RESUMO
Clathrin is considered the prototype vesicle coat protein whose self-assembly mediates sorting of membrane cargo and recruitment of lipid modifiers. Detailed knowledge of clathrin biochemistry, structure, and interacting proteins has accumulated since the first observation, almost 50 years ago, of its role in receptor-mediated endocytosis of yolk protein. This review summarizes that knowledge, and focuses on properties of the clathrin heavy and light chain subunits and interaction of the latter with Hip proteins, to address the diversity of clathrin function beyond conventional receptor-mediated endocytosis. The distinct functions of the two human clathrin isoforms (CHC17 and CHC22) are discussed, highlighting CHC22's specialized involvement in traffic of the GLUT4 glucose transporter and consequent role in human glucose metabolism. Analysis of clathrin light chain function and interaction with the actin-binding Hip proteins during bacterial infection defines a novel actin-organizing function for CHC17 clathrin. By considering these diverse clathrin functions, along with intracellular sorting roles and influences on mitosis, further relevance of clathrin function to human health and disease is established.
Assuntos
Clatrina/fisiologia , Animais , Transporte Biológico , Clatrina/química , Clatrina/genética , Vesículas Revestidas por Clatrina/metabolismo , Vesículas Revestidas por Clatrina/fisiologia , Citoesqueleto/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
Clathrin-mediated endocytosis is the endocytic portal into cells through which cargo is packaged into vesicles with the aid of a clathrin coat. It is fundamental to neurotransmission, signal transduction and the regulation of many plasma membrane activities and is thus essential to higher eukaryotic life. Morphological stages of vesicle formation are mirrored by progression through various protein modules (complexes). The process involves the formation of a putative FCH domain only (FCHO) initiation complex, which matures through adaptor protein 2 (AP2)-dependent cargo selection, and subsequent coat building, dynamin-mediated scission and finally auxilin- and heat shock cognate 70 (HSC70)-dependent uncoating. Some modules can be used in other pathways, and additions or substitutions confer cell specificity and adaptability.
Assuntos
Clatrina/fisiologia , Endocitose/fisiologia , Actinas/fisiologia , Complexo 2 de Proteínas Adaptadoras/antagonistas & inibidores , Complexo 2 de Proteínas Adaptadoras/genética , Complexo 2 de Proteínas Adaptadoras/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Animais , Clatrina/antagonistas & inibidores , Clatrina/genética , Vesículas Revestidas por Clatrina/fisiologia , Dinaminas/fisiologia , Humanos , Modelos Biológicos , Mutação , Neoplasias/etiologia , Interferência de RNA , Transdução de Sinais , Vesículas Sinápticas/fisiologiaRESUMO
Deformability of the plasma membrane, the outermost surface of metazoan cells, allows cells to be dynamic, mobile and flexible. Factors that affect this deformability, such as tension on the membrane, can regulate a myriad of cellular functions, including membrane resealing, cell motility, polarisation, shape maintenance, membrane area control and endocytic vesicle trafficking. This review focuses on mechanoregulation of clathrin-mediated endocytosis (CME). We first delineate the origins of cell membrane tension and the factors that yield to its spatial and temporal fluctuations within cells. We then review the recent literature demonstrating that tension on the membrane is a fast-acting and reversible regulator of CME. Finally, we discuss tension-based regulation of endocytic clathrin coat formation during physiological processes.
Assuntos
Membrana Celular , Clatrina/metabolismo , Endocitose , Células Eucarióticas , Animais , Membrana Celular/química , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Vesículas Revestidas por Clatrina/fisiologia , Endocitose/fisiologia , Células Eucarióticas/fisiologia , Células Eucarióticas/ultraestrutura , Exocitose/fisiologia , Humanos , Transporte Proteico , Vesículas TransportadorasRESUMO
Inside living cells, the remodelling of membrane tubules by actomyosin networks is crucial for processes such as intracellular trafficking or organelle reshaping. In this review, we first present various in vivo situations in which actin affects membrane tubule remodelling, then we recall some results on force production by actin dynamics and on membrane tubules physics. Finally, we show that our knowledge of the underlying mechanisms by which actomyosin dynamics affect tubule morphology has recently been moved forward. This is thanks to in vitro experiments that mimic cellular membranes and actin dynamics and allow deciphering the physics of tubule remodelling in biochemically controlled conditions, and shed new light on tubule shape regulation.
