The structures of natively assembled clathrin-coated vesicles.
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ABSTRACT:
SUBMITTER: Paraan M
PROVIDER: S-EPMC7375819 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
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Project description: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.
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Project description:Myosin VI is involved in membrane traffic and dynamics and is the only myosin known to move towards the minus end of actin filaments. Splice variants of myosin VI with a large insert in the tail domain were specifically expressed in polarized cells containing microvilli. In these polarized cells, endogenous myosin VI containing the large insert was concentrated at the apical domain co-localizing with clathrin- coated pits/vesicles. Using full-length myosin VI and deletion mutants tagged with green fluorescent protein (GFP) we have shown that myosin VI associates and co-localizes with clathrin-coated pits/vesicles by its C-terminal tail. Myosin VI, precipitated from whole cytosol, was present in a protein complex containing adaptor protein (AP)-2 and clathrin, and enriched in purified clathrin-coated vesicles. Over-expression of the tail domain of myosin VI containing the large insert in fibroblasts reduced transferrin uptake in transiently and stably transfected cells by >50%. Myosin VI is the first motor protein to be identified associated with clathrin-coated pits/vesicles and shown to modulate clathrin-mediated endocytosis.
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Project description:It is generally assumed that cells interrogate the mechanical properties of their environment by pushing and pulling on the extracellular matrix (ECM). For instance, acto-myosin-dependent contraction forces exerted at focal adhesions (FAs) allow the cell to actively probe substrate elasticity. Here, we report that a subset of long-lived and flat clathrin-coated structures (CCSs), also termed plaques, are contractility-independent mechanosensitive signaling platforms. We observed that plaques assemble in response to increasing substrate rigidity and that this is independent of FAs, actin and myosin-II activity. We show that plaque assembly depends on ?v?5 integrin, and is a consequence of frustrated endocytosis whereby ?v?5 tightly engaged with the stiff substrate locally stalls CCS dynamics. We also report that plaques serve as platforms for receptor-dependent signaling and are required for increased Erk activation and cell proliferation on stiff environments. We conclude that CCSs are mechanotransduction structures that sense substrate rigidity independently of cell contractility.
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Project description:Clathrin-mediated endocytosis plays an important role in the recycling of synaptic vesicle in presynaptic terminals, and in the recycling of transmitter receptors in neuronal soma/dendrites. The present study uses electron microscopy (EM) and immunogold EM to document the different categories of clathrin-coated vesicles (CCV) and pits (CCP) in axons compared to soma/dendrites, and the depolarization-induced redistribution of clathrin in these two polarized compartments of the neuron. The size of CCVs in presynaptic terminals (~ 40 nm; similar to the size of synaptic vesicles) is considerably smaller than the size of CCVs in soma/dendrites (~ 90 nm). Furthermore, neuronal stimulation induces an increase in the number of CCV/CCP in presynaptic terminals, but a decrease in soma/dendrites. Immunogold labeling of clathrin revealed that in presynaptic terminals under resting conditions, the majority of clathrin molecules are unassembled and concentrated outside of synaptic vesicle clusters. Upon depolarization with high K+, label for clathrin became scattered among de-clustered synaptic vesicles and moved closer to the presynaptic active zone. In contrast to axons, clathrin-labeled CCVs and CCPs were prominent in soma/dendrites under resting conditions, and became inconspicuous upon depolarization with high K+. Thus, EM examination suggests that the regulation and mechanism of clathrin-mediated endocytosis differ between axon and dendrite, and that clathrin redistributes differently in these two neuronal compartments upon depolarization.
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Project description:Description Adhesive clathrin-coated structures steer cell migration by accumulating on collagen fibers decorated with specific ligands. Migrating cells navigate in complex environments through sensing and interpreting biochemical and/or mechanical cues. Here, we report that recently identified tubular clathrin/AP-2 lattices (TCALs), a subset of clathrin-coated structures (CCSs) that pinch collagen fibers, mechanically control directed migration along fibers decorated with ligands of CCS cargoes in three-dimensional (3D) environments. We observed that epidermal growth factor or low-density lipoprotein bound to collagen fibers leads to increased local nucleation and accumulation of TCALs. By using engineered, mixed collagen networks, we demonstrate that this mechanism selectively increases local forces applied on ligand-decorated fibers. We show that these effects depend on the ligand’s receptors but do not rely on their ability to trigger signaling events. We propose that the preferential accumulation of TCALs along ligand-decorated fibers steers migration in 3D environments. We conclude that ligand-regulated, local TCAL accumulation results in asymmetric force distribution that orients cell migration in 3D environments.
