RESUMO
The epidermal growth factor receptor (EGFR) plays an essential role during development and diseases including cancer. Lamellipodin (Lpd) is known to control lamellipodia protrusion by regulating actin filament elongation via Ena/VASP proteins. However, it is unknown whether this mechanism supports endocytosis of the EGFR. Here, we have identified a novel role for Lpd and Mena in clathrin-mediated endocytosis (CME) of the EGFR. We have discovered that endogenous Lpd is in a complex with the EGFR and Lpd and Mena knockdown impairs EGFR endocytosis. Conversely, overexpressing Lpd substantially increases the EGFR uptake in an F-actin-dependent manner, suggesting that F-actin polymerization is limiting for EGFR uptake. Furthermore, we found that Lpd directly interacts with endophilin, a BAR domain containing protein implicated in vesicle fission. We identified a role for endophilin in EGFR endocytosis, which is mediated by Lpd. Consistently, Lpd localizes to clathrin-coated pits (CCPs) just before vesicle scission and regulates vesicle scission. Our findings suggest a novel mechanism in which Lpd mediates EGFR endocytosis via Mena downstream of endophilin.
Assuntos
Actinas/fisiologia , Aciltransferases/fisiologia , Proteínas de Transporte/fisiologia , Endocitose/genética , Receptores ErbB/metabolismo , Proteínas de Membrana/fisiologia , Proteínas dos Microfilamentos/fisiologia , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/fisiologia , Actinas/genética , Actinas/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Invaginações Revestidas da Membrana Celular/fisiologia , Células HEK293 , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Células NIH 3T3 , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Clathrin mediated endocytosis (CME) is the main route of receptor internalization in mammalian cells and this well conserved mechanism has been intensively studied for over 40yrs. In the general or 'canonical' model of CME clathrin coated pits form stochastically at the plasma membrane and coated pit curvature develops as the coated pit grows through clathrin polymerization. However, the canonical model of CME does not explain the diversity of endocytically active clathrin coated structures (CCSs) found at the plasma membrane by both electron and light microscopy. In this review we examine the canonical model of CME, highlight discrepancies with published experimental data and suggest future avenues of exploration while paying particular attention to the relationship between clathrin coated pits, plaques, sites of adhesion and the formation of endocytic 'hotspots'.
Assuntos
Invaginações Revestidas da Membrana Celular/fisiologia , Endocitose , Animais , Clatrina/metabolismo , Mamíferos , PolimerizaçãoRESUMO
Clathrin-dependent endocytosis is the major pathway for the uptake of nutrients and signaling molecules in higher eukaryotic cells. The long-held tenet that clathrin-coated vesicles are created from flat coated plasma membrane patches by a sequential process of invagination, bud formation and fission recently received strong support from the results of advanced live cell fluorescence microscopy. The data on the critical components that deform the plasma membrane locally into a coated bud suggest that membrane bending is a team effort requiring membrane-curving protein domains, actin dynamics and, last but not least, clathrin. The scission step requires the mechano-enzymatic function of dynamin, actin dynamics and possibly myosin motor proteins. Finally, a burst of auxilin/GAK initiates the uncoating of the vesicle.
Assuntos
Membrana Celular/fisiologia , Vesículas Revestidas por Clatrina/fisiologia , Clatrina/fisiologia , Invaginações Revestidas da Membrana Celular/fisiologia , Endocitose/fisiologia , Células Eucarióticas/fisiologiaRESUMO
As the basic physiological function of synapses, vesicle cycling involves in many aspects of process. Among them, vesicle recycling is the basis of synaptic vesicle cycling. Studies show that clathrin mediated endocytosis is a major pathway of vesicle recycling, in which Dynamin plays an important role. Dynamin is a GTPases with molecular weight of 100 kD, which acts as "scissors" in the endocytosis, separating the clathrin coated pits from membrane. It has been found that Dynamin is associated with epilepsy, Alzheimer's disease, centronuclear myopathy, and several other neurological diseases. In this paper, we discussed the structure, function and regulation of Dynamin, and reviewed recent advance in the studies on Dynamin related diseases.
