The Experts below are selected from a list of 270 Experts worldwide ranked by ideXlab platform
Richard G W Anderson - One of the best experts on this subject based on the ideXlab platform.
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synaptotagmin regulation of Coated Pit assembly
Journal of Biological Chemistry, 2000Co-Authors: Christine Von Poser, Jimmy Z Zhang, Thomas C Sudhof, Chieko Mineo, Wei Ding, Yunshu Ying, Richard G W AndersonAbstract:Abstract Synaptotagmins bind clathrin AP-2 with high affinity via their second C2 domain, which indicates they are involved in Coated Pit function. We now report that expression of synaptotagmins lacking either the second C2 domain or the entire cytoplasmic region potently inhibit endocytosis. Inhibition was dependent on two intramembrane cysteine residues that were found to be essential for synaptotagmin oligomerization. Cells expressing the wild-type, but not the mutant, truncated synaptotagmin fragment had a reduced number of clathrin-Coated Pits. These results suggest that the formation of synaptotagmin multimers is an important step in the regulation of Coated Pit assembly.
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a requirement for ankyrin binding to clathrin during Coated Pit budding
Journal of Biological Chemistry, 1999Co-Authors: Peter Michaely, Richard G W Anderson, Adeela Kamal, Vann BennettAbstract:Abstract Recent studies suggest that the mobility of clathrin-Coated Pits at the cell surface are restricted by an actin cytoskeleton and that there is an obligate reduction in the amount of spectrin on membranes during Coated Pit budding. The spectrin-actin cytoskeleton associates with membranes primarily through ankyrins, which interact with the cytoplasmic region of numerous integral membrane proteins. We now report that the fourth repeat domain (D4) of ankyrinR binds to the N-terminal domain of clathrin heavy chain with high affinity. Addition of peptides containing the D4 region inhibited clathrin-Coated Pit budding in vitro. In addition, microinjection of D4 containing peptides blocked the endocytosis of fluorescent low density lipoprotein (LDL). AnkyrinR peptides that contained repeat domains other than D4 had no effect on either in vitro budding or internalization of LDL. Finally, immunofluorescence shows that ankyrin is uniformly associated with endosomes that contain fluorescent LDL. These results suggest that ankyrin plays a role in the budding of clathrin-Coated Pits during endocytosis.
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annexin vi mediated loss of spectrin during Coated Pit budding is coupled to delivery of ldl to lysosomes
Journal of Cell Biology, 1998Co-Authors: Adeela Kamal, Yunshu Ying, Richard G W AndersonAbstract:Previously we reported that annexin VI is required for the budding of clathrin-Coated Pits from human fibroblast plasma membranes in vitro. Here we show that annexin VI bound to the NH 2 -terminal 28-kD portion of membrane spectrin is as effective as cytosolic annexin VI in supporting Coated Pit budding. Annexin VI–dependent budding is accompanied by the loss of ∼50% of the spectrin from the membrane and is blocked by the cysteine protease inhibitor N -acetyl-leucyl-leucyl-norleucinal (ALLN). Incubation of fibroblasts in the presence of ALLN initially blocks the uptake of low density lipoprotein (LDL), but the cells recover after 1 h and internalize LDL with normal kinetics. The LDL internalized under these conditions, however, fails to migrate to the center of the cell and is not degraded. ALLN-treated cells have twice as many Coated Pits and twofold more membrane clathrin, suggesting that new Coated Pits have assembled. Annexin VI is not required for the budding of these new Coated Pits and ALLN does not inhibit. Finally, microinjection of a truncated annexin VI that inhibits budding in vitro has the same effect on LDL internalization as ALLN. These findings suggest that fibroblasts are able to make at least two types of Coated Pits, one of which requires the annexin VI–dependent activation of a cysteine protease to disconnect the clathrin lattice from the spectrin membrane cytoskeleton during the final stages of budding.
