Stochastic model of clathrin-coated pit assembly

In recent years, fluorescence microscopy has enabled researchers to observe the dynamics of clathrin-coated pit (CCP) assembly in real time. The assembly dynamics of CCPs shows striking heterogeneity. Some CCPs are long-lived (productive CCPs); they bind cargo and grow in size to form clathrin-coated vesicles. In contrast, other CCPs (abortive CCPs) are relatively short-lived and disassemble well before reaching vesicle size. Within both populations there is significant variance in CCP lifetime. We propose a stochastic biophysical model that links these observations with the energetics of CCPs and kinetics of their assembly. We show that without cargo, CCP assembly faces a high energy barrier that is difficult to overcome. As a consequence, CCPs without cargo are almost always abortive. We suggest a mechanism by which cargo binding stabilizes CCPs and facilitates their growth. The lifetime distribution of abortive pits calculated from our model agrees well with published experimental data. We also estimate the lifetimes of productive CCPs and show that the stochastic nature of CCP assembly plays a crucial role in causing their observed wide distribution.     

Reconstitution of clathrin-coated pit budding from plasma membranes

Receptor-mediated endocytosis begins with the binding of ligand to receptors in clathrin-coated pits followed by the budding of the pits away from the membrane. We have successfully reconstituted this sequence in vitro. Highly purified plasma membranes labeled with gold were obtained by incubating cells in the presence of anti-LDL receptor IgG-gold at 4 degrees C, attaching the labeled cells to a poly-L-lysine-coated substratum at 4 degrees C and then gently sonicating them to remove everything except the adherent membrane. Initially the gold label was clustered over flat, clathrin-coated pits. After these membranes were warmed to 37 degrees C for 5-10 min in the presence of buffer that contained cytosol extract, Ca2+, and ATP, the coated pits rounded up and budded from the membrane, leaving behind a membrane that was devoid of LDL gold. Simultaneous with the loss of the ligand, the clathrin triskelion and the AP-2 subunits of the coated pit were also lost. These results suggest that the budding of a coated pit to form a coated vesicle occurs in two steps: (a) the spontaneous rounding of the flat lattice into a highly invaginated coated pit at 37 degrees C; (b) the ATP, 150 microM Ca2+, and cytosolic factors(s) dependent fusion of the adjoining membrane segments at the neck of the invaginated pit.     

The glycophospholipid-linked folate receptor internalizes folate without entering the clathrin-coated pit endocytic pathway

The folate receptor, also known as the membrane folate-binding protein, is maximally expressed on the surface of folate-depleted tissue culture cells and mediates the high affinity accumulation of 5-methyltetrahydrofolic acid in the cytoplasm of these cells. Recent evidence suggests that this receptor recycles during folate internalization and that it is anchored in the membrane by a glycosyl-phosphatidylinositol linkage. Using quantitative immunocytochemistry, we now show that (a) this receptor is highly clustered on the cell surface; (b) these clusters are preferentially associated with uncoated membrane invaginations rather than clathrin-coated pits; and (c) the receptor is not present in endosomes or lysosomes. This receptor appears to physically move in and out of the cell using a novel uncoated pit pathway that does not merge with the clathrin-coated pit endocytic machinery.     

The inositol 5-phosphatase SHIP2 regulates endocytic clathrin-coated pit dynamics

Phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P₂) and its phosphorylated product PI 3,4,5-triphosphate (PI(3,4,5)P₃) are two major phosphoinositides concentrated at the plasma membrane. Their levels, which are tightly controlled by kinases, phospholipases, and phosphatases, regulate a variety of cellular functions, including clathrin-mediated endocytosis and receptor signaling. In this study, we show that the inositol 5-phosphatase SHIP2, a negative regulator of PI(3,4,5)P₃-dependent signaling, also negatively regulates PI(4,5)P₂ levels and is concentrated at endocytic clathrin-coated pits (CCPs) via interactions with the scaffold protein intersectin. SHIP2 is recruited early at the pits and dissociates before fission. Both knockdown of SHIP2 expression and acute production of PI(3,4,5)P₃ shorten CCP lifetime by enhancing the rate of pit maturation, which is consistent with a positive role of both SHIP2 substrates, PI(4,5)P₂ and PI(3,4,5)P₃, on coat assembly. Because SHIP2 is a negative regulator of insulin signaling, our findings suggest the importance of the phosphoinositide metabolism at CCPs in the regulation of insulin signal output.     

