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1.
Sci Rep ; 9(1): 6680, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31040310

RESUMO

Bacteria show sophisticated control of their cellular organization, and many bacteria deploy different polar landmark proteins to organize the cell pole. Super-resolution microscopy, such as Photo-Activated Localization Microscopy (PALM), provides the nanoscale localization of molecules and is crucial for better understanding of organization and dynamics in single-molecule. However, analytical tools are not fully available yet, in particular for bacterial cell biology. For example, quantitative and statistical analyses of subcellular localization with multiple cells from multiple fields of view are lacking. Furthermore, brightfield images are not sufficient to get accurate contours of small and low contrast bacterial cells, compared to subpixel presentation of target molecules. Here we describe a novel analytic tool for PALM which integrates precisely drawn cell outlines, of either inner membrane or periplasm, labelled by PALM-compatible fluorescent protein fusions, with molecule data for >10,000 molecules from >100 cells by fitting each cell into an oval arc. In the vibrioid bacterium Vibrio cholerae, the polar anchor HubP constitutes a big polar complex which includes multiple proteins involved in chemotaxis and the flagellum. With this pipeline, HubP is shown to be slightly skewed towards the inner curvature side of the cell, while its interaction partners showed rather loose polar localization.


Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Imagem Molecular , Imagem Individual de Molécula , Imunofluorescência , Regulação Bacteriana da Expressão Gênica , Imagem Molecular/métodos , Imagem Individual de Molécula/métodos
2.
Nat Commun ; 7: 13582, 2016 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-27917880

RESUMO

The nucleotidyl cyclase toxin ExoY is one of the virulence factors injected by the Pseudomonas aeruginosa type III secretion system into host cells. Inside cells, it is activated by an unknown eukaryotic cofactor to synthesize various cyclic nucleotide monophosphates. ExoY-like adenylate cyclases are also found in Multifunctional-Autoprocessing Repeats-in-ToXin (MARTX) toxins produced by various Gram-negative pathogens. Here we demonstrate that filamentous actin (F-actin) is the hitherto unknown cofactor of ExoY. Association with F-actin stimulates ExoY activity more than 10,000 fold in vitro and results in stabilization of actin filaments. ExoY is recruited to actin filaments in transfected cells and alters F-actin turnover. Actin also activates an ExoY-like adenylate cyclase MARTX effector domain from Vibrio nigripulchritudo. Finally, using a yeast genetic screen, we identify actin mutants that no longer activate ExoY. Our results thus reveal a new sub-group within the class II adenylyl cyclase family, namely actin-activated nucleotidyl cyclase (AA-NC) toxins.


Assuntos
Actinas/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Glucosiltransferases/metabolismo , Pseudomonas aeruginosa/metabolismo , Actinas/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Glucosiltransferases/genética , Mutação , Ligação Proteica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo , Virulência/genética
3.
EMBO J ; 35(21): 2270-2284, 2016 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-27670760

RESUMO

The large GTPase dynamin is the first protein shown to catalyze membrane fission. Dynamin and its related proteins are essential to many cell functions, from endocytosis to organelle division and fusion, and it plays a critical role in many physiological functions such as synaptic transmission and muscle contraction. Research of the past three decades has focused on understanding how dynamin works. In this review, we present the basis for an emerging consensus on how dynamin functions. Three properties of dynamin are strongly supported by experimental data: first, dynamin oligomerizes into a helical polymer; second, dynamin oligomer constricts in the presence of GTP; and third, dynamin catalyzes membrane fission upon GTP hydrolysis. We present the two current models for fission, essentially diverging in how GTP energy is spent. We further discuss how future research might solve the remaining open questions presently under discussion.


Assuntos
Membrana Celular/fisiologia , Dinaminas/fisiologia , Animais , Guanosina Trifosfato/fisiologia , Humanos
4.
J Struct Biol ; 196(1): 48-56, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27431447

RESUMO

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 , Polimerização
5.
Nat Cell Biol ; 18(3): 347, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26911911
6.
Nat Cell Biol ; 18(2): 142-4, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26820438

RESUMO

An actin filament coat promotes cargo expulsion from large exocytosing vesicles, but the mechanisms of coat formation and force generation have been poorly characterized. Elegant imaging studies of the Drosophila melanogaster salivary gland now reveal how actin and myosin are recruited, and show that myosin II forms a contractile 'cage' that facilitates exocytosis.


