TY - JOUR. T1 - The SNARE proteins SNAP25 and synaptobrevin are involved in endocytosis at hippocampal synapses. AU - Zhang, Zhen. AU - Wang, Dongsheng. AU - Sun, Tao. AU - Xu, Jianhua. AU - Chiang, Hsueh Cheng. AU - Shin, Wonchul. AU - Wu, Ling Gang. PY - 2013/5/22. Y1 - 2013/5/22. N2 - SNAP25, an essential component of the soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor (SNARE) complex that mediates exocytosis, is not considered to play a role in endocytosis, which couples to exocytosis by retrieving a similar amount of exocytosed vesicles. By knocking down SNAP25 and imaging slow endocytosis at a conventional synapse, the rat cultured hippocampal synapse, we found that SNAP25 is involved in slow, clathrin-dependent endocytosis. With similar techniques, we found that not only SNAP25, but also synaptobrevin is involved in slow endocytosis. These results provide the first evidence showing the dual role of SNAP25 and synaptobrevin in both exocytosis and slow ...

UNC-51 overexpression can inhibit the transferrin endocytosis in the transfected COS-7 cells. We think that this inhibition of transferrin endocytosis should be caused by UNC-51-dependent...

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 ...

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 ...

The potential for transcytosis (endocytosis → intracellular transport → exocytosis) of protein and membrane events associated with fluid phase and adsorptive endocytic processes within epithelia of the choroid plexus [blood-cerebrospinal fluid (CSF) barrier] were investigated in mice injected intravenously or into the lateral cerebral ventricle with native horseradish peroxidase (HRP) or the lectin wheatgerm agglutinin (WGA) conjugated to HRP. WGA binds to specific cell surface oligosaccharides and enters cells by the process of adsorptive endocytosis; native HRP is taken into cells non-specifically by fluid phase endocytosis. The lysosomal system of organelles and the endoplasmic reticulum, identified by enzyme cytochemical markers applied to choroid epithelia, were analysed for possible participation in transcytosis and compared to epithelial organelles harbouring the exogenous tracer proteins. Blood-borne native HRP was endocytosed readily by choroid epithelia whereas WGA-HRP was not, perhaps

article{f63aac35-6350-4bf7-866a-e224429f2246, abstract = {BACKGROUND: Galectin-3 (the Mac-2 antigen) is abundantly expressed in both macrophage like cells and certain non-macrophage cells. We have studied endocytosis of galectin-3 as one important step relevant for its function, and compared it between variants of a macrophage like cell line, and non-macrophage cells. ,br/,,br, ,br/,,br, METHODS: Endocytosis of galectin-3 was observed by fluorescence microscopy and measured by flow cytometry. The endocytosis mechanism was analysed using galectin-3 mutants, galectin-3 inhibitors and endocytic pathways inhibitors in the human leukaemia THP-1 cell line differentiated into naïve (M0), classical (M1) or alternatively activated (M2) macrophage like cells, and the non-macrophage cell lines HFL-1 fibroblasts and SKBR3 breast carcinoma. ,br/,,br, ,br/,,br, RESULTS: Galectin-3 endocytosis in non-macrophage cells and M2 cells was blocked by lactose and a potent galectin-3 inhibitor TD139, and also by the ...

The animation shows calcium-stimulated exocytosis of synaptic vesicles followed by clathrin-mediated vesicle recycling. Many of the molecular components that are involved in synaptic vesicle priming, docking, fusion, and endocytosis are shown. Although the process in the animation describes synaptic vesicle cycling, similar cellular processes occur for most calcium-coupled secretory and clathrin-mediated endocytotic events. The animation could be used to help illustrate the sequence of events associated with both exocytosis and endocytosis, as well as aid in understanding the processes involved in neurotransmitter release in response to nerve stimulation.. [Resource Details] ...

The animation shows calcium-stimulated exocytosis of synaptic vesicles followed by clathrin-mediated vesicle recycling. Many of the molecular components that are involved in synaptic vesicle priming, docking, fusion, and endocytosis are shown. Although the process in the animation describes synaptic vesicle cycling, similar cellular processes occur for most calcium-coupled secretory and clathrin-mediated endocytotic events. The animation could be used to help illustrate the sequence of events as. Published by Learning Registry #GoOpen. 4 Views, 0 Likes on Docs.com. #synapse #neuron #NSDL #NSDL_SetSpec_BEN #movie #signal transduction #endocytosis #Life Science

The central finding of the present study is that Mt3 plays a key role in the clathrin-dependent endocytosis of Aβ in astrocytes. In Mt3 −/− astrocytes, clathrin-mediated endocytosis, the mechanism responsible for Aβ endocytosis, was markedly decreased, whereas caveolin-mediated endocytosis was not altered. Astrocytes are likely key players in the clearance of extracellular Aβ; thus, our results suggest that changes in the Mt3 expression in astrocytes may have clinical relevance in AD. Taken together with our previous findings that Mt3 helps to maintain lysosomal degradation in astrocytes, the reduction in Mt3 in astrocytes may aggravate Aβ accumulation in the extracellular space.. Early studies showed that AD brain extracts induce more neurite outgrowth in cell cultures than do control brain extracts [27], suggesting upregulation of a growth-inducing factor or downregulation of a growth-inhibitory factor (GIF) in AD brains. The latter was shown to be the case, and a subsequent study ...

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 ...

In light of the fact that the main myelin protein PLP is enriched in LE/Lys compartments and undergoes regulated endocytosis and exocytosis (Kramer et al., 2001; Trajkovic et al., 2006), we asked whether endocytic recycling is common to myelin proteins. We first analyzed the steady state localization of myelin proteins in endosomal compartments by co-labeling primary cultured oligodendrocytes with transferrin-FITC or antibodies against LAMP1, which serve as markers for recycling endosomes or LE/Lys, respectively. Although PLP strongly colocalized with LAMP1 in LE/Lys (Fig. 1, top row, magenta in overlay pictures), we observed only a little overlap of MAG and MOG with LAMP1 or transferrin-FITC in endocytic compartments (Fig. 1, middle and bottom row, magenta and yellow color in overlays, respectively). Thus, among the studied myelin proteins, only PLP accumulates in endosomal compartments. However, a transient endosomal localization of MAG and MOG may be missed or underestimated with this type of ...

We have demonstrated a requirement for cellular ATP in the receptor-mediated endocytosis of transferrin. This has been accomplished using a novel assay for endocytosis based on acquisition of resistance to the membrane impermeable reducing agent, glutathione (GSH). Diferric-transferrin was conjugated to biotin via a cleavable disulfide bond and iodinated. Internalization of 125I-biotin-S-S-transferrin (125I-BSST) was quantitated by adsorption to avidin-Sepharose after treatment of cells with GSH. Receptor-mediated endocytosis of 125I-BSST was severely inhibited in ATP-depleted cells. Similar results were obtained when ATP was depleted by incubation of cells either under a N2-atmosphere or in the presence of NaN3 and NaF. The latter treatment, alone, also resulted in a loss of surface transferrin receptors which could not be correlated to reductions in cellular ATP. In contrast to the acquisition of GSH resistance, the apparent internalization of 125I-BSST as assessed by inaccessibility to ...

Looking for endocytic vacuole? Find out information about endocytic vacuole. A membrane-bound cellular organelle containing extracellular particles engulfed by the mechanisms of endocytosis Explanation of endocytic vacuole

PathHunter® Receptor Tyrosine Kinase (RTK) Internalization Assays are nonimaging, non-antibody based chemiluminescent detection assays that provide a direct and quantitative measurement of internalized RTKs localized to early endosomes. Your PathHunter Receptor Tyrosine Kinase Internalization Cell Line, when used in conjunction with a PathHunter Detection Kit (DiscoveRx, Cat. #93-0001 Series), provides a cell-based functional assay for receptor tyrosine kinase internalization. ...