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synaptotagmin i is a high affinity receptor for clathrin ap 2 implications for membrane recycling
Cell, 1994Co-Authors: Jimmy Z Zhang, Bazbek Davletov, Thomas C Sudhof, Richard G W AndersonAbstract:Summary In nerve terminals, Ca 2+ -stimulated synaptic vesicle exocytosis is rapidly followed by endocytosis. Synaptic vesicle endocytosis requires clathrin-Coated Pits similar to receptor-mediated endocytosis in fibroblasts. Binding of clathrin AP-2 (adaptor complex) to an unidentified high affinity membrane receptor appears to be necessary for Coated Pit assembly in fibroblasts. We now show that synaptic vesicles have a high affinity AP-2 site (K D , ∼1 × 10 su−10 M) similar to the one observed in fibroblasts. Using a combination of competition and direct binding assays, we demonstrate that synaptotagmin I, an intrinsic membrane protein of synaptic vesicles, has all of the properties of the AP-2 receptor and that AP-2 binds to the second C 2 domain in the molecule. Thus, synaptotagmin I may be a multifunctional protein with a function in endocytosis in addition to the previously proposed role in exocytosis.
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mis assembly of clathrin lattices on endosomes reveals a regulatory switch for Coated Pit formation
Journal of Cell Biology, 1993Co-Authors: Lihsien Wang, Karen G Rothberg, Richard G W AndersonAbstract:The clathrin-Coated Pit lattice is held onto the plasma membrane by an integral membrane protein that binds the clathrin AP-2 subunit with high affinity. In vitro studies have suggested that this protein controls the assembly of the Pit because membrane bound AP-2 is required for lattice assembly. If so, the AP-2 binding site must be a resident protein of the Coated Pit and recycle with other receptors that enter cells through this pathway. Proper recycling, however, would require the switching off of AP-2 binding to allow the binding site to travel through the endocytic pathway unencumbered. Evidence for this hypothesis has been revealed by the cationic amphiphilic class of drugs (CAD), which have previously been found to inhibit receptor recycling. Incubation of human fibroblasts in the presence of these drugs caused clathrin lattices to assemble on endosomal membranes and at the same time prevented Coated Pit assembly at the cell surface. These effects suggest that CADs reverse an on/off switch that controls AP-2 binding to membranes. We conclude that cells have a mechanism for switching on and off AP-2 binding during the endocytic cycle.
Sandra L Schmid - One of the best experts on this subject based on the ideXlab platform.
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phosphatidylinositol 4 5 bisphosphate regulates clathrin Coated Pit initiation stabilization and size
Molecular Biology of the Cell, 2011Co-Authors: Costin N Antonescu, Gaudenz Danuser, Francois Aguet, Sandra L SchmidAbstract:Clathrin-mediated endocytosis (CME) is the major mechanism for internalization in mammalian cells. CME initiates by recruitment of adaptors and clathrin to form clathrin-Coated Pits (CCPs). Nearly half of nascent CCPs abort, whereas others are stabilized by unknown mechanisms and undergo further maturation before pinching off to form clathrin-Coated vesicles (CCVs). Phosphatidylinositol-(4,5)-bisphosphate (PIP2), the main lipid binding partner of endocytic proteins, is required for CCP assembly, but little is currently known about its contribution(s) to later events in CCV formation. Using small interfering RNA (siRNA) knockdown and overexpression, we have analyzed the effects of manipulating PIP2 synthesis and turnover on CME by quantitative total internal reflection fluorescence microscopy and computational analysis. Phosphatidylinositol-4-phosphate-5-kinase cannot be detected within CCPs but functions in initiation and controls the rate and extent of CCP growth. In contrast, the 5′-inositol phosphatase synaptojanin 1 localizes to CCPs and controls early stabilization and maturation efficiency. Together these results suggest that the balance of PIP2 synthesis in the bulk plasma membrane and its local turnover within CCPs control multiple stages of CCV formation.
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local clustering of transferrin receptors promotes clathrin Coated Pit initiation
Journal of Cell Biology, 2010Co-Authors: Francois Aguet, Gaudenz Danuser, Sandra L SchmidAbstract: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.
