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.     

Evidence from lateral mobility studies for dynamic interactions of a mutant influenza hemagglutinin with coated pits

Replacement of cysteine at position 543 by tyrosine in the influenza virus hemagglutinin (HA) protein enables the endocytosis of the mutant protein (Tyr 543) through coated pits (Lazarovits, J., and M. G. Roth. 1988. Cell. 53:743-752). To investigate the interactions between Tyr 543 and the clathrin coats in the plasma membrane of live cells, we performed fluorescence photobleaching recovery measurements comparing the lateral mobilities of Tyr 543 (which enters coated pits) and wild-type HA (HA wt, which is excluded from coated pits), following their expression in CV-1 cells by SV-40 vectors. While both proteins exhibited the same high mobile fractions, the lateral diffusion rate of Tyr 543 was significantly slower than that of HA wt. Incubation of the cells in a sucrose-containing hypertonic medium, a treatment that disperses the membrane-associated coated pits, resulted in similar lateral mobilities for Tyr 543 and HA wt. These findings indicate that the lateral motion of Tyr 543 (but not of HA wt) is inhibited by transient interactions with coated pits (which are essentially immobile on the time scale of the lateral mobility measurements). Acidification of the cytoplasm by prepulsing the cells with NH4Cl (a treatment that arrests the pinching-off of coated vesicles from the plasma membrane and alters the clathrin lattice morphology) led to immobilization of a significant part of the Tyr 543 molecules, presumably due to their entrapment in coated pits for the entire duration of the lateral mobility measurement. Furthermore, in both untreated and cytosol-acidified cells, the restrictions on Tyr 543 mobility were less pronounced in the cold, suggesting that the mobility-restricting interactions are temperature dependent and become weaker at low temperatures. From these studies we conclude the following. (a) Lateral mobility measurements are capable of detecting interactions of transmembrane proteins with coated pits in intact cells. (b) The interactions of Tyr 543 with coated pits are dynamic, involving multiple entries of Tyr 543 molecules into and out of coated pits. (c) Alterations in the clathrin lattice structure can modulate the above interactions.     

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.     

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.     

A glycolipid-anchored prion protein is endocytosed via clathrin-coated pits

The cellular prion protein (PrPc) is a glycolipid-anchored, cell surface protein of unknown function, a posttranslationally modified isoform of which PrPSc is involved in the pathogenesis of Creutzfeldt- Jakob disease, scrapie, and other spongiform encephalopathies. We have shown previously that chPrP, a chicken homologue of mammalian PrPC, constitutively cycles between the cell surface and an endocytic compartment, with a transit time of approximately 60 min in cultured neuroblastoma cells. We now report that endocytosis of chPrP is mediated by clathrin-coated pits. Immunogold labeling of neuroblastoma cells demonstrates that the concentration of chPrP within 0.05 microns of coated pits is 3-5 times higher than over other areas of the plasma membrane. Moreover, gold particles can be seen within coated vesicles and deeply invaginated coated pits that are in the process of pinching off from the plasma membrane. ChPrP is also localized to coated pits in primary cultures of neurons and glia, and is found in coated vesicles purified from chicken brain. Finally, internalization of chPrP is reduced by 70% after neuroblastoma cells are incubated in hypertonic medium, a treatment that inhibits endocytosis by disrupting clathrin lattices. Caveolae, plasmalemmal invaginations in which several other glycolipid-anchored proteins are concentrated, are not seen in neuroblastoma cells analyzed by thin-section or deep-etch electron microscopy. Moreover, these cells do not express detectable levels of caveolin, a caveolar coat protein. Since chPrP lacks a cytoplasmic domain that could interact directly with the intracellular components of clathrin-coated pits, we propose that the polypeptide chain of chPrP associates with the extracellular domain of a transmembrane protein that contains a coated pit internalization signal.     

Single-molecule live-cell imaging of clathrin-based endocytosis

Clathrin-coated vesicles carry traffic from the plasma membrane to endosomes. We report here the first real-time visualization of cargo sorting and endocytosis by clathrin-coated pits in living cells. We have visualized the formation of coats by monitoring the incorporation of fluorescently tagged clathrin or its adaptor AP-2 (adaptor protein 2), and have followed clathrin-mediated uptake of transferrin, single LDL (low-density lipoprotein) and single 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 preferred nucleation sites. A proportion of the nucleation events appear to be abortive. Cargo incorporation occurs primarily or exclusively in a newly formed coated pit, and loading appears to commit that pit to finish assembly. Our data led to a model in which coated pits initiate randomly, but collapse with high likelihood unless stabilized, presumably by cargo capture.     