Assuntos
Actomiosina/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Exocitose , Proteínas do Grude Salivar de Drosophila/metabolismo , Proteínas de Membrana/metabolismo , Glândulas Salivares/metabolismo , Vesículas Secretórias/metabolismo , Animais
7.
Dev Cell ; 33(2): 163-75, 2015 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-25898166

RESUMO

The size of endocytic clathrin-coated vesicles (CCVs) is remarkably uniform, suggesting that it is optimized to achieve the appropriate levels of cargo and lipid internalization. The three most abundant proteins in mammalian endocytic CCVs are clathrin and the two cargo-selecting, clathrin adaptors, CALM and AP2. Here we demonstrate that depletion of CALM causes a substantial increase in the ratio of "open" clathrin-coated pits (CCPs) to "necked"/"closed" CCVs and a doubling of CCP/CCV diameter, whereas AP2 depletion has opposite effects. Depletion of either adaptor, however, significantly inhibits endocytosis of transferrin and epidermal growth factor. The phenotypic effects of CALM depletion can be rescued by re-expression of wild-type CALM, but not with CALM that lacks a functional N-terminal, membrane-inserting, curvature-sensing/driving amphipathic helix, the existence and properties of which are demonstrated. CALM is thus a major factor in controlling CCV size and maturation and hence in determining the rates of endocytic cargo uptake.


Assuntos
Forma Celular/genética , Vesículas Revestidas por Clatrina/fisiologia , Invaginações Revestidas da Membrana Celular/fisiologia , Proteínas de Ligação a Ácido Graxo/genética , Proteínas Monoméricas de Montagem de Clatrina/genética , Proteínas Monoméricas de Montagem de Clatrina/fisiologia , Linhagem Celular Tumoral , Membrana Celular/fisiologia , Endocitose , Fator de Crescimento Epidérmico/metabolismo , Células HeLa , Humanos , Lipossomos/metabolismo , Estrutura Terciária de Proteína , Proteínas R-SNARE/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Transferrina/metabolismo
8.
Cold Spring Harb Perspect Biol ; 6(11): a016733, 2014 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-25280766

RESUMO

Up to 60 different proteins are recruited to the site of clathrin-mediated endocytosis in an ordered sequence. These accessory proteins have roles during all the different stages of clathrin-mediated endocytosis. First, they participate in the initiation of the endocytic event, thereby determining when and where endocytic vesicles are made; later they are involved in the maturation of the clathrin coat, recruitment of specific cargo molecules, bending of the membrane, and finally in scission and uncoating of the nascent vesicle. In addition, many of the accessory components are involved in regulating and coupling the actin cytoskeleton to the endocytic membrane. We will discuss the different accessory components and their various roles. Most of the data comes from studies performed with cultured mammalian cells or yeast cells. The process of endocytosis is well conserved between these different organisms, but there are also many interesting differences that may shed light on the mechanistic principles of endocytosis.


Assuntos
Endocitose/fisiologia , Modelos Biológicos , Animais , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Vesículas Revestidas por Clatrina/metabolismo , Vesículas Revestidas por Clatrina/fisiologia , Mamíferos/metabolismo , Leveduras/citologia , Leveduras/metabolismo
9.
Mol Biol Cell ; 25(19): 3070-80, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25079691

RESUMO

The dynamic relationship between constitutive and ligand-triggered clathrin-mediated endocytosis is only poorly characterized, and it remains controversial whether clathrin-coated pits specialize to internalize particular receptor cargo. Here we analyzed the ligand-triggered endocytosis of the model G-protein-coupled receptors (GPCRs) ß2-adrenergic receptor (ß2AR) and Mu-opioid receptor (MOR) at the level of individual endocytic events using a total internal reflection fluorescence microscopy (TIRFM)-based assay. Similar to the constitutive endocytosis of transferrin receptor (TfR), ligand- triggered endocytosis of ß2AR occurs via quantized scission events hosted by clathrin spots and plaques of variable size and persistence. To address whether clathrin-coated structures (CCSs) specialize to internalize particular GPCRs, we adapted the TIRFM imaging assay to simultaneously quantify the internalization of TfR and the ligand- triggered endocytosis of the ß2AR or MOR. Agonist-triggered ß2AR or MOR endocytosis extended the maturation time of CCSs, as shown previously, but did not affect the rate of constitutive TfR endocytosis or loading of TfR into individual endocytic vesicles. Both the ß2AR and the MOR receptors entered cells in the same vesicles as TfR, and the overall evidence for CCS specialization was weak. These data support a simple model in which different cargoes internalize through common CCSs.


