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Sample records for activity-dependent bulk endocytosis

  1. The Molecular Physiology of Activity-Dependent Bulk Endocytosis of Synaptic Vesicles

    Clayton, Emma L.; Cousin, Michael A.

    2010-01-01

    Central nerve terminals release neurotransmitter in response to a wide variety of stimuli. Since maintenance of neurotransmitter release is dependent on the continual supply of synaptic vesicles (SVs), nerve terminals possess an array of endocytosis modes to retrieve and recycle SV membrane and proteins. During mild stimulation conditions single SV retrieval modes such as clathrin-mediated endocytosis (CME) predominate. However during increased neuronal activity additional SV retrieval capacity is required, which is provided by activity-dependent bulk endocytosis (ADBE). ADBE is the dominant SV retrieval mechanism during elevated neuronal activity. It is a high capacity SV retrieval mode that is immediately triggered during such stimulation conditions. This review will summarise the current knowledge regarding the molecular mechanism of ADBE, including molecules required for its triggering and subsequent steps, including SV budding from bulk endosomes. The molecular relationship between ADBE and the SV reserve pool will also be discussed. It is becoming clear that an understanding of the molecular physiology of ADBE will be of critical importance in attempts to modulate both normal and abnormal synaptic function during intense neuronal activity. PMID:19765184

  2. The molecular physiology of activity-dependent bulk endocytosis of synaptic vesicles.

    Clayton, E. L.; Cousin, M. A.

    2009-01-01

    Central nerve terminals release neurotransmitter in response to a wide variety of stimuli. Because maintenance of neurotransmitter release is dependent on the continual supply of synaptic vesicles (SVs), nerve terminals possess an array of endocytosis modes to retrieve and recycle SV membrane and proteins. During mild stimulation conditions, single SV retrieval modes such as clathrin-mediated endocytosis predominate. However, during increased neuronal activity, additional SV retrieval capacit...

  3. The Molecular Physiology of Activity-Dependent Bulk Endocytosis of Synaptic Vesicles

    Clayton, Emma L.; Cousin, Michael A

    2009-01-01

    Central nerve terminals release neurotransmitter in response to a wide variety of stimuli. Since maintenance of neurotransmitter release is dependent on the continual supply of synaptic vesicles (SVs), nerve terminals possess an array of endocytosis modes to retrieve and recycle SV membrane and proteins. During mild stimulation conditions single SV retrieval modes such as clathrin-mediated endocytosis (CME) predominate. However during increased neuronal activity additional SV retrieval capaci...

  4. Activity-dependent acceleration of endocytosis at a central synapse.

    Wu, Wei; Xu, Jianhua; Wu, Xin-Sheng; Wu, Ling-Gang

    2005-12-14

    Accumulated evidence indicates the existence of rapid and slow endocytosis at many synapses. It has been proposed that rapid endocytosis is activated by intense stimulation when vesicle recycling needs to be speeded up to supply vesicles at hippocampal synapses. However, the evidence, as obtained with imaging techniques, which are somewhat indirect in indicating rapid endocytosis, is controversial. Furthermore, a slower time course of endocytosis is often found after more intense nerve activity, casting doubt on the role of rapid endocytosis at synapses. Here, we addressed this issue at a mammalian central synapse, the calyx of Held, using a capacitance measurement technique that provides a higher time resolution than imaging techniques. We found that rapid endocytosis with a time constant of approximately 1-2 s was activated during intense nerve activity. Reducing the presynaptic calcium current or buffering the intracellular calcium with EGTA significantly inhibited rapid endocytosis, suggesting that calcium triggers rapid endocytosis. During intense stimulation, rapid endocytosis retrieved up to approximately eight vesicles per second per active zone, approximately eightfold larger than reported in the hippocampus, and thus played a dominant role during and within 3 s after intense stimulation. Slow endocytosis became dominant 3 s after intense stimulation likely because of the fall of the intracellular calcium level that deactivated rapid endocytosis. These results underscore the importance of calcium-triggered rapid endocytosis, which offers the nerve terminal the plasticity to speed up vesicle cycling during intense nerve activity. PMID:16354926

  5. CPG2 Recruits Endophilin B2 to the Cytoskeleton for Activity-Dependent Endocytosis of Synaptic Glutamate Receptors.

    Loebrich, Sven; Benoit, Marc Robert; Konopka, Jaclyn Aleksandra; Cottrell, Jeffrey Richard; Gibson, Joanne; Nedivi, Elly

    2016-02-01

    Internalization of glutamate receptors at the postsynaptic membrane via clathrin-mediated endocytosis (CME) is a key mechanism for regulating synaptic strength. A role for the F-actin cytoskeleton in CME is well established, and recently, PKA-dependent association of candidate plasticity gene 2 (CPG2) with the spine-cytoskeleton has been shown to mediate synaptic glutamate receptor internalization. Yet, how the endocytic machinery is physically coupled to the actin cytoskeleton to facilitate glutamate receptor internalization has not been demonstrated. Moreover, there has been no distinction of endocytic-machinery components that are specific to activity-dependent versus constitutive glutamate receptor internalization. Here, we show that CPG2, through a direct physical interaction, recruits endophilin B2 (EndoB2) to F-actin, thus anchoring the endocytic machinery to the spine cytoskeleton and facilitating glutamate receptor internalization. Regulation of CPG2 binding to the actin cytoskeleton by protein kinase A directly impacts recruitment of EndoB2 and clathrin. Specific disruption of EndoB2 or the CPG2-EndoB2 interaction impairs activity-dependent, but not constitutive, internalization of both NMDA- and AMPA-type glutamate receptors. These results demonstrate that, through direct interactions with F-actin and EndoB2, CPG2 physically bridges the spine cytoskeleton and the endocytic machinery, and this tripartite association is critical specifically for activity-dependent CME of synaptic glutamate receptors. PMID:26776730

  6. Endocytosis in filamentous fungi

    Kalkman, Edward R I C

    2007-01-01

    Endocytosis is little understood in filamentous fungi. For some time it has been controversial as to whether endocytosis occurs in filamentous fungi. A comparative genomics analysis between Saccharomyces cerevisiae and 10 genomes of filamentous fungal species showed that filamentous fungi possess complex endocytic machineries. The use of the endocytic marker dye FM4-64, and various vesicle trafficking inhibitors revealed many similarities between endocytosis in the filamentous ...