Assuntos
Dinaminas/fisiologia , Endocitose , Vesículas Sinápticas/fisiologia , Clatrina/fisiologia , Invaginações Revestidas da Membrana Celular/fisiologia , Humanos , Sinapses/fisiologia , Transmissão SinápticaRESUMO
Using Caenorhabditis elegans genetic screens, we identified receptor-mediated endocytosis (RME)-4 and RME-5/RAB-35 as important regulators of yolk endocytosis in vivo. In rme-4 and rab-35 mutants, yolk receptors do not accumulate on the plasma membrane as would be expected in an internalization mutant, rather the receptors are lost from cortical endosomes and accumulate in dispersed small vesicles, suggesting a defect in receptor recycling. Consistent with this, genetic tests indicate the RME-4 and RAB-35 function downstream of clathrin, upstream of RAB-7, and act synergistically with recycling regulators RAB-11 and RME-1. We find that RME-4 is a conserved DENN domain protein that binds to RAB-35 in its GDP-loaded conformation. GFP-RME-4 also physically interacts with AP-2, is enriched on clathrin-coated pits, and requires clathrin but not RAB-5 for cortical association. GFP-RAB-35 localizes to the plasma membrane and early endocytic compartments but is lost from endosomes in rme-4 mutants. We propose that RME-4 functions on coated pits and/or vesicles to recruit RAB-35, which in turn functions in the endosome to promote receptor recycling.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Proteínas de Transporte/fisiologia , Invaginações Revestidas da Membrana Celular/fisiologia , Endocitose/fisiologia , Proteínas de Membrana/fisiologia , Proteínas rab de Ligação ao GTP/fisiologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/genética , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Invaginações Revestidas da Membrana Celular/metabolismo , Gema de Ovo/metabolismo , Endossomos/metabolismo , Endossomos/fisiologia , Proteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas rab de Ligação ao GTP/genéticaRESUMO
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.
Assuntos
Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/fisiologia , Endocitose/fisiologia , Células 3T3 , Actinas/fisiologia , Animais , Auxilinas/metabolismo , Células Cultivadas , Invaginações Revestidas da Membrana Celular/ultraestrutura , Células HeLa , Humanos , Camundongos , Microscopia Confocal , Microscopia Eletrônica , Microscopia de Fluorescência , RatosRESUMO
Viruses utilize cellular lipids and manipulate host lipid metabolism to ensure their replication and spread. Therefore, the identification of lipids and metabolic pathways that are suitable targets for antiviral development is crucial. Using a library of compounds targeting host lipid metabolic factors and testing them for their ability to block pseudorabies virus (PRV) and vesicular stomatitis virus (VSV) infection, we found that U18666A, a specific inhibitor of Niemann-Pick C1 (NPC1), is highly potent in suppressing the entry of diverse viruses including pseudotyped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). NPC1 deficiency markedly attenuates viral growth by decreasing cholesterol abundance in the plasma membrane, thereby inhibiting the dynamics of clathrin-coated pits (CCPs), which are indispensable for clathrin-mediated endocytosis. Significantly, exogenous cholesterol can complement the dynamics of CCPs, leading to efficient viral entry and infectivity. Administration of U18666A improves the survival and pathology of PRV- and influenza A virus-infected mice. Thus, our studies demonstrate a unique mechanism by which NPC1 inhibition achieves broad antiviral activity, indicating a potential new therapeutic strategy against SARS-CoV-2, as well as other emerging viruses.
Assuntos
Androstenos/farmacologia , Clatrina/fisiologia , Invaginações Revestidas da Membrana Celular/fisiologia , Vírus de DNA/efeitos dos fármacos , Proteína C1 de Niemann-Pick/fisiologia , Vírus de RNA/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Vírus de DNA/fisiologia , Proteína C1 de Niemann-Pick/antagonistas & inibidores , Vírus de RNA/fisiologiaRESUMO
How soluble cargo molecules concentrate into budding vesicles is the subject of intensive current research. Clathrin-based vesiculation from the plasma membrane and the trans-Golgi network constitutes the best described system that supports this sorting process. Soluble ligands bind to specific transmembrane receptors which have been shown to interact directly with clathrin adaptor complexes, components of clathrin coats. At the same time, these clathrin adaptors facilitate clathrin coat assembly and probably regulate the recruitment of the rest of the coat components. Recent studies have looked at both the interaction of receptor tails with adaptors and the assembly of the clathrin coat. Progress has also been made in elucidating how soluble cargo molecules may be concentrated for exit from the endoplasmic reticulum.
