin adaptor protein complex AP-2 is the major mechanism of cargo sorting into coated pits in mammalian cells. Recent studies have revealed a variety of alternative mechanisms of cargo recruitment involving additional adaptor proteins. These alternative mechanisms appear to be particularly important during clathrin-mediated endocytosis of signaling receptors.     

Regulating the clathrin-coated vesicle cycle by AP2 subunit phosphorylation

Clathrin-coated pits form from the concerted assembly of many different protein components that interact with each other and with the lipid bilayer. When the coated pit pinches off to form a coated vesicle, the coat is removed to allow fusion of the endocytic vesicle with an early endosome, and the disassembled coat components can then recycle through the cytosol to participate in new rounds of coated pit formation. One potential mechanism to regulate the cycles of coat protein assembly and disassembly is reversible phosphorylation, particularly given that many of the coat components, such as the AP2 adaptor complex, are phosphorylated on multiple sites both in vitro and in vivo. Recent studies have provided particular insight into the role of phosphorylation of the cargo-binding micro2 subunit of AP2.     

Role of lipids and actin in the formation of clathrin-coated pits

Assembly of clathrin-coated pits and their maturation into coated vesicles requires coordinated interactions between specific lipids and several structural and regulatory proteins. In the presence of primary alcohols, phospholipase D generates phosphatidylalcohols instead of PA, reducing stimulation of phosphatidyl inositol 5-kinase (PI5K) and hence decreasing formation of phosphoinositide-4,5-biphosphate (PIP(2)). Using live-cell imaging, we have shown that acute treatment of cells with 1-butanol or other small primary alcohols induces rapid disassembly of coated pits at the plasma membrane and blocks appearance of new ones. Addition of exogenous PIP(2) reverses this effect. Coated pits and vesicles reappear synchronously upon removal of 1-butanol; we have used this synchrony to assess the role of actin in coated vesicle assembly. Prolonged inhibition of actin polymerization by latrunculin A or cytochalasin D reduced by approximately 50% the frequency of coated pit formation without affecting maturation into coated vesicles. As in control cells, removal of 1-butanol in the continued presence of an actin depolymerizer led to synchronous appearance of new pits, which matured normally. Thus, remodeling of the actin cytoskeleton is not essential for clathrin-coated vesicle assembly but may indirectly affect the nucleation of clathrin-coated pits.     

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.     

Role of the AP2 β-Appendage Hub in Recruiting Partners for Clathrin-Coated Vesicle Assembly

Adaptor protein complex 2 α and β-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of β-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the β-appendage (the “top” and “side” sites) that bind motifs distinct from those previously identified on the α-appendage. We solved the structure of the β-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor β-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the β-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability (“matricity”). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as β-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.     

Role of the AP2 β-Appendage Hub in Recruiting Partners for Clathrin-Coated Vesicle Assembly

Adaptor protein complex 2 α and β-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of β-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the β-appendage (the “top” and “side” sites) that bind motifs distinct from those previously identified on the α-appendage. We solved the structure of the β-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor β-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the β-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability (“matricity”). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as β-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.     

Using quantum dots to visualize clathrin associations

Our current understanding of clathrin-mediated endocytosis proposes that the process is initiated at a specialized anatomical structure called a coated pit. Electron microscopy has been required for elucidation of the morphology of coated pits and the vesicles produced therein, and the presence of a bristle coat has been taken as suggestive of clathrin surrounding these vesicles. More recently, immunocytochemical methods have confirmed that endocytic vesicles are surrounded by clathrin and its adaptor proteins, but there is a need to identify precisely and to follow the fate of the cellular organelles seen by fluorescence microscopy. We used quantum immune-electron microscopy to localize clathrin in a human adrenal cortical cell line (SW-13). Clathrin was shown to associate with a variety of vesicle types including the classic clathrin-coated vesicles and pits used in receptor internalization, pentilaminar annular gap junction vesicles, and multivesicular bodies. The images obtained with quantum dot technology allow accurate and specific localization of clathrin and the clathrin adaptor protein, AP-2, with cellular organelles and suggest that some of the structures classified as typical coated vesicles by immunocytochemical light microscopic techniques actually may be membrane bound pits.     

Adaptor self-aggregation, adaptor-receptor recognition and binding of alpha-adaptin subunits to the plasma membrane contribute to recruitment of adaptor (AP2) components of clathrin-coated pits.