  7. Exocytosis and Endocytosis: Modes, Functions, and Coupling Mechanisms*

    Wu, Ling-Gang; Hamid, Edaeni; Shin, Wonchul; Chiang, Hsueh-Cheng

    2016-01-01

    Vesicle exocytosis releases content to mediate many biological events, including synaptic transmission essential for brain functions. Following exocytosis, endocytosis is initiated to retrieve exocytosed vesicles within seconds to minutes. Decades of studies in secretory cells reveal three exocytosis modes coupled to three endocytosis modes: (a) full-collapse fusion, in which vesicles collapse into the plasma membrane, followed by classical endocytosis involving membrane invagination and vesicle reformation; (b) kiss-and-run, in which the fusion pore opens and closes; and (c) compound exocytosis, which involves exocytosis of giant vesicles formed via vesicle-vesicle fusion, followed by bulk endocytosis that retrieves giant vesicles. Here we review these exo- and endocytosis modes and their roles in regulating quantal size and synaptic strength, generating synaptic plasticity, maintaining exocytosis, and clearing release sites for vesicle replenishment. Furthermore, we highlight recent progress in understanding how vesicle endocytosis is initiated and is thus coupled to exocytosis. The emerging model is that calcium influx via voltage-dependent calcium channels at the calcium microdomain triggers endocytosis and controls endocytosis rate; calmodulin and synaptotagmin are the calcium sensors; and the exocytosis machinery, including SNARE proteins (synaptobrevin, SNAP25, and syntaxin), is needed to coinitiate endocytosis, likely to control the amount of endocytosis. PMID:24274740

  8. Exocytosis and endocytosis: modes, functions, and coupling mechanisms.

    Wu, Ling-Gang; Hamid, Edaeni; Shin, Wonchul; Chiang, Hsueh-Cheng

    2014-01-01

    Vesicle exocytosis releases content to mediate many biological events, including synaptic transmission essential for brain functions. Following exocytosis, endocytosis is initiated to retrieve exocytosed vesicles within seconds to minutes. Decades of studies in secretory cells reveal three exocytosis modes coupled to three endocytosis modes: (a) full-collapse fusion, in which vesicles collapse into the plasma membrane, followed by classical endocytosis involving membrane invagination and vesicle reformation; (b) kiss-and-run, in which the fusion pore opens and closes; and (c) compound exocytosis, which involves exocytosis of giant vesicles formed via vesicle-vesicle fusion, followed by bulk endocytosis that retrieves giant vesicles. Here we review these exo- and endocytosis modes and their roles in regulating quantal size and synaptic strength, generating synaptic plasticity, maintaining exocytosis, and clearing release sites for vesicle replenishment. Furthermore, we highlight recent progress in understanding how vesicle endocytosis is initiated and is thus coupled to exocytosis. The emerging model is that calcium influx via voltage-dependent calcium channels at the calcium microdomain triggers endocytosis and controls endocytosis rate; calmodulin and synaptotagmin are the calcium sensors; and the exocytosis machinery, including SNARE proteins (synaptobrevin, SNAP25, and syntaxin), is needed to coinitiate endocytosis, likely to control the amount of endocytosis. PMID:24274740

  9. Myosin light chain kinase facilitates endocytosis of synaptic vesicles at hippocampal boutons.

    Li, Lin; Wu, Xiaomei; Yue, Hai-Yuan; Zhu, Yong-Chuan; Xu, Jianhua

    2016-07-01

    At nerve terminals, endocytosis efficiently recycles vesicle membrane to maintain synaptic transmission under different levels of neuronal activity. Ca(2+) and its downstream signal pathways are critical for the activity-dependent regulation of endocytosis. An activity- and Ca(2+) -dependent kinase, myosin light chain kinase (MLCK) has been reported to regulate vesicle mobilization, vesicle cycling, and motility in different synapses, but whether it has a general contribution to regulation of endocytosis at nerve terminals remains unknown. We investigated this issue at rat hippocampal boutons by imaging vesicle endocytosis as the real-time retrieval of vesicular synaptophysin tagged with a pH-sensitive green fluorescence protein. We found that endocytosis induced by 200 action potentials (5-40 Hz) was slowed by acute inhibition of MLCK and down-regulation of MLCK with RNA interference, while the total amount of vesicle exocytosis and somatic Ca(2+) channel current did not change with MLCK down-regulation. Acute inhibition of myosin II similarly impaired endocytosis. Furthermore, down-regulation of MLCK prevented depolarization-induced phosphorylation of myosin light chain, an effect shared by blockers of Ca(2+) channels and calmodulin. These results suggest that MLCK facilitates vesicle endocytosis through activity-dependent phosphorylation of myosin downstream of Ca(2+) /calmodulin, probably as a widely existing mechanism among synapses. Our study suggests that MLCK is an important activity-dependent regulator of vesicle recycling in hippocampal neurons, which are critical for learning and memory. The kinetics of vesicle membrane endocytosis at nerve terminals has long been known to depend on activity and Ca(2+) . This study provides evidence suggesting that myosin light chain kinase increases endocytosis efficiency at hippocampal neurons by mediating Ca(2+) /calmodulin-dependent phosphorylation of myosin. The authors propose that this signal cascade may serve as

  10. A neurotoxic phospholipase A2 impairs yeast amphiphysin activity and reduces endocytosis.

    Mojca Mattiazzi

    Full Text Available BACKGROUND: Presynaptically neurotoxic phospholipases A(2 inhibit synaptic vesicle recycling through endocytosis. PRINCIPAL FINDINGS: Here we provide insight into the action of a presynaptically neurotoxic phospholipase A(2 ammodytoxin A (AtxA on clathrin-dependent endocytosis in budding yeast. AtxA caused changes in the dynamics of vesicle formation and scission from the plasma membrane in a phospholipase activity dependent manner. Our data, based on synthetic dosage lethality screen and the analysis of the dynamics of sites of endocytosis, indicate that AtxA impairs the activity of amphiphysin. CONCLUSIONS: We identified amphiphysin and endocytosis as the target of AtxA intracellular activity. We propose that AtxA reduces endocytosis following a mechanism of action which includes both a specific protein-protein interaction and enzymatic activity, and which is applicable to yeast and mammalian cells. Knowing how neurotoxic phospholipases A(2 work can open new ways to regulate endocytosis.

  11. Endocytosis of influenza viruses

    Lakadamyali, Melike; Rust, Michael J.; Zhuang, Xiaowei

    2004-01-01

    Receptor-mediated endocytosis is known to play an important role in the entry of many viruses into host cells. However, the exact internalization mechanism has, until recently, remained poorly understood for many medically important viruses, including influenza. Developments in real-time imaging of single viruses as well as the use of dominant negative mutants to selectively block specific endocytic pathways, have improved our understanding of the influenza infection process.

  12. Endosome-mediated endocytic mechanism replenishes the majority of synaptic vesicles at mature CNS synapses in an activity-dependent manner.

    Park, Joohyun; Cho, Oh Yeon; Kim, Jung Ah; Chang, Sunghoe

    2016-01-01

    Whether synaptic vesicles (SVs) are recovered via endosome-mediated pathways is a matter of debate; however, recent evidence suggests that clathrin-independent bulk endocytosis (CIE) via endosomes is functional and preferentially replenishes SV pools during strong stimulation. Here, using brefeldin-A (BFA) to block CIE, we found that CIE retrieved a minority of SVs at developing CNS synapses during strong stimulation, but its contribution increased up to 61% at mature CNS synapses. Contrary to previous views, BFA not only blocked SV formation from the endosome but also blocked the endosome formation at the plasma membrane. Adaptor protein 1 and 3 (AP-1/3) have key roles in SV reformation from endosomes during CIE, and AP-1 also affects bulk endosome formation from the plasma membrane. Finally, temporary blocking of chronic or acute neuronal activity with tetrodotoxin in mature neurons redirected most SV retrieval to endosome-independent pathways. These results show that during high neuronal activity, CIE becomes the major endocytic pathway at mature CNS synapses. Moreover, mature neurons use clathrin-mediated endocytosis and the CIE pathway to different extents depending on their previous activity; this may result in activity-dependent alterations of the SV composition which ultimately influence transmitter release and contribute to synaptic plasticity. PMID:27534442

  13. Endosomes Derived from Clathrin-Independent Endocytosis Serve as Precursors for Endothelial Lumen Formation

    Porat-Shliom, Natalie; Weigert, Roberto; Donaldson, Julie G.