Assuntos
Clatrina/fisiologia , Invaginações Revestidas da Membrana Celular/fisiologia , Vesículas Revestidas/fisiologia , Receptores de Superfície Celular/fisiologia , Sequência de Aminoácidos , Animais , Arrestinas/fisiologia , Clatrina/biossíntese , Proteínas de Ligação a DNA/metabolismo , Endocitose , Complexo de Golgi/fisiologia , Mamíferos , Modelos Biológicos , Dados de Sequência Molecular , Receptores de Superfície Celular/química , Saccharomyces cerevisiae/fisiologia , Transdução de Sinais , Fator de Transcrição AP-2 , Fatores de Transcrição/metabolismo , beta-ArrestinasRESUMO
There is accumulating evidence that lateral assemblies (rafts) of sphingolipids and cholesterol form platforms that serve to support numerous cellular events in membrane traffic and signal transduction. Raft membrane microdomains are thought to function by preferentially associating with specific proteins while excluding others. The basic forces driving raft formation are lipid interactions which are, per se, weak and transient. Sphingolipid rafts should therefore be considered to be dynamic structures in which cholesterol plays an important role as a linker. Caveolins influence these dynamics by forming stabilized raft domains in intracellular membranes as well as at the plasma membrane. Recent data suggest that clustering of raft components could regulate raft dynamics and therefore represents an important feature in the function of these membrane microdomains.
Assuntos
Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Colesterol/metabolismo , Invaginações Revestidas da Membrana Celular/fisiologia , Glicolipídeos/fisiologia , Membranas Intracelulares/fisiologia , Lipídeos de Membrana/fisiologia , Esfingolipídeos/metabolismo , Animais , Anexinas/fisiologia , Detergentes , Humanos , Proteínas de Membrana/fisiologia , Modelos Biológicos , Modelos Estruturais , Transdução de SinaisRESUMO
Amphiphysin, a protein that is highly concentrated in nerve terminals, has been proposed to function as a linker between the clathrin coat and dynamin in the endocytosis of synaptic vesicles. Here, using a cell-free system, we provide direct morphological evidence in support of this hypothesis. Unexpectedly, we also find that amphiphysin-1, like dynamin-1, can transform spherical liposomes into narrow tubules. Moreover, amphiphysin-1 assembles with dynamin-1 into ring-like structures around the tubules and enhances the liposome-fragmenting activity of dynamin-1 in the presence of GTP. These results show that amphiphysin binds lipid bilayers, indicate a potential function for amphiphysin in the changes in bilayer curvature that accompany vesicle budding, and imply a close functional partnership between amphiphysin and dynamin in endocytosis.
Assuntos
Clatrina/metabolismo , Endocitose/fisiologia , GTP Fosfo-Hidrolases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Encéfalo/metabolismo , Bovinos , Sistema Livre de Células , Clatrina/química , Clatrina/ultraestrutura , Invaginações Revestidas da Membrana Celular/fisiologia , Invaginações Revestidas da Membrana Celular/ultraestrutura , Dimerização , Dinamina I , Dinaminas , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/ultraestrutura , Humanos , Cinética , Lipossomos , Microscopia Eletrônica , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/ultraestrutura , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestruturaRESUMO
Here we visualize new aspects of the dynamics of endocytotic clathrin-coated pits and vesicles in mammalian cells by using a fusion protein consisting of green fluorescent protein and clathrin light chain a. Clathrin-coated pits invaginating from the plasma membrane show definite, but highly limited, mobility within the membrane that is relaxed upon treatment with latrunculin B, an inhibitor of actin assembly, indicating that an actin-based framework may be involved in the mobility of these pits. Transient, motile coated vesicles that originate from coated pits can be detected, with multiple vesicles occasionally appearing to emanate from a single pit. Despite their seemingly random distribution, coated pits tend to form repeatedly at defined sites while excluding other regions. This spatial regulation of coated-pit assembly and function is attributable to the attachment of the coated pits to the membrane skeleton.