Initiation of receptor-mediated endocytosis by nucleation of clathrin-coated pits involves binding of AP2 adaptor molecules to the plasma membrane. This process was reconstituted in vitro, using plasma membrane fragments, prepared by freeze-thaw lysis of cells, and stripped of their endogenous coat proteins, as targets for binding of purified adaptor molecules and their dissociated subunits. The dissociated alpha-adaptin subunit of AP2 bound to plasma membrane fragments, while the dissociated beta-adaptin subunit did not, suggesting that plasma membrane localization of AP2 adaptors is mediated by alpha-adaptin. Membrane binding of intact AP2 adaptor molecules was enhanced by adaptor self-aggregation, which can be modulated by physiological concentrations of inositol phosphates, and may therefore be sensitive to receptor signaling. Adaptor binding was partially inhibited by soluble peptides representing the cytoplasmic domains of the asialoglycoprotein receptor and the polymeric immunoglobulin receptor. These results indicate that direct binding of adaptors to the cytoplasmic domains of receptors contributes to coated pit nucleation but this appears to be a weak interaction, suggesting that an additional recognition signal could be required for high affinity adaptor binding.     

A Highlights from MBoC Selection: Dynamin recruitment and membrane scission at the neck of a clathrin-coated pit

Dynamin, the GTPase required for clathrin-mediated endocytosis, is recruited to clathrin-coated pits in two sequential phases. The first is associated with coated pit maturation; the second, with fission of the membrane neck of a coated pit. Using gene-edited cells that express dynamin2-EGFP instead of dynamin2 and live-cell TIRF imaging with single-molecule EGFP sensitivity and high temporal resolution, we detected the arrival of dynamin at coated pits and defined dynamin dimers as the preferred assembly unit. We also used live-cell spinning-disk confocal microscopy calibrated by single-molecule EGFP detection to determine the number of dynamins recruited to the coated pits. A large fraction of budding coated pits recruit between 26 and 40 dynamins (between 1 and 1.5 helical turns of a dynamin collar) during the recruitment phase associated with neck fission; 26 are enough for coated vesicle release in cells partially depleted of dynamin by RNA interference. We discuss how these results restrict models for the mechanism of dynamin-mediated membrane scission.     

Cbl-dependent ubiquitination is required for progression of EGF receptors into clathrin-coated pits

Ligand binding causes the EGF receptor (EGFR) to become ubiquitinated by Cbl upon association with the adaptor protein Grb2. We have investigated the role of ubiquitin and Grb2 in ligand-induced endocytosis of the EGFR. Incubation of cells with EGF on ice caused translocation of Grb2 and Cbl from the cytosol to the rim of coated pits. Grb2 with point mutations in both SH3 domains inhibited recruitment of the EGFR to clathrin-coated pits, in a Ras-independent manner. On overexpression of the Cbl-binding protein Sprouty, ubiquitination of the EGFR was inhibited, the EGFR was recruited only to the rim of coated pits, and endocytosis of the EGFR was inhibited. Conjugation-defective ubiquitin similarly inhibited recruitment of EGF-EGFR to clathrin-coated pits. Even though this does not prove that cargo must be ubiquitinated, this indicates the importance of interaction of ubiquitinated protein(s) with proteins harboring ubiquitin-interacting domains. We propose that Grb2 mediates transient anchoring of the EGFR to an Eps15-containing molecular complex at the rim of coated pits and that Cbl-induced ubiquitination of the EGFR allows relocation of EGFR from the rim to the center of clathrin-coated pits.     

Multiple GTP-binding proteins participate in clathrin-coated vesicle- mediated endocytosis

We have examined the effects of various agonists and antagonists of GTP- binding proteins on receptor-mediated endocytosis in vitro. Stage- specific assays which distinguish coated pit assembly, invagination, and coat vesicle budding have been used to demonstrate requirements for GTP-binding protein(s) in each of these events. Coated pit invagination and coated vesicle budding are both stimulated by addition of GTP and inhibited by GDP beta S. Although coated pit invagination is resistant to GTP gamma S, A1F4-, and mastoparan, late events involved in coated vesicle budding are inhibited by these antagonists of G protein function. Earlier events involved in coated pit assembly are also inhibited by GTP gamma S, A1F4-, and mastoparan. These results demonstrate that multiple GTP-binding proteins, including heterotrimeric G proteins, participate at discrete stages in receptor- mediated endocytosis via clathrin-coated pits.     