Local clustering of transferrin receptors promotes clathrin-coated pit initiation

Clathrin-mediated endocytosis (CME) is the major pathway for concentrative uptake of receptors and receptor-ligand complexes (cargo). Although constitutively internalized cargos are known to accumulate into maturing clathrin-coated pits (CCPs), whether and how cargo recruitment affects the initiation and maturation of CCPs is not fully understood. Previous studies have addressed these issues by analyzing the global effects of receptor overexpression on CME or CCP dynamics. Here, we exploit a refined approach using expression of a biotinylated transferrin receptor (bTfnR) and controlling its local clustering using mono- or multivalent streptavidin. We show that local clustering of bTfnR increased CCP initiation. By tracking cargo loading in individual CCPs, we found that bTfnR clustering preceded clathrin assembly and confirmed that bTfnR-containing CCPs mature more efficiently than bTfnR-free CCPs. Although neither the clustering nor the related changes in cargo loading altered the rate of CCP maturation, bTfnR-containing CCPs exhibited significantly longer lifetimes than other CCPs within the same cell. Together these results demonstrate that cargo composition is a key source of the differential dynamics of CCPs.     

Divergent modes for cargo-mediated control of clathrin-coated pit dynamics

Clathrin-mediated endocytosis has long been viewed as a process driven by core endocytic proteins, with internalized cargo proteins being passive. In contrast, an emerging view suggests that signaling receptor cargo may actively control its fate by regulating the dynamics of clathrin-coated pits (CCPs) that mediate their internalization. Despite its physiological implications, very little is known about such “cargo-mediated regulation” of CCPs by signaling receptors. Here, using multicolor total internal reflection fluorescence microscopy imaging and quantitative analysis in live cells, we show that the μ-opioid receptor, a physiologically relevant G protein–coupled signaling receptor, delays the dynamics of CCPs in which it is localized. This delay is mediated by the interactions of two critical leucines on the receptor cytoplasmic tail. Unlike the previously known mechanism of cargo-mediated regulation, these residues regulate the lifetimes of dynamin, a key component of CCP scission. These results identify a novel means for selectively controlling the endocytosis of distinct cargo that share common trafficking components and indicate that CCP regulation by signaling receptors can operate via divergent modes.     

Caenorhabditis elegans RME-6 is a novel regulator of RAB-5 at the clathrin-coated pit

Here we identify a new regulator of endocytosis called RME-6. RME-6 is evolutionarily conserved among metazoans and contains Ras-GAP (GTPase-activating protein)-like and Vps9 domains. Consistent with the known catalytic function of Vps9 domains in Rab5 GDP/GTP exchange, we found that RME-6 binds specifically to Caenorhabditis elegans RAB-5 in the GDP-bound conformation, and rme-6 mutants have phenotypes that indicate low RAB-5 activity. However, unlike other Rab5-associated proteins, a rescuing green fluorescent protein (GFP)-RME-6 fusion protein primarily localizes to clathrin-coated pits, physically interacts with alpha-adaptin, a clathrin adaptor protein, and requires clathrin to achieve its cortical localization. In rme-6 mutants, transport from the plasma membrane to endosomes is defective, and small 110-nm endocytic vesicles accumulate just below the plasma membrane. These results suggest a mechanism for the activation of Rab5 in clathrin-coated pits or clathrin-coated vesicles that is essential for the delivery of endocytic cargo to early endosomes.     

Haemodynamics in the first seconds after coronary artery occlusion.