Assuntos
Citoesqueleto de Actina , Membrana Celular , Células Eucarióticas , Citoesqueleto de Actina/fisiologia , Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Cavéolas/fisiologia , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Vesículas Revestidas por Clatrina/fisiologia , Endocitose/fisiologia , Células Eucarióticas/fisiologia , Células Eucarióticas/ultraestrutura , Transporte ProteicoRESUMO
Clathrin-mediated endocytosis (CME) proceeds through a series of morphological changes of the plasma membrane induced by a number of protein components. Although the spatiotemporal assembly of these proteins has been elucidated by fluorescence-based techniques, the protein-induced morphological changes of the plasma membrane have not been fully clarified in living cells. Here, we visualize membrane morphology together with protein localizations during CME by utilizing high-speed atomic force microscopy (HS-AFM) combined with a confocal laser scanning unit. The plasma membrane starts to invaginate approximately 30 s after clathrin starts to assemble, and the aperture diameter increases as clathrin accumulates. Actin rapidly accumulates around the pit and induces a small membrane swelling, which, within 30 s, rapidly covers the pit irreversibly. Inhibition of actin turnover abolishes the swelling and induces a reversible open-close motion of the pit, indicating that actin dynamics are necessary for efficient and irreversible pit closure at the end of CME.
Assuntos
Vesículas Revestidas por Clatrina/fisiologia , Endocitose , Actinas/metabolismo , Animais , Células COS , Chlorocebus aethiops , Dinaminas/metabolismo , Microscopia de Força Atômica , Microscopia ConfocalRESUMO
Phosphatidylserine (PS), a negatively charged phospholipid present predominantly at the inner leaflet of the plasma membrane, has been widely implicated in many cellular processes including membrane trafficking. Along this line, PS has been demonstrated to be important for endocytosis, however, the involved mechanisms remain uncertain. By monitoring clathrin-mediated endocytosis (CME) of single vesicles in mouse chromaffin cells using cell-attached capacitance measurements that offer millisecond time resolution, we demonstrate in the present study that the fission-pore duration is reduced by PS addition, indicating a stimulatory role of PS in regulating the dynamics of vesicle fission during CME. Furthermore, our results show that the PS-mediated effect on the fission-pore duration is Ca2+ -dependent and abolished in the absence of synaptotagmin 1 (Syt1), implying that Syt1 is necessary for the stimulatory role of PS in vesicle fission during CME. Consistently, a Syt1 mutant with a defective PS-Syt1 interaction increases the fission-pore duration. Taken together, our study suggests that PS-Syt1 interaction may be critical in regulating fission dynamics during CME.
Assuntos
Células Cromafins/fisiologia , Vesículas Revestidas por Clatrina/fisiologia , Clatrina/fisiologia , Fosfatidilserinas/fisiologia , Animais , Células Cultivadas , Endocitose/fisiologia , Exocitose/fisiologia , Feminino , Técnicas de Inativação de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Sinaptotagmina I/genética , Sinaptotagmina I/fisiologiaRESUMO
A critical step in cellular-trafficking pathways is the budding of membranes by protein coats, which recent experiments have demonstrated can be inhibited by elevated membrane tension. The robustness of processes like clathrin-mediated endocytosis (CME) across a diverse range of organisms and mechanical environments suggests that the protein machinery in this process has evolved to take advantage of some set of physical design principles to ensure robust vesiculation against opposing forces like membrane tension. Using a theoretical model for membrane mechanics and membrane protein interaction, we have systematically investigated the influence of membrane rigidity, curvature induced by the protein coat, area covered by the protein coat, membrane tension, and force from actin polymerization on bud formation. Under low tension, the membrane smoothly evolves from a flat to budded morphology as the coat area or spontaneous curvature increases, whereas the membrane remains essentially flat at high tensions. At intermediate, physiologically relevant, tensions, the membrane undergoes a "snap-through instability" in which small changes in the coat area, spontaneous curvature or membrane tension cause the membrane to "snap" from an open, U-shape to a closed bud. This instability can be smoothed out by increasing the bending rigidity of the coat, allowing for successful budding at higher membrane tensions. Additionally, applied force from actin polymerization can bypass the instability by inducing a smooth transition from an open to a closed bud. Finally, a combination of increased coat rigidity and force from actin polymerization enables robust vesiculation even at high membrane tensions.
