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Clathrin-coated vesicles (CCVs) facilitate the transport of cargo between the trans-Golgi network, endosomes, and the plasma membrane. This study presents the first comparative proteomics investigation of CCVs. A CCV-enriched fraction was isolated from HeLa cells and a "mock CCV" fraction from clathrin-depleted cells. We used a combination of 2D difference gel electrophoresis and isobaric tags for relative and absolute quantification (iTRAQ) in conjunction with mass spectrometry to analyze and compare the two fractions. In total, 63 bona fide CCV proteins were identified, including 28 proteins whose association with CCVs had not previously been established. These include numerous post-Golgi SNAREs; subunits of the AP-3, retromer, and BLOC-1 complexes; lysosomal enzymes; CHC22; and five novel proteins of unknown function. The strategy outlined in this paper should be widely applicable as a means of distinguishing genuine organelle components from contaminants.
Clathrin-coated vesicles mediate trafficking of proteins and nutrients in the cell and between organelles. Proteins included in the clathrin-coated vesicles (CCVs) category include clathrin heavy chain (CHC), clathrin light chain (CLC), and a variety of adaptor protein complexes. Much is known about the structures of the individual CCV components, but data are lacking about the structures of the fully assembled complexes together with membrane and in complex with cargo. Here, we determined the structures of natively assembled CCVs in a variety of geometries. We show that the adaptor β2 appendages crosslink adjacent CHC β-propellers and that the appendage densities are enriched in CCV hexagonal faces. We resolve how adaptor protein 2 and other associated factors in hexagonal faces form an assembly hub with an extensive web of interactions between neighboring β-propellers and propose a structural model that explains how adaptor binding can direct the formation of pentagonal and hexagonal faces.
Clathrin-mediated endocytosis (CME) is crucial for modulating the protein composition of a cell's plasma membrane. Clathrin forms a cage-like, polyhedral outer scaffold around a vesicle, to which cargo-selecting clathrin adaptors are attached. Adaptor protein complex (AP2) is the key adaptor in CME. Crystallography has shown AP2 to adopt a range of conformations. Here, we used cryo-electron microscopy, tomography, and subtomogram averaging to determine structures, interactions, and arrangements of clathrin and AP2 at the key steps of coat assembly, from AP2 in solution to membrane-assembled clathrin-coated vesicles (CCVs). AP2 binds cargo and PtdIns(4,5)P 2 (phosphatidylinositol 4,5-bisphosphate)-containing membranes via multiple interfaces, undergoing conformational rearrangement from its cytosolic state. The binding mode of AP2 β2 appendage into the clathrin lattice in CCVs and buds implies how the adaptor structurally modulates coat curvature and coat disassembly.
In searching for binding partners of the intracellular domain of the immunoglobulin superfamily adhesion molecule CHL1, we identified the clathrin-uncoating ATPase Hsc70. CHL1 gene ablation resulted in reduced targeting of Hsc70 to the synaptic plasma membrane and synaptic vesicles, suggesting CHL1 as a synapse-targeting cue for Hsc70. CHL1 accumulates in presynaptic membranes and, in response to synapse activation, is targeted to synaptic vesicles by endocytosis. CHL1 deficiency or disruption of the CHL1/Hsc70 complex results in accumulation of abnormally high levels of clathrin-coated synaptic vesicles with a reduced ability to release clathrin. Generation of new clathrin-coated synaptic vesicles in an activity-dependent manner is inhibited when the CHL1/Hsc70 complex is disrupted, resulting in impaired uptake and release of FM dyes in synaptic boutons. Abnormalities in clathrin-dependent synaptic vesicle recycling may thus underlie brain malfunctions in humans and mice that carry mutations in the CHL1 gene.
