BACKGROUND: Clathrin is a multimeric protein involved in vesicle coat assembly. Recently clathrin distribution was reported to change during the cell cycle and was found to associate with the mitotic spindle. Here we test whether the recruitment of clathrin to the spindle is indicative of a critical functional contribution to mitosis. METHODOLOGY/PRINCIPAL FINDINGS: Previously a chicken pre-B lymphoma cell line (DKO-R) was developed in which the endogenous clathrin heavy chain alleles were replaced with the human clathrin heavy chain under the control of a tetracycline-regulatable promoter. Receptor-mediated and fluid-phase endocytosis were significantly inhibited in this line following clathrin knockout, and we used this to explore the significance of clathrin heavy chain expression for cell cycle progression. We confirmed using confocal microscopy that clathrin colocalised with tubulin at mitotic spindles. Using a propidium iodide flow cytometric assay we found no statistical difference in the cell
The heterotetrameric adaptor protein complex AP2 is one of the best-studied components of the endocytic machinery. The AP2 complex consists of four different subunits, α, β2, σ2, and μ2, which assemble into a core domain with two appendages (Fig. 2; Collins et al., 2002; Jackson et al., 2010). AP2 has multiple binding partners, including phosphatidylinositol 4,5-bisphosphate (PIP2), clathrin, several endocytic accessory proteins, and two signaling motifs present on some cargo receptors (see Traub, 2009 for a detailed review). The AP2 complex has classically been considered to be the master initiator of clathrin-mediated endocytosis through its role in recruiting clathrin molecules to the membrane. However, several lines of evidence question this idea.. If the AP2 complex has an essential role in initiation then its presence would be required for the formation of endocytic sites. However, in yeast the endocytosis of mating pheromone α-factor is unaffected in strains lacking functional AP2 ...
Coat proteins appear to play a general role in intracellular protein trafficking by coordinating a membrane budding event with cargo selection. Here we show that the AP-2 adaptor, a clathrin-associated coat-protein complex that nucleates clathrin-coated vesicle formation at the cell surface, can also initiate the assembly of normal polyhedral clathrin coats on dense lysosomes under physiological conditions in vitro. Clathrin coat formation on lysosomes is temperature dependent, displays an absolute requirement for ATP, and occurs in both semi-intact cells and on purified lysosomes, suggesting that clathrin-coated vesicles might regulate retrograde membrane traffic out of the lysosomal compartment. ...
Clathrin-mediated endocytosis plays an important role in the recycling of synaptic vesicle in presynaptic terminals, and in the recycling of transmitter receptors in neuronal soma/dendrites. The present study uses electron microscopy (EM) and immunogold EM to document the different categories of clathrin-coated vesicles (CCV) and pits (CCP) in axons compared to soma/dendrites, and the depolarization-induced redistribution of clathrin in these two polarized compartments of the neuron. The size of CCVs in presynaptic terminals (~ 40 nm; similar to the size of synaptic vesicles) is considerably smaller than the size of CCVs in soma/dendrites (~ 90 nm). Furthermore, neuronal stimulation induces an increase in the number of CCV/CCP in presynaptic terminals, but a decrease in soma/dendrites. Immunogold labeling of clathrin revealed that in presynaptic terminals under resting conditions, the majority of clathrin molecules are unassembled and concentrated outside of synaptic vesicle clusters. Upon
To assess the role of clathrin in the bulk endocytic flow of rat foetal fibroblasts, the rate of internalization of fluid-phase and membrane-lipid tracers were compared, under control conditions and after inhibition of endocytic clathrin-coated pit formation. After intracellular potassium depletion or upon cell transfer into 0.35 M NaCl, the rate of internalization of receptor-bound transferrin and the residual membrane area of plasmalemmal clathrin-coated pits and vesicles were similarly decreased by approximately 90%. In contrast, the initial rate (, 5 min) of intracellular accumulation of the fluid-phase tracer HRP was not affected. Both in control and treated cells, the rate of HRP accumulation declined after approximately 5 min, and was twofold lower in treated cells, due to enhanced regurgitation. After correction for regurgitation, the endocytic rate constant was similar to measurements at shorter intervals and identical in control and treated cells. Similarly, the rate of internalization ...
Clathrin-mediated endocytosis is exploited by bacterial and viral pathogens during internalization. Humphries and Way review recent studies which highlight the fact that, in addition to a structural role, clathrin can function as a signalling platform during pathogen entry, and other studies revealing that, in conjunction with actin, clathrin is involved in pathogen cell-cell spread and release. The role of clathrin in pathogen entry has received much attention and has highlighted the adaptability of clathrin during internalization. Recent studies have now uncovered additional roles for clathrin and have
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article{1864472, abstract = {Endocytosis is a crucial mechanism by which eukaryotic cells internalize extracellular and plasma membrane material, and it is required for a multitude of cellular and developmental processes in unicellular and multicellular organisms. In animals and yeast, the best characterized pathway for endocytosis depends on the function of the vesicle coat protein clathrin. Clathrin-mediated endocytosis has recently been demonstrated also in plant cells, but its physiological and developmental roles remain unclear. Here, we assessed the roles of the clathrin-mediated mechanism of endocytosis in plants by genetic means. We interfered with clathrin heavy chain (CHC) function through mutants and dominant-negative approaches in Arabidopsis thaliana and established tools to manipulate clathrin function in a cell type-specific manner. The chc2 single mutants and dominant-negative CHC1 (HUB) transgenic lines were defective in bulk endocytosis as well as in internalization of ...