Assuntos
Membrana Celular/química , Vesículas Revestidas por Clatrina/fisiologia , Clatrina/fisiologia , Simulação por Computador , Endocitose/fisiologia , Proteínas de Membrana/fisiologia , Modelos Químicos , Estresse Mecânico , Algoritmos , Fenômenos Biomecânicos , Membrana Celular/ultraestrutura , Fluidez de Membrana , Proteínas de Membrana/química , Propriedades de SuperfícieRESUMO
BACKGROUND: Inhibition of the renin-angiotensin system remains a cornerstone in reducing proteinuria and progression of kidney failure, effects believed to be the result of reduction in BP and glomerular hyperfiltration. However, studies have yielded conflicting results on whether podocyte-specific angiotensin II (AngII) signaling directly induces podocyte injury. Previous research has found that after AngII stimulation, ß-arrestin-bound angiotensin II receptor type 1 (AT1R) is internalized in a clathrin- and dynamin-dependent manner, and that Dynamin1 and Dynamin2 double-knockout mice exhibit impaired clathrin-mediated endocytosis. METHODS: We used podocyte-specific Dyn double-knockout mice to examine AngII-stimulated AT1R internalization and signaling in primary podocytes and controls. We also examined the in vivo effect of AngII in these double-knockout mice through renin-angiotensin system blockers and through deletion of Agtr1a (which encodes the predominant AT1R isoform expressed in kidney, AT1aR). We tested calcium influx, Rac1 activation, and lamellipodial extension in control and primary podocytes of Dnm double-knockout mice treated with AngII. RESULTS: We confirmed augmented AngII-stimulated AT1R signaling in primary Dnm double-knockout podocytes resulting from arrest of clathrin-coated pit turnover. Genetic ablation of podocyte Agtr1a in Dnm double-knockout mice demonstrated improved albuminuria and kidney function compared with the double-knockout mice. Isolation of podocytes from Dnm double-knockout mice revealed abnormal membrane dynamics, with increased Rac1 activation and lamellipodial extension, which was attenuated in Dnm double-knockout podocytes lacking AT1aR. CONCLUSIONS: Our results indicate that inhibiting aberrant podocyte-associated AT1aR signaling pathways has a protective effect in maintaining the integrity of the glomerular filtration barrier.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Vesículas Revestidas por Clatrina/fisiologia , Podócitos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/efeitos dos fármacos , Albuminúria/fisiopatologia , Angiotensina II/farmacologia , Animais , Sinalização do Cálcio , Células Cultivadas , Creatinina/sangue , Creatinina/urina , Dinamina I/deficiência , Dinamina I/fisiologia , Dinamina II/deficiência , Dinamina II/fisiologia , Endocitose , Glomerulonefrite/genética , Glomerulonefrite/fisiopatologia , Hemodinâmica , Glomérulos Renais/patologia , Masculino , Camundongos , Camundongos Knockout , Neuropeptídeos/fisiologia , Podócitos/efeitos dos fármacos , Podócitos/ultraestrutura , Pseudópodes/fisiologia , Receptor Tipo 1 de Angiotensina/deficiência , Proteínas rac1 de Ligação ao GTP/fisiologiaRESUMO
Stoichiometric balance, or dosage balance, implies that proteins that are subunits of obligate complexes (e.g. the ribosome) should have copy numbers expressed to match their stoichiometry in that complex. Establishing balance (or imbalance) is an important tool for inferring subunit function and assembly bottlenecks. We show here that these correlations in protein copy numbers can extend beyond complex subunits to larger protein-protein interactions networks (PPIN) involving a range of reversible binding interactions. We develop a simple method for quantifying balance in any interface-resolved PPINs based on network structure and experimentally observed protein copy numbers. By analyzing such a network for the clathrin-mediated endocytosis (CME) system in yeast, we found that the real protein copy numbers were significantly more balanced in relation to their binding partners compared to randomly sampled sets of yeast copy numbers. The observed balance is not perfect, highlighting both under and overexpressed proteins. We evaluate the potential cost and benefits of imbalance using two criteria. First, a potential cost to imbalance is that 'leftover' proteins without remaining functional partners are free to misinteract. We systematically quantify how this misinteraction cost is most dangerous for strong-binding protein interactions and for network topologies observed in biological PPINs. Second, a more direct consequence of imbalance is that the formation of specific functional complexes depends on relative copy numbers. We therefore construct simple kinetic models of two sub-networks in the CME network to assess multi-protein assembly of the ARP2/3 complex and a minimal, nine-protein clathrin-coated vesicle forming module. We find that the observed, imperfectly balanced copy numbers are less effective than balanced copy numbers in producing fast and complete multi-protein assemblies. However, we speculate that strategic imbalance in the vesicle forming module allows cells to tune where endocytosis occurs, providing sensitive control over cargo uptake via clathrin-coated vesicles.