Assuntos
Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/fisiologia , Invaginações Revestidas da Membrana Celular/ultraestrutura , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Células COS , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Invaginações Revestidas da Membrana Celular/efeitos dos fármacos , Endocitose , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Fluorescência Verde , Proteínas Luminescentes/metabolismo , Toxinas Marinhas/farmacologia , Camundongos , Placenta/metabolismo , Gravidez , Proteínas Recombinantes de Fusão/metabolismo , Tiazóis/farmacologia , TiazolidinasRESUMO
Numb is an endocytic protein that is proposed to influence clathrin-coated pit assembly, although its mode of action and the mechanisms that regulate its activity are unknown. In this study, we show that Numb binds to and is phosphorylated by adaptor-associated kinase 1 (AAK1), a key endocytic kinase. We find that AAK1 redistributes Numb to perinuclear endosomes when overexpressed, while kinase depletion causes Numb to accumulate at the plasma membrane. Overexpression of a Numb point mutant (T102A) that lacks the AAK1 phosphorylation site potently disrupts transferrin and low-density lipoprotein internalization but does not impact EGF uptake. Consistent with Numb redistribution results, we find that T102A Numb no longer localizes to perinuclear endosomes. Instead, it is enriched at the plasma membrane where it shows elevated levels of colocalization with coated pit markers. Collectively, these observations demonstrate that Numb endocytic activity is regulated by AAK1 and that phosphorylation may be a critical step in promoting coated pit maturation.
Assuntos
Clatrina/fisiologia , Invaginações Revestidas da Membrana Celular/fisiologia , Endocitose/fisiologia , Endossomos/fisiologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/enzimologia , Invaginações Revestidas da Membrana Celular/metabolismo , Endossomos/enzimologia , Endossomos/metabolismo , Imunofluorescência , Células HeLa , Humanos , Imunoprecipitação , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Transporte Proteico , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Epithelial cells form a barrier against the environment, but are also required for the regulated exchange of molecules between an organism and its surroundings. Epithelial cells are characterised by a remarkable polarization of their plasma membrane, evidenced by the appearance of structurally, compositionally, and functionally distinct surface domains. Here we consider the (in)dependence of epithelial cell polarisation and the function of smaller plasma membrane domains (e.g. adherens junctions, gap junctions, tight junctions, apical lipid rafts, caveolae, and clathrin-coated pits) in the development and maintenance of cell surface polarity. Recent evidence of cross-talk and/or overlap between the different cell-cell junction components and alternate functions of junction components, including gene expression regulation, are discussed in the context of cell surface polarity.
Assuntos
Membrana Celular/fisiologia , Polaridade Celular/fisiologia , Células Epiteliais/fisiologia , Junções Comunicantes/fisiologia , Junções Íntimas/fisiologia , Junções Aderentes , Animais , Cavéolas/fisiologia , Adesão Celular/fisiologia , Invaginações Revestidas da Membrana Celular/fisiologia , Desmossomos/fisiologia , Humanos , Bicamadas Lipídicas/metabolismo , Microdomínios da Membrana/fisiologia , Transdução de SinaisRESUMO
Dynamin is a large GTPase involved in several distinct modes of cell endocytosis. In this study, we examined the possible role of dynamin in UT-A1 internalization. The direct relationship of UT-A1 and dynamin was identified by coimmunoprecipitation. UT-A1 has cytosolic NH(2) and COOH termini and a large intracellular loop. Dynamin specifically binds to the intracellular loop of UT-A1, but not the NH(2) and COOH termini. In cell surface biotinylation experiments, coexpression of dynamin and UT-A1 in HEK293 cells resulted in a decrease of UT-A1 cell surface expression. Conversely, cells expressing dynamin mutant K44A, which is deficient in GTP binding, showed an increased accumulation of UT-A1 protein on the cell surface. Cell plasma membrane lipid raft fractionation experiments revealed that blocking endocytosis with dynamin K44A causes UT-A1 protein accumulation in both the lipid raft and nonlipid raft pools, suggesting that both caveolae- and clathrin-mediated mechanisms may be involved in the internalization of UT-A1. This was further supported by 1) small interfering RNA to knock down either caveolin-1 or µ2 reduced UT-A1 internalization in HEK293 cells and 2) inhibition of either the caveolae pathway by methyl-ß-cyclodextrin or the clathrin pathway by concanavalin A caused UT-A1 cell membrane accumulation. Functionally, overexpression of dynamin, caveolin, or µ2 decreased UT-A1 urea transport activity and decreased UT-A1 cell surface expression. We conclude that UT-A1 endocytosis is dynamin-dependent and mediated by both caveolae- and clathrin-coated pit pathways.