EpsinR is a clathrin-coated vesicle (CCV) enriched 70-kD protein that binds to phosphatidylinositol-4-phosphate, clathrin, and the gamma appendage domain of the adaptor protein complex 1 (AP1). In cells, its distribution overlaps with the perinuclear pool of clathrin and AP1 adaptors. Overexpression disrupts the CCV-dependent trafficking of cathepsin D from the trans-Golgi network to lysosomes and the incorporation of mannose-6-phosphate receptors into CCVs. These biochemical and cell biological data point to a role for epsinR in AP1/clathrin budding events in the cell, just as epsin1 is involved in the budding of AP2 CCVs. Furthermore, we show that two gamma appendage domains can simultaneously bind to epsinR with affinities of 0.7 and 45 microM, respectively. Thus, potentially, two AP1 complexes can bind to one epsinR. This high affinity binding allowed us to identify a consensus binding motif of the form DFxDF, which we also find in gamma-synergin and use to predict that an uncharacterized EF-hand
The early events of viral infection usually involve the attachment of virus to cellular receptor molecules on the plasma membrane of host cells. This is followed by internalization, uncoating, and subsequent virus gene transcription and/or translation at specific locations in cells. Studies have clearly demonstrated that animal viruses can utilize different internalization and trafficking pathways that allow specific localization within the cells upon entry for a successful infection (15). For enveloped viruses, the entry process can occur either via the fusion of virus envelope glycoproteins at the plasma membrane at neutral pH to promote the internalization of viral nucleocapsids or virus particles undergoing endocytosis prior to fusion with endocytic membrane. For the latter, conformational change of the virus fusion protein to expose the hydrophobic fusion peptide is induced by an acidic pH for the release of the viral nucleocapsids into the cytoplasm (14).. In the present study, a variety ...
Link to Pubmed [PMID] - 20486136. Bioessays 2010 Jun;32(6):496-504. Clathrin and the endocytosis machinery has recently been described as being required in mammalian cells for the internalization of large particles including pathogenic bacteria, fungi, and large viruses. These apparently unexpected observations, within the framework of the classical mechanisms for the formation of clathrin-coated vesicles, are now considered as examples of a new non-classical function of clathrin, which can promote the internalization of membrane domains associated to planar clathrin lattices. The role of actin downstream of clathrin seems to be critical for this still poorly characterized process. The historical frontier between endocytosis and phagocytosis is vanishing in the light of this new role for clathrin.. http://www.ncbi.nlm.nih.gov/pubmed/20486136 ...
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Our study provides evidence for a new role of Src in the phosphorylation of the clathrin adaptor complex AP-2 (via the phosphorylation of the β-subunit, i.e. β2-adaptin). We identify Y737 in the ear domain of β2-adaptin as an important Src target. We show that this residue represents a regulatory site for controlling the dissociation of β-arrestin from AP-2 in clathrin-coated pits (CCPs). Our results not only extend the pleiotropic function of Src in GPCR internalization, but also highlight the importance of receptor-dependent signaling in the clathrin-mediated endocytosis of AT1R.. β-arrestins are multifunctional adaptors involved in the endocytosis and signaling of many GPCRs (Claing et al., 2002; Lefkowitz, 1998). They have been shown to target receptors to CCPs through binding to clathrin and the β-subunit of AP-2 (Goodman, Jr et al., 1996; Laporte et al., 1999), and to act as signal transducers by recruiting different kinases to GPCRs (Luttrell and Luttrell, 2004). We have previously ...
set by micropipette aspiration to be less than 1 over a wide range of spontaneous curvatures, indicating a high membrane-tension regime in their set up. Thus, our model is consistent with their observations of shallow buds observed in isotonic conditions. One result that our model cannot explain is the lack of any clathrin assembly observed under hypotonic conditions. It is possible that at extremely high membrane tensions, the coat is simply unable to stay bound to the membrane at the extremely flat morphology that would be expected.. Avinoam et al. (46) found that the size of the clathrin coat does not change substantially during membrane deformation in CME in human skin melanoma (SK-MEL-2) cells. This observation is in contrast to the canonical view that the clathrin coat should directly impose its preferred curvature on the underlying membrane (8). There are two possible explanations for this observation in the context of our study. One is that the membrane tension is too high for the coat ...
VOLUME 21 ISSUE 12 12 2011 1655 1661 Novel functions of endocytic player clathrin in mitosis Wenxiang Fu Qing Jiang and Chuanmao Zhang The MOE Key Laboratory of Cell Proliferation and Differentiation and the State Key Laboratory of Bio membrane and Membrane Biotechnology College of Life Sciences Peking University Beijing 100871 China Correspondence Chuanmao Zhang Tel 86 10 62757173 E mail zhangcm pku edu cn Clathrin has been widely recognized as a pivotal player in endocytosis in which several adaptors and accessory proteins are involved Recent studies suggested that clathrin is also essential for cell division Here this review mainly focuses on the clathrin dependent mechanisms involved in spindle assembly and chromosome alignment In mitosis clathrin forms a complex with phosphorylated TACC3 to ensure spindle stability and proper chromosome alignment The clathrin regulated mechanism in mitosis requires the crosstalk among clathrin spindle assembly factors SAFs Ran GTP and mitotic kinases ...
The structure of Clathrin is known as a triskeleion structure with there being three bent legs extending from a central point known as the central trimerization domain. Clathrin is made up of six chains of protein that are braided together in such a way to form to form its distinct shape. Three of these chains are known as heavy chains and form the backbone of Clathrin, they consist of two sub domains a N-terminal zone and a proximal leg domain. A seven bladed β-propeller structure is what the N-terminal domain consists off. While the proximal leg consists of a super Helix (Conner and Schmid, 2002). The other three chains are known as light chains and regulate formation and disassembly of the Clathrin and can be found connected to the proximal portion of the heavy chains. Multiple Clathrin molecules have the ability to form a variety of complex shapes when they interact, they can form 5 or 6 sided rings with the 5 sided kind having a greature curvature and when enough get together they can form ...