The changes in cardiac and in total haemodynamics, occurring during the first seconds of occlusion and the subsequent desocclusion of coronary arteries were studied on 28 dogs. The most intensive changes were observed after the trunk occlusion of the left coronary artery. Simultaneously with decreasing blood inflow into the myocardium its contractility and the systolic pressure in the left ventricle and the outflow from the coronary sinus began to fall rapidly. The systolic pressure in the left ventricle decreased within the first 10 s from 24 to 13-15 kPa (180 to 100-110 mm Hg), which means that the systolic pressure fell about 1 kPa (7-8 mm Hg) per second, or 0.5-0.6 kPa (4-5 mm Hg) per systole. At the same time the end-diastolic pressure in the left ventricle also increased from zero to 3-4 kPa (25-30 mm Hg). After the trunk desocclusion of the left coronary artery the systolic pressure in the left ventricle proceeded to fall by about 2-3 kPa (15-22 mm Hg). Only then, 20-25 s after the desocclusion, blood flow in the left coronary artery began to rise intensively and 4-6 s later the myocardial contractility and the systolic pressure in the left ventricle also increased. After unclamping (50-60 s), there was an overshoot of haemodynamic values above preocclusive values and then followed the compensatory phase. This phase lasted 80-90 s and on its peak the pressure and flow parameters increased by about 50-60% above preocclusive values. During the occlusion of ramus interventricularis anterior or ramus circumflexus for 30-60 s the haemodynamic parameters changed only slightly. The same was observed during trunk occlusion of the right coronary artery (30-60 s), but in that case many extrasystoles occurred.     

Rhythmic cycle of clathrin-coated pit formation at the trans-golgi network in human MDA-MB-435 cells

We studied the in vivo dynamics of enhanced green fluorescent protein-tagged clathrin light chain a (GFP-CLCa) at the trans-Golgi network (TGN) in MDA-MB-435 cells. The intensity of fluorescence signals of GFP-CLCa periodically increased and decreased at the TGN approximately every 100 s. This suggests that the formation of clathrin-coated pits occurs synchronously and periodically at the TGN.     

An alternative mechanism of clathrin-coated pit closure revealed by ion conductance microscopy

Current knowledge of the structural changes taking place during clathrin-mediated endocytosis is largely based on electron microscopy images of fixed preparations and x-ray crystallography data of purified proteins. In this paper, we describe a study of clathrin-coated pit dynamics in living cells using ion conductance microscopy to directly image the changes in pit shape, combined with simultaneous confocal microscopy to follow molecule-specific fluorescence. We find that 70% of pits closed with the formation of a protrusion that grew on one side of the pit, covered the entire pit, and then disappeared together with pit-associated clathrin-enhanced green fluorescent protein (EGFP) and actin-binding protein-EGFP (Abp1-EGFP) fluorescence. This was in contrast to conventionally closing pits that closed and cleaved from flat membrane sheets and lacked accompanying Abp1-EGFP fluorescence. Scission of both types of pits was found to be dynamin-2 dependent. This technique now enables direct spatial and temporal correlation between functional molecule-specific fluorescence and structural information to follow key biological processes at cell surfaces.     

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.     

Endocytosis of activated receptors and clathrin-coated pit formation: deciphering the chicken or egg relationship

The fundamental mechanisms by which receptors once targeted for endocytosis are found in coated pits is an important yet unresolved question. Specifically, are activated receptors simply trapped on encountering preexisting coated pits, subsequently being rapidly internalized? Or do the receptors themselves, by active recruitment, gather soluble coat and cytosolic components and initiate the rapid assembly of new coated pits that then mediate their internalization? To explore this question, we studied the relationship between activation of IgE-bound high affinity Fc receptors (FCepsilonRI) and coated pit formation. Because these receptors are rapidly internalized via clathrin-coated pits only when cross-linked by the binding of multivalent antigens, we were able to separate activation from internalization by using an immobilized antigen. The FCepsilonRIs, initially uniformly distributed over the cell surface. relocalized and aggregated on the antigen-exposed membrane. The process was specific for the antigen, and temperature- and time-dependent. This stimulation initiated a cascade of cellular responses typical of FCepsilonRI signaling including membrane ruffling, cytoskeletal rearrangements, and, in the presence of Ca2+, exocytosis. Despite these responses, no change in coated pit disposition or in the distribution of clathrin and assembly protein AP2 was detected, as monitored by immunoblotting and by quantitative (vertical sectioning) confocal microscopy analysis of immunofluorescently stained cells. Specifically, there was no decrease in the density of clathrin-coated pits in regions of the cell membrane not in contact with the antigen, and there was no apparent increase in clathrin-coated pits in proximity to stimulated FCepsilonRI receptors as would have been expected if the receptors were inducing formation of new pits by active recruitment. These results indicate that de novo formation of clathrin-coated pits is not a prerequisite for rapid internalization or a direct response to stimulation of FCepsilonRI receptors. Therefore, increases in coated pits reported to occur in response to activation of some signaling receptors must be consequences of the signal transduction processes, rather than strictly serving the purpose of the internalization of the receptors.     