Assuntos
Vesículas Revestidas por Clatrina/metabolismo , Endocitose/fisiologia , Receptores Adrenérgicos beta 2/metabolismo , Receptores Opioides mu/metabolismo , Receptores da Transferrina/metabolismo , Linhagem Celular , Invaginações Revestidas da Membrana Celular/metabolismo , Células HEK293 , Humanos , Microscopia de Fluorescência
10.
J Cell Biol ; 197(4): 499-508, 2012 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-22564416

RESUMO

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


Assuntos
Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Animais , Células COS , Chlorocebus aethiops , Clatrina/química , Dinamina II/metabolismo , Endocitose , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia
11.
Nat Cell Biol ; 14(5): 452-4, 2012 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-22552146

RESUMO

The membrane-curvature-inducing protein Fcho was proposed to be part of a ubiquitous nucleation mechanism for clathrin-coated pits. However, studies in developing zebrafish embryos now indicate a role for Fcho as a receptor-specific adaptor in bone morphogenetic protein (BMP) signalling, rather than a global coated-pit nucleator.


Assuntos
Complexo 2 de Proteínas Adaptadoras/fisiologia , Padronização Corporal , Clatrina/metabolismo , Endocitose , Proteínas/fisiologia , Proteínas de Ligação a Ácido Graxo , Humanos , Proteínas de Membrana
12.
PLoS Biol ; 10(4): e1001302, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22505844

RESUMO

Clathrin-mediated endocytosis proceeds by a sequential series of reactions catalyzed by discrete sets of protein machinery. The final reaction in clathrin-mediated endocytosis is membrane scission, which is mediated by the large guanosine triophosphate hydrolase (GTPase) dynamin and which may involve the actin-dependent recruitment of N-terminal containing BIN/Amphiphysin/RVS domain containing (N-BAR) proteins. Optical microscopy has revealed a detailed picture of when and where particular protein types are recruited in the ∼20-30 s preceding scission. Nevertheless, the regulatory mechanisms and functions that underpin protein recruitment are not well understood. Here we used an optical assay to investigate the coordination and interdependencies between the recruitment of dynamin, the actin cytoskeleton, and N-BAR proteins to individual clathrin-mediated endocytic scission events. These measurements revealed that a feedback loop exists between dynamin and actin at sites of membrane scission. The kinetics of dynamin, actin, and N-BAR protein recruitment were modulated by dynamin GTPase activity. Conversely, acute ablation of actin dynamics using latrunculin-B led to a ∼50% decrease in the incidence of scission, an ∼50% decrease in the amplitude of dynamin recruitment, and abolished actin and N-BAR recruitment to scission events. Collectively these data suggest that dynamin, actin, and N-BAR proteins work cooperatively to efficiently catalyze membrane scission. Dynamin controls its own recruitment to scission events by modulating the kinetics of actin and N-BAR recruitment to sites of scission. Conversely actin serves as a dynamic scaffold that concentrates dynamin and N-BAR proteins at sites of scission.