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The lipid mediator sphingosine 1-phosphate (S1P) regulates a wide range of cellular activities, including vascular maturation, angiogenesis, and immune-cell trafficking. Among the five known receptors for S1P (S1PR1-S1PR5), S1PR1 is a critical regulator of lymphocyte trafficking: its signaling is required for lymphocyte egress from lymphoid organs, while its down-modulation by agonist-induced internalization is a prerequisite for lymphocyte entry into lymphoid organs from the bloodstream. Despite the importance of S1PR1 down-regulation in determining lymphocyte behavior, the molecular mechanism of its internalization in lymphocytes has not been defined. Here we show that agonist-induced S1PR1 internalization in T cells occurs via clathrin-mediated endocytosis and is regulated by moesin, an ezrin-radixin-moesin (ERM) family member. In S1P-stimulated T cells, S1PR1 relocalized within clathrin-coated vesicles (CCVs) and early endosomes, and S1PR1 internalization was blocked when clathrin was
The lectin isolated from Xerocomus chrysenteron (XCL) displays a toxic activity towards insects. In order to assess its possible mode of action and to gather useful data for its potential use in insect-resistant transgenic plants, we investigated the
We found that lipid vesicles are internalized by both clathrin-mediated and clathrin-independent endocytosis pathways which require an acidification step for liposomes destabilization and fusion with the endosomes. The fusion event possibly triggers a microtubule driven pathway which avoids classical sorting endosomes and favours an ER pathway. This is an alternate to the SV40 virus caveolar route using a pH dependent, direct plasma membrane-to-ER pathway to efficiently deliver extracellular encapsulated cargo to the ER. Whilst further studies are needed to dissect this novel pathway in more detail, the finding of a second major ER targeting route leads to the idea that the import of molecules from the outside cell into the ER could be as important for the cell function as its secretion processes ...
Clathrin-mediated endocytosis (CME) is a key metabolic pathway that plays a central role in the delivery of nutrients and drug carriers into cells. In this study, we model the interactions of lipid membranes with different types of protein scaffolds and active forces to provide mechanistic insights into CME. To this end, we develop and employ an extended theoretical framework of lipid membranes that entertains spatial heterogeneity and local anisotropy that could arise from membrane-protein interactions. We show that a departure from homogeneity and isotropy can lead to a variable surface tension field, conventionally assumed to be a constant parameter. We model the impact of resting tension in a cell and discuss its consequences on the minimal protein machinery needed to complete vesicle formation. Based on our quantitative model and findings, we highlight the physical principles that unify CME in apparently distinct yeast and mammalian cells.
SMAP2 immunoprecipitated clathrin and AP-1 through a putative clathrin-binding domain and a CALM-binding domain, and SMAP2 mutants that did not interact with clathrin or AP-1 could not localize to recycling ...
mmmmm cow brains the concept centers on a particular protein called clathrin which has a unique knack for assembling itself into versatile structures that foster the formation of complex molecules clathrin is present in every cell in the human body but cows possess a vast wealth of it in their bovine brains that make them an ideal source for the stuff and given the right biochemical directions researchers think they can coax clathrin into creating better batteries and solar cells http www popsci com science article - protein-cow-brains-could-build-better-batteries-solar-cells
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Reactome is pathway database which provides intuitive bioinformatics tools for the visualisation, interpretation and analysis of pathway knowledge.
Control of E-cadherin apical localisation and morphogenesis by a SOAP-1/AP-1/clathrin pathway in C. elegans epidermal cells. ...
Reactome is pathway database which provides intuitive bioinformatics tools for the visualisation, interpretation and analysis of pathway knowledge.
Thermodynamics of protein-mediated membrane deformation â application to clathrin dependent and clathrin independent endocytosis (2009 ...
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SSO mediated functional uptake in MHT cells is AP2M1 dependent and clathrin independent. (A) MHT cells were treated with 25 nM control, AP2M1, clathrin or RNA
Function: Phosphorylates the AP2M1/mu2 subunit of the adaptor protein complex 2 (AP-2). May play a role in regulating aspects of clathrin-mediated endocytosis (By similarity ...
Studies for the virusCcell connections have proven dear in elucidating vital cellular procedures. small and restricted fitting, as opposed to the uptake via covered vesicles, where in fact the getting into virus could be associated with detectable levels of liquid (Marsh and Helenius 1980). The endocytic uptake, via covered vesicle, relays on particular proteins which are turned on upon ATP or GTP binding or hydrolysis. Both recycling from the clathrin layer as well as the dynamin-directed pinching from the covered vesicle in the PM are ATP/GTP-dependent procedures (Gao et al. 1991; Hinshaw and Schmid 1995). The acidic milieu in endosomes is necessary for the translocation from the viral particle with the membrane of the endocytic vesicle (Marsh and Helenius 1989). Nevertheless, it has additionally been suggested (Carrasco 1994, Carrasco 1995) which the proton motive drive (p), instead of acidic milieu by itself, drives this technique. enveloped bacteriophage 6 is normally a unique trojan within ...
The vesicle simulation is available in two forms, one having enough for about one and a half vesicles (vesicle.obj) and the other having enough for about three (vesicle2.obj). Both can use either the vesicle_hub.tmpl or the vesicle_no_hub.tmpl template files (the first contains a sphere at the centre of each clathrin and is more polished visually, the second is faster). Note that in some runs of the simulation many fragments of vesicles form, rather than the few complete structures ...