A Highlights from MBoC Selection: Asymmetric formation of coated pits on dorsal and ventral surfaces at the leading edges of motile cells and on protrusions of immobile cells

Clathrin/AP2-coated vesicles are the principal endocytic carriers originating at the plasma membrane. In the experiments reported here, we used spinning-disk confocal and lattice light-sheet microscopy to study the assembly dynamics of coated pits on the dorsal and ventral membranes of migrating U373 glioblastoma cells stably expressing AP2 tagged with enhanced green fluorescence (AP2-EGFP) and on lateral protrusions from immobile SUM159 breast carcinoma cells, gene-edited to express AP2-EGFP. On U373 cells, coated pits initiated on the dorsal membrane at the front of the lamellipodium and at the approximate boundary between the lamellipodium and lamella and continued to grow as they were swept back toward the cell body; coated pits were absent from the corresponding ventral membrane. We observed a similar dorsal/ventral asymmetry on membrane protrusions from SUM159 cells. Stationary coated pits formed and budded on the remainder of the dorsal and ventral surfaces of both types of cells. These observations support a previously proposed model that invokes net membrane deposition at the leading edge due to an imbalance between the endocytic and exocytic membrane flow at the front of a migrating cell.     

Cholinergic stimulation of chromaffin cells induces rapid coating of the plasma membrane

Stimulation of primary bovine adrenal chromaffin cells by carbachol produced a 6-fold increase in cell surface coated pits within 30 s. This coat appeared not to be recruited from a preformed pool at the plasma membrane, but from some pool transparent to electron microscopy. The number of coated pits appeared to decrease rapidly after 1 to 2 min stimulation, but processing for electron microscopy using tannic acid to enhance contrast indicated that both coated pits and closed coated vesicles were increased relative to unstimulated cells for up to 30 min. Analyses of purified adrenal medulla coated vesicles showed a lipid composition close to that expected for cell surface membrane, but there were only trace levels of plasma membrane marker enzymes. Coated vesicles contained significant amounts of both membrane and content proteins characteristic of the chromaffin granule, suggesting that medulla coated vesicles preferentially carry secretion granule proteins. The kinetics of stimulus-dependent formation of coated membrane in the cortical zone of chromaffin cells is closely similar to that observed for secretion granule membrane retrieval.     

Induction of mutant dynamin specifically blocks endocytic coated vesicle formation

Dynamin is the mammalian homologue to the Drosophila shibire gene product. Mutations in this 100-kD GTPase cause a pleiotropic defect in endocytosis. To further investigate its role, we generated stable HeLa cell lines expressing either wild-type dynamin or a mutant defective in GTP binding and hydrolysis driven by a tightly controlled, tetracycline- inducible promoter. Overexpression of wild-type dynamin had no effect. In contrast, coated pits failed to become constricted and coated vesicles failed to bud in cells overexpressing mutant dynamin so that endocytosis via both transferrin (Tfn) and EGF receptors was potently inhibited. Coated pit assembly, invagination, and the recruitment of receptors into coated pits were unaffected. Other vesicular transport pathways, including Tfn receptor recycling, Tfn receptor biosynthesis, and cathepsin D transport to lysosomes via Golgi-derived coated vesicles, were unaffected. Bulk fluid-phase uptake also continued at the same initial rates as wild type. EM immunolocalization showed that membrane-bound dynamin was specifically associated with clathrin-coated pits on the plasma membrane. Dynamin was also associated with isolated coated vesicles, suggesting that it plays a role in vesicle budding. Like the Drosophila shibire mutant, HeLa cells overexpressing mutant dynamin accumulated long tubules, many of which remained connected to the plasma membrane. We conclude that dynamin is specifically required for endocytic coated vesicle formation, and that its GTP binding and hydrolysis activities are required to form constricted coated pits and, subsequently, for coated vesicle budding.     

Light and electron microscopic immunocytochemical localization of clathrin in rat cerebellum and kidney

The precise cellular and subcellular locations of coated vesicle protein, clathrin, in rat kidney and cerebellum have been visualized by immunocytochemical techniques. In the renal tubular epithelia, clathrin-positive products were found on both free ribosomes and on those attached to rough endoplasmic reticulum (RER) and the nuclear envelope. No clathrin was observed in the cisternae of RER or the Golgi apparatus. Clathrin-positive reaction products could also be seen on coated pits, coated vesicles, Golgi-associated vesicles, basolateral cell membrane, the ground substance, and in the autophagic vacuoles. In cerebellar Purkinje and granule cell bodies, reaction products were seen localized on coated vesicles, on the budding areas from the Golgi-associated membrane and Golgi-associated vesicles. Furthermore, the membrane of the multivesicular body, the bound-ribosomes, and the ground substance were also stained. In the myelinated axon, the clathrin appeared to be concentrated on certain segments and seemed to fill in the space between neurotubules and some vesicles. In certain synaptic terminals clathrin was often seen attached to presynaptic vesicles, presynaptic membrane, and post-synaptic membrane. However, in most mossy fibers, some synaptic vesicles were not stained. These observations suggest that clathrin is synthesized on bound and free ribosomes and discharged into the cytosol where it becomes associated with a variety of ground substances and assembles on coated pits, coated vesicles, Golgi-associated vesicles, presynaptic vesicles, and pre- and postsynaptic membranes. Clathrin may be finally degraded in autophagic vacuoles.     