Assuntos
Cavéolas/fisiologia , Invaginações Revestidas da Membrana Celular/fisiologia , Dinaminas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Clatrina/fisiologia , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Microdomínios da Membrana/metabolismo , Interferência de RNA , beta-Ciclodextrinas/farmacologia , Transportadores de UreiaRESUMO
The endocytosis of the T cell differentiation antigen CD4 has been investigated in CD4-transfected HeLa cells, the promyelocytic HL-60 cell line, and in a number of leukemia- or lymphoma-derived T cell lines. CD4 internalization was followed using radioiodinated antibodies in an acid-elution endocytosis assay, or by covalently modifying cell surface proteins with biotin and analyzing CD4 distributions by immunoprecipitation; both approaches gave equivalent results. The assays demonstrated that in transfected HeLa cells and in HL-60 cells CD4 was constitutively internalized and recycled in the absence of ligand. Immunogold labeling and electron microscopy demonstrated that CD4 enters cells through coated pits. In contrast to the nonlymphocytic cells, T cell lines showed very little endocytosis of CD4. Measurements of fluid phase endocytosis and morphometric analysis of the endosome compartment indicated that the endocytic capacities of HeLa and lymphoid cells are equivalent and suggested that the low level of CD4 uptake in lymphocytic cells is due to exclusion of CD4 from coated pits. This conclusion was supported by experiments using truncated CD4 molecules, lacking the bulk of the cytoplasmic domain, which were internalized equally efficiently in both transfected lymphocytes and HeLa cells. Together, these results indicate that the cytoplasmic domain of CD4 mediates the different interactions with the endocytic apparatus in lymphoid and nonlymphoid cells. We suggest that the CD4-associated lymphocyte-specific protein tyrosine kinase p56lck may be involved in preventing CD4 endocytosis in T cells.
Assuntos
Antígenos CD4 , Endocitose , Antígenos CD4/genética , Linhagem Celular , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Invaginações Revestidas da Membrana Celular/fisiologia , Invaginações Revestidas da Membrana Celular/ultraestrutura , Células HeLa/fisiologia , Humanos , Cinética , Leucemia , Linfoma , Microscopia Eletrônica , Organelas/fisiologia , Organelas/ultraestrutura , Linfócitos T , TransfecçãoRESUMO
Three-dimensional "deep-etch" electron microscopy (DEEM) resolves a longstanding controversy concerning poxvirus morphogenesis. By avoiding fixative-induced membrane distortions that confounded earlier studies, DEEM shows that the primary poxvirus envelope is a single membrane bilayer coated on its external surface by a continuous honeycomb lattice. Freeze fracture of quick-frozen poxvirus-infected cells further shows that there is only one fracture plane through this primary envelope, confirming that it consists of a single lipid bilayer. DEEM also illustrates that the honeycomb coating on this envelope is completely replaced by a different paracrystalline coat as the poxvirus matures. Correlative thin section images of infected cells freeze substituted after quick-freezing, plus DEEM imaging of Tokuyasu-type cryo-thin sections of infected cells (a new application introduced here) all indicate that the honeycomb network on immature poxvirus virions is sufficiently continuous and organized, and tightly associated with the envelope throughout development, to explain how its single lipid bilayer could remain stable in the cytoplasm even before it closes into a complete sphere.
Assuntos
Invaginações Revestidas da Membrana Celular/fisiologia , Vaccinia virus/fisiologia , Animais , Linhagem Celular , Chlorocebus aethiops , Técnica de Congelamento e Réplica , Microscopia Eletrônica de VarreduraRESUMO
Clathrin-mediated endocytosis (CME) in mammalian cells is driven by resilient machinery that includes >70 endocytic accessory proteins (EAP). Accordingly, perturbation of individual EAPs often results in minor effects on biochemical measurements of CME, thus providing inconclusive/misleading information regarding EAP function. Live-cell imaging can detect earlier roles of EAPs preceding cargo internalization; however, this approach has been limited because unambiguously distinguishing abortive coats (ACs) from bona fide clathrin-coated pits (CCPs) is required but unaccomplished. Here, we develop a thermodynamics-inspired method, "disassembly asymmetry score classification (DASC)", that resolves ACs from CCPs based on single channel fluorescent movies. After extensive verification, we use DASC-resolved ACs and CCPs to quantify CME progression in 11 EAP knockdown conditions. We show that DASC is a sensitive detector of phenotypic variation in CCP dynamics that is uncorrelated to the variation in biochemical measurements of CME. Thus, DASC is an essential tool for uncovering EAP function.