Mills IG, Praefcke GJ, Vallis Y, Peter BJ, Olesen LE, Gallop JL, Butler PJ, Evans PR, McMahon HT. EpsinR: an AP1/clathrin interacting protein involved in vesicle trafficking. J Cell Biol. 2003 Jan 20;160(2):213-22. Epub 2003 Jan 21. PMID:12538641 doi:http://dx.doi.org/10.1083/jcb.200208023 ...
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Cell-surface viral proteins most frequently enter the cell through clathrin or caveolae endocytosis. Respiratory syncytial virus antigen internalization by immune cells is via caveolin, however, uptake of paramyxovirus cell membrane proteins by non-immune cells is done through clathrin-coated pits. In this work, the uptake of respiratory syncytial virus cell surface glycoproteins by non-immune human epithelial cells was investigated through indirect immunofluorescence with polyclonal anti-RSV antibody and confocal lasser-scanner microscopy. Clathrin and caveolae internalization pathways were monitored through specific inhibitors monodansylcadaverine (MDC) and methyl-beta-cyclodextrin (MBCD), respectively. Internalization of RSV antigens was inhibited by MDC but not by MBCD, implying that clathrin-mediated endocytosis is the major uptake route of RSV antigens by an epithelial human cell line.
TY - JOUR. T1 - A dynamic actin cytoskeleton functions at multiple stages of clathrin-mediated endocytosis. AU - Yarar, Defne. AU - Waterman-Storer, Clare M.. AU - Schmid, Sandra L.. N1 - Copyright: Copyright 2008 Elsevier B.V., All rights reserved.. PY - 2005/2. Y1 - 2005/2. N2 - Clathrin-mediated endocytosis in mammalian cells is critical for a variety of cellular processes including nutrient uptake and cell surface receptor down-regulation. Despite the findings that numerous endocytic accessory proteins directly or indirectly regulate actin dynamics and that actin assembly is spatially and temporally coordinated with endocytosis, direct functional evidence for a role of actin during clathrin-coated vesicle formation is lacking. Here, we take parallel biochemical and microscopic approaches to address the contribution of actin polymerization/depolymerization dynamics to clathrin-mediated endocytosis. When measured using live-cell fluorescence microscopy, disruption of the F-actin assembly and ...
In this report we have addressed the biological consequence of endofin-mediated TOM1 recruitment onto endosomes. Our results collectively suggest that TOM1 serves as an adaptor for endofin to recruit clathrin heavy chain onto the endosomes. This conclusion is supported by several lines of evidence as described in our study.. First, via large-scale pull-down experiments using immobilized GST-TOM1(300-492), we have recovered clathrin heavy chain as the major and specific partner for TOM1. This conclusion was corroborated by analytical pull-down experiments showing that clathrin heavy chain was very efficiently retained by immobilized GST-TOM1(300-492), so much so that it was depleted from the cytosol. The specific interaction between TOM1 and clathrin heavy chain was further defined by our identification of three sites in the carboxyl-terminal region of TOM1, which seem to act together for efficient interaction with clathrin. Moreover, the specific blockage of interaction between TOM1 and clathrin ...
Clathrin, a three-legged triskelion composed of three clathrin heavy chains (CHCs) and three light chains (CLCs), plays a critical role in clathrin-mediated endocytosis (CME) in eukaryotic cells. In this study, the genes ZmCHC1 and ZmCHC2 encoding clathrin heavy chain in maize were cloned and characterized for the first time in monocots. ZmCHC1 encodes a 1693-amino acid-protein including 29 exons and 28 introns, and ZmCHC2 encodes a 1746-amino acid-protein including 28 exons and 27 introns. The high similarities of gene structure, protein sequences and 3D models among ZmCHC1, and Arabidopsis AtCHC1 and AtCHC2 suggest their similar functions in CME. ZmCHC1 gene is predominantly expressed in maize roots instead of ubiquitous expression of ZmCHC2. Consistent with a typical predicted salicylic acid (SA)-responsive element and four predicted ABA-responsive elements (ABREs) in the promoter sequence of ZmCHC1, the expression of ZmCHC1 instead of ZmCHC2 in maize roots is significantly up-regulated by SA or ABA,
Assembly protein recruiting clathrin and adapter protein complex 2 (AP2) to cell membranes at sites of coated-pit formation and clathrin-vesicle assembly. May be required to determine the amount of membrane to be recycled, possibly by regulating the size of the clathrin cage. Involved in AP2-dependent clathrin-mediated endocytosis at the neuromuscular junction. Plays a crucial role in fetal and adult hematopoiesis, and normal prenatal and postnatal growth and viability.
Fingerprint Dive into the research topics of CAP-1A is a novel linker that binds clathrin and the voltage-gated sodium channel Na,sub,v,/sub,1.8. Together they form a unique fingerprint. ...
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Clathrin-coated vesicles are the most prominent carriers of membrane traffic from cell surface to endosomes (endocytosis), a pathway by which hormones, transferrin, immunoglobulins, LDL, viruses, and their receptors enter cells. They are also important for traffic between endosomes and the trans-Golgi network. In this presentation, I will discuss (i) technological and analytical advances that I developed to directly visualize clathrin-mediated membrane traffic in three dimensions and in living cells; (ii) data obtained using these advances that defined a role for actin filament polymerization in counteracting membrane tension during clathrin-coated vesicle budding at the apical surface of polarized epithelial cells; and (iii) how these advances can be used to study a wide variety of biological processes that occur in living cells and tissues. ...