Effect of membrane flow on the capture of receptors by coated pits. Theoretical results.

Coated pits trap cell surface receptors and mediate their internalization. Once internalized, many receptors recycle back to the cell surface. When recycled receptors are inserted into the plasma membrane, they move until they are again trapped in coated pits. The mechanisms for moving receptors from their insertion sites to coated pits are unknown. Unaided diffusion as the transport mechanism is consistent with the observed kinetics of receptor recycling. Another candidate for the transport mechanism is convection. For receptors that recycle to random positions on the cell surface, or to restricted regions about coated pits, we assess the importance of convective flow in the transport of receptors to coated pits. First we consider local flows set up by the formation of coated pits and their transformation into coated vesicles. As coated pits form and round into coated vesicles, surrounding membrane is drawn inward, creating flows directed toward the coated pit centers. We show that unless the lifetime of a coated pit is very short, 10 s or less, such local flows have a negligible effect on the time it takes receptors to reach coated pits. We also show that they are unlikely to be the mechanism that keeps receptors that have reached coated pits trapped within coated pits until they are internalized. Finally we calculate the mean time tau for a diffusing receptor to reach a coated pit in the presence of membrane flow that is constant in magnitude and direction, as may occur on moving cells. We show that for typical membrane flow velocities, tau can be reduced significantly from its value in the absence of flow. For example, a velocity v = 2.8 micron/min cuts the mean transport time in half.     

Annexin VI is required for budding of clathrin-coated pits.

Isolated plasma membranes attached to a solid substratum at 4 degrees C have numerous clathrin-coated pits. These pits initially are flat but become deeply invaginated after warming to 37 degrees C. The pits remain tethered to the membrane in this rounded condition unless supplied with ATP, Ca2+, and cytosol. We now show that when cytosol is treated to remove the Ca(2+)-dependent, phospholipid-binding protein annexin VI, coated pit budding no longer takes place. Addition of purified annexin VI back to the annexin VI-depleted cytosol restores budding activity to normal. Purified annexin VI alone shows only a modest budding activity, suggesting that the cytosol contains a factor(s) in addition to annexin VI that is required for full activity. Cytosol-dependent activation of annexin VI requires both ATP and Ca2+. Annexin VI appears to be not only an active component in the detachment of coated pits from the membrane but also a site for regulating the formation of coated vesicles.     

The mechanism of receptor-mediated endocytosis: more questions than answers.

Receptor-mediated endocytosis occurs via clathrin-coated pits and is therefore coupled to the dynamic cycle of assembly and disassembly of the coat constituents. These coat proteins comprise part, but certainly not all, of the machinery involved in the recognition of membrane receptors and their selective packaging into transport vesicles for internalization. Despite considerable knowledge about the biochemistry of coated vesicles and purified coat proteins, little is known about the mechanisms of coated pit assembly, receptor-sorting and coated vesicle formation. Cell-free assays which faithfully reconstitute these events provide powerful new tools with which to elucidate the overall mechanism of receptor-mediated endocytosis.     

Activation of MAPK by TRH requires clathrin-dependent endocytosis and PKC but not receptor interaction with beta-arrestin or receptor endocytosis

To determine whether the interaction of the TRH receptor with beta-arrestin is necessary for TRH activation of MAPK, cells expressing either intact or truncated, internalization-defective TRH receptors were transfected with a beta-arrestin-green fluorescent protein conjugate. In cells expressing the wild-type pituitary TRH receptor, TRH caused translocation of the beta-arrestin-green fluorescent protein conjugate from the cytosol to the plasma membrane within 30 sec. After 5 min, the beta-arrestin-green fluorescent protein conjugate was visible in vesicles, where it colocalized with rhodamine-labeled TRH. In hypertonic sucrose, the beta-arrestin-green fluorescent protein conjugate translocated to the plasma membrane after TRH addition but did not internalize. In cells expressing the truncated TRH receptor, TRH did not cause translocation of the beta-arrestin-green fluorescent protein conjugate. TRH activated MAPK strongly in cells expressing intact or truncated TRH receptors, indicating that the receptor does not need to bind beta-arrestin or internalize. MAPK activation by TRH, epidermal growth factor, and phorbol ester was strongly inhibited by hypertonic sucrose and concanavalin A, which block movement of proteins into coated pits and coated pit assembly. Hypertonic sucrose did not affect MAPK activation in cells overexpressing MAPK kinase 1. Dominant negative dynamin, which blocks conversion of coated pits to vesicles, also reduced receptor internalization and TRH activation of MAPK. TRH activation of MAPK required PKC but was insensitive to pertussis toxin and did not require ras, epidermal growth factor receptor kinase, or PI3K. These results show that the TRH receptor itself does not need to bind beta-arrestin or undergo sequestration to activate MAPK but that the endocytic pathway must be intact.     