Assuntos
Perfilação da Expressão Gênica/métodos , Mapeamento de Interação de Proteínas/métodos , Proteostase/fisiologia , Clatrina , Vesículas Revestidas por Clatrina/fisiologia , Endocitose/fisiologia , Dosagem de Genes/fisiologia , Cinética , Modelos Biológicos , Ligação Proteica , Mapas de Interação de Proteínas/fisiologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologiaRESUMO
Protein transport between organelles is an essential process in all eukaryotic cells and is mediated by the regulation of processes such as vesicle formation, transport, docking, and fusion. In animals, SCY1-LIKE2 (SCYL2) binds to clathrin and has been shown to play roles in trans-Golgi network-mediated clathrin-coated vesicle trafficking. Here, we demonstrate that SCYL2A and SCYL2B, which are Arabidopsis (Arabidopsis thaliana) homologs of animal SCYL2, are vital for plant cell growth and root hair development. Studies of the SCYL2 isoforms using multiple single or double loss-of-function alleles show that SCYL2B is involved in root hair development and that SCYL2A and SCYL2B are essential for plant growth and development and act redundantly in those processes. Quantitative reverse transcription-polymerase chain reaction and a ß-glucuronidase-aided promoter assay show that SCYL2A and SCYL2B are differentially expressed in various tissues. We also show that SCYL2 proteins localize to the Golgi, trans-Golgi network, and prevacuolar compartment and colocalize with Clathrin Heavy Chain1 (CHC1). Furthermore, bimolecular fluorescence complementation and coimmunoprecipitation data show that SCYL2B interacts with CHC1 and two Soluble NSF Attachment Protein Receptors (SNAREs): Vesicle Transport through t-SNARE Interaction11 (VTI11) and VTI12. Finally, we present evidence that the root hair tip localization of Cellulose Synthase-Like D3 is dependent on SCYL2B. These findings suggest the role of SCYL2 genes in plant cell developmental processes via clathrin-mediated vesicle membrane trafficking.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Vesículas Revestidas por Clatrina/fisiologia , Desenvolvimento Vegetal , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Arabidopsis/genética , Cadeias Pesadas de Clatrina/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Qb-SNARE/metabolismo , Rede trans-Golgi/metabolismoRESUMO
Precise and efficient endocytosis is essential for vesicle recycling during a sustained neurotransmission. The regulation of endocytosis has been extensively studied, but inhibitors have rarely been found. Here, we show that synaptotagmin-11 (Syt11), a non-Ca(2+)-binding Syt implicated in schizophrenia and Parkinson's disease, inhibits clathrin-mediated endocytosis (CME) and bulk endocytosis in dorsal root ganglion neurons. The frequency of both types of endocytic event increases in Syt11 knockdown neurons, while the sizes of endocytosed vesicles and the kinetics of individual bulk endocytotic events remain unaffected. Specifically, clathrin-coated pits and bulk endocytosis-like structures increase on the plasma membrane in Syt11-knockdown neurons. Structural-functional analysis reveals distinct domain requirements for Syt11 function in CME and bulk endocytosis. Importantly, Syt11 also inhibits endocytosis in hippocampal neurons, implying a general role of Syt11 in neurons. Taken together, we propose that Syt11 functions to ensure precision in vesicle retrieval, mainly by limiting the sites of membrane invagination at the early stage of endocytosis.