Assuntos
Clatrina/fisiologia , Endocitose/fisiologia , Vesículas Revestidas por Clatrina/fisiologia , Invaginações Revestidas da Membrana Celular/fisiologia , Humanos , TermodinâmicaRESUMO
Clathrin-mediated endocytosis occurs via the assembly of clathrin-coated pits (CCPs) that invaginate and pinch off to form clathrin-coated vesicles (CCVs). It is well known that adaptor protein 2 (AP2) complexes trigger clathrin assembly on the plasma membrane, and biochemical and structural studies have revealed the nature of these interactions. Numerous endocytic accessory proteins collaborate with clathrin and AP2 to drive CCV formation. However, many questions remain as to the molecular events involved in CCP initiation, stabilization, and curvature generation. Indeed, a plethora of recent evidence derived from cell perturbation, correlative light and EM tomography, live-cell imaging, modeling, and high-resolution structural analyses has revealed more complexity and promiscuity in the protein interactions driving CCP maturation than anticipated. After briefly reviewing the evidence supporting prevailing models, we integrate these new lines of evidence to develop a more dynamic and flexible model for how redundant, dynamic, and competing protein interactions can drive endocytic CCV formation and suggest new approaches to test emerging models.
Assuntos
Vesículas Revestidas por Clatrina/metabolismo , Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Invaginações Revestidas da Membrana Celular/fisiologia , Complexo 2 de Proteínas Adaptadoras/metabolismo , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Endocitose/fisiologia , HumanosRESUMO
We have examined, by analyzing thin (15-20 nm) serial sections, whether coated pits involved in adsorptive pinocytosis in cultured fibroblasts give rise to free coated vesicles or represent permanently surface-associated structures from the neck of which uncoated receptosomes pinch off and carry ligand into the cell. Human skin fibroblasts and mouse L-929 fibroblasts were incubated with cationized ferritin (CF), a ligand known to bind to coated pit regions, at 37 degrees C before fixation. In thin sections, CF was found in coated vesicular profiles within the cytoplasm. Serial sections revealed that whereas many of these coated profiles communicated with the cell surface, thus representing pits, about 10% in L-cells and 36% in skin fibroblasts were actually free coated vesicles. Moreover, evidence for uncoated vesicular structures (receptosomes) budding off from the coated pits was not obtained. We therefore conclude that coated pits do pinch off from the plasma membrane to form free, coated vesicles (pinosomes).
Assuntos
Invaginações Revestidas da Membrana Celular/ultraestrutura , Endossomos/ultraestrutura , Pinocitose , Adsorção , Animais , Células Cultivadas , Invaginações Revestidas da Membrana Celular/fisiologia , Ferritinas , Fibroblastos , Humanos , Células L , Camundongos , Microscopia EletrônicaRESUMO
Interactions between membrane proteins are believed to be important for the induction of transmembrane signaling. Endocytosis is one of the responses which is regulated by both intracellular and extracellular signals. To study such interactions, we have measured the lateral mobility and rate of endocytosis of epidermal growth factor receptor in three transfected NIH-3T3 cell lines (HER84, HER22, and HER82) expressing 2 X 10(4), 2 X 10(5) and 1.5 X 10(6) EGF-receptors per cell, respectively. Using rhodamine-labeled EGF (Rh-EGF) and rhodamine-labeled monoclonal anti-EGF-receptor antibody (Rh-mAb-108), we measured twofold decreases in the lateral diffusion coefficients for each approximately 10-fold increase in EGF-receptor concentration. Since steric effects cannot account for such dependence, we propose that protein mobility within the membrane, which is determined by the rate of motion between immobile barriers, decreases due to aggregate formation. The rate of endocytosis also decreases twofold between the HER84 (2 X 10(4) receptors/cell) and HER22 (2 X 10(5) receptors/cell) cell lines, suggesting that it is diffusion limited. The comparable rates of endocytosis of the HER82 and HER22 cell lines suggest that at high receptor density endocytosis may be limited by the total number of sites for receptors in coated-pits and by their rate of recycling.