During clathrin-mediated endocytosis, it has been thought that the sensing and binding of the clathrin adaptor protein AP2 to cargo and lipids leads to the recruitment of clathrin, nucleating the formation of a clathrin-coated pit. Henne et al. have now found that this process of AP2 binding may not in fact represent either the first or the nucleation event of endocytosis. Instead, ubiquitous proteins called FCHo1/2 (F-BAR proteins) bind to the plasma membrane and define the sites of endocytosis independently of AP2. The F-BAR protein can generate very low curvatures and, at higher concentrations, generates higher curvatures like those required at the neck of budding vesicles. The C terminus of the protein has a μ-homology domain (with homology to the μ domain of the AP2 complex) that interacts with Eps15 and intersectin and via these proteins recruits AP2, which further recruits clathrin. Thus, a curvature-inducing protein can act to nucleate clathrin-coated pit assembly during ...
Steps in CCV assembly and links to structures and information around clathrin-coated vesicle formation and other forms of vesicle budding
PICALM, the gene encoding phosphatidylinositol-binding clathrin assembly (picalm) protein, was recently shown to be associated with risk of Alzheimer disease (AD). Picalm is a key component of clathrin-mediated endocytosis. It recruits clathrin and adaptor protein 2 (AP-2) to the plasma membrane and, along with, AP-2 recognizes target proteins. The attached clathrin triskelions cause membrane deformation around the target proteins enclosing them within clathrin-coated vesicles to be processed in lysosomes or endosomes. We examined the distribution of picalm in control and AD brain tissue and measured levels of picalm messenger RNA (mRNA) by real-time polymerase chain reaction. Immunolabeling of brain tissue showed that picalm is predominately present in endothelial cells. This was further supported by the demonstration of picalm in human cerebral microvascular cells grown in culture. Picalm mRNA was elevated in relation to glyceraldehyde-3-phosphate dehydrogenase but not factor VIII-related ...
Toxoplasma gondii possesses a highly polarized secretory system, which efficiently assembles de novo micronemes and rhoptries during parasite replication. These apical secretory organelles release their contents into host cells promoting parasite invasion and survival. Using a CreLox-based inducible knock-out strategy and the ddFKBP over-expression system, we unraveled novel functions of the clathrin adaptor complex TgAP1. First, our data indicate that AP1 in T. gondii likely functions as a conserved heterotetrameric complex composed of the four subunits γ, β, μ1, σ1 and interacts with known regulators of clathrin-mediated vesicular budding such as the unique ENTH-domain containing protein, which we named Epsin-like protein (TgEpsL). Disruption of the μ1 subunit resulted in the mis-sorting of microneme proteins at the level of the Trans-Golgi-Network (TGN). Furthermore, we demonstrated that TgAP1 regulates rhoptry biogenesis by activating rhoptry protein exit from the TGN, but also ...
Clathrin-mediated endocytosis is a critical process through which a wide variety of extracellular material is internalized. The primary component, clathrin, forms a cargo-selective lattice at the plasma membrane, as well as on endosomes and the TGN, though the cargo-selective components are incompletely defined. An ideal tool for understanding the spatio-temporal dynamics of both the clathrin coat and the cargo selected is total internal reflection fluorescence microscopy (TIR-FM), which permits selective imaging of events closely apposed to the ventral plasma membrane. Previously, observation of the clathrin coat has shown both static and dynamic populations, with some dynamic structures undergoing microtubule-dependent motion; the 70-110 nm decay constant of the TIR-FM field has led to the assumption that these are all representative of coated pits. Here, I demonstrate that the dynamic population of clathrin is primarily endosomal, as it lacks colocalization with the plasma membrane-specific ...
Bicaudal-D (Bic-D), Egalitarian (Egl), microtubules and their motors form a transport machinery that localizes a remarkable diversity of mRNAs to specific cellular regions during oogenesis and embryogenesis. Bic-D family proteins also promote dynein-dependent transport of Golgi vesicles, lipid droplets, synaptic vesicles and nuclei. However, the transport of these different cargoes is still poorly understood. We searched for novel proteins that either mediate Bic-D-dependent transport processes or are transported by them. Clathrin heavy chain (Chc) co-immunopurifies with Bic-D in embryos and ovaries, and a fraction of Chc colocalizes with Bic-D. Both proteins control posterior patterning of the Drosophila oocyte and endocytosis. Although the role of Chc in endocytosis is well established, our results show that Bic-D is also needed for the elevated endocytic activity at the posterior of the oocyte. Apart from affecting endocytosis indirectly by its role in osk mRNA localization, Bic-D is also ...
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Endocytosis is an essential phenomenon in eukaryotic cells. In animal cells, dynamin and clathrin play central roles in vesicle formation in the process of endocytosis, but the roles of similar proteins in plants are less well understood. Here, we observed the localization pattern and behavior of GFP-labeled ,i,Arabidopsis,/i, dynamin-related proteins (DRP1A and DRP2B), and clathrin light chain (AtCLC) around the plasma membrane in tobacco suspension cells by using variable incidence angle fluorescence microscopy (VIAFM). GFP fusions of DRP1A, DRP2B and AtCLC were observed as dot-like puncta 200-500 nm in diameter. The puncta moved to and away from the cell surface or also assembled and disassembled. The localization pattern and behavior of the puncta were similar to those of animal dynamin and clathrin signals reported previously. These results raise the possibility that DRP1A, DRP2B and AtCLC are involved in membrane trafficking around the plasma membrane, including endocytosis.. ...