    2013-01-01

    Clathrin-independent endocytosis (CIE) is a form of bulk plasma membrane (PM) endocytosis that allows cells to sample and evaluate PM composition. Once in endosomes, the internalized proteins and lipids can be recycled back to the PM or delivered to lysosomes for degradation. Endosomes arising from CIE contain lipid and signaling molecules suggesting that they might be involved in important biological processes. During vasculogenesis, new blood vessels are formed from precursor cells in a pro...

  14. Ricin transport into cells: studies of endocytosis and intercellular transport

    Sandvig, Kirsten; Grimmer, S.; Iversen, T.G.; Rodal, S.K.; Torgersen, Maria Lyngaas; Nicoziani, Paolo; van Deurs, Bo

    Cell Biology, ricin, endocytosis, Golgi apparatus, cholesterol, clathrin, toxin, Rab, endoplasmic reticulum......Cell Biology, ricin, endocytosis, Golgi apparatus, cholesterol, clathrin, toxin, Rab, endoplasmic reticulum...

  15. Multiple Functions of Sterols in Yeast Endocytosis

    Heese-Peck, Antje; Pichler, Harald; Zanolari, Bettina; Watanabe, Reika; Daum, Günther; Riezman, Howard

    2002-01-01

    Sterols are essential factors for endocytosis in animals and yeast. To investigate the sterol structural requirements for yeast endocytosis, we created a variety of ergΔ mutants, each accumulating a distinct set of sterols different from ergosterol. Mutant erg2Δerg6Δ and erg3Δerg6Δ cells exhibit a strong internalization defect of the α-factor receptor (Ste2p). Specific sterol structures are necessary for pheromone-dependent receptor hyperphosphorylation, a prerequisite for internalization. Th...

  16. Activity Dependent Regulation of Inhibitory Circuitry

    Sharma, Nikhil

    2015-01-01

    Inhibition controls information flow through a neural circuit by modulating synaptic integration, restricting action potentials, and coordinating the activity of ensembles of neurons. These functions are mediated by a diverse array of inhibitory neuron subtypes that synapse on defined domains of a postsynaptic neuron. Activity-dependent transcription controls inhibitory synapse number and function, but how this transcription program affects the inhibitory inputs that form on di...

  17. Nanomechanics of magnetically driven cellular endocytosis

    Zablotskyy, Vitaliy A.; Lunov, O.; Dejneka, Alexandr; Jastrabík, Lubomír; Polyakova, T.; Syrovets, T.; Simmet, T.

    2011-01-01

    Roč. 99, č. 18 (2011), 183701/1-183701/3. ISSN 0003-6951 R&D Projects: GA AV ČR KAN301370701; GA MŠk(CZ) 1M06002 Institutional research plan: CEZ:AV0Z10100522 Keywords : magnetically controled endocytosis * cell membranes * iron oxide nanoparticles Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 3.844, year: 2011

  18. Endosomes derived from clathrin-independent endocytosis serve as precursors for endothelial lumen formation.

    Natalie Porat-Shliom

    Full Text Available Clathrin-independent endocytosis (CIE is a form of bulk plasma membrane (PM endocytosis that allows cells to sample and evaluate PM composition. Once in endosomes, the internalized proteins and lipids can be recycled back to the PM or delivered to lysosomes for degradation. Endosomes arising from CIE contain lipid and signaling molecules suggesting that they might be involved in important biological processes. During vasculogenesis, new blood vessels are formed from precursor cells in a process involving internalization and accumulation of endocytic vesicles. Here, we found that CIE has a role in endothelial lumen formation. Specifically, we found that human vascular endothelial cells (HUVECs utilize CIE for internalization of distinct cargo molecules and that in three-dimensional cultures CIE membranes are delivered to the newly formed lumen.

  19. Interaction among Saccharomyces cerevisiae pheromone receptors during endocytosis

    Chien-I Chang

    2014-03-01

    Full Text Available This study investigates endocytosis of Saccharomyces cerevisiae α-factor receptor and the role that receptor oligomerization plays in this process. α-factor receptor contains signal sequences in the cytoplasmic C-terminal domain that are essential for ligand-mediated endocytosis. In an endocytosis complementation assay, we found that oligomeric complexes of the receptor undergo ligand-mediated endocytosis when the α-factor binding site and the endocytosis signal sequences are located in different receptors. Both in vitro and in vivo assays suggested that ligand-induced conformational changes in one Ste2 subunit do not affect neighboring subunits. Therefore, recognition of the endocytosis signal sequence and recognition of the ligand-induced conformational change are likely to be two independent events.

  20. An Abp1-dependent route of endocytosis functions when the classical endocytic pathway in yeast is inhibited.

    Soheil Aghamohammadzadeh

    Full Text Available Clathrin-mediated endocytosis (CME is a well characterized pathway in both yeast and mammalian cells. An increasing number of alternative endocytic pathways have now been described in mammalian cells that can be both clathrin, actin, and Arf6- dependent or independent. In yeast, a single clathrin-mediated pathway has been characterized in detail. However, disruption of this pathway in many mutant strains indicates that other uptake pathways might exist, at least for bulk lipid and fluid internalization. Using a combination of genetics and live cell imaging, here we show evidence for a novel endocytic pathway in S. cerevisiae that does not involve several of the proteins previously shown to be associated with the 'classic' pathway of endocytosis. This alternative pathway functions in the presence of low levels of the actin-disrupting drug latrunculin-A which inhibits movement of the proteins Sla1, Sla2, and Sac6, and is independent of dynamin function. We reveal that in the absence of the 'classic' pathway, the actin binding protein Abp1 is now essential for bulk endocytosis. This novel pathway appears to be distinct from another described alternative endocytic route in S. cerevisiae as it involves at least some proteins known to be associated with cortical actin patches rather than being mediated at formin-dependent endocytic sites. These data indicate that cells have the capacity to use overlapping sets of components to facilitate endocytosis under a range of conditions.