EphA4, a receptor tyrosine kinase, is expressed in various pre-, post- and peri-synaptic organelles and implicated in the regulation of morphological and physiological properties of synapses. It regulates synaptic plasticity by acting as a binding partner for glial ephrin-A3 and possibly other pre- or post-synaptic ephrins. Now, its trafficking mechanisms remain unknown. In this study, we examine the association of EphA4 with transport, clathrin-coated and synaptic vesicles using cell fractionation, vesicle immunoisolation and electron microscopy. EphA4 was found in highly purified fractions of clathrin-coated or synaptic vesicles. It was also detected in vesicles immuno-isolated with antibodies anti-synaptophysin, anti-vesicular glutamate transporter or anti-vesicular GABA transporter; demonstrating its presence in synaptic vesicles. However, it was not detected in immuno-isolated piccolo-bassoon transport vesicles. In vivo and in dissociated cultures, EphA4 was localized by immunoelectron microscopy in vesicular glutamate transporter 1-positive terminals of hippocampal neurons. Remarkably, the cell surface immunofluorescence of EphA4 increased markedly in cultured hippocampal neurons following KCl depolarization. These observations indicate that EphA4 is present in subsets of synaptic vesicles, can be externalized during depolarization, and internalized within clathrin-coated vesicles. This trafficking itinerary may serve to regulate the levels of EphA4 in the synaptic plasma membrane and thereby modulate signaling events that contribute to synaptic plasticity.
The transport of the two mannose 6-phosphate receptors (MPRs) from the secretory pathway to the endocytic pathway is mediated by carrier vesicles coated with the AP-1 Golgi-specific assembly protein and clathrin. Using an in vitro assay that reconstitutes the ARF-1-dependent translocation of cytosolic AP-1 onto membranes of the TGN, we have previously reported that the MPRs are key components for the efficient recruitment of AP-1 (Le Borgne, R., G. Griffiths, and B. Hoflack. 1996. J. Biol. Chem. 271:2162-2170). Using a polyclonal antibody against the mouse gamma-adaptin, we have now examined the steady state distribution of AP-1 after subcellular fractionation of mouse fibroblasts lacking both MPRs or reexpressing physiological levels of either MPR. We report that the amount of AP-1 bound to membranes and associated with clathrin-coated vesicles depends on the expression level of the MPRs and on the integrity of their cytoplasmic domains. Thus, these results indicate that the concentration of the MPRs, i.e., the major transmembrane proteins sorted toward the endosomes, determines the number of clathrin-coated vesicles formed in the TGN.
Here we investigate the role of rab5 and its cognate exchange factors rabex-5 and hRME-6 in the regulation of AP2 uncoating from endocytic clathrin-coated vesicles (CCVs). In vitro, we show that the rate of AP2 uncoating from CCVs is dependent on the level of functional rab5. In vivo, overexpression of dominant-negative rab5(S34N), or small interfering RNA (siRNA)-mediated depletion of hRME-6, but not rabex-5, resulted in increased steady-state levels of AP2 associated with endocytic vesicles, which is consistent with reduced uncoating efficiency. hRME-6 guanine nucleotide exchange factor activity requires hRME-6 binding to alpha-adaptin ear, which displaces the ear-associated mu2 kinase AAK1. siRNA-mediated depletion of hRME-6 increases phospho-mu2 levels, and expression of a phosphomimetic mu2 mutant increases levels of endocytic vesicle-associated AP2. Depletion of hRME-6 or rab5(S35N) expression also increases the levels of phosphoinositide 4,5-bisphosphate (PtdIns(4,5)P(2)) associated with endocytic vesicles. These data are consistent with a model in which hRME-6 and rab5 regulate AP2 uncoating in vivo by coordinately regulating mu2 dephosphorylation and PtdIns(4,5)P(2) levels in CCVs.
It has been proposed that, after agonist binding, the thyrotropin-releasing hormone receptor (TRHR) becomes internalised associated with Gq, as part of a TRH-TRHR-Gq ternary complex [13]. We tested this hypothesis directly by examining the intracellular distribution of the TRHR and Gq/11 after agonist binding. The localisation of the TRH-TRHR complex and Gq/11alpha was studied by the biochemical isolation of clathrin-coated vesicles (CCVs). The internalised TRH-TRHR complex was localised in CCVs. The CCVs, which had internalised [3H]MeTRH, contained 4-fold higher levels of radiolabelled ligand than did CCVs from cells incubated with [3H]MeTRH at 4 degrees C. Like the receptor-ligand (RL) complex, G11alpha also translocated to these endocytic vesicles. For example, CCVs from cells with internalised TRH-TRHR complexes contained G11alpha, whereas CCVs from cells without internalised RL complexes lacked G11alpha. We conclude that, after agonist-induced TRHR-G11alpha coupling, both the TRH-TRHR complex and G11alpha are internalised in CCVs.