Assuntos
Vesículas Revestidas por Clatrina/fisiologia , Clatrina/metabolismo , Endocitose , Neurônios/metabolismo , Sinaptotagminas/genética , Sinaptotagminas/metabolismo , Animais , Exocitose , Gânglios Espinais/citologia , Técnicas de Silenciamento de Genes , Neurônios/ultraestrutura , Ratos , Transmissão Sináptica/genética , Transmissão Sináptica/fisiologiaRESUMO
The functions of the minor phospholipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] during vegetative plant growth remain obscure. Here, we targeted two related phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) PIP5K1 and PIP5K2, which are expressed ubiquitously in Arabidopsis thaliana. A pip5k1 pip5k2 double mutant with reduced PtdIns(4,5)P2 levels showed dwarf stature and phenotypes suggesting defects in auxin distribution. The roots of the pip5k1 pip5k2 double mutant had normal auxin levels but reduced auxin transport and altered distribution. Fluorescence-tagged auxin efflux carriers PIN-FORMED (PIN1)-green fluorescent protein (GFP) and PIN2-GFP displayed abnormal, partially apolar distribution. Furthermore, fewer brefeldin A-induced endosomal bodies decorated by PIN1-GFP or PIN2-GFP formed in pip5k1 pip5k2 mutants. Inducible overexpressor lines for PIP5K1 or PIP5K2 also exhibited phenotypes indicating misregulation of auxin-dependent processes, and immunolocalization showed reduced membrane association of PIN1 and PIN2. PIN cycling and polarization require clathrin-mediated endocytosis and labeled clathrin light chain also displayed altered localization patterns in the pip5k1 pip5k2 double mutant, consistent with a role for PtdIns(4,5)P2 in the regulation of clathrin-mediated endocytosis. Further biochemical tests on subcellular fractions enriched for clathrin-coated vesicles (CCVs) indicated that pip5k1 and pip5k2 mutants have reduced CCV-associated PI4P 5-kinase activity. Together, the data indicate an important role for PtdIns(4,5)P2 in the control of clathrin dynamics and in auxin distribution in Arabidopsis.
Assuntos
Arabidopsis/metabolismo , Transporte Biológico , Vesículas Revestidas por Clatrina/fisiologia , Fosfatidilinositol 4,5-Difosfato/fisiologia , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/análise , Proteínas de Arabidopsis/metabolismo , Polaridade Celular , Endocitose , Proteínas de Fluorescência Verde/análise , Ácidos Indolacéticos/metabolismo , Proteínas de Membrana Transportadoras/análise , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Fenótipo , Fosfotransferases (Aceptor do Grupo Álcool)/genéticaRESUMO
Successful macrophage colonization by Coxiella burnetii, the cause of human Q fever, requires pathogen-directed biogenesis of a large, growth-permissive parasitophorous vacuole (PV) with phagolysosomal characteristics. The vesicular trafficking pathways co-opted by C. burnetii for PV development are poorly defined; however, it is predicted that effector proteins delivered to the cytosol by a defective in organelle trafficking/intracellular multiplication (Dot/Icm) type 4B secretion system are required for membrane recruitment. Here, we describe involvement of clathrin-mediated vesicular trafficking in PV generation and the engagement of this pathway by the C. burnetii type 4B secretion system substrate Coxiella vacuolar protein A (CvpA). CvpA contains multiple dileucine [DERQ]XXXL[LI] and tyrosine (YXXΦ)-based endocytic sorting motifs like those recognized by the clathrin adaptor protein (AP) complexes AP1, AP2, and AP3. A C. burnetii ΔcvpA mutant exhibited significant defects in replication and PV development, confirming the importance of CvpA in infection. Ectopically expressed mCherry-CvpA localized to tubular and vesicular domains of pericentrosomal recycling endosomes positive for Rab11 and transferrin receptor, and CvpA membrane interactions were lost upon mutation of endocytic sorting motifs. Consistent with CvpA engagement of the endocytic recycling system, ectopic expression reduced uptake of transferrin. In pull-down assays, peptides containing CvpA-sorting motifs and full-length CvpA interacted with AP2 subunits and clathrin heavy chain. Furthermore, depletion of AP2 or clathrin by siRNA treatment significantly inhibited C. burnetii replication. Thus, our results reveal the importance of clathrin-coated vesicle trafficking in C. burnetii infection and define a role for CvpA in subverting these transport mechanisms.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Sistemas de Secreção Bacterianos/fisiologia , Vesículas Revestidas por Clatrina/fisiologia , Coxiella burnetii/crescimento & desenvolvimento , Proteínas de Membrana Transportadoras/metabolismo , Transdução de Sinais/fisiologia , Análise de Variância , Vesículas Revestidas por Clatrina/metabolismo , Clonagem Molecular , Biologia Computacional , Coxiella burnetii/metabolismo , Células HeLa , Humanos , Microscopia de Fluorescência , Transporte Proteico/fisiologia , RNA Interferente Pequeno/genéticaRESUMO