Efficient non-viral gene delivery often involves the conjugation of a cell-specific ligand to the vector, which directs the vector to its intended target through binding to a cellular receptor followed by internalization via endocytosis. However, little is known in terms of how the various endocytosis pathways affect the performance of the delivery vehicle. Previously, the Pack lab has demonstrated that caveolin-mediated endocytosis is important to in vitro polyethylenimine (PEI)- and polyamidoamine (PAMAM)-mediated gene delivery in HeLa cells through the use of small molecule drugs and small-interfering RNA [1-2]. The goal of this thesis is to further elucidate the effects of cellular uptake mechanism on non-viral gene delivery in vitro and in vivo utilizing a small hairpin RNA (shRNA) Tet-on system. Specifically, we have investigated the effects of clathrin-dependent endocytosis and caveolin-dependent endocytosis on the efficacy of PEI and PEI-derivative gene delivery in HeLa and MDA-MB-231 ...

Adapter protein that functions as clathrin-associated sorting protein (CLASP) required for clathrin-mediated endocytosis of selected cargo proteins. Can bind and assemble clathrin, and binds simultaneously to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and cargos containing non-phosphorylated NPXY internalization motifs, such as the LDL receptor, to recruit them to clathrin-coated pits. Can function in clathrin-mediated endocytosis independently of the AP-2 complex. Involved in endocytosis of integrin beta-1; this function seems to redundant with the AP-2 complex and seems to require DAB2 binding to endocytosis accessory EH domain-containing proteins such as EPS15, EPS15L1 and ITSN1. Involved in endocytosis of cystic fibrosis transmembrane conductance regulator/CFTR. Involved in endocytosis of megalin/LRP2 lipoprotein receptor during embryonal development. Required for recycling of the TGF-beta receptor. Involved in CFTR trafficking to the late endosome. Involved in several receptor-mediated

Membrane vesicle traffic to and from the plasma membrane is essential for cellular homeostasis in all eukaryotes. In plants, constitutive traffic to and from the plasma membrane has been implicated in maintaining the population of integral plasma-membrane proteins and its adjustment to a variety of hormonal and environmental stimuli. However, direct evidence for evoked and selective traffic has been lacking. Here, we report that the hormone abscisic acid (ABA), which controls ion transport and transpiration in plants under water stress, triggers the selective endocytosis of the KAT1 K+ channel protein in epidermal and guard cells. Endocytosis of the K+ channel from the plasma membrane initiates in concert with changes in K+ channel activities evoked by ABA and leads to sequestration of the K+ channel within an endosomal membrane pool that recycles back to the plasma membrane over a period of hours. Selective K+ channel endocytosis, sequestration, and recycling demonstrates a tight and dynamic ...

Recently, several theoretical and computational studies have also been performed to investigate the membrane wrapping efficiency of elastic NPs during endocytosis, which has been shown regulated synergistically by NP size, shape, and elasticity (22, 23, 33). However, despite the fact that the effects of NP elasticity on different endocytic uptake mechanisms have been experimentally explored in this work, the theoretical modeling of these uptake mechanisms remains challenging. More thorough and realistic modelings with explicit consideration of the actin network and adaptor proteins underneath the cell membrane are called for. Clathrin- and caveolin-independent endocytosis can be modeled using a membrane wrapping based on the Helfrich-Canham membrane theory (34), suggesting that stiff NPs are energetically more prone to full wrapping than soft ones (21). This elasticity effect on the simple membrane wrapping of an NP gradually becomes less significant as the NP elasticity increases. For isotropic ...

Thank you for sharing this Journal of Virology article.. NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.. ...

Endocytotic uptake of HPMA-based polymers by different cancer cells: impact of extracellular acidosis and hypoxia Daniel Gündel,1 Mareli Allmeroth,2 Sarah Reime,1 Rudolf Zentel,2 Oliver Thews1 1Institute of Physiology, Martin Luther University Halle-Wittenberg, Halle (Saale), 2Institute of Organic Chemistry, Johannes Gutenberg-University, Mainz, Germany Background: Polymeric nanoparticles allow to selectively transport chemotherapeutic drugs to the tumor tissue. These nanocarriers have to be taken up into the cells to release the drug. In addition, tumors often show pathological metabolic characteristics (hypoxia and acidosis) which might affect the polymer endocytosis.Materials and methods: Six different N-(2-hydroxypropyl)methacrylamide (HPMA)-based polymer structures (homopolymer as well as random and block copolymers with lauryl methacrylate containing hydrophobic side chains) varying in molecular weight and size were analyzed in two different tumor models. The cellular uptake of fluorescence

The ClC-5 chloride channel resides mainly in vesicles of the endocytotic pathway and contributes to their acidification. Its disruption in mice entails a broad defect in renal endocytosis and causes secondary changes in calciotropic hormone levels. Inactivating mutations in Dents disease lead to proteinuria and kidney stones. Possibly by recycling, a small fraction of ClC-5 also reaches the plasma membrane. Here we identify a carboxyl-terminal internalization motif in ClC-5. It resembles the PY motif, which is crucial for the endocytosis and degradation of epithelial Na+ channels. Mutating this motif increases surface expression and currents about 2-fold. This is probably because of interactions with WW domains, because dominant negative mutants of the ubiquitin-protein ligase WWP2 increased surface expression and currents of ClC-5 only when its PY motif was intact. Stimulating endocytosis by expressing rab5 or its GTPase-deficient Q79L mutant decreased WT ClC-5 currents but did not affect ...

All corticosteroid hormones share cholesterol as a common precursor. Therefore, the first step in steroidogenesis is cholesterol uptake or synthesis. Cells that produce steroid hormones can acquire cholesterol through two paths. The main source is through dietary cholesterol transported via the blood as cholesterol esters within low density lipoproteins (LDL). LDL enters the cells through receptor-mediated endocytosis. The other source of cholesterol is synthesis in the cells endoplasmic reticulum. Synthesis can compensate when LDL levels are abnormally low.[4] In the lysosome, cholesterol esters are converted to free cholesterol, which is then used for steroidogenesis or stored in the cell.[29]. The initial part of conversion of cholesterol into steroid hormones involves a number of enzymes of the cytochrome P450 family that are located in the inner membrane of mitochondria. Transport of cholesterol from the outer to the inner membrane is facilitated by steroidogenic acute regulatory protein ...

Nano-meter sized particles of de-aluminated zeolite Y has a high adsorption capacity of both low molecular weight bio-molecules and macromolecules. In this study we used de-aluminated zeolite Y as a novel approach to study the mechanisms of endocytosis in immature human peripheral blood dendritic cells (DCs). Probes detecting pH neutral and acidic endosomes were adsorbed to the zeolite and used as a tracer of the endosomal pathway of a cell in the form of acidification and lysosomal function. Both the myeloid (M-DCs) and the plasmacytoid (P-DCs) dendritic cell subsets showed an endocytosing capacity comparable to peripheral blood monocytes but only the M-DCs were able to form acidic endosomes after internalization of zeolite particles. Furthermore, during lipopolysaccharide (LPS) stimulation of the DCs population, an enhanced induction of acidic endosomes was only seen in the M-DC population. Proteolytic degradation of endocytosed proteins was detected using self-quenched DQ-ovalbumin adsorbed ...