A phosphatidylinositol (4,5)-bisphosphate binding site within mu2-adaptin regulates clathrin-mediated endocytosis

The clathrin adaptor complex AP-2 serves to coordinate clathrin-coated pit assembly with the sorting of transmembrane cargo proteins at the plasmalemma. How precisely AP-2 assembly and cargo protein recognition at sites of endocytosis are regulated has remained unclear, but recent evidence implicates phosphoinositides, in particular phosphatidylinositol (4,5)-bisphosphate (PI[4,5]P2), in these processes. Here we have identified and functionally characterized a conserved binding site for PI(4,5)P2 within mu2-adaptin, the medium chain of the clathrin adaptor complex AP-2. Mutant mu2 lacking a cluster of conserved lysine residues fails to bind PI(4,5)P2 and to compete the recruitment of native clathrin/AP-2 to PI(4,5)P2-containing liposomes or to presynaptic membranes. Moreover, we show that expression of mutant mu2 inhibits receptor-mediated endocytosis in living cells. We suggest that PI(4,5)P2 binding to mu2-adaptin regulates clathrin-mediated endocytosis and thereby may contribute to structurally linking cargo recognition to coat formation.     

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.     

Clathrin Hub Expression Dissociates the Actin-Binding Protein Hip1R from Coated Pits and Disrupts Their Alignment with the Actin Cytoskeleton

The actin cytoskeleton has been implicated in the maintenance of discrete sites for clathrin-coated pit formation during receptor-mediated endocytosis in mammalian cells, and its function is intimately linked to the endocytic pathway in yeast. Here we demonstrate that staining for mammalian endocytic clathrin-coated pits using a monoclonal antibody against the AP2 adaptor complex revealed a linear pattern that correlates with the organization of the actin cytoskeleton. This vesicle organization was disrupted by treatment of cells with cytochalasin D, which disassembles actin, or with 2,3-butanedione monoxime, which prevents myosin association with actin. The linear AP2 staining pattern was also disrupted in HeLa cells that were induced to express the Hub fragment of the clathrin heavy chain, which acts as a dominant-negative inhibitor of receptor-mediated endocytosis by direct interference with clathrin function. Additionally, Hub expression caused the actin-binding protein Hip1R to dissociate from coated pits. These findings indicate that proper function of clathrin is required for coated pit alignment with the actin cytoskeleton and suggest that the clathrin–Hip1R interaction is involved in the cytoskeletal organization of coated pits.     

Diffusion-limited forward rate constants in two dimensions. Application to the trapping of cell surface receptors by coated pits.

A variety of receptors are known to aggregate in specialized cell surface structures called coated pits, prior to being internalized when the coated pits close off. At 37 degrees C on human fibroblasts, as well as on other cell types, a recycling process maintains a constant number of coated pits on the cell surface. In this paper, we explore implications for receptor aggregation and internalization of the two types of recycling models that have been proposed for the maintenance of the coated pit concentration. In one model, coated pits alternate between accessible and inaccessible states at fixed locations on the cell surface, while in the other model, coated pits recycle to random locations on the cell surface. We consider receptors that are randomly inserted in the membrane, move by pure diffusion with diffusion coefficient D, and are instantly and irreversibly trapped when they reach a coated pit boundary (the diffusion limit). For such receptors, we calculate for each of the two models: the mean time tau to reach a coated pit, the forward rate constant k+ for the interaction of a receptor with a coated pit, and the fraction phi of receptors aggregated in coated pits. We show that for the parameters that characterize coated pits on human fibroblasts, the way in which coated pits return to the surface has a negligible effect on the values of tau, k+, and phi for mobile receptors, D greater than or equal to 1.0 X 10(-11) cm2/s, but has a substantial effect for "immobile" receptors, D much less than 1 X 10(-11) cm2/s. We present numerical examples to show that it may be possible to distinguish between these models if one can monitor slowly diffusing receptors (D less than 1 X 10(-11) cm2/s) on cells whose coated pits have relatively short lifetimes (less than or equal to 1 min). Finally, we show that for the low-density lipoprotein (LDL) receptor on human fibroblasts (D = 4.5 X 10(-11) cm2/s), the predicted and observed values of K+ and phi are in close agreement. Therefore, even for slowly diffusing LDL receptor, unaided diffusion as the transport mechanism of receptors to coated pits is consistent with measured rates of LDL internalization.