  1. An Abp1-dependent route of endocytosis functions when the classical endocytic pathway in yeast is inhibited.

    Aghamohammadzadeh, Soheil; Smaczynska-de Rooij, Iwona I; Ayscough, Kathryn R

    2014-01-01

    Clathrin-mediated endocytosis (CME) is a well characterized pathway in both yeast and mammalian cells. An increasing number of alternative endocytic pathways have now been described in mammalian cells that can be both clathrin, actin, and Arf6- dependent or independent. In yeast, a single clathrin-mediated pathway has been characterized in detail. However, disruption of this pathway in many mutant strains indicates that other uptake pathways might exist, at least for bulk lipid and fluid internalization. Using a combination of genetics and live cell imaging, here we show evidence for a novel endocytic pathway in S. cerevisiae that does not involve several of the proteins previously shown to be associated with the 'classic' pathway of endocytosis. This alternative pathway functions in the presence of low levels of the actin-disrupting drug latrunculin-A which inhibits movement of the proteins Sla1, Sla2, and Sac6, and is independent of dynamin function. We reveal that in the absence of the 'classic' pathway, the actin binding protein Abp1 is now essential for bulk endocytosis. This novel pathway appears to be distinct from another described alternative endocytic route in S. cerevisiae as it involves at least some proteins known to be associated with cortical actin patches rather than being mediated at formin-dependent endocytic sites. These data indicate that cells have the capacity to use overlapping sets of components to facilitate endocytosis under a range of conditions. PMID:25072293

  2. Cell mobility after endocytosis of carbon nanotubes

    Pirbhai, Massooma; Flores, Thomas; Jedlicka, Sabrina; Rotkin, Slava

    2013-03-01

    Directed cell movement plays a crucial role in cellular behaviors such as neuronal cell division, cell migration, and cell differentiation. There is evidence in preclinical in vivo studies that small fields have successfully been used to enhance regrowth of damages spinal cord axons but with a small success rate. Fortunately, the evolution of functional biomaterials and nanotechnology may provide promising solutions for enhancing the application of electric fields in guiding neuron migration and neurogenesis within the central nervous system. In this work, we studied how endocytosis and subsequent retention of carbon nanotubes affects the mobility of cells under the influence of an electric field, including the directed cell movement.

  3. Nanomechanics of magnetically driven cellular endocytosis

    Zablotskii, V.; Lunov, O.; Dejneka, A.; Jastrabík, L.; Polyakova, T.; Syrovets, T.; Simmet, Th.

    2011-10-01

    Being essential for many pharmacodynamic and pharmacokinetic processes and playing a crucial role in regulating substrate detachment that enables cellular locomotion, endocytotic mechanisms in many aspects still remain a mystery and therefore can hardly be controlled. Here, we report on experimental and modeling studies of the magnetically assisted endocytosis of functionalized superparamagnetic iron oxide nanoparticles by prostate cancer cells (PC-3) and characterize the time and force scales of the cellular uptake machinery. The results indicate how the cellular uptake rate could be controlled by applied magnetic field, membrane elasticity, and nanoparticle magnetic moment.

  4. Yeast Exocytic v-SNAREs Confer Endocytosis

    Gurunathan, Sangiliyandi; Chapman-Shimshoni, Daphne; Trajkovic, Selena; Gerst, Jeffrey E.

    2000-01-01

    In yeast, homologues of the synaptobrevin/VAMP family of v-SNAREs (Snc1 and Snc2) confer the docking and fusion of secretory vesicles at the cell surface. As no v-SNARE has been shown to confer endocytosis, we examined whether yeast lacking the SNC genes, or possessing a temperature-sensitive allele of SNC1 (SNC1ala43), are deficient in the endocytic uptake of components from the cell surface. We found that both SNC and temperature-shifted SNC1ala43 yeast are d...

  5. Exocytosis and endocytosis in juxtaglomerular cells.

    Friis, U G; Jensen, B L; Hansen, P B; Andreasen, D; Skøtt, O

    2000-01-01

    The cellular events related to secretion of renin are not well understood. Here we review some of the evidence that has led to the current understanding of renin secretion as a process that involves exocytosis as the predominant mode of secretion. This is based on the observation of occasional fusion events between secretory granules and cell membrane and measurement of intermittent secretion of renin from single afferent arterioles, with a renin content of each secretion episode that corresponds to the renin content of one secretory granule. More recently it has been demonstrated that the afferent arterioles lose a large number of renin granules after acute stimulation without changing the average granular volume. Current electrophysiological techniques have now permitted direct measurements of cell membrane capacitance in juxtaglomerular (JG) cells as a measure of net addition (exocytosis) or removal (endocytosis) of membrane material. With this technique we have shown that cAMP, which is a vasodilator and stimulates renin secretion, enhances net exocytosis at low concentrations, while at higher concentrations membrane retrieval processes are also stimulated. We suggest that both exocytosis and endocytosis are regulated processes in the JG-cells and both may be important for the long-term control of renin secretion at the single cell level. PMID:10691785

  6. Endocytosis of Integrin-Binding Human Picornaviruses

    Pirjo Merilahti

    2012-01-01

    Full Text Available Picornaviruses that infect humans form one of the largest virus groups with almost three hundred virus types. They include significant enteroviral pathogens such as rhino-, polio-, echo-, and coxsackieviruses and human parechoviruses that cause wide range of disease symptoms. Despite the economic importance of picornaviruses, there are no antivirals. More than ten cellular receptors are known to participate in picornavirus infection, but experimental evidence of their role in cellular infection has been shown for only about twenty picornavirus types. Three enterovirus types and one parechovirus have experimentally been shown to bind and use integrin receptors in cellular infection. These include coxsackievirus A9 (CV-A9, echovirus 9, and human parechovirus 1 that are among the most common and epidemic human picornaviruses and bind to αV-integrins via RGD motif that resides on virus capsid. In contrast, echovirus 1 (E-1 has no RGD and uses integrin α2β1 as cellular receptor. Endocytosis of CV-A9 has recently been shown to occur via a novel Arf6- and dynamin-dependent pathways, while, contrary to collagen binding, E-1 binds inactive β1 integrin and enters via macropinocytosis. In this paper, we review what is known about receptors and endocytosis of integrin-binding human picornaviruses.

  7. Endocytosis and Membrane Turnover in the Germ Tube of Uromyces fabae

    Hoffmann, Jochen; Mendgen, Kurt

    1998-01-01

    We have used the fluorescent dye FM4-64 as a tracer to demonstrate bulk membrane internalization (endocytosis) and redistribution of the dye within the cytoplasm of the germ tube of the rust fungus Uromyces fabae. Staining of the hyphal membrane was detected 4 s after application of FM4-64 and reached a maximum after 1 min. The highest fluorescence intensity occurred in the apex. Subsequently, staining of the plasma membrane decreased and a subapical region of the fungal protoplast (5 20 µm f...

  8. Differential requirements for actin during yeast and mammalian endocytosis.

    Aghamohammadzadeh, Soheil; Ayscough, Kathryn R

    2009-08-01

    Key features of clathrin-mediated endocytosis have been conserved across evolution. However, endocytosis in Saccharomyces cerevisiae is completely dependent on a functional actin cytoskeleton, whereas actin appears to be less critical in mammalian cell endocytosis. We reveal that the fundamental requirement for actin in the early stages of yeast endocytosis is to provide a strong framework to support the force generation needed to direct the invaginating plasma membrane into the cell against turgor pressure. By providing osmotic support, pressure differences across the plasma membrane were removed and this reduced the requirement for actin-bundling proteins in normal endocytosis. Conversely, increased turgor pressure in specific yeast mutants correlated with a decreased rate of endocytic patch invagination. PMID:19597484

  9. Exocytosis and endocytosis in juxtaglomerular cells

    Friis, U G; Jensen, B L; Hansen, Pernille B. Lærkegaard;

    2000-01-01

    that the afferent arterioles lose a large number of renin granules after acute stimulation without changing the average granular volume. Current electrophysiological techniques have now permitted direct measurements of cell membrane capacitance in juxtaglomerular (JG) cells as a measure of net addition (exocytosis...... fusion events between secretory granules and cell membrane and measurement of intermittent secretion of renin from single afferent arterioles, with a renin content of each secretion episode that corresponds to the renin content of one secretory granule. More recently it has been demonstrated...... and endocytosis are regulated processes in the JG-cells and both may be important for the long-term control of renin secretion at the single cell level....