Given the importance of functional SV recycling and the role of synaptotagmin 1 in coupling exo- and endocytosis in neuro secretory cells, we first set out to identify the domains required for its internalization. Expression of stonin 2 in human embryonic kidney (HEK) 293 cells stably transfected with FLAG-synaptotagmin 1 leads to redistribution of synaptotagmin 1 from the plasma membrane to internal compartments (Diril et al., 2006). Internalization assays based on anti-FLAG antibody uptake showed that endocytosed synaptotagmin localizes to a subset of AP-2-coated puncta and to a perinuclear endosomal compartment (Fig. 1 A; Diril et al., 2006), suggesting that stonin 2 targets synaptotagmin 1 to clathrin/AP-2-coated pits. To confine the region within synaptotagmin 1 required for stonin 2-mediated internalization deletion, constructs lacking one or both of the C2 domains were generated and analyzed by antibody uptake experiments. After antibody chase for 20 min at 37°C, surface-stranded ...

GPI-anchored surface proteins mediate many important functions, including transport, signal transduction, adhesion, and protection against complement. They cluster into glycolipid-based membrane domains and caveolae, plasmalemmal vesicles involved in the transcytosis and endocytosis of these surface proteins. However, in lymphocytes, neither the characteristic flask shaped caveolae nor caveolin, a transmembrane protein typical of caveolae, have been observed. Here, we show that the GPI-anchored CD59 molecule on Jurkat T cells is internalized after cross-linking, a process inhibited by nystatin, a sterol chelating agent. Clustered CD59 molecules mostly accumulate in non-coated invaginations of the lymphocyte membrane before endocytosis, in marked contrast with the pattern of CD3-TCR internalization. Cytochalasin H blocked CD59 internalization in lymphocytes, but neither CD3 internalization nor transferrin uptake. Confocal microscopy analysis of F-actin distribution within lymphocytes showed that ...

Quantitative fluorescence spectroscopy and flow cytometry analyses of cell-penetrating peptides internalization pathways: optimization, pitfalls, comparison with mass spectrometry quantification, Scientific Reports 6, 36938 (2016). Since their discovery twenty years ago, cell-penetrating peptides (CPPs) or protein transduction domains (PTDs) have been described as promising drug delivery systems. There are increasing numbers of successful applications of CPPs/PTDs in vivo. However, one of the limitations to their wide and diverse application is the diversity of their uptake pathways. The CPP and its cargo may end up free in the cytosol and reach their biological target only following translocation, but remain confined in intracellular vesicles after endocytosis, unless subsequent events such as endosomal rupture occur. Along with the development of CPPs/PTDs as vectors to carry various macromolecules for targeted cellular therapies, engineering new CPPs/PTDs with optimized transport and ...

Degradation and removal of ECM proteins is a cell-mediated process, which is involved in a number of physiological processes, such as development, postnatal tissue remodeling and tissue repair (Clark, 1996; Holmbeck et al., 1999; Vu et al., 1998). Impaired or abnormal degradation and removal of ECM components contributes to the onset and progression of many diseases, including fibrosis, arthritis and cancer (Hotary et al., 2003; Kurban et al., 2006; Liotta and Kohn, 2001; Mutsaers et al., 1997; Poole et al., 2003). Two principal molecular mechanisms are believed to be involved in the turnover of ECM. One is extracellular degradation of ECM proteins by matrix metalloproteases (MMPs), plasmin and other proteases (Marchina and Barlati, 1996; Shapiro, 1998). The other pathway occurs via internalization of ECM proteins and intracellular degradation in lysosomes (Godyna et al., 1995; Memmo and McKeown-Longo, 1998; Murphy-Ullrich and Mosher, 1987; Wienke et al., 2003). These two pathways are not ...

Endocytosis by African trypanosomes. I. Three dimensional structure of the endocytic organelles in Trypanosoma brucei and Trypanosoma ...

Clathrin-mediated endocytosis regulates the signalling activity and turnover of multiple plasma membrane proteins. Interfering with endocytosis can therefore have complex effects on developmental processes. William Sessa and colleagues (p. 1465) investigate the role of the Dynamin 2 (Dnm2) GTPase - a key component of the endocytic machinery - during angiogenesis in mice. Using endothelial cells in culture, they find that downregulation of Dnm2 impairs angiogenesis, even though vascular endothelial growth factor (VEGF) signalling is enhanced - due to increased surface levels of the receptor. This is in contrast to previous work suggesting that endocytosis might promote VEGF signalling. The authors ascribe the Dnm2 knockdown-induced defect in vessel formation at least partially to disrupted integrin turnover: focal adhesion size is increased and inactive integrin receptors appear to accumulate on the cell surface. Importantly, these observations hold true in vivo: conditional deletion of Dnm2 in ...

TY - JOUR. T1 - Ligand-bound quantum dot probes for studying the molecular scale dynamics of receptor endocytic trafficking in live cells. AU - Sundara Rajan, Sujata. AU - Liu, Hong Yan. AU - Vu, Tania Q.. PY - 2008/6/1. Y1 - 2008/6/1. N2 - Endocytic receptor trafficking is a complex, dynamic process underlying fundamental cell function. An integrated understanding of endocytosis at the level of single or small numbers of ligand bound-receptor complexes inside live cells is currently hampered by technical limitations. Here, we develop and test ligand nerve growth factor-bound quantum dot (NGF-QD) bioconjugates for imaging discrete receptor endocytic events inside live NGF-responsive PC12 cells. Using single particle tracking, QD hybrid gel coimmunoprecipitation, and immunocolocalization, we illustrate and validate the use of QD-receptor complexes for imaging receptor trafficking at synchronized time points after QD-ligand - receptor binding and internalization (t = 15-150 min). The unique value ...

On Some Aspects of the Thermodynamic of Membrane Recycling Mediated by Fluid Phase Endocytosis: Evaluation of Published Data and ...

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

Ligand-induced endocytosis of receptor tyrosine kinases (RTKs) is a dynamic process governed by numerous protein-protein and protein-lipid interactions. This is a major mechanism of signal termination and is also frequently impaired in cancer. The Cbl family of ubiquitin ligases has been shown to play a key role in downregulation of RTKs, by directing their ligand-induced ubiquitination and subsequent lysosomal degradation. My thesis work has led to the identification of novel, ubiquitin-ligase independent, functions of Cbl in receptor endocytosis. We demonstrated that the adaptor protein CIN85 links Cbl with epidermal growth factor receptor (EGFR) internalization. The three SH3 domains of CIN85 interact with Cbl/Cbl-b in a phosphotyrosine dependent manner, whereas its proline-rich region constitutively binds endophilins, known regulators of plasma membrane invagination. The SH3 domains of CIN85 recognize an atypical proline-arginine (PxxxPR) motif present in Cbl and Cbl-b. Moreover, we showed ...

The adaptor protein beta-arrestin2 enhances endocytosis of the low density lipoprotein receptor.: Endocytosis of the low density lipoprotein (LDL) receptor (LDL

Regulation of synaptic transmission is a widespread means for dynamic alterations in nervous system function. In several cases, this regulation targets vesicular recycling in presynaptic terminals and may result in substantial changes in efficiency of synaptic transmission. Traditionally, experimental accessibility of the synaptic vesicle cycle in central neuronal synapses has been largely limited to the exocytotic side, which can be monitored with electrophysiological responses to neurotransmitter release. Recently, physiological measurements on the endocytotic portion of the cycle have been made possible by the introduction of styryl dyes such as FM1-43 as fluorescent markers for recycling synaptic vesicles. Here we demonstrate the existence of fast endocytosis in hippocampal nerve terminals and derive its kinetics from fluorescence measurements using dyes with varying rates of membrane departitioning. The rapid mode of vesicular retrieval was greatly speeded by exposure to staurosporine or ...