The adaptor protein complex-1 (AP-1) sorts and packages membrane proteins into clathrin-coated vesicles (CCVs) at the TGN and endosomes. Here we show that this process is highly regulated by phosphorylation of AP-1 subunits. Cell fractionation studies revealed that membrane-associated AP-1 differs from cytosolic AP-1 in the phosphorylation status of its beta1 and mu1 subunits. AP-1 recruitment onto the membrane is associated with protein phosphatase 2A (PP2A)-mediated dephosphorylation of its beta1 subunit, which enables clathrin assembly. This Golgi-associated isoform of PP2A exhibits specificity for phosphorylated beta1 compared with phosphorylated mu1. Once on the membrane, the mu1 subunit undergoes phosphorylation, which results in a conformation change, as revealed by increased sensitivity to trypsin. This conformational change is associated with increased binding to sorting signals on the cytoplasmic tails of cargo molecules. Dephosphorylation of mu1 (and mu2) by another PP2A-like phosphatase reversed the effect and resulted in adaptor release from CCVs. Immunodepletion and okadaic acid inhibition studies demonstrate that PP2A is the cytosolic cofactor for Hsc-70-mediated adaptor uncoating. A model is proposed where cyclical phosphorylation/dephosphorylation of the subunits of AP-1 regulate its function from membrane recruitment until its release into cytosol.
Human transferrin receptors (TR) and receptors for polymeric immunoglobulins (pIgR) expressed in polarized MDCK cells maintain steady-state, asymmetric distributions on the separate basolateral and apical surfaces even though they are trafficking continuously into and across these cells. The intracellular mechanisms required to maintain these asymmetric distributions have not been located. Here we show that TR and pIgR internalize from both surfaces to a common interconnected endosome compartment that includes tubules with buds coated with clathrin lattices. These buds generate vesicles that carry TR to the basolateral border. The lattices contain gamma-adaptin and are dispersed by treatment with brefeldin A (BFA). Since BFA treatment abrogates the vectorial trafficking of TR in polarized MDCK cells, we propose that the clathrin-coated domains of the endosome tubules contain the polarized sorting mechanism responsible for their preferential basolateral distribution.
Many viruses that enter cells by clathrin-dependent endocytosis are significantly larger than the dimensions of a typical clathrin-coated vesicle. The mechanisms by which viruses co-opt the clathrin machinery for efficient internalization remain uncertain. Here we examined how clathrin-coated vesicles accommodate vesicular stomatitis virus (VSV) during its entry into cells. Using high-resolution imaging of the internalization of single viral particles into cells expressing fluorescent clathrin and adaptor molecules, we show that VSV enters cells through partially clathrin-coated vesicles. We found that on average, virus-containing vesicles contain more clathrin and clathrin adaptor molecules than conventional vesicles, but this increase is insufficient to permit full coating of the vesicle. We further show that virus-containing vesicles depend upon the actin machinery for their internalization. Specifically, we found that components of the actin machinery are recruited to virus-containing vesicles, and chemical inhibition of actin polymerization trapped viral particles in vesicles at the plasma membrane. By analysis of multiple independent virus internalization events, we show that VSV induces the nucleation of clathrin for its uptake, rather than depending upon random capture by formation of a clathrin-coated pit. This work provides new mechanistic insights into the process of virus internalization as well as uptake of unconventional cargo by the clathrin-dependent endocytic machinery.
Coated vesicles prepared from bovine brain cerebral cortex exhibited [3H]5-hydroxytryptamine (5-HT, serotonin) and [3H]spiperone binding activities. The binding activities were localized in the inner core vesicles. Binding reached an equilibrium level by 30-45 min at 30 degreesC, and was reversed by the addition of 100 microM 5-HT for [3H]5-HT binding or 10 microM ketanserin for [3H]spiperone binding. The saturation binding experiments indicated a single class of binding sites for [3H]5-HT and [3H]spiperone with apparent Kd values of 2.4 and 1.75 nM, respectively. The binding of [3H]5-HT was displaced by 5-HT and 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT), but not by ketanserin. The binding of [3H]spiperone was displaced by spiperone and ketanserin but not by 5-HT or 8-OH-DPAT even at 1 mM. The coated vesicles were shown by immunoblotting assay to contain alpha-subunits of GTP-binding proteins, Galphas, Galphai2, Galphai3, Galphao and Galphaq/11. Forskolin-stimulated adenylate cyclase activity in the coated vesicles was inhibited to 80% of the control level by 5-HT or 8-OH-DPAT. These results suggested that 5-HT1A and 5-HT2A receptors are present in bovine brain coated vesicles and that the 5-HT1A receptors are coupled to adenylate cyclase activity via GTP binding proteins.