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Project description:Clathrin-coated vesicles mediate vesicular traffic in cells. Three-dimensional image reconstructions of homogenous populations of in vitro assembled clathrin coats have yielded a molecular model for clathrin and its interactions with some of its partners. The intrinsic averaging required for those calculations has precluded detailed analysis of heterogeneous populations of clathrin-coated vesicles isolated from cells. We have therefore used cryo-electron tomography to study the lattice organization of individual clathrin-coated vesicles and the disposition of the captured vesicle with respect to the surrounding coat. We find a wide range of designs for the clathrin lattice, with different patterns of pentagonal, hexagonal, and occasionally heptagonal facets. Many coats, even smaller ones, enclose membrane vesicles, which are generally offset from the center of the clathrin shell. The electron density distribution between the coat and the underlying vesicle is not uniform, and the number of apparent contacts that anchor the clathrin lattice to the vesicle membrane is significantly less than the number of clathrin heavy chains in the assembly. We suggest that the eccentric position of the vesicle reflects the polarity of assembly, from initiation of coat formation to membrane pinching.
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Project description:Endocytic organelles maintain their acidic pH using the V-type ATPase proton pump. However, proton accumulation across the membrane generates a voltage and requires the movement of an additional ion, known as a counterion, to dissipate charge buildup. The role of counterion movement in endosomes is not clear, but a subpopulation of early endosomes, clathrin-coated vesicles (CCVs), has previously been shown to use external chloride (Cl-) to allow V-ATPase-dependent acidification. We aimed to determine the identity and function of this presumed Cl- transporting protein. Our sample of highly enriched bovine brain CCVs exhibited V-type ATPase-facilitated acidification in the presence of external Cl-, independent of the monovalent cations present. While unsuccessful at identifying the mechanism of anion transport, we used glutamate-facilitated acidification, density gradients, and mass spectrometry to show that most brain CCVs are synaptic vesicles, complementing results from earlier studies that argued similarity only on the basis on protein content. The source of Cl--dependent acidification in brain CCVs may be vGLUT1, a synaptic vesicle glutamate transporter with known Cl- permeability, although CCVs in other tissues are likely to utilize different proteins to facilitate acidification.
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Project description:BackgroundClathrin-mediated endocytosis (CME) plays a fundamental role in podocyte health. Genetic ablation of genes implicated in CME has been shown to cause severe proteinuria and foot process effacement in mice. However, little is known about the cargo of clathrin-coated vesicles (CCVs) in podocytes. The goal of this study was to isolate CCVs from podocytes and identify their cargo by proteomic analysis.MethodsGlomeruli isolated from Podocin-Cre Rosa-DTR flox mouse kidneys were seeded and treated with diphtheria toxin to obtain pure primary podocyte cultures. CCVs were isolated by differential gradient ultracentrifugation, and enrichment of CCVs was assessed by immunoblotting and electron microscopy (EM). Liquid chromatography-mass spectrometry (LC-MS) was performed for proteomic analysis. Proteins with higher abundance than transferrin receptor protein 1 were evaluated for CCV cargo potential against previously published literature. Immunofluorescence staining of identified cargo proteins and CCVs was performed in podocytes for further verification.ResultsImmunoblotting for multiple protein markers of CME revealed enrichment in the CCV fraction. Enrichment of CCVs among other small vesicles was observed via EM. Proteomics yielded a total of >1200 significant proteins. Multiple-step data analysis revealed 36 CCV-associated proteins, of which 10 represent novel, highly abundant cargo proteins in podocytes. Colocalization of cargo proteins and CCVs on immunostaining was observed.ConclusionsOur identification of podocyte CCV cargo proteins helps to elucidate the importance of endocytic trafficking for podocyte health and maintenance of the glomerular environment.
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Project description:?-Arrestins critically regulate G-protein-coupled receptor (GPCR) signalling, not only 'arresting' the G protein signal but also modulating endocytosis and initiating a discrete G-protein-independent signal through MAP kinase. Despite enormous recent progress towards understanding biophysical aspects of arrestin function, arrestin cell biology remains relatively poorly understood. Two key tenets underlie the prevailing current view: ?-arrestin accumulates in clathrin-coated structures (CCSs) exclusively in physical complex with its activating GPCR, and MAP kinase activation requires endocytosis of formed GPCR-?-arrestin complexes. We show here, using ?1-adrenergic receptors, that ?-arrestin-2 (arrestin 3) accumulates robustly in CCSs after dissociating from its activating GPCR and transduces the MAP kinase signal from CCSs. Moreover, inhibiting subsequent endocytosis of CCSs enhances the clathrin- and ?-arrestin-dependent MAP kinase signal. These results demonstrate ?-arrestin 'activation at a distance', after dissociating from its activating GPCR, and signalling from CCSs. We propose a ?-arrestin signalling cycle that is catalytically activated by the GPCR and energetically coupled to the endocytic machinery.
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