Probable lipid-binding protein with higher affinity for phosphatidic acid, a lipid enriched in recycling endosome membranes. On endosome membranes, may act as a downstream effector of Rab proteins recruiting cytosolic proteins to regulate membrane tubulation. May be involved in a late step of receptor-mediated endocytosis regulating for instance endocytosed-EGF receptor trafficking. Alternatively, may regulate slow endocytic recycling of endocytosed proteins back to the plasma membrane. May indirectly play a role in neurite outgrowth.

Drug-dependent neural plasticity related to drug addiction and schizophrenia can be modeled in animals as behavioral sensitization, which is induced by repeated noncontingent or self-administration of many drugs of abuse. Molecular mechanisms that are critical for behavioral sensitization have yet to be specified. Long-term depression (LTD) of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptor (AMPAR)-mediated synaptic transmission in the brain has been proposed as a cellular substrate for learning and memory. The expression of LTD in the nucleus accumbens (NAc) required clathrin-dependent endocytosis of postsynaptic AMPARs. NAc LTD was blocked by a dynamin-derived peptide that inhibited clathrin-mediated endocytosis or by a GluR2-derived peptide that blocked regulated AMPAR endocytosis. Systemic or intra-NAc infusion of the membrane-permeable GluR2 peptide prevented the expression of amphetamine-induced behavioral sensitization in the rat. ...

Image:exo_endo_cytosis.jpg,300px,thumb,right,Exocytosis and Endocytosis cartoon]] == Cell Import - Endocytosis == [[Image:Endocytosis types.png,thumb,300px,Endocytosis types]] This lecture introduces how substances that are imported (endocytosis) and processed by the cell. Notes in Preparation (notice removed when complete) There are a number of processes by which the cell can absorb substances. The two main forms are by endocytosis or phagocytosis. Absorbed substances include: substances required for cell growth, cell signaling toxic chemicals and drugs, bacteria and viruses. In addition, this is the main method for membrane removal and recycling. We will study this topic at the level of the cellular components and organelles involved in the process: endosomes, lysosomes, peroxisomes, transport vesicles, Golgi apparatus and endoplasmic reticulum. :Movie [[Media:JCB200109030.v2.mov,Vesicles Display Bidirectional Motility along Microtubules]] Visualization of Rab9-mediated vesicle transport ...

Clathrin-mediated endocytosis involves cargo selection and membrane budding into vesicles with the aid of a protein coat. Formation of invaginated pits on the plasma membrane and subsequent budding of vesicles is an energetically demanding process that involves the cooperation of clathrin with many different proteins. Here we investigate the role of the brain-enriched protein epsin 1 in this process. Epsin is targeted to areas of endocytosis by binding the membrane lipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)). We show here that epsin 1 directly modifies membrane curvature on binding to PtdIns(4,5)P(2) in conjunction with clathrin polymerization. We have discovered that formation of an amphipathic alpha-helix in epsin is coupled to PtdIns(4,5)P(2) binding. Mutation of residues on the hydrophobic region of this helix abolishes the ability to curve membranes. We propose that this helix is inserted into one leaflet of the lipid bilayer, inducing curvature. On lipid monolayers epsin alone is

Parkinsons disease (PD) is linked to impaired degradation and accumulation of the misfolded protein, alpha-synuclein. Therefore, accelerating the degradation of alpha-synuclein is of therapeutic interest. Our lab has tested the hypothesis that α-synuclein uses endocytosis as a route to the lysosome for its degradation. We have found evidence that several endocytosis genes regulate α-synucleins PD related properties. Most importantly, the absence of vps28 increases α-synuclein aggregation and cellular accumulation, and confers toxicity in a yeast PD model. I, specifically, found that increasing the concentration of α-synuclein in yeast that lack vps28 further enhances each of these pathological characteristics.

Here, we describe an approach to directly visualize the activity of proteases that are incorporated into the phagosome at different stages of maturation. Only small numbers of phagocytes are required and there is no need to isolate individual endosomal compartments before analysis. This method is sensitive enough to allow an examination of primary cultures of professional APC, including DCs. Furthermore, the use of covalent active site-directed probes in conjunction with electrophoresis ensures specificity. Methods that employ fluorogenic substrates to detect protease activity suffer from the drawback that more than one enzyme can usually cleave a given peptide substrate. Analysis of the delivery of active hydrolases to the phagosome helped clarify both the distribution of cysteine protease activities among the different endocytic organelles and the dynamics of phagosomal maturation in primary cultures of professional APCs.. We validated our method of in vivo labeling of phagosomal proteolytic ...

PubMed comprises more than 30 million citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.

Failure of trabecular myocytes to undergo appropriate cell cycle withdrawal leads to ventricular noncompaction and heart failure. Signaling of growth factor receptor ERBB2 is critical for myocyte proliferation and trabeculation. However, the mechanisms underlying appropriate downregulation of trabecular ERBB2 signaling are little understood. Here, we have found that the endocytic adaptor proteins NUMB and NUMBL were required for downregulation of ERBB2 signaling in maturing trabeculae. Loss of NUMB and NUMBL resulted in a partial block of late endosome formation, resulting in sustained ERBB2 signaling and STAT5 activation. Unexpectedly, activated STAT5 overrode Hippo-mediated inhibition and drove YAP1 to the nucleus. Consequent aberrant cardiomyocyte proliferation resulted in ventricular noncompaction that was markedly rescued by heterozygous loss of function of either ERBB2 or YAP1. Further investigations revealed that NUMB and NUMBL interacted with small GTPase Rab7 to transition ERBB2 from ...

One unique aspect of VEGFR2 endocytosis is its regulation by several transmembrane and cytosolic interacting proteins, which play a role in either VEGFR2 internalization or degradation. A group of such proteins involved in VEGFR2 internalization is Dab2, ephrin-B2, and PAR-3. Dab2,46 a clathrin-associated sorting protein, and the cell polarity regulator PAR-3 interact with the transmembrane protein ephrin-B2 and VEGFR2. Disruption of this interaction by silencing of Dab2 or PAR-3 causes reduced VEGFR2 internalization and impaired VEGF-induced angiogenesis. After RTKs are internalized into early endosomes, a proportion of the receptors is modified by ubiquitin and then sorted for lysosomal degradation. CCM347 and myoferlin,48 respectively, associate with VEGFR2 in ECs and serve to enhance VEGFR2 stability by preventing receptor degradation.. Apart from internalization and degradation, VEGFR2 signaling is regulated by protein tyrosine phosphatases (PTPs), such as VE-PTP and PTP1b. VE-PTP is a ...

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 ...

The early events in the infection of normal B lymphocytes and B lymphoblastoid cells by Epstein-Barr virus (EBV) were examined by electron and immunoelectron microscopy and by infectivity and inhibition studies. Purified EBV remained on the cell surface at 4 degrees and appeared as 250-nm ovoid particles in contact with the cell membrane through 50-nm envelope projections. Internalization of EBV in normal B lymphocytes into large (300-500 nm) uncoated vacuoles was initiated within 2 to 5 min at 37 degrees. At this stage approximately 1/3 of cell-associated virus was located in cellular invaginations while another 1/3 was in cell vacuoles. Direct fusion of EBV with the outer cell membrane was not observed. Instead, viral deenvelopment and nucleocapsid transit into the cytoplasm occurred from the large endocytic vesicles within 15 to 30 min at 37 degrees and did not involve lysosomal enzymes. During this time, the viral envelope became amorphous and its separation from the nucleocapsid was ...