Assuntos
Actinas/metabolismo , Clatrina/metabolismo , Dinamina I/metabolismo , Endocitose , Retroalimentação Fisiológica , Actinas/antagonistas & inibidores , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Membrana Celular/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Vesículas Revestidas por Clatrina/ultraestrutura , Proteínas do Citoesqueleto/metabolismo , Dinamina I/genética , Cinética , Camundongos , Mutação de Sentido Incorreto , Células NIH 3T3 , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Tiazolidinas/farmacologia
13.
Mol Biol Cell ; 23(7): 1267-82, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22323285

RESUMO

Water expulsion by the contractile vacuole (CV) in Dictyostelium is carried out by a giant kiss-and-run focal exocytic event during which the two membranes are only transiently connected but do not completely merge. We present a molecular dissection of the GTPase Rab8a and the exocyst complex in tethering of the contractile vacuole to the plasma membrane, fusion, and final detachment. Right before discharge, the contractile vacuole bladder sequentially recruits Drainin, a Rab11a effector, Rab8a, the exocyst complex, and LvsA, a protein of the Chédiak-Higashi family. Rab8a recruitment precedes the nucleotide-dependent arrival of the exocyst to the bladder by a few seconds. A dominant-negative mutant of Rab8a strongly binds to the exocyst and prevents recruitment to the bladder, suggesting that a Rab8a guanine nucleotide exchange factor activity is associated with the complex. Absence of Drainin leads to overtethering and blocks fusion, whereas expression of constitutively active Rab8a allows fusion but blocks vacuole detachment from the plasma membrane, inducing complete fragmentation of tethered vacuoles. An indistinguishable phenotype is generated in cells lacking LvsA, implicating this protein in postfusion detethering. Of interest, overexpression of a constitutively active Rab8a mutant reverses the lvsA-null CV phenotype.


Assuntos
Dictyostelium/fisiologia , Proteínas de Protozoários/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Dictyostelium/genética , Dictyostelium/ultraestrutura , Exocitose/genética , Exocitose/fisiologia , Genes de Protozoários , Fusão de Membrana/genética , Fusão de Membrana/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Biológicos , Mutação , Fenótipo , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vacúolos/fisiologia , Proteínas rab de Ligação ao GTP/genética
14.
PLoS Biol ; 9(3): e1000604, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21445324

RESUMO

Dual colour total internal reflection fluorescence microscopy is a powerful tool for decoding the molecular dynamics of clathrin-mediated endocytosis (CME). Typically, the recruitment of a fluorescent protein-tagged endocytic protein was referenced to the disappearance of spot-like clathrin-coated structure (CCS), but the precision of spot-like CCS disappearance as a marker for canonical CME remained unknown. Here we have used an imaging assay based on total internal reflection fluorescence microscopy to detect scission events with a resolution of ∼ 2 s. We found that scission events engulfed comparable amounts of transferrin receptor cargo at CCSs of different sizes and CCS did not always disappear following scission. We measured the recruitment dynamics of 34 types of endocytic protein to scission events: Abp1, ACK1, amphiphysin1, APPL1, Arp3, BIN1, CALM, CIP4, clathrin light chain (Clc), cofilin, coronin1B, cortactin, dynamin1/2, endophilin2, Eps15, Eps8, epsin2, FBP17, FCHo1/2, GAK, Hip1R, lifeAct, mu2 subunit of the AP2 complex, myosin1E, myosin6, NECAP, N-WASP, OCRL1, Rab5, SNX9, synaptojanin2ß1, and syndapin2. For each protein we aligned ∼ 1,000 recruitment profiles to their respective scission events and constructed characteristic "recruitment signatures" that were grouped, as for yeast, to reveal the modular organization of mammalian CME. A detailed analysis revealed the unanticipated recruitment dynamics of SNX9, FBP17, and CIP4 and showed that the same set of proteins was recruited, in the same order, to scission events at CCSs of different sizes and lifetimes. Collectively these data reveal the fine-grained temporal structure of CME and suggest a simplified canonical model of mammalian CME in which the same core mechanism of CME, involving actin, operates at CCSs of diverse sizes and lifetimes.


Assuntos
Clatrina/metabolismo , Endocitose , Mamíferos/metabolismo , Simulação de Dinâmica Molecular , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Invaginações Revestidas da Membrana Celular/metabolismo , Dinaminas/metabolismo , Camundongos , Miosinas/metabolismo , Células NIH 3T3 , Polimerização , Ligação Proteica , Estrutura Terciária de Proteína , Fatores de Tempo
15.
J Cell Sci ; 118(Pt 22): 5269-77, 2005 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-16263762