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global and local regulation of clathrin Coated Pit dynamics detected on patterned substrates
Biophysical Journal, 2009Co-Authors: Dinah Loerke, Sandra L Schmid, Gaudenz DanuserAbstract:Live-cell imaging of individual clathrin-Coated Pit (CCP) dynamics has revealed a broad variation in their internalization kinetics, but the functional significance and mechanistic underpinnings of this heterogeneity remain unknown. One contributing factor may be the spatial variations in the underlying actin cortex. To test this, we cultured cells on fibronectin (Fn) micropatterned substrates to vary the cortical actin mechanics in a defined manner. Under these conditions, stress fibers became organized to bridge adhesive islands, creating spatial heterogeneity in the cortical actin architecture. CCP lifetimes within the Fn-Coated islands were selectively prolonged. This differential effect was not due to adherence to Fn-Coated surfaces, and was not observed in cells grown on patterned surfaces that did not induce organized stress fiber assembly. Pharmacological agents that lower cortical tension selectively lowered CCP lifetimes within Fn islands, thus abolishing the spatial heterogeneity in the CCP dynamics. Although we cannot rule out the possibility that other factors might locally affect CCP dynamics at Fn islands, our data suggest that localized modulation in cortical tension may spatially regulate clathrin-mediated endocytosis.
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cortical tension affects the spatial heterogeneity of clathrin Coated Pit dynamics
Biophysical Journal, 2009Co-Authors: Dinah Loerke, Sandra L Schmid, Gaudenz DanuserAbstract:Clathrin-mediated endocytosis (CME) in mammalian cells is critical for many cellular processes including cell surface receptor down-regulation and nutrient uptake. From analyses of protein interaction networks, the actin polymerization machinery is a modular component within the endocytic interactome. However, the precise role of actin in CME is still under debate. Live cell microscopy has revealed a wide variation in the dynamics of clathrin-Coated Pits (CCPs). To gain insight of the heterogeneity of CCP dynamics and how cortical actin might influence this heterogeneity, we applied total internal reflection fluorescence microscopy to live cells grown on micro-fabricated substrates patterned with adhesive and non-adhesive regions. Cells on patterns showed overall longer CCP lifetimes compared to cells on chemically uniform surfaces, possibly the result of increased cortical tension. CCP lifetime distributions were also significantly different between adhesive and non-adhesive regions. When the structure of cortical actin is weakened by application of an actin monomer sequestering drug latrunculin A (latA), we found that the CCP lifetimes were homogenized to the level of the non-adherent regions. The decrease in CCP lifetime on adherent regions suggests that cortical actin filaments act as barriers at the adherent surface in CME.
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Cargo and Dynamin Regulate Clathrin-Coated Pit Maturation
PLoS Biology, 2009Co-Authors: Dinah Loerke, Marcel Mettlen, Defne Yarar, Henry Jaqaman, Khuloud Jaqaman, Gaudenz Danuser, Sandra L SchmidAbstract:Total internal reflection fluorescence microscopy (TIR-FM) has become a powerful tool for studying clathrin-mediated endocytosis. However, due to difficulties in tracking and quantifying their heterogeneous dynamic behavior, detailed analyses have been restricted to a limited number of selected clathrin-Coated Pits (CCPs). To identify intermediates in the formation of clathrin-Coated vesicles and factors that regulate progression through these stages, we used particle-tracking software and statistical methods to establish an unbiased and complete inventory of all visible CCP trajectories. We identified three dynamically distinct CCP subpopulations: two short-lived subpopulations corresponding to aborted intermediates, and one longer-lived productive subpopulation. In a manner dependent on AP2 adaptor complexes, increasing cargo concentration significantly enhances the maturation efficiency of productive CCPs, but has only minor effects on their lifetimes. In contrast, small interfering RNA (siRNA) depletion of dynamin-2 GTPase and reintroduction of wild-type or mutant dynamin-1 revealed dynamin's role in controlling the turnover of abortive intermediates and the rate of CCP maturation. From these data, we infer the existence of an endocytic restriction or checkpoint, responsive to cargo and regulated by dynamin.