Eukaryotic cells require selective sorting and transport of cargo between intracellular compartments. This is accomplished at least in part by vesicles that bud from a donor compartment, sequestering a subset of resident protein "cargos" destined for transport to an acceptor compartment. A key step in vesicle formation and targeting is the recruitment of specific proteins that form a coat on the outside of the vesicle in a process requiring the activation of regulatory GTPases of the ARF family. Like all such GTPases, ARFs cycle between inactive, GDP-bound, and membrane-associated active, GTP-bound, conformations. And like most regulatory GTPases the activating step is slow and thought to be rate limiting in cells, requiring the use of ARF guanine nucleotide exchange factor (GEFs). ARF GEFs are characterized by the presence of a conserved, catalytic Sec7 domain, though they also contain motifs or additional domains that confer specificity to localization and regulation of activity. These domains have been used to define and classify five different sub-families of ARF GEFs. One of these, the BIG/GBF1 family, includes three proteins that are each key regulators of the secretory pathway. GEF activity initiates the coating of nascent vesicles via the localized generation of activated ARFs and thus these GEFs are the upstream regulators that define the site and timing of vesicle production. Paradoxically, while we have detailed molecular knowledge of how GEFs activate ARFs, we know very little about how GEFs are recruited and/or activated at the right time and place to initiate transport. This review summarizes the current knowledge of GEF regulation and explores the still uncertain mechanisms that position GEFs at "budding ready" membrane sites to generate highly localized activated ARFs.
Assuntos
Fatores de Ribosilação do ADP/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Transporte Biológico , Vesículas Revestidas por Clatrina/fisiologia , Complexo I de Proteína do Envoltório/metabolismo , Complexo I de Proteína do Envoltório/fisiologia , Fatores de Troca do Nucleotídeo Guanina/análise , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Metabolismo dos Lipídeos , Transdução de SinaisRESUMO
Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2-specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation.
Assuntos
Caenorhabditis elegans , Proteínas Ativadoras de GTPase/metabolismo , Complexo de Golgi , Vesículas Secretórias , Proteína rab2 de Ligação ao GTP/genética , Animais , Autoantígenos/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Vesículas Revestidas por Clatrina/genética , Vesículas Revestidas por Clatrina/fisiologia , Endossomos/genética , Endossomos/metabolismo , Proteínas Ativadoras de GTPase/genética , Regulação da Expressão Gênica , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Humanos , Neurônios Motores/metabolismo , Transporte Proteico/genética , Vesículas Secretórias/genética , Vesículas Secretórias/fisiologia , Proteína rab2 de Ligação ao GTP/metabolismo , Proteínas rab3 de Ligação ao GTP/genética , Proteínas rab3 de Ligação ao GTP/metabolismoRESUMO
Clathrin has previously been implicated in Drosophila male fertility and spermatid individualization. To understand further the role of membrane transport in this process, we analyzed the phenotypes of mutations in Drosophila auxilin (aux), a regulator of clathrin function, in spermatogenesis. Like partial loss-of-function Clathrin heavy chain (Chc) mutants, aux mutant males are sterile and produce no mature sperm. The reproductive defects of aux males were rescued by male germ cell-specific expression of aux, indicating that auxilin function is required autonomously in the germ cells. Furthermore, this rescue depends on both the clathrin-binding and J domains, suggesting that the ability of Aux to bind clathrin and the Hsc70 ATPase is essential for sperm formation. aux mutant spermatids show a deficit in formation of the plasma membrane during elongation, which probably disrupts the subsequent coordinated migration of investment cones during individualization. In wild-type germ cells, GFP-tagged clathrin localized to clusters of vesicular structures near the Golgi. These structures also contained the Golgi-associated clathrin adaptor AP-1, suggesting that they were Golgi-derived. By contrast, in aux mutant cells, clathrin localized to abnormal patches surrounding the Golgi and its colocalization with AP-1 was disrupted. Based on these results, we propose that Golgi-derived clathrin-positive vesicles are normally required for sustaining the plasma membrane increase necessary for spermatid differentiation. Our data suggest that Aux participates in forming these Golgi-derived clathrin-positive vesicles and that Aux, therefore, has a role in the secretory pathway.