CHC22 clathrin plays a key role in intracellular membrane traffic of the insulin-responsive glucose transporter GLUT4 in humans. We performed population genetic and phylogenetic analyses of the CHC22-encoding CLTCL1 gene, revealing independent gene loss in at least two vertebrate lineages, after arising from gene duplication. All vertebrates retained the paralogous CLTC gene encoding CHC17 clathrin, which mediates endocytosis. For vertebrates retaining CLTCL1 , strong evidence for purifying selection supports CHC22 functionality. All human populations maintained two high frequency CLTCL1 allelic variants, encoding either methionine or valine at position 1316. Functional studies indicated that CHC22-V1316, which is more frequent in farming populations than in hunter-gatherers, has different cellular dynamics than M1316-CHC22 and is less effective at controlling GLUT4 membrane traffic, attenuating its insulin-regulated response. These analyses suggest that ancestral human dietary change influenced ...
The general objective of our lab is to understand the functions of clathrin-coated structures (CCSs) during the different steps of cancer development. CCSs recruit specific cell surface receptors and progressively shape the plasma membrane in receptor-containing vesicles that are released in the cytosol. This endocytosis machinery allows for nutrient uptake but also for the fine-tuned control of signaling pathways triggered by cell surface receptors. As a consequence, deregulation of endocytosis has been linked to many pathological situations, including cancers.. Tumor development is accompanied by dramatic changes in the mechanical characteristics of tissues. Also, when cancer cells invade the stroma to establish distant metastases, they migrate in an environment with different topological features than the tumor mass. However, it is not known how the physical parameters of the environment impact on CCSs and what are the consequences for the cell.. Our team addresses this general question by ...
TY - JOUR. T1 - An ATP-driven proton pump in clathrin-coated vesicles.. AU - Stone, D. K.. AU - Xie, X. S.. AU - Racker, E.. PY - 1983/4/10. Y1 - 1983/4/10. N2 - Clathrin containing coated vesicles prepared from bovine brain catalyzed ATP-driven proton translocation and a 32Pi-ATP exchange reaction. Both activities were measured in the presence of 5 micrograms of oligomycin/mg of protein which completely inhibited these reactions catalyzed by submitochondrial particles. Analyses performed during the purification procedure demonstrated that the oligomycin-resistant pump was concentrated and highly purified in the fractions containing coated vesicles. Moreover, vesicles precipitated by either monoclonal or polyclonal antibodies against clathrin contained the H+ pump activity. Dicyclohexylcarbodiimide (0.5 mM) and N-ethylmaleimide (1 mM) added to the assay mixture inhibited the pump completely, whereas neither vanadate, sodium azide, efrapeptin, or mitochondrial ATPase inhibitor had an ...
Immunogen = synthetic peptide: E E D P A A A F L A Q Q E S E I A G I E N D, corresp. to amino acids 23-44 of Cow Clathrin light chain. ...
Both Gn and Gc proteins are implicated in virion host-cell attachment (Kingsford et al., 1983, Ludwig et al., 1989, Hacker et al., 1995). However, the Gc protein appears to be the primary attachment protein for orthobunyaviruses (Plassmeyer et al., 2005b). Following attachment, the virion enters the cell through clathrin-mediated endocytosis and acidification leads to pH-induced insertion of the viral Gc fusion peptide into the endosomal membranes (Shi and Elliott 2007, Plassmeyer et al., 2005, Jacoby et al., 1993). This insertion facilitates the membrane-to-membrane fusion and subsequent release of ribonucleocapsids into the cytoplasm. Only encapsidated RNA segments serve as templates for both primary transcription and genomic replication (Reguera et al., 2013). Complementary 5′- and 3′-termini are promoters for both mRNA and antigenome synthesis (Kohl et al., 2004). The Gn and Gc proteins, and the NSm protein in the case of some viruses, are translated from a single ORF as a polyprotein ...
Zwiewka, M; Nodzynski, T; Robert, S; Vanneste, S; Friml, J, 2015: Osmotic Stress Modulates the Balance between Exocytosis and Clathrin-Mediated Endocytosis in Arabidopsis thaliana. MOLECULAR PLANT 8(8), p. 1175 - 1187, doi: 10.1016/j.molp.2015.03.007. Research Groups:. ...
Glyvuk et al (2010) argue that the CME of SV membranes represents a kinetic bottleneck of the recycling pathway. Under conditions of sustained activity BE provides a compensatory mechanism to balance high exocytic load with matching endocytic activity. Vacuolar membrane invaginations are then consumed by undefined budding events that chop these membranes into small vesicles that may re‐enter the SV cycle (Figure 1A). It is this consumption step that the authors envision to depend on AP‐1/σ1B (Figure 1B). The experimental evidence for this model at present remains indirect. AP‐1, as its relative AP‐2, is one of the major recruitment factors for clathrin and loss of either protein complex results in depletion of clathrin‐coated pits from TGN/endosomes or the plasmalemma, respectively. Why then do AP‐1/σ1B‐KO mice accumulate clathrin‐coated pits on endosome‐like vacuoles? One possibility is that other σ1 isoproteins such as σ1A do a poor job in functionally replacing σ1B on ...