Gaudenz Danuser - One of the best experts on this subject based on the ideXlab platform.
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phosphatidylinositol 4 5 bisphosphate regulates clathrin Coated Pit initiation stabilization and size
Molecular Biology of the Cell, 2011Co-Authors: Costin N Antonescu, Gaudenz Danuser, Francois Aguet, Sandra L SchmidAbstract:Clathrin-mediated endocytosis (CME) is the major mechanism for internalization in mammalian cells. CME initiates by recruitment of adaptors and clathrin to form clathrin-Coated Pits (CCPs). Nearly half of nascent CCPs abort, whereas others are stabilized by unknown mechanisms and undergo further maturation before pinching off to form clathrin-Coated vesicles (CCVs). Phosphatidylinositol-(4,5)-bisphosphate (PIP2), the main lipid binding partner of endocytic proteins, is required for CCP assembly, but little is currently known about its contribution(s) to later events in CCV formation. Using small interfering RNA (siRNA) knockdown and overexpression, we have analyzed the effects of manipulating PIP2 synthesis and turnover on CME by quantitative total internal reflection fluorescence microscopy and computational analysis. Phosphatidylinositol-4-phosphate-5-kinase cannot be detected within CCPs but functions in initiation and controls the rate and extent of CCP growth. In contrast, the 5′-inositol phosphatase synaptojanin 1 localizes to CCPs and controls early stabilization and maturation efficiency. Together these results suggest that the balance of PIP2 synthesis in the bulk plasma membrane and its local turnover within CCPs control multiple stages of CCV formation.
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local clustering of transferrin receptors promotes clathrin Coated Pit initiation
Journal of Cell Biology, 2010Co-Authors: Francois Aguet, Gaudenz Danuser, Sandra L SchmidAbstract: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.
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global and local regulation of clathrin Coated Pit dynamics detected on patterned substrates
Biophysical Journal, 2009Co-Authors: Dinah Loerke, Sandra L Schmid, Gaudenz DanuserAbstract:Live-cell imaging of individual clathrin-Coated Pit (CCP) dynamics has revealed a broad variation in their internalization kinetics, but the functional significance and mechanistic underpinnings of this heterogeneity remain unknown. One contributing factor may be the spatial variations in the underlying actin cortex. To test this, we cultured cells on fibronectin (Fn) micropatterned substrates to vary the cortical actin mechanics in a defined manner. Under these conditions, stress fibers became organized to bridge adhesive islands, creating spatial heterogeneity in the cortical actin architecture. CCP lifetimes within the Fn-Coated islands were selectively prolonged. This differential effect was not due to adherence to Fn-Coated surfaces, and was not observed in cells grown on patterned surfaces that did not induce organized stress fiber assembly. Pharmacological agents that lower cortical tension selectively lowered CCP lifetimes within Fn islands, thus abolishing the spatial heterogeneity in the CCP dynamics. Although we cannot rule out the possibility that other factors might locally affect CCP dynamics at Fn islands, our data suggest that localized modulation in cortical tension may spatially regulate clathrin-mediated endocytosis.
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cortical tension affects the spatial heterogeneity of clathrin Coated Pit dynamics
Biophysical Journal, 2009Co-Authors: Dinah Loerke, Sandra L Schmid, Gaudenz DanuserAbstract:Clathrin-mediated endocytosis (CME) in mammalian cells is critical for many cellular processes including cell surface receptor down-regulation and nutrient uptake. From analyses of protein interaction networks, the actin polymerization machinery is a modular component within the endocytic interactome. However, the precise role of actin in CME is still under debate. Live cell microscopy has revealed a wide variation in the dynamics of clathrin-Coated Pits (CCPs). To gain insight of the heterogeneity of CCP dynamics and how cortical actin might influence this heterogeneity, we applied total internal reflection fluorescence microscopy to live cells grown on micro-fabricated substrates patterned with adhesive and non-adhesive regions. Cells on patterns showed overall longer CCP lifetimes compared to cells on chemically uniform surfaces, possibly the result of increased cortical tension. CCP lifetime distributions were also significantly different between adhesive and non-adhesive regions. When the structure of cortical actin is weakened by application of an actin monomer sequestering drug latrunculin A (latA), we found that the CCP lifetimes were homogenized to the level of the non-adherent regions. The decrease in CCP lifetime on adherent regions suggests that cortical actin filaments act as barriers at the adherent surface in CME.