The B cell coreceptor CD22 associates with AP50, a clathrin-coated pit adapter protein,via tyrosine-dependent interaction

The B cell coreceptor CD22 plays an important role in regulating signal transduction via the B cell Ag receptor. Studies have shown that surface expression of CD22 can be modulated in response to binding of ligand (i.e., mAb). Thus, it is possible that alterations in the level of CD22 expression following binding of natural ligand(s) may affect its ability to modulate the Ag receptor signaling threshold at specific points during B cell development and differentiation. Therefore, it is important to delineate the physiologic mechanism by which CD22 expression is controlled. In the current study, yeast two-hybrid analysis was used to demonstrate that CD22 interacts with AP50, the medium chain subunit of the AP-2 complex, via tyrosine-based internalization motifs in its cytoplasmic domain. This interaction was further characterized using yeast two-hybrid analysis revealing that Tyr(843) and surrounding amino acids in the cytoplasmic tail of CD22 comprise the primary binding site for AP50. Subsequent studies using transfectant Jurkat cell lines expressing wild-type or mutant forms of CD22 demonstrated that either Tyr(843) or Tyr(863) is sufficient for mAb-mediated internalization of CD22 and that these motifs are involved in its interaction with the AP-2 complex, as determined by coprecipitation of alpha-adaptin. Finally, experiments were performed demonstrating that treatment of B cells with either intact anti-Ig Ab or F(ab')(2) blocks ligand-mediated internalization of CD22. In conclusion, these studies demonstrate that internalization of CD22 is dependent on its association with the AP-2 complex via tyrosine-based internalization motifs.     

Dual interaction of synaptotagmin with mu2- and alpha-adaptin facilitates clathrin-coated pit nucleation

The synaptic vesicle protein synaptotagmin was proposed to act as a major docking site for the recruitment of clathrin coats implicated in endocytosis, including the recycling of synaptic vesicles. We show here that the C2B domain of synaptotagmin binds mu2- and alpha-adaptin, two of the four subunits of the endocytic adaptor complex AP-2. mu2 represents the major interacting subunit of AP-2 within this complex. Its binding to synaptotagmin is mediated by a site in subdomain B that is distinct from the binding site for tyrosine-based sorting motifs located in subdomain A. The presence of the C2B domain of synaptotagmin at the surface of liposomes enhances the recruitment of AP-2 and clathrin. Conversely, perturbation of the interaction between synaptotagmin and AP-2 by synprint, the cytoplasmic synaptotagmin-binding domain of N-type calcium channels, inhibits transferrin internalization in living cells. We conclude that a dual interaction of synaptotagmin with the clathrin adaptor AP-2 plays a key physiological role in the nucleation of endocytic clathrin-coated pits.     

Recruitment of endophilin to clathrin-coated pit necks is required for efficient vesicle uncoating after fission

Endophilin is a membrane-binding protein with curvature-generating and -sensing properties that participates in clathrin-dependent endocytosis of synaptic vesicle membranes. Endophilin also binds the GTPase dynamin and the phosphoinositide phosphatase synaptojanin and is thought to coordinate constriction of coated pits with membrane fission (via dynamin) and subsequent uncoating (via synaptojanin). We show that although synaptojanin is recruited by endophilin at bud necks before fission, the knockout of all three mouse endophilins results in the accumulation of clathrin-coated vesicles, but not of clathrin-coated pits, at synapses. The absence of endophilin impairs but does not abolish synaptic transmission and results in perinatal lethality, whereas partial endophilin absence causes severe neurological defects, including epilepsy and neurodegeneration. Our data support a model in which endophilin recruitment to coated pit necks, because of its curvature-sensing properties, primes vesicle buds for subsequent uncoating after membrane fission, without being critically required for the fission reaction itself.     