Phospholipase D (PLD) has been implicated in many cellular functions, such as vesicle trafficking, exocytosis, differentiation, and proliferation. The aim of this study was to characterize the role of PLD in HSY cells, a human cell line originating from the intercalated duct of the parotid gland. As the function and intracellular localization of PLD varies according to cell type, initially, the intracellular localization of PLD1 and PLD2 was determined. By immunofluorescence, PLD1 and PLD2 both showed a punctate cytoplasmic distribution with extensive co-localization with TGN-46. PLD1 was also found in the nucleus, while PLD2 was associated with the plasma membrane. Treatment of cells with the primary alcohol 1-butanol inhibits the hydrolysis of phosphatidylcoline by PLD thereby suppressing phosphatidic acid (PA) production. In untreated HSY cells, there was only a slight co-localization of PLD with the clathrin coated vesicles. When HSY cells were incubated with 1-butanol the total number of clathrin coated vesicles increased, especially in the juxtanuclear region and the co-localization of PLD with the clathrin coated vesicles was augmented. Transmission electron microscopy confirmed that the number of Golgi-associated coated vesicles was greater. Treatment with 1-butanol also affected the Golgi apparatus, increasing the volume of the Golgi saccules. The decrease in PA levels after treatment with 1-butanol likewise resulted in an accumulation of enlarged lysosomes in the perinuclear region. Therefore, in HSY cells PLD appears to be involved in the formation of Golgi associated clathrin coated vesicles as well as in the structural maintenance of the Golgi apparatus.
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.
SNAREs provide the specificity and energy for the fusion of vesicles with their target membrane, but how they are sorted into the appropriate vesicles on post-Golgi trafficking pathways is largely unknown. We demonstrate that the clathrin-mediated endocytosis of the SNARE VAMP7 is directly mediated by Hrb, a clathrin adaptor and ArfGAP. Hrb wraps 20 residues of its unstructured C-terminal tail around the folded VAMP7 longin domain, demonstrating that unstructured regions of clathrin adaptors can select cargo. Disrupting this interaction by mutation of the VAMP7 longin domain or depletion of Hrb causes VAMP7 to accumulate on the cell's surface. However, the SNARE helix of VAMP7 binds back onto its longin domain, outcompeting Hrb for binding to the same groove and suggesting that Hrb-mediated endocytosis of VAMP7 occurs only when VAMP7 is incorporated into a cis-SNARE complex. These results elucidate the mechanism of retrieval of a postfusion SNARE complex in clathrin-coated vesicles.
Intracellular dynamics of an abnormal isoform of prion protein (PrPSc) are tightly associated with prion propagation. However, the machineries involved in the intracellular trafficking of PrPSc are not fully understood. Our previous study suggested that PrPSc in persistently prion-infected cells dynamically circulates between endocytic-recycling compartments (ERCs) and peripheral regions of the cells. To investigate these machineries, we focused on retrograde transport from endosomes to the trans-Golgi network, which is one of the pathways involved in recycling of molecules. PrPSc was co-localized with components of clathrin-coated vesicles (CCVs) as well as those of the retromer complex, which are known as machineries for retrograde transport. Fractionation of intracellular compartments by density gradient centrifugation showed the presence of PrPSc and the components of CCVs in the same fractions. Furthermore, PrPSc was detected in CCVs isolated from intracellular compartments of prion-infected cells. Knockdown of clathrin interactor 1, which is one of the clathrin adaptor proteins involved in retrograde transport, did not change the amount of PrPSc, but it altered the distribution of PrPSc from ERCs to peripheral regions, including late endosomes/lysosomes. These data demonstrated that some PrPSc is transported from endosomes to ERCs by CCVs, which might be involved in the recycling of PrPSc.