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Cargo and Dynamin Regulate Clathrin-Coated Pit Maturation
PLoS Biology, 2009Co-Authors: Dinah Loerke, Marcel Mettlen, Defne Yarar, Henry Jaqaman, Khuloud Jaqaman, Gaudenz Danuser, Sandra L SchmidAbstract:Total internal reflection fluorescence microscopy (TIR-FM) has become a powerful tool for studying clathrin-mediated endocytosis. However, due to difficulties in tracking and quantifying their heterogeneous dynamic behavior, detailed analyses have been restricted to a limited number of selected clathrin-Coated Pits (CCPs). To identify intermediates in the formation of clathrin-Coated vesicles and factors that regulate progression through these stages, we used particle-tracking software and statistical methods to establish an unbiased and complete inventory of all visible CCP trajectories. We identified three dynamically distinct CCP subpopulations: two short-lived subpopulations corresponding to aborted intermediates, and one longer-lived productive subpopulation. In a manner dependent on AP2 adaptor complexes, increasing cargo concentration significantly enhances the maturation efficiency of productive CCPs, but has only minor effects on their lifetimes. In contrast, small interfering RNA (siRNA) depletion of dynamin-2 GTPase and reintroduction of wild-type or mutant dynamin-1 revealed dynamin's role in controlling the turnover of abortive intermediates and the rate of CCP maturation. From these data, we infer the existence of an endocytic restriction or checkpoint, responsive to cargo and regulated by dynamin.
P Verroust - One of the best experts on this subject based on the ideXlab platform.
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comparative immunochemistry and ontogeny of two closely related Coated Pit proteins the 280 kd target of teratogenic antibodies and the 330 kd target of nephritogenic antibodies
American Journal of Pathology, 1993Co-Authors: Djillali Sahali, N Mulliez, F Chatelet, C Laurentwinter, D Citadelle, Charles Roux, P Ronco, J C Sabourin, P VerroustAbstract:We have previously shown that monoclonal antibodies specific for a 280-kd protein (gp280) concentrated within the Coated Pits of renal and yolk sac brush border-induced fetal malformations, whereas antibodies specific for gp330, another Coated Pit protein with a similar distribution, had no deleterious effect on embryonic development. In this study, we show that gp280 and gp330 are closely related proteins, as indicated by: 1) similarities in peptide maps obtained after cyanogen bromide cleavage, 2) immunological cross-reactivity related to a minor contingent of antibodies that do not have teratogenic activity, and 3) asynchronous but related expressions during ontogenesis. During the early stages of development, the expression of the two glycoproteins was limited to (gp330) or predominant in (gp280) the clathrin-Coated Pits and intermicrovillar areas. In the pre-implantation embryo, gp330 was expressed by trophectodermal cells, which became negative in day-6 embryos trapped in endometrial infoldings. At this stage, gp280 and gp330 were both simultaneously detectable at the apical pole of the first entoblastic cells and remained expressed by the brush border of visceral yolk sac ePithelial cells until the end of pregnancy. In addition, gp330 was expressed by amniotic cells and neurectodermal structures. During nephrogenesis, in contrast, the expression of gp280 and gp330 by the intermicrovillar areas of the proximal tubule cell was the result of a complex maturation process. gp280 and gp330 were diffusely distributed in S-shaped bodies in the presumptive areas of the glomerulus, proximal tubule, and distal tubule (gp330). During development of the nephron, the pattern of expression became progressively restricted to the proximal tubule and glomerulus (gp330), and selective localization in the intermicrovillar areas was only achieved in filtrating nephrons.