Dynamin 1 is a neuron-specific guanosine triphosphatase thought to be critically required for the fission reaction of synaptic vesicle endocytosis. Unexpectedly, mice lacking dynamin 1 were able to form functional synapses, even though their postnatal viability was limited. However, during spontaneous network activity, branched, tubular plasma membrane invaginations accumulated, capped by clathrin-coated pits, in synapses of dynamin 1-knockout mice. Synaptic vesicle endocytosis was severely impaired during strong exogenous stimulation but resumed efficiently when the stimulus was terminated. Thus, dynamin 1-independent mechanisms can support limited synaptic vesicle endocytosis, but dynamin 1 is needed during high levels of neuronal activity.

Gangliosides are sialic acid-containing glycosphingolipids mainly expressed at the outer leaflet of the plasma membrane. Sialidase NEU3 is a key enzyme in the catabolism of gangliosides with its up-regulation having been observed in human cancer cells. In the case of CME (clathrin-mediated endocytosis), although this has been widely studied, the role of NEU3 and gangliosides in this cellular process has not yet been established. In the present study, we found an increased internalization of Tf (transferrin), the archetypical cargo for CME, in cells expressing complex gangliosides with high levels of sialylation. The ectopic expression of NEU3 led to a drastic decrease in Tf endocytosis, suggesting the participation of gangliosides in this process. However, the reduction in Tf endocytosis caused by NEU3 was still observed in glycosphingolipid-depleted cells, indicating that NEU3 could operate in a way that is independent of its action on gangliosides. Additionally, internalization of ...

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 ...

Human glomerulonephritis (GN) is characterized by sustained proteinuria, sodium retention, hypertension, and edema formation. Increasing quantities of filtered protein enter the renal tubule, where they may alter epithelial transport functions. Exaggerated endocytosis and consequent protein overload may affect proximal tubules, but intrinsic malfunction of distal epithelia has also been reported. A straightforward assignment to a particular tubule segment causing salt retention in GN is still controversial. We hypothesized that 1) trafficking and surface expression of major transporters and channels involved in volume regulation were altered in GN, and 2) proximal tubular endocytosis may influence locally as well as downstream expressed tubular transporters and channels. Effects of anti-glomerular basement membrane GN were studied in controls and megalin-deficient mice with blunted proximal endocytosis. Mice displayed salt retention and elevated systolic blood pressure when proteinuria had ...

Differentiation of oligodendrocytes is associated with dramatic changes in plasma membrane structure, culminating in the formation of myelin membrane sheaths. Previous results have provided evidence that regulation of endocytosis may represent a mechanism to control myelin membrane growth. Immature oligodendrocytes have a high rate of clathrin-independent endocytosis for the transport of membrane to late endosomes/lysosomes (LE/Ls). After maturation and receiving signals from neurons, endocytosis is reduced and transport of membrane from LE/Ls to the plasma membrane is triggered. Here, we show that changes in Rho GTPase activity are responsible for switching between these two modes of membrane transport. Strikingly, Rho inactivation did not only reduce the transport of cargo to LE/L but also increased the dynamics of LE/L vesicles. Furthermore, we provide evidence that Rho inactivation results in the condensation of the plasma membrane in a polarized manner. In summary, our data reveal a novel ...

TY - JOUR. T1 - Asialoglycoprotein receptor phosphorylation and receptor-mediated endocytosis in hepatoma cells. Effect of phorbol esters. AU - Fallon, R. J.. AU - Schwartz, A. L.. PY - 1988/1/1. Y1 - 1988/1/1. N2 - The asialoglycoprotein (ASGP) receptor on Hep G2 cells undergoes constitutive recycling and ligand endocytosis in the presence of phorbol dibutyrate, at a 50% reduced rate relative to control cells (Fallon, R. J., and Schwartz, A. L. (1986) J. Biol. Chem. 261, 15081-15089). The relevance of receptor phosphorylation to these events was investigated by selective immunoprecipitation of surface receptors with polyclonal anti-human ASGP antiserum and pulse-chase labeling with [32P]orthophosphate to identify subcellular locations of initial receptor phosphorylation events as well as the eventual fate of phosphorylated receptor during recycling. The surface immunoprecipitation method recovers greater than 95% of surface ASGP receptors and only 5% or less of intracellular ...

TY - JOUR. T1 - Vps9p CUE domain ubiquitin binding is required for efficient endocytic protein traffic. AU - Davies, Brian A.. AU - Topp, Justin D.. AU - Sfeir, Agnel J.. AU - Katzmann, David J.. AU - Carney, Darren S.. AU - Tall, Gregory G.. AU - Friedberg, Andrew S.. AU - Deng, Li. AU - Chen, Zhijian. AU - Horazdovsky, Bruce F.. PY - 2003/5/30. Y1 - 2003/5/30. N2 - Rab5 GTPases are key regulators of protein trafficking through the early stages of the endocytic pathway. The yeast Rab5 ortholog Vps21p is activated by its guanine nucleotide exchange factor Vps9p. Here we show that Vps9p binds ubiquitin and that the CUE domain is necessary and sufficient for this interaction. Vps9p ubiquitin binding is required for efficient endocytosis of Ste3p but not for the delivery of the biosynthetic cargo carboxypeptidase Y to the vacuole. In addition, Vps9p is itself monoubiquitylated. Ubiquitylation is dependent on a functional CUE domain and Rsp5p, an E3 ligase that participates in cell surface receptor ...

The endocytosis of horseradish peroxidase (HRP) by the vascular cells of retinal and choroidal blood vessels was compared in immersion and perfusion fixed eyes from individual rats. The mechanisms of endocytosis of HRP appeared identical in both retinal and choroidal vessels. The bulk of internalised tracer occurred in macropinosomes 300-400 nm in diameter. Tracer was localised to a 20-30 nm layer on the internal aspect of the limiting membrane. This layer was coincident with the glycocalyx of the luminal plasma membrane as revealed by ruthenium redosmium tetroxide staining. Horseradish peroxidase was also internalised by a small scattered population of vesicles (100-130 nm in diameter). The size of these vesicles suggested that they may have arisen from clathrin coated regions of the plasma membrane. It is suggested that the endocytosis of HRP in retinal and choroidal vascular endothelium occurs as a function of plasma membrane recycling. Horseradish peroxidase may also be internalised as a ...

Oocyte maturation in oviparous animals is characterized by a vitellogenic period during which all maternally derived yolk protein precursors are sequestered in the oocyte, usually through receptor-mediated endocytosis (Bu and Schwartz, 1994; Sappington and Raikhel, 1995, 1998; Raikhel and Snigirevskaya, 1998; Burmester and Scheller, 1999). Large amounts of maternal lipids are also sequestered (Speake and Thompson, 1999), and lipid accumulation in eggs has been reported in many insects (for reviews, see Engelmann, 1970; Kunkel and Nordin, 1985). Important egg lipids are phospholipids for membrane formation during embryogenesis, and triacylglycerol is used mainly as an energy source. Surprisingly though, there are few quantitative studies on apolar lipids in insect eggs, even though both embryos and neonates in most insects require extensive waterproofing. Nelson and Sukkestad (1970) found complex mixtures of branched HCs, including mono-, di- and trimethyl HCs in eggs of the moth Manduca sexta ...

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 ...