RESUMO

Dishevelled is a crucial effector upstream in the Wnt signalling pathway, but the molecular mechanism by which it transduces the Wnt signal remains elusive. Dishevelled is a cytoplasmic protein with a strong tendency to form puncta, which correlates with its potent activity in stimulating Wnt signal transduction when overexpressed. These puncta are thought to reflect cytoplasmic vesicles. However, we show here that the mammalian Dishevelled protein Dvl2 does not colocalise with known vesicle markers for clathrin-mediated or clathrin-independent endocytic pathways. Furthermore, Dvl2 puncta do not stain with lipid dyes, indicating that these puncta do not contain membranes. Instead, our evidence from live imaging by TIRF microscopy of Dvl2 tagged with green fluorescent protein (GFP-Dvl2) revealed that these puncta move in and out of the evanescent field near the plasma membrane in an undirected fashion, and that they can grow by collision and fusion. Furthermore, high-resolution confocal microscopy and photobleaching experiments indicate that the GFP-Dvl2 puncta are protein assemblies; there is a constant exchange of GFP-Dvl2 between puncta and a diffuse cytoplasmic pool, which, therefore, are in a dynamic equilibrium with each other. The same is true for the DIX domain of Dvl2 itself and also for Axin-GFP, which equilibrates between the punctate and cytosolic pools. Our evidence indicates that Dvl2 and Axin puncta are dynamic protein assemblies rather than cytoplasmic vesicles.


Assuntos
Vesículas Citoplasmáticas/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteína Axina , Biomarcadores , Células COS , Chlorocebus aethiops , Clatrina/metabolismo , Vesículas Citoplasmáticas/efeitos dos fármacos , Citosol/metabolismo , Proteínas Desgrenhadas , Endocitose , Genes Reporter , Complexo de Golgi/metabolismo , Lipídeos/análise , Lipídeos/química , Fosfoproteínas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Repressoras/metabolismo
16.
Dev Cell ; 9(5): 581-92, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16256734

RESUMO

Clathrin-mediated endocytosis is the main path for receptor internalization in metazoans and is essential for controlling cell integrity and signaling. It is driven by a large array of protein and lipid interactions that have been deciphered mainly by biochemical and genetic means. To place these interactions into context, and ultimately build a fully operative model of endocytosis at the molecular level, it is necessary to know the kinetic details of the role of each protein in this process. In this review, we describe the recent efforts made, by using live cell imaging, to define clear steps in the formation of endocytic vesicles and to observe the recruitment of key proteins during membrane invagination, the scission of a newly formed vesicle, and its movement away from the plasma membrane.


Assuntos
Clatrina/metabolismo , Vesículas Revestidas/metabolismo , Endocitose/fisiologia , Animais , Membrana Celular/metabolismo
17.
Cell ; 121(4): 593-606, 2005 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-15907472

RESUMO

During clathrin-mediated endocytosis, membrane scission marks the isolation of a cargo-laden clathrin-coated pit (CCP) from the cell exterior. Here we used live-cell imaging of a pH-sensitive cargo to visualize the formation of clathrin-coated vesicles (CCVs) at single CCPs with a time resolution of seconds. We show that CCPs are highly dynamic and can produce multiple vesicles in succession. Using alternating evanescent field and epifluorescence illumination, we show that CCP invagination and scission are tightly coupled, with scission coinciding with maximal displacement of CCPs from the plasma membrane and with peak recruitment of cortactin-DsRed, a dynamin and F-actin binding protein. Finally, perturbing actin polymerization with latrunculin-B drastically reduces the efficiency of membrane scission and affects many aspects of CCP dynamics. We propose that CCP invagination, actin polymerization, and CCV formation are highly coordinated for efficient endocytosis.