  10. Clathrin-independent endocytosis: mechanisms and function

    Sandvig, Kirsten; Pust, Sascha; Skotland, Tore;

    2011-01-01

    It is now about 20 years since we first wrote reviews about clathrin-independent endocytosis. The challenge at the time was to convince the reader about its existence. Then the suggestion came up that caveolae might be responsible for the uptake. However, clearly this could not be the case since a...... large fraction of the clathrin-independent uptake is dynamin-independent. Today, two decades later, the field has developed considerably. New techniques have enabled a detailed analysis of several clathrin-independent endocytic mechanisms, and caveolae have been found to be mostly stable structures...... having several functions of their own. This article aims at providing a brief update on the importance of clathrin-independent endocytic mechanisms, how the processes are regulated differentially, for instance on the poles of polarized cells, and the challenges in studying them....

  11. Imaging the Dynamics of Endocytosis in Live Mammalian Tissues

    Weigert, Roberto

    2014-01-01

    In mammalian cells, endocytosis plays a pivotal role in regulating several basic cellular functions. Up to now, the dynamics and the organization of the endocytic pathways have been primarily investigated in reductionist model systems such as cell and organ cultures. Although these experimental models have been fully successful in unraveling the endocytic machinery at a molecular level, our understanding of the regulation and the role of endocytosis in vivo has been limited. Recently, advance...

  12. Rapid endocytosis is triggered upon imbibition in Arabidopsis seeds

    Pagnussat, Luciana; Burbach, Christian; Baluška, František; de la Canal, Laura

    2012-01-01

    During seed imbibition and embryo activation, rapid change from a metabolically resting state to the activation of diverse extracellular and/or membrane bound molecules is essential and, hence, endocytosis could be activated too. In fact, we have documented endocytic internalization of the membrane impermeable endocytic tracer FM4–64 already upon 30 min of imbibition of Arabidopsis seeds. This finding suggest that endocytosis is activated early during seed imbibition in Arabidopsis. Immunoloc...

  13. Ankyrin-G Inhibits Endocytosis of Cadherin Dimers.

    Cadwell, Chantel M; Jenkins, Paul M; Bennett, Vann; Kowalczyk, Andrew P

    2016-01-01

    Dynamic regulation of endothelial cell adhesion is central to vascular development and maintenance. Furthermore, altered endothelial adhesion is implicated in numerous diseases. Therefore, normal vascular patterning and maintenance require tight regulation of endothelial cell adhesion dynamics. However, the mechanisms that control junctional plasticity are not fully understood. Vascular endothelial cadherin (VE-cadherin) is an adhesive protein found in adherens junctions of endothelial cells. VE-cadherin mediates adhesion through trans interactions formed by its extracellular domain. Trans binding is followed by cis interactions that laterally cluster the cadherin in junctions. VE-cadherin is linked to the actin cytoskeleton through cytoplasmic interactions with β- and α-catenin, which serve to increase adhesive strength. Furthermore, p120-catenin binds to the cytoplasmic tail of cadherin and stabilizes it at the plasma membrane. Here we report that induced cis dimerization of VE-cadherin inhibits endocytosis independent of both p120 binding and trans interactions. However, we find that ankyrin-G, a protein that links membrane proteins to the spectrin-actin cytoskeleton, associates with VE-cadherin and inhibits its endocytosis. Ankyrin-G inhibits VE-cadherin endocytosis independent of p120 binding. We propose a model in which ankyrin-G associates with and inhibits the endocytosis of VE-cadherin cis dimers. Our findings support a novel mechanism for regulation of VE-cadherin endocytosis through ankyrin association with cadherin engaged in lateral interactions. PMID:26574545

  14. Clathrin-mediated endocytosis: the gateway into plant cells.

    Chen, Xu; Irani, Niloufer G; Friml, Jiří

    2011-12-01

    Endocytosis in plants has an essential role not only for basic cellular functions but also for growth and development, hormonal signaling and communication with the environment including nutrient delivery, toxin avoidance, and pathogen defense. The major endocytic mechanism in plants depends on the coat protein clathrin. It starts by clathrin-coated vesicle formation at the plasma membrane, where specific cargoes are recognized and packaged for internalization. Recently, genetic, biochemical and advanced microscopy studies provided initial insights into mechanisms and roles of clathrin-mediated endocytosis in plants. Here we summarize the present state of knowledge and compare mechanisms of clathrin-mediated endocytosis in plants with animal and yeast paradigms as well as review plant-specific regulations and roles of this process. PMID:21945181

  15. Bulk undercooling

    Kattamis, T. Z.

    1984-01-01

    Bulk undercooling methods and procedures will first be reviewed. Measurement of various parameters which are necessary to understand the solidification mechanism during and after recalescence will be discussed. During recalescence of levitated, glass-encased large droplets (5 to 8 mm diam) high speed temperature sensing devices coupled with a rapid response oscilloscope are now being used at MIT to measure local thermal behavior in hypoeutectic and eutectic binary Ni-Sn alloys. Dendrite tip velocities were measured by various investigators using thermal sensors or high speed cinematography. The confirmation of the validity of solidification models of bulk-undercooled melts is made difficult by the fineness of the final microstructure, the ultra-rapid evolution of the solidifying system which makes measurements very awkward, and the continuous modification of the microstructure which formed during recalescence because of precipitation, remelting and rapid coarsening.

  16. A novel fluorescence-activated cell sorter-based screen for yeast endocytosis mutants identifies a yeast homologue of mammalian eps15

    1996-01-01

    A complete understanding of the molecular mechanisms of endocytosis requires the discovery and characterization of the protein machinery that mediates this aspect of membrane trafficking. A novel genetic screen was used to identify yeast mutants defective in internalization of bulk lipid. The fluorescent lipophilic styryl dye FM4-64 was used in conjunction with FACS to enrich for yeast mutants that exhibit internalization defects. Detailed characterization of two of these mutants, dim1-1 and ...

  17. Cdk5 is essential for synaptic vesicle endocytosis

    Tan, Timothy C; Valova, Valentina A; Malladi, Chandra S;

    2003-01-01

    Synaptic vesicle endocytosis (SVE) is triggered by calcineurin-mediated dephosphorylation of the dephosphin proteins. SVE is maintained by the subsequent rephosphorylation of the dephosphins by unidentified protein kinases. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates dynami...