Human cytomegalovirus (HCMV) is a widespread pathogen and a member of the Herpesviridae family. HCMV has a large genome that encodes many genes that are non-essential for virus replication but instead play roles in manipulation of the host immune environment. One of these is the US27 gene, which encodes a protein with homology to the chemokine receptor family of G protein-coupled receptors (GPCRs). The US27 protein has no known chemokine ligands but can modulate the signaling activity of host receptor CXCR4. We investigated the mechanism for enhanced CXCR4 signaling in the presence of US27 using a novel biosensor system comprised of fluorogen activating proteins (FAPs). FAP-tagged CXCR4 and US27 were used to explore receptor internalization and recovery dynamics, and the results demonstrate that significantly more CXCR4 internalization was observed in the presence of US27 compared to CXCR4 alone upon stimulation with CXCL12. While ligand-induced endocytosis rates were higher, steady state ...

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 ...

Endocytic vacuoles can be up to 10 μm in diameter and appear specifically in pathological conditions as a result of aberrant retrieval of structures formed by compound endocytosis [7]. The co-localization hypothesis of trypsinogen activation suggests intracellular/intra-organellar activation of trypsinogen and the formation of active (and potentially damaging) trypsin as a result of fusion of trypsinogen containing organelles with organelles containing lysosomal proteases (reviewed in [31]). In our previous study, we have indeed detected trypsin activity in the endocytic vacuoles and observed that some of the vacuoles have lysosomal markers [7]. In the present study, we specifically addressed the question about the role of SOCE in the formation of these vacuoles. The results of our experiments revealed that the SOCE-mediated Ca2+ plateaus produced by CCK or TLC-S (two commonly used inducers of experimental pancreatitis) are effectively suppressed by the Orai1 inhibitor GSK-7975A and that this ...

This study suggests that, unlike the mechanisms of β2 adrenoceptor regulation (Zhang et al., 1997b; Oakley et al., 1999; Gainetdinov et al., 2004), MOR resensitization in LC neurons does not require βarr-2-dependent mechanisms. When MOR endocytosis was presumably impaired by deletion of βarr-2, disruption of GRK2 function, or disruption of dynamin function, MOR resensitization was similarly accelerated rather than inhibited. Furthermore, intracellular application of a phosphatase inhibitor in the absence of βarrestin-dependent receptor trafficking (GRK2 inhibition in βarr-2 k.o.) prevented full recovery of MOR function, suggesting that MOR dephosphorylation is required for MOR resensitization and that this can occur independently of βarr-2-dependent MOR trafficking and endocytosis. The ability of MOR to dephosphorylate and resensitize rapidly when GRK2 and βarr-2 processes are disrupted is consistent with the observation that blocking endocytosis with concanavalin A in cultured LC neurons ...

A defining characteristic of HIV-1 infection is the ability of the virus to persist within the host. Specifically, MHC-I downregulation by the HIV-1 accessory protein Nef is of critical importance in preventing infected cells from cytotoxic T-cell mediated killing. Nef downregulates MHC-I by modulating the host membrane trafficking machinery, resulting in the endocytosis and eventual sequestration of MHC-I within the cell. In the current report, we utilized the intracellular protein-protein interaction reporter system, bimolecular fluorescence complementation (BiFC), in combination with super-resolution microscopy, to track the Nef/MHC-I interaction and determine its subcellular localization in cells. We demonstrate that this interaction occurs upon Nef binding the MHC-I cytoplasmic tail early during endocytosis in a Rab5-positive endosome. Disruption of early endosome regulation inhibited Nef-dependent MHC-I downregulation, demonstrating that Nef hijacks the early endosome to sequester MHC-I within the

Synaptic vesicles fuse with the plasma membrane for neurotransmission. Subsequently, these vesicles are retrieved, and new vesicles are reconstituted locally at synapses. However, how exocytosis and endocytosis are controlled spatially and temporally remains enigmatic. We developed novel techniques in electron microscopy to capture membrane and protein dynamics with millisecond temporal resolution. In the seminar, I will discuss our recent progress on when, where, and how vesicles are consumed and recycled at synaptic terminals ...

Hyphal tip growth in fungi is important because of the economic and medical importance of fungi, and because it may be a useful model for polarized growth in other organisms. We have investigated the central questions of the roles of cytoskeletal elements and of the precise sites of exocytosis and endocytosis at the growing hyphal tip by using the model fungus Aspergillus nidulans. Time-lapse imaging of fluorescent fusion proteins reveals a remarkably dynamic, but highly structured, tip growth apparatus. Live imaging of SYNA, a synaptobrevin homologue, and SECC, an exocyst component, reveals that vesicles accumulate in the Spitzenkörper (apical body) and fuse with the plasma membrane at the extreme apex of the hypha. SYNA is recycled from the plasma membrane by endocytosis at a collar of endocytic patches, 1-2 μm behind the apex of the hypha, that moves forward as the tip grows. Exocytosis and endocytosis are thus spatially coupled. Inhibitor studies, in combination with observations of ...

Pruning, referred to as selective removal of unnecessary neurites without cell death, occurs in both vertebrates and invertebrates. The Drosophila dorsal class IV dendritic arborization neuron (ddaC) can serve as an excellent model to study the mechanisms of dendrite pruning. To identify novel molecules orchestrating this developmental degeneration process, I performed an RNAi screen, from which a previously uncharacterized gene named pruning defect 1(prd1) was isolated. It binds to Adaptor Protein (AP)-2 complex and regulates dendrite pruning in a cell-autonomous manner. Consistently, AP-2 complex dependent endocytic degradation pathway is also important for dendrite pruning. Interestingly, Prd1 also complexes with a Kinesin-3 family member Immaculate connections (Imac), which plays a critical role in regulating dendrite pruning as well. With the help of Prd1, Imac transports AP-2 enriched Clathrin Coated Vesicles (CCVs) or endocytic vesicles from plasma membrane to early endosomes along ...

Endophilins have been proposed to have an enzymatic activity (a lysophosphatidic acid acyl transferase or LPAAT activity) that can make phosphatidic acid in membranes1,2,3. This activity is thought to change the bilayer asymmetry in such a way that negative membrane curvature at the neck of a budding vesicle will be stabilized. An LPAAT activity has also been proposed for CtBP/BARS (carboxy-terminal binding protein/brefeldin A-ribosylated substrate), a transcription co-repressor that is implicated in dynamin-independent endocytosis and fission of the Golgi in mitosis4,5,6. Here we show that the LPAAT activity associated with endophilin is a contaminant of the purification procedure and can be also found associated with the pleckstrin homology domain of dynamin. Likewise, the LPAAT activity associated with CtBP/BARS is also a co-purification artefact. The proposed locus of activity in endophilins includes the BAR domain, which has no catalytic site but instead senses positive membrane curvature. These

Steps in CCV assembly and links to structures and information around clathrin-coated vesicle formation and other forms of vesicle budding

Non-injectable delivery of peptides and proteins is not feasible due to the limitations of large molecular mass, high hydrophilic properties, and gastrointestinal degradation. Therefore, proposing a new method to solve this problem is a burning issue. The objective of this study was to propose a novel protein delivery strategy to overcome the poor efficacy and irritation of buccal insulin delivery. In this study, we applied a conjugate of cell-penetrating peptides (LMWP) and insulin (INS-PEG-LMWP) for buccal delivery. INS-PEG-LMWP was prepared using insulin solution and mixture as references. The transport behaviour, in vivo bioactivity, hypoglycaemic effect, and safety of INS-PEG-LMWP were systematically characterised. An in vitro study demonstrated that the uptake and transportation of INS-PEG-LMWP across buccal mucosal multilayers significantly increased. By comparing the effects of different endocytic inhibitors on INS-PEG-LMWP uptake, the conjugate might be delivered via an energy ...