The Slit diaphragm (SD) is the key filtration structure in human glomerular kidney that is affected in many types of renal diseases. SD proteins are known to undergo endocytosis and recycling to maintain the integrity of the filtration structure. However, the key components of this pathway remain unclear. Using the Drosophila nephrocyte as a genetic screen platform, we screened most genes involved in endocytosis and cell trafficking, and identified the key components of the cell trafficking pathway required for SD protein endocytosis and recycling. We discovered that the SD protein endocytosis and recycling pathway contains clathrin, dynamin, AP-2 complex, like-AP180 (Lap), auxilin and Hsc70-4 (the endocytosis part) followed by Rab11 and the exocyst complex (the recycling part). Disrupting any component in this pathway led to disrupted SD on the cell surface and intracellular accumulation of mislocalized SD proteins. We also showed the first in vivo evidence of trapped SD proteins in clathrin-coated
Researchers of the group of cellular and molecular neurobiology of the Bellvitge Biomedical Research Institute and the University of Barcelona, led by researcher Artur Llobet, have shown that synaptic levels of the protein clathrin are a determinant factor for synaptic plasticity of neurons.
Clathrin (see MIM 118955)-mediated endocytosis is a major mechanism for internalization of proteins and lipids. Members of the connecdenn family, such as DENND1A, function as guanine nucleotide exchange factors (GEFs) for the early endosomal small GTPase RAB35 (MIM 604199) and bind to clathrin and clathrin adaptor protein-2 (AP2; see MIM 601024). Thus, connecdenns link RAB35 activation with the clathrin machinery (Marat and McPherson, 2010 [PubMed 20154091]).[supplied by OMIM, Nov 2010 ...
Video articles in JoVE about clathrin coated vesicles include Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast, Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy, Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons, The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins, Pulling Membrane Nanotubes from Giant Unilamellar Vesicles, In vivo and in vitro Studies of Adaptor-clathrin Interaction, Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers, Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell, Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes, Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the
Within the clathrin-coated vesicle (CCV) cycle, coat assembly drives the internalization of receptors from the cell surface and disassembly allows for the processing of internalized ligands. The heat shock cognate protein, hsc70, has been implicated in regulating coat disassembly. We find that in cells overexpressing ATPase-deficient hsc70 mutants, uncoating of CCVs is inhibited in vivo, and the majority of unassembled cytosolic clathrin shifts to an assembled pool that cofractionates with AP1 and AP2. Surprisingly, this assembled pool of coat proteins accumulates in the absence of cargo receptors, suggesting that disruption of hsc70 activity may cause misassembly of empty clathrin cages. The strongest effect of overexpression of hsc70 mutants is a block in transferrin receptor (TfnR) recycling, which cannot be accounted for by the degree of inhibition of uncoating of endocytic CCVs. These results suggest that hsc70 participates in multiple transport and/or sorting events between endosomal ...
Component of the adaptor complexes which link clathrin to receptors in coated vesicles. Clathrin-associated protein complexes are believed to interact with the cytoplasmic tails of membrane proteins, leading to their selection and concentration. AP50 is a subunit of the plasma membrane adaptor. The complex binds polyphosphoinositide-containing lipids. [-] ...
Expression of α-Synuclein and clathrin in Z310 cells with or without a-Syn treatment in a typical experiment (n = 5). Z310 cells are immortalized rat choro
E. M. Wenzel, S. W. Schultz, K. O. Schink, N. M. Pedersen, V. Nähs, A. Carlson, A. Brech, H. Stenmark and C. Raiborg, Concerted ESCRT and clathrin recruitment waves define the timing and morphology of intraluminal vesicle formation, Nature Communications, 9, 2018.. W. Wang, J. V. I. Timonen, A. Carlson, D.-M. Drotlef, C. T. Zhang, S. Kolle, A. Grinthal, T.-S. Wong, B. Hatton, S. H. Kang, S. Kennedy, J. Chi, R. T. Blough, M. Sitti, L. Mahadevan and J. Aizenberg, Multifunctional ferrofluid-infused surfaces with reconfigurable multiscale topography, Nature, 559, 2018.. A. Carlson, Fluctuation assisted spreading of a fluid filled elastic blister, Journal of Fluid Mechanics, 846, 2018.. Y. Wang, G. Amberg and A. Carlson, Local dissipation limits the dynamics of impacting droplets on smooth and rough substrates, Physical Review Fluids, 2, 2017.. A. Carlson and L. Mahadevan, Singularity and self-similarity of elastohydrodynamic touchdown, Physics of Fluids (letter), 28, 2016.. P. Johansson, A. Carlson ...
Endocytosis is required for internalization of micronutrients and turnover of membrane components. Endophilin has been assigned as a component of clathrin-mediated endocytosis. Here we show in mammalian cells that endophilin marks and controls a fast-acting tubulovesicular endocytic pathway that is independent of AP2 and clathrin, activated upon ligand binding to cargo receptors, inhibited by inhibitors of dynamin, Rac, phosphatidylinositol-3-OH kinase, PAK1 and actin polymerization, and activated upon Cdc42 inhibition. This pathway is prominent at the leading edges of cells where phosphatidylinositol-3,4-bisphosphate-produced by the dephosphorylation of phosphatidylinositol-3,4,5-triphosphate by SHIP1 and SHIP2-recruits lamellipodin, which in turn engages endophilin. This pathway mediates the ligand-triggered uptake of several G-protein-coupled receptors such as α2a- and β1-adrenergic, dopaminergic D3 and D4 receptors and muscarinic acetylcholine receptor 4, the receptor tyrosine kinases ...
Ybe and Niu used X-ray crystallography to look at an area of interest on the surface of HIP1, which works in concert with clathrin to traffic nutrients into a cell, and has long been implicated as playing an important role in the development of Huntingtons disease. They learned that the potential binding surface of HIPPI in HIP1 has an unexpected shape for a binding site, a spiraling spiral called a coiled coil. This finding was contrary to predicted results that the binding surface that receives HIPPI is folded into a so-called death effector domain. Using the information from the published molecular structure of HIP1, IU biologists hope to be able to test which protein connections are ultimately responsible for triggering the chain of interactions leading to Huntingtons disease and how to block them. For example, they observed that clathrin, protein involved in bringing nutrients to the cell, binds with HIP1 right next door to where HIPPI binds. While clathrin packages nutrients for a ...