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.     

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.     

Uptake of ferritin by the mosaic egg surface of Brachydanio

The internalization of membrane from the mosaic egg surface of the zebra fish, Brachydanio, was investigated using anionic ferritin and transmission electron microscopy. The cortical cytoplasm of the 5-min activated egg showed numerous membrane-bound vesicles not found in the unactivated egg cortex. Two types of vesicles were identified: uncoated (smooth) and coated. Coated vesicles measured about 0.7 to 0.9 micrometer in diameter. Coated pits, considered to be precursors to the formation of coated vesicles, were frequently observed at the base of membrane-lined cortical granule crypts. Anionic ferritin was localized over coated pits and in both smooth and coated vesicles. The absence of any morphological evidence of a surface origin for smooth vesicles suggested these ferritin-labeled organelles might be formed by coated vesicle fusion. Our results indicate that the plasma membrane redundancy created by the exocytosis of cortical granules in Brachydanio appears to be resolved in part by the internalization of membrane through endocytosis.     

Receptors compete for adaptors found in plasma membrane coated pits.

An affinity matrix of LDL receptor cytoplasmic tails binds the HA-II 100/50/16 kd complexes found in plasma membrane coated pits. Other receptors (or their cytoplasmic domains), which are localized in coated pits during endocytosis, inhibit this binding. This includes an 8 residue peptide containing tyrosine, corresponding to the cytoplasmic portion of a mutant influenza haemagglutinin. In contrast, the equivalent peptide lacking tyrosine (like the tail of the native haemagglutinin, a protein excluded from coated pits) does not compete. These results imply that the HA-II complex has a recognition site for a common signal, probably involving a tyrosine residue, carried by the LDL receptor and competing receptors also found in plasma membrane coated pits. The HA-II complex therefore fulfils the role of an 'adaptor', the name proposed for the structural units which mediate the binding of clathrin to receptors in coated vesicles. Another related complex, the HA-I adaptor, which is restricted to Golgi coated pits, probably does not recognize the 'tyrosine signal' on the LDL receptor tail. The HA-I adaptor is likely to contain a recognition site for a different signal carried by receptors, e.g. the mannose-6-phosphate receptor, which are found in Golgi coated pits.     

Identification of a putative yeast homolog of the mammalian beta chains of the clathrin-associated protein complexes.

The clathrin-associated protein complexes are heterotetrameric structures believed to interact with clathrin and with membrane components of mammalian coated pits and coated vesicles. I have identified a yeast homolog of the mammalian beta-type large chains, suggesting the existence in yeast cells of clathrin-associated protein complexes. A sequence comparison between the putative yeast beta-type chain and its mammalian counterparts shows that their amino-terminal domains are related over their entire length and that their carboxyl-terminal domains diverge completely. This observation is consistent with our earlier proposal (T. Kurchhausen et al., Proc. Natl. Acad. Sci. USA 86:2612-2616, 1989) for the bifunctional-domain organization of the large chains, in which the invariant amino-terminal region interacts with conserved proteins of the coat while the variable carboxyl-terminal domain interacts with different membrane components of coated pits and coated vesicles.     

Cytosol- and clathrin-dependent stimulation of endocytosis in vitro by purified adaptors

Using stage-specific assays for receptor-mediated endocytosis of transferrin (Tfn) into perforated A431 cells we show that purified adaptors stimulate coated pit assembly and ligand sequestration into deeply invaginated coated pits. Late events in endocytosis involving membrane fission and coated vesicle budding which lead to the internalization of Tfn are unaffected. AP2, plasma membrane adaptors, are active at physiological concentrations, whereas AP1, Golgi adaptors, are inactive. Adaptor-dependent stimulation of Tfn sequestration requires cytosolic clathrin, but is unaffected by clathrin purified from coated vesicles suggesting that soluble and assembled clathrin pools are functionally distinct. In addition to adaptors and cytosolic clathrin other, as yet unidentified, cytosolic factors are also required for efficient coated pit invagination. These results provide new insight into the mechanisms and regulation of coated pit assembly and invagination.