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coexpression in humans by kidney and fetal envelopes of a 280 kda Coated Pit restricted protein similarity with the murine target of teratogenic antibodies
American Journal of Pathology, 1992Co-Authors: Djillali Sahali, N Mulliez, F Chatelet, C Laurentwinter, D Citadelle, Charles Roux, P Ronco, P VerroustAbstract:Experimental studies performed in the rat over the last three decades have shown that antibodies raised against kidney or yolk sac, which, in the rat, surrounds the embryo and serves as a placenta during the major part of pregnancy, induced fetal resorptions or malformations. It is generally considered that the teratogenic antibodies decrease internalization and degradation of maternal proteins by yolk sac ePithelial cells leading to an inadequate supply of nutriments to the embryo. These observations demonstrating the pathogenic role of antibodies to fetal envelopes are of great potential interest in clinical pathology since most cases of fetal malformations in humans are of unknown cause. The authors have recently shown that the key teratogenic antibodies in the murine system were directed against a 280 kDa-Coated Pit protein (gp280) specific for the brush border of ePithelial cells lining the renal proximal tubule and the yolk sac. This observation allows for the unique opportunity to search for a similar system in humans. In this study, the presence in humans of a protein closely related to murine gp280 is shown, as indicated by extensive immunologic crossreactivity, close apparent molecular weights, strong homology of bidimensional peptide maps, and restricted distribution at the organ and subcellular level. In addition to kidney and yolk sac, human gp280 was also detected within the Coated Pits of the placental syncytiotrophoblastic cells. When introduced in an in vitro rat embryo culture system, antibodies to human gp280-induced developmental anomalies in a dose-dependent manner. These observations indicate that the antigenic component of the murine model is present in humans and can give rise to heterologous antibodies that cause developmental anomalies, suggesting that the experimental model might be of significance in human pathology.
Elizabeth Smythe - One of the best experts on this subject based on the ideXlab platform.
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cargo regulates clathrin Coated Pit invagination via clathrin light chain phosphorylation
Journal of Cell Biology, 2018Co-Authors: Hannes Maib, Stéphane Vassilopoulos, Filipe Ferreira, Elizabeth SmytheAbstract:Clathrin light chains (CLCs) control selective uptake of a range of G protein–coupled receptors (GPCRs), although the mechanism by which this occurs has remained elusive thus far. In particular, site-specific phosphorylation of CLCb controls the uptake of the purinergic GPCR P2Y12, but it is dispensable for the constitutive uptake of the transferrin receptor (TfR). We demonstrate that phosphorylation of CLCb is required for the maturation of clathrin-Coated Pits (CCPs) through the transition of flat lattices into invaginated buds. This transition is dependent on efficient clathrin exchange regulated by CLCb phosphorylation and mediated through auxilin. Strikingly, this rearrangement is required for the uptake of P2Y12 but not TfR. These findings link auxilin-mediated clathrin exchange to early stages of CCP invagination in a cargo-specific manner. This supports a model in which CCPs invaginate with variable modes of curvature depending on the cargo they incorporate.
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the role of dynamin and its binding partners in Coated Pit invagination and scission
Journal of Cell Biology, 2001Co-Authors: Elaine Hill, Jeroen Van Der Kaay, Peter C Downes, Elizabeth SmytheAbstract: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.