Catalytic domain of Yeast Protein Kinase 1-like Protein Serine/Threonine Kinases. Serine/Threonine Kinases (STKs), Yeast protein kinase 1 (YPK1)-like subfamily, catalytic (c) domain. STKs catalyze the transfer of the gamma-phosphoryl group from ATP to serine/threonine residues on protein substrates. The YPK1-like subfamily is part of a larger superfamily that includes the catalytic domains of other protein STKs, protein tyrosine kinases, RIO kinases, aminoglycoside phosphotransferase, choline kinase, and phosphoinositide 3-kinase. This subfamily is composed of fungal proteins with similarity to the AGC STKs, Saccharomyces cerevisiae YPK1 and Schizosaccharomyces pombe Gad8p. YPK1 is required for cell growth and acts as a downstream kinase in the sphingolipid-mediated signaling pathway of yeast. It also plays a role in efficient endocytosis and in the maintenance of cell wall integrity. Gad8p is a downstream target of Tor1p, the fission yeast homolog of mTOR. It plays a role in cell growth and ...

Overactivity of platelet-derived growth factor receptor (PDGFR) is a frequent event in many types of solid tumors. Therefore, it is of great importance to uncover the mechanisms that regulate PDGF/PDGFR signalling, to develop efficient inhibitors targeting this pathway. The first step of downregulation of PDGFR activity upon ligand binding is internalization; thus we investigated how endocytosis pathways affect PDGFR signaling. We showed that in Ras-transformed fibroblasts, the internalization of PDGFR is shifted from the routine clathrin-dependent endocytosis to macropinocytosis, which results in enhanced PDGFR activity and subsequent downstream signalling, promoting anchorage-independent growth.. We were also interested in how intracellular trafficking regulates signalling attenuation of PDGFR. We found that His-domain containing protein tyrosine phosphatase (HD-PTP) positively regulates phosphorylation level of the ubiquitin-ligases c-Cbl and Cbl-b; consistently, silencing of HD-PTP led to a ...

Regardless of the type of endocytosis, vesicles are fused with an internal membranous compartment known as endosome. Intramecellular compartments formed after phagocytosis and macropinocytosis beccome particular types of endosomes known as phagosome and macropinosome, respectively. Endosomes show an irregular shape, like large bags, although sometimes they form tubular structures. Like the TGN of the Golgi complex , endosomes are stations for receiving and distributing molecules packed in vesicles. Vesicles arrive to endosomes from the plasma membrane and from the TGN of the Golgi complex. From endosomes, vesicles leave toward the plasma membrane or TGN of the Golgi complex, both are pathways for recycling membrane receptors and lipids. However, most of the molecules in the endosomes are transported to lysosomes for degradation. Two ways of endosomal organization have been proposed: a) Cells would contain several types of endosomes. Early endosomes close to the plasma membrane that receive ...

In cell biology, an endosome is a membrane-bounded compartment inside eukaryotic cells. It is a compartment of the endocytic membrane transport pathway originating from the trans Golgi membrane. Molecules or ligands internalized from the plasma membrane can follow this pathway all the way to lysosomes for degradation, or they can be recycled back to the plasma membrane. Molecules are also transported to endosomes from the trans-Golgi network and either continue to lysosomes or recycle back to the Golgi. Endosomes can be classified as early, sorting, or late depending on their stage post internalization. Endosomes represent a major sorting compartment of the endomembrane system in cells. In HeLa cells, endosomes are approximately 500 nm in diameter when fully mature. Endosomes provide an environment for material to be sorted before it reaches the degradative lysosome. For example, LDL is taken into the cell by binding to the LDL receptor at the cell surface. Upon reaching early endosomes, the LDL ...

Ekanayake G, LaMontagne ED, Heese A. (2019). Never Walk Alone: Clathrin-Coated Vesicle (CCV) Components in Plant Immunity. Annu Rev Phytopathol. 57:387-409. doi: 10.1146/annurev-phyto-080417-045841. [PubMed]. LaMontagne ED, Heese A. (2017). Trans-Golgi network/early endosome: a central sorting station for cargo proteins in plant immunity. Curr Opin Plant Biol. 40:114-121. doi: 10.1016/j.pbi.2017.08.012. Review. [PubMed]. Leslie ME, Heese A. (2017). Quantitative Analysis of Ligand-Induced Endocytosis of FLAGELLIN-SENSING 2 Using Automated Image Segmentation. Methods Mol Biol. 1578:39-54. doi: 10.1007/978-1-4939-6859-6_4. [PubMed]. Collins CA, Leslie ME, Peck SC, Heese A. (2017). Simplified Enrichment of Plasma Membrane Proteins from Arabidopsis thaliana Seedlings Using Differential Centrifugation and Brij-58 Treatment. Methods Mol Biol. 1564:155-168. doi: 10.1007/978-1-4939-6813-8_13. [PubMed]. Leslie ME, Rogers SW, Heese A. (2016). Increased callose deposition in plants lacking DYNAMIN-RELATED ...

Studies with recombinant receptors in cell lines and cultured neurons have defined rules for the trafficking of kainate receptors to the plasma membrane. The relative level of their surface expression depends on subunits and alternative splicing of their C-terminal domain, and on subunit composition of heteromeric receptors. Some subunit splice variants are endowed with a forward trafficking motif, whereas others are retained in the ER (endoplasmic reticulum) due to retention signals.. KA2 was initially thought to require the presence of other subunits to form receptor complexes, but KA2 can in fact form homomeric assemblies in heterologous cells, although it is retained in the ER in the absence of GluR5, GluR6 or GluR7 [3-5]. This subunit possesses an ER retention motif in its C-terminus (RRRRR), and a di-leucine endocytic motif that may mediate its rapid, clathrin-dependent endocytosis and low steady-state plasma membrane expression. The ER retention/retrieval signal in KA2 is sterically ...

Exosomes, small-membrane vesicles, are secreted by cells and include several types of proteins and nucleic acids. Exosomes transfer cellular information derived from donor cells and are involved in various physiological and pathological events, such as organ-specific metastasis. Elucidating the exosome uptake mechanisms is important for understanding the progression processes of organ-specific metastasis. However, whether the exosomes secreted by the donor cells are selectively or non-selectively incorporated into the recipient cells is unknown. In this study, three human carcinoma cell lines, A549 (lung), HCT116 and COLO205 (colon), were used. The exosome isolation efficiency was compared between three methods: ultracentrifugation, ExoQuick-TC and Total Exosome Isolation kits. Recipient cells were treated with Pitstop 2, an inhibitor of clathrin-dependent endocytosis, or genistein, an inhibitor of caveolae-dependent endocytosis, and then incubated with DiO-labeled exosomes. Among the three methods

The new edition of Hepatology is considerably revised and updated. The content has been expanded through the rewriting of most of the chapters and by the addition of new contributors and several new chapters that are based on achievements in basic and clinical hepatology research done after publication of the first edition. Now in two volumes, the second edition is indeed more comprehensive, authoritative, and useful to readers than the previous one.. The first volume covers liver physiology, biochemistry, abnormal hepatic functions and manifestations, and then their evaluation. A new chapter, Receptor-Mediated Endocytosis Mechanisms: Biologic Function and Molecular Properties, is ...