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Coated pit: …of the membrane called a coated pit, which is lined by a special protein known as clathrin. As the coated pit invaginates, it is pinched off in the cytoplasm to form a coated vesicle. The coated vesicle fuses with cytoplasmic endosomes (membrane-enclosed vesicles) and then with cell organelles called lysosomes,…
Abstract In metazoans, intracellular trafficking is mediated by many conserved pathways. The main trafficking pathways involve the Adaptor Protein complexes (AP) AP-1, AP-2 and AP-3, Clathrin and Clathrin Associated Sorting Proteins (CLASPS). These proteins direct cargoes to and from the Golgi, the endosome, the lysosome and the plasma membrane. Lipoprotein receptors are one class of proteins that use these pathways and are endocytosed by AP-2 and Disabled family proteins. Disabled family proteins are ~ conserved family of monomeric adaptor proteins and consist of a single PTB domain and have a variety of motifs associated with trafficking. C. elegans has one Disabled family member, DAB-1.1 show here that .DAB-1 is a generally expressed regulator of membrane trafficking. Loss of dab-1 function, as well as an array of molecules involved membrane trafficking, perturbs cadherin-catenin function, though I have not been able to determine the precise basis of these genetic interactions. I show that ...
Involved in cell growth regulation. May be involved in the regulation of mitogenic signals and control of cell proliferation. Involved in the internalization of ligand-inducible receptors of the receptor tyrosine kinase (RTK) type, in particular EGFR. Plays a role in the assembly of clathrin-coated pits (CCPs). Acts as a clathrin adapter required for post-Golgi trafficking. Seems to be involved in CCPs maturation including invagination or budding. Involved in endocytosis of integrin beta-1 (ITGB1) and transferrin receptor (TFR); internalization of ITGB1 as DAB2-dependent cargo but not TFR seems to require association with DAB2. ...
Ischebeck, T.; Werner , S.; Krishnamoorthy, P.; Lerche, J.; Meijón, M.; Stenzel, I.; Löfke, C.; Wiessner, T.; Im , Y. J.; Perera, I. Y. et al.; Iven , T.; Feussner, I.; Busch, W.; Boss, W. F.; Teichmann, T.; Hause, B.; Persson, S.; Heilmann, I.: Phosphatidylinositol 4,5-Bisphosphate Influences PIN Polarization by Controlling Clathrin-Mediated Membrane Trafficking in Arabidopsis. The Plant Cell (2013 ...
In light of the presented data, we suggest that LRRK2 is part of a functional protein network that controls SV trafficking within the recycling pool by interacting with a subset of presynaptic proteins. A role of LRRK2 in vesicle trafficking involving Rab5b had already been suggested (Shin et al., 2008), but we can now show for the first time that electrophysiological properties as well as vesicular trafficking in the presynaptic pool depend on the presence of LRRK2 as an integral part of presynaptic protein complex. We have in fact identified presynaptic proteins-NSF, AP-2 complex subunits, SV2A, synapsing, and syntaxin 1-as well as actin as putative LRRK2 interactors. These proteins have been described previously as key elements of synaptic vesicle trafficking (Takamori et al., 2006). NSF catalyzes the release of the SNARE complex (SNAP-25, syntaxin 1, and Vamp) and allows the first step of the endocytic cycle (Littleton et al., 1998, 2001). The clathrin complex [clathrin, AP-2 adaptor ...
Quasi-enveloped hepatitis A virions undergo clathrin-mediated endocytosis, followed by ALIX-dependent trafficking to lysosomes where the quasi-envelope is degraded, triggering uncoating of the RNA genome in association with lysosomal membrane rupture.
I found myself taking some photos of red winter coats recently. I figured Id make a little theme out of them. Theres something quite splendid about a red coat moving across the urban landscape on a bicycle. A slow, flirting rush of intense colour which, in the dull winter light, is remarkable to behold, day or night ...
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The interaction between cytoplasmic coat proteins and specific signals in the cytoplasmic domains of integral membrane proteins is a general mechanism controlling protein sorting. The best-characterized sorting signal is the tyrosine-based sorting motif that is involved in various sorting events by interaction with adaptor proteins (Trowbridge et al., 1993; Mellman, 1996; Marks et al., 1997). The YRSLE sequence present in the cytoplasmic domain of the neuronal form of L1 conforms to the tyrosine-based sorting motif. We have found that the AP-2 adaptor specifically recognizes and interacts with the YRSLE sequence, resulting in clathrin-mediated endocytosis of L1 (H. Kamiguchi, K. E. Long, M. Pendergast, A. W. Schaefer, I. Rapoport, T. Kirchhausen, and V. Lemmon, unpublished observations). These observations indicate that the YRSLE sequence of L1 actually functions as a tyrosine-based sorting signal. The L1 cytoplasmic domain contains three other tyrosine residues (Hlavin and Lemmon, 1991), ...
The Berro lab is interested in how forces are produced and sensed at the molecular scale in cells. Our research is currently focused on unraveling how the molecular machinery of clathrin-mediated endocytosis generates forces to deform the plasma membrane and conversely how this machinery senses membrane tension and adapts to it. To answer these questions, the lab develops new experimental and computational quantitative methods for cell biology, biochemistry and biophysics.. ...