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A novel role for Rab5-GDI in ligand sequestration into clathrin-Coated Pits
1998Co-Authors: Hilary Mclauchlan, Jane Newell, Nick Morrice, Ar Osborne, Michele A. West, Elizabeth SmytheAbstract:BACKGROUND: Clathrin-Coated Pits are formed at the plasma membrane by the assembly of the coat components, namely clathrin and adaptors from the cytosol. Little is known about the regulation and mechanism of this assembly process. RESULTS: We have used an in vitro assay for clathrin-Coated Pit assembly to identify a novel component required for the invagination of newly formed Coated Pits. We have purified this cytosolic component and shown it to be a complex of Rab5 and GDI (guanine-nucleotide dissociation inhibitor), that was previously demonstrated to be involved in downstream processing of endocytic vesicles. Using a combination of quantitative electron microscopy and in vitro endocytosis assays, we have demonstrated that although coat proteins and ATP are sufficient to increase the number of new Coated Pits at the cell surface in permeabilised cells, the Rab5-GDI complex is required for ligand sequestration into clathrin-Coated Pits. CONCLUSIONS: We have identified Rab5 as a critical cytosolic component required for clathrin-Coated Pit function. Given the well-established role of Rab5 in the fusion of endocytic vesicles with endosomes, our results suggest that recruitment of essential components of the targeting and fusion machinery is coupled to the formation of functional transport vesicles.
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A novel role for Rab5-GDI in ligand sequestration into clathrin-Coated Pits
Current Biology, 1998Co-Authors: Hilary Mclauchlan, Jane Newell, Nick Morrice, Ar Osborne, Michele A. West, Elizabeth SmytheAbstract:Abstract Background: Clathrin-Coated Pits are formed at the plasma membrane by the assembly of the coat components, namely clathrin and adaptors from the cytosol. Little is known about the regulation and mechanism of this assembly process. Results: We have used an in vitro assay for clathrin-Coated Pit assembly to identify a novel component required for the invagination of newly formed Coated Pits. We have purified this cytosolic component and shown it to be a complex of Rab5 and GDI (guanine-nucleotide dissociation inhibitor), that was previously demonstrated to be involved in downstream processing of endocytic vesicles. Using a combination of quantitative electron microscopy and in vitro endocytosis assays, we have demonstrated that although coat proteins and ATP are sufficient to increase the number of new Coated Pits at the cell surface in permeabilised cells, the Rab5–GDI complex is required for ligand sequestration into clathrin-Coated Pits. Conclusions: We have identified Rab5 as a critical cytosolic component required for clathrin-Coated Pit function. Given the well-established role of Rab5 in the fusion of endocytic vesicles with endosomes, our results suggest that recruitment of essential components of the targeting and fusion machinery is coupled to the formation of functional transport vesicles.
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stage specific assays for Coated Pit formation and Coated vesicle budding in vitro
Journal of Cell Biology, 1991Co-Authors: Sandra L Schmid, Elizabeth SmytheAbstract:Internalization of biotin-S-S-125I-transferrin (125I-BSST) into semiintact A431 cells were assessed by two different criteria which have allowed us to distinguish partial reactions in the complex overall process of receptor-mediated endocytosis. Early events resulting in the sequestration of ligand into deeply invaginated Coated Pits were measured by inaccessibility of 125I-BSST to exogenously added antibodies. Later events involving Coated vesicle budding and membrane fission were measured by resistance of 125I-BSST to reduction by the membrane impermeant-reducing agent, MesNa. Acquisition of Ab inaccessibility occurred very efficiently in this cell-free system (approximately 50% of total cell-associated 125I-BSST became inaccessible) and could be inhibited by anti-clathrin mAbs and by antibodies directed against the cytoplasmic domain of the transferrin-receptor. In contrast, acquisition of MesNa resistance occurred less efficiently (approximately 10-20% of total cell-associated 125I-BSST) and showed differential sensitivity to inhibition by anti-clathrin and anti-transferrin receptor mAbs. Both partial reactions were stimulated by ATP and cytosol; indicating at least two ATP-requiring events in receptor-mediated endocytosis. The temperature dependence of both reactions was similar to that for 125I-BSST internalization in intact cells with no activity being observed below 10 degrees C. Morphological studies using gold-labeled ligands confirmed that internalization of transferrin receptors into semiintact A431 cell occurred via Coated Pits and Coated vesicles and resulted in delivery of ligand to endosomal structures.