S. Majumder*, J. Garamella*, Y.-L. Wang, M. DeNies, V. Noireaux, A.P. Liu, Cell-sized mechanosensitive and biosensing compartment programmed by DNA, ChemComm, 52, 7349-7352, 2017. [article link] * equal contribution; A.P. Liu and V. Noireaux are co-corresponding authors.. Y. Zheng*, S. Wang*, X. Xue, A. Xu, W. Liao, A. Deng, G. Dai, A.P. Liu, J. Fu, Dll4-Notch signaling in regulating angiogenesis in a 3D biomimetic environment, Lab on a Chip, 17. 1948-1959, 2017. [article link] * equal contribution; A.P. Liu and J. Fu are co-corresponding authors.. A.P. Liu*, O. Chaudhuri*, S. Parekh*, New advances in probing cell-extracellular matrix interactions, Integrative Biology, 9, 383-405, 2017. [article link] * equal contribution and corresponding author (top 10 downloaded article published in Integrative Biology in 2017). K.K.Y. Ho, J.W. Lee, G. Durand, S. Majumder, A.P. Liu, Protein aggregation with poly(vinyl) alcohol surfactant reduces double emulsion-encpasulated mammalian cell-free expression, ...

The mechanisms by which altered processing, distribution and secretion of proteolytic enzymes occur, facilitating degradation of the extracellular matrix in invasive and metastatic cells, are not fully understood. Studies on the MCF-10 A breast epithelial cell line and its premalignant, c-Ha-ras-transfected MCF-10AneoT counterpart have shown that the ras-transfected cell line has a more alkaline pH. The objective of this study was to determine which organelles of the endosome-lysosome route were alkalinized and shifted to the cell periphery after ras-transfection. Antibodies to the hapten 2,4-dinitrophenyl (DNP), required for pH studies, were raised in rabbits and chickens using DNP-ovalbumin (DNP-OVA) as immunogen. Cationised DNP-OVA (DNP-catOVA) was also inoculated to increase antibody titres. Anti-hapten and carrier antibody titres were assessed. In rabbits, cationisation seems useful to increase anti-DNP titres if a non-self carrier protein (OVA) is used. In chickens, cationisation of ...

Video explaining Endocytosis & Exocytosis for Biochemistry. This is one of many videos provided by Clutch Prep to prepare you to succeed in your college

PubMed comprises more than 30 million citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.

In a new study, a team of scientists based at The Picower Institute for Learning and Memory at MIT and the Whitehead Institute for Biomedical Research reveals evidence showing that the most prominent Alzheimers disease risk gene may disrupt a fundamental process in a key type of brain cell. Moreover, in a sign of how important it is to delve into the complex ways that genes intersect in disease, they found that increasing the expression of another Alzheimers-associated gene in those cells could help alleviate the problem.. About 25 percent of people have the APOE4 variant of the APOE gene, which puts them at substantially greater risk for Alzheimers disease than those with the more common APOE3 version. Scientists have been working for decades to understand why this is so. The new study in Cell Reports finds that in astrocytes, which are the most common non-neuron cell in the brain, the variant hampers the process of endocytosis, which is a major way that cells bring materials in from ...

TY - JOUR. T1 - Subtype-specific regulation of receptor internalization and recycling by the carboxyl-terminal domains of the human A1 and rat A3 adenosine receptors. T2 - consequences for agonist-stimulated translocation of arrestin3. AU - Ferguson, Gail. AU - Watterson, Kenneth R. AU - Palmer, Timothy M. PY - 2002/12/17. Y1 - 2002/12/17. N2 - In this study, we have characterized the differential effects on inhibitory adenosine receptor (AR) trafficking of disrupting predicted sites for palmitoylation and phosphorylation within each receptors carboxyl terminus. While a Cys(302,305)Ala-mutated rat A(3)AR mutant internalizes significantly faster than the wild-type (WT) receptor in response to agonist exposure, analogous mutation of the human A(1)AR (Cys(309)Ala) had no effect on receptor internalization. Moreover, unlike the WT A(3)AR, the entire pool of internalized mutant A(3)AR is able to recycle back to the plasma membrane following agonist removal. These properties do not reflect ...

Hotsing, some sorting nexins appear to be involved in endosomal signalling (image taken from Cullen: Nat. Regardless of the type of website mnaager own, we can help you with high quality hosting. As a result of VPS hostings hybrid nature, its cost isnt quite as low as shared hosts fees, but its not nearly as high as whm access key free hosting manager hosts fees. ThoughВ other websites may be hosted on the same physical system, yours would be the only website(s) hosted in the virtual compartment allocated - with independent server resources (CPU, RAM, disk space, etc) to you. Gengyo-Ando K, Kuroyanagi H, Kobayashi T, Murate M, Fujimoto K, Okabe Mail hosting solutions Mitani S (2007) The SM protein VPS-45 is required for RAB-5-dependent endocytic transport in Caenorhabditis elegans. LunarVPS is a pretty well-diversified European VPS specialist. Even the best proxy service will only secure traffic via an internet browser using the proxy server settings. Our highly trained staff is ready to ...

In addition to initiating signaling events, the activation of cell surface receptors also triggers regulatory processes that restrict the duration of signaling. Acute attenuation of signaling can be accomplished either via ligand-induced internalization of receptors (endocytic downregulation) or via ligand-induced receptor desensitization. These phenomena have traditionally been viewed in the context of adaptation wherein the receptor system enters a refractory state in the presence of sustained ligand stimuli and thereby prevents the cell from over-responding to the ligand. Here we use the epidermal growth factor receptor (EGFR) and G-protein coupled receptors (GPCR) as model systems to respectively examine the effects of downregulation and desensitization on the ability of signaling receptors to decode time-varying ligand stimuli. Using a mathematical model, we show that downregulation and desensitization mechanisms can lead to tight and efficient input-output coupling thereby ensuring synchronous

Peptides , Cell Permeable Peptides (CPP) , Drug Delivery Peptides , TAT (47-57); This is the most characterized fragment of the HIV transactivator protein (TAT). This arginine-rich TAT peptide penetrates plasma membrane directly, but not through endocytosis.; YGRKKRRQRRR; H-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-OH

Growth cone-driven axonal extension and guidance are essential for the formation of neural circuits, and thus for correct neural functioning. In addition to signaling pathways that lead to the stabilization of cytoskeletal components, mechanisms controlling membrane dynamics and extension through exocytosis and endocytosis are essential for axonal growth. Thus, it has been proposed that while axonal chemoattraction requires exocytosis, growth cone collapse and chemorepulsion rely mainly on endocytotic events (Tojima et al., 2007, 2010, 2011; Cotrufo et al., 2011, 2012; Zylbersztejn et al., 2012). Membrane markers (e.g., FM1-43 and Dextran) have allowed end point determination of cone exocytosis (Pfenninger et al., 2003; Cotrufo et al., 2011). Furthermore, recent studies using pHluorins have reported eventual single vesicle fusion events in growth cones (Tojima et al., 2007, 2010; Burgo et al., 2012; Nakazawa et al., 2012). However, how and where these exocytotic events occur in these structures ...

Ubiquitin has two functional surfaces which are critical for various signaling processes. The hydrophobic patch (Leu8, Ile44 and Val70) is important for binding to the proteasome, UIM (ubiquitin interacting motif) and UBA (ubiquitin associated) domains. This multifunctional surface thus influences many ubiquitination and deubiquitination reactions. The other surface defined by Phe4 is required specifically for non-proteasome-dependent functions such as endocytosis and internalization, which often involves mono-ubiquitination. It is thought that Phe4 may be involved in specific protein-protein interactions that facilitate endocytosis. In addition, Ile44 forms a di-leucine signal with Leu43 that may be involved in mediating endocytosis of substrate proteins that are mono-ubiquitinated. Ubiquitin F4A can form an E1-catalyzed active thioester at the C-terminus allowing the molecule to be transferred to the lysines of substrate proteins.. ...