Febrile convulsions in a national cohort followed up from birth. I--Prevalence and recurrence in the first five years of life.

Of 13 135 children followed up from birth to the age of 5 years, 303 (2.3%) had febrile convulsions. Prior neurological abnormality had been noted in 13. Of the 290 remaining children, 57 (20%) presented with a complex convulsion, and 103 children (35%) went on to have further febrile convulsions. The risk of further febrile convulsions varied with the age at first convulsion and the presence of a history of convulsive disorders in relatives. There were no significant differences between the sexes.     

Changes in the spectral properties of a plasma membrane lipid analog during the first seconds of endocytosis in living cells.

N-[5-(5, 7-dimethyl Bodipy)-1-pentanoyl]-D-erythro-sphingosylphosphorylcholine (C5-DMB-SM), a fluorescent analog of sphingomyelin, has been used in a study of the formation of very early endosomes in human skin fibroblasts. This lipid exhibits a shift in its fluorescence emission maximum from green (approximately 515 nm) to red (approximately 620 nm) wavelengths with increasing concentrations in membranes. When cells were incubated with 5 microM C5-DMB-SM at 4 degrees C and washed, only plasma membrane fluorescence (yellow-green) was observed. When these cells were briefly (< or = 1 min) warmed to 37 degrees C to allow internalization to occur, and then incubated with defatted bovine serum albumin (back-exchanged) at 11 degrees C to remove fluorescent lipids from the plasma membrane, C5-DMB-SM was distributed in a punctate pattern throughout the cytoplasm. Interestingly, within the same cell some endosomes exhibited green fluorescence, whereas others emitted red-orange fluorescence. Furthermore, the red-orange endosomes were usually seen at the periphery of the cell, while the green endosomes were more uniformly distributed throughout the cytoplasm. This mixed population of endosomes was seen after internalization times as short as 7 s and was also seen over a wide range of C5-DMB-SM concentrations (1-25 microM). Control experiments established that the variously colored endosomes were not induced by changes in pH, membrane potential, vesicle size, or temperature. Quantitative fluorescence microscopy demonstrated that the apparent concentration of the lipid analog in the red-orange endosomes was severalfold higher than its initial concentration at the plasma membrane, suggesting selective internalization (sorting) of the lipid into a subset of early endosomes. Colocalization studies using C5-DMB-SM and either anti-transferrin receptor antibodies or fluorescently labeled low-density lipoprotein further demonstrated that this subpopulation of endosomes resulted from receptor-mediated endocytosis. We conclude that the spectral properties of C5-DMB-SM can be used to distinguish unique populations of early endosomes from one another and to record dynamic changes in their number and distribution within living cells.     

Effect of tyrosine kinase inhibitors on clathrin-coated pit recruitment and internalization of epidermal growth factor receptor

Several inhibitors of epidermal growth factor receptor (EGFR) kinase and Src family kinases (SFK) were employed to study the role of these kinases in EGFR internalization through clathrin-coated pits. The EGFR kinase-specific compound PD158780 substantially diminished EGFR internalization. PP2, an inhibitor of SFK, had a moderate effect on EGFR internalization in several types of cells, including cells lacking SFK, indicating that the inhibition of endocytosis by PP2 is mediated by kinases other than SFK. In contrast, SU6656, a more specific inhibitor of SFK, did not affect EGFR internalization. To examine what stage of internalization requires receptor kinase activity, we established a quantitative assay based on three-dimensional fluorescence microscopy that measures co-localization of an EGF-rhodamine conjugate and a fluorescently tagged clathrin adaptor protein complex, AP-2. Interestingly, recruitment of EGFR into coated pits did not require physiological temperature because the maximal accumulation of EGFR in coated pits was observed at 4 degrees C. Pretreatment of the cells with PD158780 prevented EGFR recruitment into coated pits, whereas the inhibitor did not block the internalization of receptors that had first been allowed to enter the coated pits at 4 degrees C. These data demonstrate that the activation of receptor kinase is essential for the initial, coated pit recruitment step of endocytosis.