  18. OUABAIN AND INSULIN INDUCE SODIUM PUMP ENDOCYTOSIS IN RENAL EPITHELIUM

    Gupta, Shalini; Yan, Yanling; Malhotra, Deepak; Liu, Jiang; Xie, Zijian; Najjar, Sonia M.; Shapiro, Joseph I

    2012-01-01

    Cardiotonic steroids signaling through the basolateral sodium pump (Na/KATPase) have been shown to alter renal salt handling in intact animals. As the relationship between renal salt handling and blood pressure is a key determinant of hypertension, and patients with insulin resistance are frequently hypertensive, we chose to examine whether there might be competition for resources necessary for receptor mediated endocytosis.

  19. Bladder uptake of liposomes after intravesical administration occurs by endocytosis.

    Bharathi Raja Rajaganapathy

    Full Text Available Liposomes have been used therapeutically and as a local drug delivery system in the bladder. However, the exact mechanism for the uptake of liposomes by bladder cells is unclear. In the present study, we investigated the role of endocytosis in the uptake of liposomes by cultured human UROtsa cells of urothelium and rat bladder. UROtsa cells were incubated in serum-free media with liposomes containing colloidal gold particles for 2 h either at 37°C or at 4°C. Transmission Electron Microscopy (TEM images of cells incubated at 37°C found endocytic vesicles containing gold inside the cells. In contrast, only extracellular binding was noticed in cells incubated with liposomes at 4°C. Absence of liposome internalization at 4°C indicates the need of energy dependent endocytosis as the primary mechanism of entry of liposomes into the urothelium. Flow cytometry analysis revealed that the uptake of liposomes at 37°C occurs via clathrin mediated endocytosis. Based on these observations, we propose that clathrin mediated endocytosis is the main route of entry for liposomes into the urothelial layer of the bladder and the findings here support the usefulness of liposomes in intravesical drug delivery.

  20. Constitutive Endocytosis of VEGFR2 Protects the Receptor against Shedding.

    Basagiannis, Dimitris; Christoforidis, Savvas

    2016-08-01

    VEGFR2 plays a fundamental role in blood vessel formation and in life threatening diseases, such as cancer angiogenesis and cardiovascular disorders. Although inactive growth factor receptors are mainly localized at the plasma membrane, VEGFR2 undergoes constitutive endocytosis (in the absence of ligand) and recycling. Intriguingly, the significance of these futile transport cycles of VEGFR2 remains unclear. Here we found that, unexpectedly, the function of constitutive endocytosis of VEGFR2 is to protect the receptor against plasma membrane cleavage (shedding), thereby preserving the functional state of the receptor until the time of activation by VEGF. Inhibition of constitutive endocytosis of VEGFR2, by interference with the function of clathrin, dynamin, or Rab5, increases dramatically the cleavage/shedding of VEGFR2. Shedding of VEGFR2 produces an N-terminal soluble fragment (100 kDa, s100), which is released in the extracellular space, and a residual C-terminal part (130 kDa, p130) that remains integrated at the plasma membrane. The released soluble fragment (s100) co-immunoprecipitates with VEGF, in line with the topology of the VEGF-binding domain at the N terminus of VEGFR2. Increased shedding of VEGFR2 (via inhibition of constitutive endocytosis) results in reduced response to VEGF, consistently with the loss of the VEGF-binding domain from the membrane remnant of VEGFR2. These data suggest that constitutive internalization of VEGFR2 protects the receptor against shedding and provides evidence for an unprecedented mechanism via which endocytosis can regulate the fate and activity of growth factor receptors. PMID:27298320

  1. Activity-dependent plasticity of hippocampal place maps.

    Schoenenberger, Philipp; O'Neill, Joseph; Csicsvari, Jozsef

    2016-01-01

    Hippocampal neurons encode a cognitive map of space. These maps are thought to be updated during learning and in response to changes in the environment through activity-dependent synaptic plasticity. Here we examine how changes in activity influence spatial coding in rats using halorhodopsin-mediated, spatially selective optogenetic silencing. Halorhoposin stimulation leads to light-induced suppression in many place cells and interneurons; some place cells increase their firing through disinhibition, whereas some show no effect. We find that place fields of the unaffected subpopulation remain stable. On the other hand, place fields of suppressed place cells were unstable, showing remapping across sessions before and after optogenetic inhibition. Disinhibited place cells had stable maps but sustained an elevated firing rate. These findings suggest that place representation in the hippocampus is constantly governed by activity-dependent processes, and that disinhibition may provide a mechanism for rate remapping. PMID:27282121

  2. Activity-dependent modulation of neural circuit synaptic connectivity

    Charles R Tessier

    2009-07-01

    Full Text Available In many nervous systems, the establishment of neural circuits is known to proceed via a two-stage process; 1 early, activity-independent wiring to produce a rough map characterized by excessive synaptic connections, and 2 subsequent, use-dependent pruning to eliminate inappropriate connections and reinforce maintained synapses. In invertebrates, however, evidence of the activity-dependent phase of synaptic refinement has been elusive, and the dogma has long been that invertebrate circuits are “hard-wired” in a purely activity-independent manner. This conclusion has been challenged recently through the use of new transgenic tools employed in the powerful Drosophila system, which have allowed unprecedented temporal control and single neuron imaging resolution. These recent studies reveal that activity-dependent mechanisms are indeed required to refine circuit maps in Drosophila during precise, restricted windows of late-phase development. Such mechanisms of circuit refinement may be key to understanding a number of human neurological diseases, including developmental disorders such as Fragile X syndrome (FXS and autism, which are hypothesized to result from defects in synaptic connectivity and activity-dependent circuit function. This review focuses on our current understanding of activity-dependent synaptic connectivity in Drosophila, primarily through analyzing the role of the fragile X mental retardation protein (FMRP in the Drosophila FXS disease model. The particular emphasis of this review is on the expanding array of new genetically-encoded tools that are allowing cellular events and molecular players to be dissected with ever greater precision and detail.

  3. Activity-dependent modulation of neural circuit synaptic connectivity

    Tessier, Charles R.; Kendal Broadie

    2009-01-01

    In many nervous systems, the establishment of neural circuits is known to proceed via a two-stage process; 1) early, activity-independent wiring to produce a rough map characterized by excessive synaptic connections, and 2) subsequent, use-dependent pruning to eliminate inappropriate connections and reinforce maintained synapses. In invertebrates, however, evidence of the activity-dependent phase of synaptic refinement has been elusive, and the dogma has long been that invertebrate circ...

  4. Activity-dependent neuronal signalling and autism spectrum disorder

    Ebert, Daniel H.; Greenberg, Michael E.

    2013-01-01

    Neuronal activity induces the post-translational modification of synaptic molecules, promotes localized protein synthesis within dendrites and activates gene transcription, thereby regulating synaptic function and allowing neuronal circuits to respond dynamically to experience. Evidence indicates that many of the genes that are mutated in autism spectrum disorders are crucial components of the activity-dependent signalling networks that regulate synapse development and plasticity. Dysregulati...