Assuntos
Auxilinas/fisiologia , Vesículas Revestidas por Clatrina/fisiologia , Drosophila/fisiologia , Complexo de Golgi/fisiologia , Espermatogênese/fisiologia , Animais , Animais Geneticamente Modificados , Auxilinas/genética , Auxilinas/metabolismo , Células Cultivadas , Vesículas Revestidas por Clatrina/metabolismo , Citocinese/genética , Citocinese/fisiologia , Drosophila/embriologia , Drosophila/genética , Drosophila/metabolismo , Embrião não Mamífero , Feminino , Fertilidade/genética , Fertilidade/fisiologia , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Masculino , Modelos Biológicos , Via Secretória/genética , Via Secretória/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Espermatogênese/genéticaRESUMO
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
Endocytic clathrin-coated vesicles arise through the deformation of a small region of plasma membrane encapsulated by a cytosol-oriented clathrin lattice. The coat assembles from soluble protomers in a rapid and highly cooperative process, and invagination is tightly linked to the selective enrichment of cargo molecules within the nascent bud. Recent structural and functional studies demonstrate that coat assembly, membrane deformation, local actin dynamics and the final scission event are intricately coupled, and begin to reveal how key multifunctional, modular proteins are responsible for this linkage. An emerging mechanistic theme is how sequential engagement of common interaction surfaces or network hubs can evict prior binding partners from the assembly zone to ensure vectorial progression of the coat assembly process.
Assuntos
Vesículas Revestidas por Clatrina/química , Vesículas Revestidas por Clatrina/fisiologia , Proteínas/química , Proteínas/fisiologia , Animais , Membrana Celular/fisiologia , Modelos BiológicosRESUMO
Endocytosis is increasingly understood to play crucial roles in most signaling pathways, from determining which signaling components are activated, to how the signal is subsequently transduced and/or terminated. Whether a receptor-ligand complex is internalized via a clathrin-dependent or clathrin-independent endocytic route, and the complexes' subsequent trafficking through specific endocytic compartments, to then be recycled or degraded, has profound effects on signaling output. This review discusses the roles of endocytosis in three markedly different signaling pathways: the Wnt, Notch, and Eph/Ephrin pathways. These offer fundamentally different signaling systems: (1) diffusible ligands inducing signaling in one cell, (2) membrane-tethered ligands inducing signaling in a contacting receptor cell, and (3) bi-directional receptor-ligand signaling in two contacting cells. In each of these systems, endocytosis controls signaling in fascinating ways, and comparison of their similarities and dissimilarities will help to expand our understanding of endocytic control of signal transduction across multiple signaling pathways.
Assuntos
Endocitose/fisiologia , Modelos Biológicos , Transdução de Sinais , Comunicação Celular , Vesículas Revestidas por Clatrina/fisiologia , Ligantes , Receptores da Família Eph/metabolismo , Receptores da Família Eph/fisiologia , Receptores Notch/metabolismo , Receptores Notch/fisiologia , Proteínas Wnt/metabolismo , Proteínas Wnt/fisiologiaRESUMO
It has long been known that the maintenance of fast communication between neurons requires that presynaptic terminals recycle the small vesicles from which neurotransmitter is released. But the mechanisms that retrieve vesicles from the cell surface are still not understood. Although we have a wealth of information about the molecular details of endocytosis in non-neuronal cells, it is clear that endocytosis at the synapse is faster and regulated in distinct ways. A satisfying understanding of these processes will require molecular events to be manipulated while observing endocytosis in living synapses. Here, we review recent work that seeks to bridge the gap between physiology and molecules to unravel the endocytic machinery operating at the synaptic terminal.