In retinal photoreceptors, vectorial transport of cargo is critical for transduction of visual signals, and defects in intracellular trafficking can lead to photoreceptor degeneration and vision impairment. Molecular signatures associated with routing of transport vesicles in photoreceptors are poorly understood. We previously reported the identification of a novel rod photoreceptor specific isoform of Receptor Expression Enhancing Protein (REEP) 6, which belongs to a family of proteins involved in intracellular transport of receptors to the plasma membrane. Here we show that loss of REEP6 in mice (Reep6-/-) results in progressive retinal degeneration. Rod photoreceptor dysfunction is observed in Reep6-/- mice as early as one month of age and associated with aberrant accumulation of vacuole-like structures at the apical inner segment and reduction in selected rod phototransduction proteins. We demonstrate that REEP6 is detected in a subset of Clathrin-coated vesicles and interacts with the t-SNARE, Syntaxin3. In concordance with the rod degeneration phenotype in Reep6-/- mice, whole exome sequencing identified homozygous REEP6-E75K mutation in two retinitis pigmentosa families of different ethnicities. Our studies suggest a critical function of REEP6 in trafficking of cargo via a subset of Clathrin-coated vesicles to selected membrane sites in retinal rod photoreceptors.
Liver sinusoidal endothelial cells (LSECs) are highly active professional scavenger cells using clathrin-mediated endocytosis to clear the blood from macromolecular waste products. Using confocal microscopy, we observed a remarkable net-like distribution of clathrin heavy chain (CHC) in LSECs while all other cell types examined including various primary endothelial cells and cell lines showed the well-known punctuate staining pattern representing clathrin-coated vesicles (CCV). The net-like distribution of CHC in LSECs co-localized fully with microtubules, but not with actin. Upon 3D imaging, the net-like distribution of CHC resolved into numerous CCVs organized along the microtubules. The CCVs only partially co-localized with early endosome antigen 1 (EEA1) and adaptor protein 2 (AP-2). Endocytic vesicles containing ligand destined for degradation (FITC-AHGG) were organized along the clathrin/tubulin net-like structures, whereas transferrin-containing recycling vesicles co-localized to a much lower extent. Disruption of the microtubules by nocodazole treatment caused a collapse of the net-like organization of CCVs as well as a profound redistribution of EEA1, AP-2 and FITC-AHGG-containing vesicles, while transferrin internalization and recycling remained unaffected.
Clathrin light chains (CLCa and CLCb) are major constituents of clathrin-coated vesicles. Unique functions for these evolutionary conserved paralogs remain elusive, and their role in clathrin-mediated endocytosis in mammalian cells is debated. Here, we find and structurally characterize a direct and selective interaction between CLCa and the long isoform of the actin motor protein myosin VI, which is expressed exclusively in highly polarized tissues. Using genetically-reconstituted Caco-2 cysts as proxy for polarized epithelia, we provide evidence for coordinated action of myosin VI and CLCa at the apical surface where these proteins are essential for fission of clathrin-coated pits. We further find that myosin VI and Huntingtin-interacting protein 1-related protein (Hip1R) are mutually exclusive interactors with CLCa, and suggest a model for the sequential function of myosin VI and Hip1R in actin-mediated clathrin-coated vesicle budding.
The actin cytoskeleton has been implicated in endocytosis, yet few molecules that link these systems have been identified. Here, we have cloned and characterized mHip1R, a protein that is closely related to huntingtin interacting protein 1 (Hip1). These two proteins are mammalian homologues of Sla2p, an actin binding protein important for actin organization and endocytosis in yeast. Sequence alignments and secondary structure predictions verified that mHip1R belongs to the Sla2 protein family. Thus, mHip1R contains an NH(2)-terminal domain homologous to that implicated in Sla2p's endocytic function, three predicted coiled-coils, a leucine zipper, and a talin-like actin-binding domain at the COOH terminus. The talin-like domain of mHip1R binds to F-actin in vitro and colocalizes with F-actin in vivo, indicating that this activity has been conserved from yeast to mammals. mHip1R shows a punctate immunolocalization and is enriched at the cell cortex and in the perinuclear region. We concluded that the cortical localization represents endocytic compartments, because mHip1R colocalizes with clathrin, AP-2, and endocytosed transferrin, and because mHip1R fractionates biochemically with clathrin-coated vesicles. Time-lapse video microscopy of mHip1R-green fluorescence protein (GFP) revealed a blinking behavior similar to that reported for GFP-clathrin, and an actin-dependent inward movement of punctate structures from the cell periphery. These data show that mHip1R is a component of clathrin-coated pits and vesicles and suggest that it might link the endocytic machinery to the actin cytoskeleton.
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