  5. Membrane Mechanics of Endocytosis in Cells with Turgor

    Dmitrieff, Serge

    2015-01-01

    Endocytosis is an essential process by which cells internalize a piece of plasma membrane and material from the outside. In cells with turgor, pressure opposes membrane defor- mations, and increases the amount of force that has to be generated by the endocytic machinery. To determine this force, and calculate the shape of the membrane, we used physical theory to model an elastic surface under pressure. Accurate fits of experimental profiles are obtained assuming that the coated membrane is highly rigid and preferentially curved at the endocytic site. The forces required from the actin machinery peaks at the onset of deformation, indicating that once invagination has been initiated, endocytosis is unlikely to stall before completion. Coat proteins do not lower the initiation force but may affect the process by the curvature they induce. In the presence of isotropic curvature inducers, pulling the tip of the invagination can trigger the formation of a neck at the base of the invagination. Hence direct neck cons...

  6. Targeted gene delivery via N-acetylglucosamine receptor mediated endocytosis.

    Singh, Bijay; Maharjan, Sushila; Kim, You-Kyoung; Jiang, Tai; Islam, Mohammad Ariful; Kang, Sang-Kee; Cho, Myung-Haing; Choi, Yun-Jaie; Cho, Chong-Su

    2014-11-01

    Receptor-mediated endocytosis is a promising approach of gene delivery into the target cells via receptor-ligand interaction. Vimentins at the cell surface are recently known to bind N-acetylglucosamine (GlcNAc) residue, therefore, the cell surfaces of vimentin-expressing cells could be targeted by using the GlcNAc residue as a specific ligand for receptor-mediated gene delivery. Here, we have developed polymeric gene delivery vectors, based on poly(ethylene oxide)(PEO) and poly(aspartamide), namely poly[(aspartamide)(diethylenetriamine)]-b-[PEO-(GlcNAc)] (PADPG) and poly[(aspartamide)(diethylenetriamine)]-b-[PEO] (PADP) to elucidate the efficiency of GlcNAc ligand for gene delivery through receptor mediated endocytosis. To determine the efficiency of these polymeric vectors for specific gene delivery, the DNA condensation ability of PADPG and PADP and the subsequent formation of polymeric nanoparticles were confirmed by gel retardation assay and transmission electron microscopy respectively. Both PADPG and PADP had lower cytotoxicity than polyethylenimine 25 K (PEI 25 K). However, their transfection efficiency was comparatively lower than PEI 25 K due to hydrophilic property of PEO in the vectors. To observe the stability of polymeric nanoparticles, the transfection of PADPG and PADP was carried out in the presence of serum. Favorably, the interfering effect of serum on the transfection efficiency of PADPG and PADP was also very low. Finally, when the cell specificity of these polymeric vectors was investigated, PADPG had high gene transfection in vimentin-expressing cells than vimentin-deficiency cells. The high transfection efficiency of PADPG was attributed to the GlcNAc in the polymeric vector which interact specifically with vimentin in the cells for the receptor-mediated endocytosis. The competitive inhibition assay further proved the receptor-mediated endocytosis of PADPG. Thus, this study demonstrates that conjugation of GlcNAc is an effective and rational

  7. Signaling induced by hop/STI-1 depends on endocytosis

    The co-chaperone hop/STI-1 is a ligand of the cell surface prion protein (PrPC), and their interaction leads to signaling and biological effects. Among these, hop/STI-1 induces proliferation of A172 glioblastoma cells, dependent on both PrPC and activation of the Erk pathway. We tested whether clathrin-mediated endocytosis affects signaling induced by hop/STI-1. Both hyperosmolarity induced by sucrose and monodansyl-cadaverine blocked Erk activity induced by hop/STI-1, without affecting the high basal Akt activity typical of A172. The endocytosis inhibitors also affected the sub-cellular distribution of phosphorylated Erk, consistent with blockade of the latter's activity. The data indicate that signaling induced by hop/STI-1 depends on endocytosis. These findings are consistent with a role of sub-cellular trafficking in signal transduction following engagement by PrPC by ligands such as hop/STI-1, and may help help unravel both the functions of the prion protein, as well as possible loss-of-function components of prion diseases

  8. A memristor SPICE model accounting for synaptic activity dependence.

    Qingjiang Li

    Full Text Available In this work, we propose a new memristor SPICE model that accounts for the typical synaptic characteristics that have been previously demonstrated with practical memristive devices. We show that this model could account for both volatile and non-volatile memristance changes under distinct stimuli. We then demonstrate that our model is capable of supporting typical STDP with simple non-overlapping digital pulse pairs. Finally, we investigate the capability of our model to simulate the activity dependence dynamics of synaptic modification and present simulated results that are in excellent agreement with biological results.

  9. A role for the dynamin-like protein Vps1 during endocytosis in yeast

    Rooij, Iwona I. Smaczynska-de; Allwood, Ellen G.; Aghamohammadzadeh, Soheil; Hettema, Ewald H.; Goldberg, Martin W.; Ayscough, Kathryn R.

    2010-01-01

    Dynamins are a conserved family of proteins involved in membrane fusion and fission. Although mammalian dynamins are known to be involved in several membrane-trafficking events, the role of dynamin-1 in endocytosis is the best-characterised role of this protein family. Despite many similarities between endocytosis in yeast and mammalian cells, a comparable role for dynamins in yeast has not previously been demonstrated. The reported lack of involvement of dynamins in yeast endocytosis has rai...

  10. Endocytosis Is Crucial for Cell Polarity and Apical Membrane Recycling in the Filamentous Fungus Aspergillus oryzae▿

    Higuchi, Yujiro; Shoji, Jun-ya; Arioka, Manabu; Kitamoto, Katsuhiko

    2008-01-01

    Establishing the occurrence of endocytosis in filamentous fungi was elusive in the past mainly due to the lack of reliable indicators of endocytosis. Recently, however, it was shown that the fluorescent dye N-(3-triethylammoniumpropyl)-4-(p-diethyl-aminophenyl-hexatrienyl)pyridinium dibromide (FM4-64) and the plasma membrane protein AoUapC (Aspergillus oryzae UapC) fused to enhanced green fluorescent protein (EGFP) were internalized from the plasma membrane by endocytosis. Although the occurr...