Assuntos
Actinas/metabolismo , Membrana Celular/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Endocitose/fisiologia , Proteínas dos Microfilamentos/metabolismo , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Membrana Celular/efeitos dos fármacos , Vesículas Revestidas por Clatrina/efeitos dos fármacos , Invaginações Revestidas da Membrana Celular/efeitos dos fármacos , Cortactina , Endocitose/efeitos dos fármacos , Camundongos , Proteínas dos Microfilamentos/efeitos dos fármacos , Microscopia de Fluorescência , Microscopia de Vídeo , Polímeros , Células Swiss 3T3 , Tiazóis/farmacologia , Tiazolidinas
18.
J Neurosci ; 24(44): 9752-9, 2004 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-15525760

RESUMO

Visual and auditory information is encoded by sensory neurons that tonically release neurotransmitter at high rates. The synaptic ribbon is an essential organelle in nerve terminals of these neurons. Its precise function is unknown, but if the ribbon could be visualized in a living terminal, both its own dynamics and its relation to calcium and vesicle dynamics could be studied. We designed a short fluorescent peptide with affinity for a known binding domain of RIBEYE, a protein unique to the ribbon. When introduced via a whole-cell patch pipette, the peptide labeled structures at the presynaptic plasma membrane of ribbon-type terminals. The fluorescent spots match in size, location, number, and distribution the known features of synaptic ribbons. Furthermore, fluorescent spots mapped by confocal microscopy directly match the ribbons identified by electron microscopy in the same cell. Clearly the peptide binds to the synaptic ribbon, but even at saturating concentrations it affects neither the morphology of the ribbon nor its tethering of synaptic vesicles. It also does not inhibit exocytosis. Using the peptide label, we observed that the ribbon is immobile over minutes and that calcium influx is concentrated at the ribbon. Finally, we find that each ribbon in a retinal bipolar cell contains approximately 4000 molecules of RIBEYE, indicating that it is the major component of the synaptic ribbon.


Assuntos
Neurônios Aferentes/ultraestrutura , Organelas/ultraestrutura , Sinapses/ultraestrutura , Motivos de Aminoácidos , Animais , Sítios de Ligação , Canais de Cálcio/ultraestrutura , Proteínas do Olho/análise , Corantes Fluorescentes , Carpa Dourada , Hipocampo/citologia , Cinética , Neurônios Aferentes/química , Organelas/química , Fragmentos de Peptídeos , Terminações Pré-Sinápticas/ultraestrutura , Ligação Proteica , Ratos , Retina/citologia , Sinapses/química , Vesículas Sinápticas/ultraestrutura
19.
Trends Cell Biol ; 14(7): 352-8, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15246428

RESUMO

Endocytosis is characterized by movement and precisely controlled changes in membrane geometry during vesicle formation. Recent developments in live-cell imaging have enabled such movements to be monitored in vivo and correlated with the recruitment and dismissal of fluorescently labeled proteins. This experimental strategy has revealed the sequential recruitment of proteins that are involved in actin polymerization, and actin to single sites of endocytosis in both yeast and mammalian cells. Actin polymerization is correlated with the inward movements of endocytic organelles, which suggests that actin polymerization has a conserved role in this process. In this article, I will discuss three models for the role of actin polymerization in endocytosis.


Assuntos
Actinas/fisiologia , Endocitose/fisiologia , Animais , Clatrina/química , Fibroblastos/fisiologia , Modelos Biológicos , Organelas/fisiologia , Leveduras/fisiologia
20.
Eur J Cell Biol ; 83(1): 13-8, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15085951

RESUMO

Several findings suggest that actin-mediated motility can play a role in clathrin-mediated endocytosis but it remains unclear whether and when key proteins required for this process are recruited to endocytic sites. Here we investigate this question in live Swiss 3T3 cells using two-colour evanescent field (EF) microscopy. We find that Arp3, a component of the Arp2/3 complex, appears transiently while single clathrin-coated pits internalize. There is also additional recruitment of Neural-Wiskott Aldrich Syndrome Protein (N-WASP), a known activator of the Arp2/3 complex. Both proteins appear at about the same time as actin. We suggest that N-WASP and the Arp2/3 complex trigger actin polymerization during a late step in clathrin-mediated endocytosis, and propel clathrin-coated pits or vesicles from the plasma membrane into the cytoplasm.


Assuntos
Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Endocitose , Fibroblastos/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Proteína 2 Relacionada a Actina , Proteína 3 Relacionada a Actina , Actinas/metabolismo , Animais , Membrana Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Dinaminas/metabolismo , Proteínas de Fluorescência Verde , Processamento de Imagem Assistida por Computador , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia de Fluorescência , Células Swiss 3T3 , Fatores de Tempo , Proteína Neuronal da Síndrome de Wiskott-Aldrich
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