  11. Activity-dependent neural plasticity from bench to bedside.

    Ganguly, Karunesh; Poo, Mu-Ming

    2013-10-30

    Much progress has been made in understanding how behavioral experience and neural activity can modify the structure and function of neural circuits during development and in the adult brain. Studies of physiological and molecular mechanisms underlying activity-dependent plasticity in animal models have suggested potential therapeutic approaches for a wide range of brain disorders in humans. Physiological and electrical stimulations as well as plasticity-modifying molecular agents may facilitate functional recovery by selectively enhancing existing neural circuits or promoting the formation of new functional circuits. Here, we review the advances in basic studies of neural plasticity mechanisms in developing and adult nervous systems and current clinical treatments that harness neural plasticity, and we offer perspectives on future development of plasticity-based therapy. PMID:24183023

  12. Activity-dependent neuronal model on complex networks.

    de Arcangelis, Lucilla; Herrmann, Hans J

    2012-01-01

    Neuronal avalanches are a novel mode of activity in neuronal networks, experimentally found in vitro and in vivo, and exhibit a robust critical behavior: these avalanches are characterized by a power law distribution for the size and duration, features found in other problems in the context of the physics of complex systems. We present a recent model inspired in self-organized criticality, which consists of an electrical network with threshold firing, refractory period, and activity-dependent synaptic plasticity. The model reproduces the critical behavior of the distribution of avalanche sizes and durations measured experimentally. Moreover, the power spectra of the electrical signal reproduce very robustly the power law behavior found in human electroencephalogram (EEG) spectra. We implement this model on a variety of complex networks, i.e., regular, small-world, and scale-free and verify the robustness of the critical behavior. PMID:22470347

  13. Human SCARB2-mediated entry and endocytosis of EV71.

    Yi-Wen Lin

    Full Text Available Enterovirus (EV 71 infection is known to cause hand-foot-and-mouth disease (HFMD and in severe cases, induces neurological disorders culminating in fatality. An outbreak of EV71 in South East Asia in 1997 affected over 120,000 people and caused neurological disorders in a few individuals. The control of EV71 infection through public health interventions remains minimal and treatments are only symptomatic. Recently, human scavenger receptor class B, member 2 (SCARB2 has been reported to be a cellular receptor of EV71. We expressed human SCARB2 gene in NIH3T3 cells (3T3-SCARB2 to study the mechanisms of EV71 entry and infection. We demonstrated that human SCARB2 serves as a cellular receptor for EV71 entry. Disruption of expression of SCARB2 using siRNAs can interfere EV71 infection and subsequent inhibit the expression of viral capsid proteins in RD and 3T3-SCARB2 but not Vero cells. SiRNAs specific to clathrin or dynamin or chemical inhibitor of clathrin-mediated endocytosis were all capable of interfering with the entry of EV71 into 3T3-SCARB2 cells. On the other hand, caveolin specific siRNA or inhibitors of caveolae-mediated endocytosis had no effect, confirming that only clathrin-mediated pathway was involved in EV71 infection. Endocytosis of EV71 was also found to be pH-dependent requiring endosomal acidification and also required intact membrane cholesterol. In summary, the mechanism of EV71 entry through SCARB2 as the receptor for attachment, and its cellular entry is through a clathrin-mediated and pH-dependent endocytic pathway. This study on the receptor and endocytic mechanisms of EV71 infection is useful for the development of effective medications and prophylactic treatment against the enterovirus.

  14. Clostridial Glucosylating Toxins Enter Cells via Clathrin-Mediated Endocytosis

    Papatheodorou, Panagiotis; Zamboglou, Constantinos; Genisyuerek, Selda; Guttenberg, Gregor; Aktories, Klaus

    2010-01-01

    Clostridium difficile toxin A (TcdA) and toxin B (TcdB), C. sordellii lethal toxin (TcsL) and C. novyi α-toxin (TcnA) are important pathogenicity factors, which represent the family of the clostridial glucosylating toxins (CGTs). Toxin A and B are associated with antibiotic-associated diarrhea and pseudomembraneous colitis. Lethal toxin is involved in toxic shock syndrome after abortion and α-toxin in gas gangrene development. CGTs enter cells via receptor-mediated endocytosis and require an ...

  15. Clathrin-mediated endocytosis in budding yeast at a glance.

    Lu, Rebecca; Drubin, David G; Sun, Yidi

    2016-04-15

    Clathrin-mediated endocytosis is an essential cellular process that involves the concerted assembly and disassembly of many different proteins at the plasma membrane. In yeast, live-cell imaging has shown that the spatiotemporal dynamics of these proteins is highly stereotypical. Recent work has focused on determining how the timing and functions of endocytic proteins are regulated. In this Cell Science at a Glance article and accompanying poster, we review our current knowledge of the timeline of endocytic site maturation and discuss recent works focusing on how phosphorylation, ubiquitylation and lipids regulate various aspects of the process. PMID:27084361

  16. The use of FM dyes to analyze plant endocytosis

    Malínská, Kateřina; Jelínková, Adriana; Petrášek, Jan

    Vol. 1209. Totowa: Humana Press, 2014 - (Otegui, M.), s. 1-11. (Methods in Molecular Biology). ISBN 978-1-4939-1419-7 R&D Projects: GA ČR(CZ) GAP305/11/2476; GA ČR GPP305/11/P797 Institutional support: RVO:61389030 Keywords : Endocytosis * Endomembranes * FM 1-43 Subject RIV: EB - Genetics ; Molecular Biology http://gateway.isiknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=Alerting&SrcApp=Alerting&DestApp=MEDLINE&DestLinkType=FullRecord&UT=25117271

  17. Ricin A chain reaches the endoplasmic reticulum after endocytosis

    Ricin is a potent ribosome inactivating protein and now has been widely used for synthesis of immunotoxins. To target ribosome in the mammalian cytosol, ricin must firstly retrograde transport from the endomembrane system to reach the endoplasmic reticulum (ER) where the ricin A chain (RTA) is recognized by ER components that facilitate its membrane translocation to the cytosol. In the study, the fusion gene of enhanced green fluorescent protein (EGFP)-RTA was expressed with the pET-28a (+) system in Escherichia coli under the control of a T7 promoter. The fusion protein showed a green fluorescence. The recombinant protein can be purified by metal chelated affinity chromatography on a column of NTA. The rabbit anti-GFP antibody can recognize the fusion protein of EGFP-RTA just like the EGFP protein. The cytotoxicity of EGFP-RTA and RTA was evaluated by the MTT assay in HeLa and HEP-G2 cells following fluid-phase endocytosis. The fusion protein had a similar cytotoxicity of RTA. After endocytosis, the subcellular location of the fusion protein can be observed with the laser scanning confocal microscopy and the immuno-gold labeling Electro Microscopy. This study provided important evidence by a visualized way to prove that RTA does reach the endoplasmic reticulum

  18. Rapid endocytosis is triggered upon imbibition in Arabidopsis seeds.

    Pagnussat, Luciana; Burbach, Christian; Baluška, František; de la Canal, Laura

    2012-03-01

    During seed imbibition and embryo activation, rapid change from a metabolically resting state to the activation of diverse extracellular and/or membrane bound molecules is essential and, hence, endocytosis could be activated too. In fact, we have documented endocytic internalization of the membrane impermeable endocytic tracer FM4-64 already upon 30 min of imbibition of Arabidopsis seeds. This finding suggest that endocytosis is activated early during seed imbibition in Arabidopsis. Immunolocalization of rhamnogalacturonan-II (RG-II) complexed with boron showed that whereas this pectin is localized only in the cell walls of dry seed embryos, it starts to be intracellular once the imbibition started. Brefeldin A (BFA) exposure resulted in recruitment of the intracellular RG-II pectin complexes into the endocytic BFA-induced compartments, confirming the endocytic origin of the RG-II signal detected intracellularly. Finally, germination was significantly delayed when Arabidopsis seeds were germinated in the presence of inhibitors of endocytic pathways, suggesting that trafficking of extracellular molecules might play an important role in the overcome of germination. This work constitutes the first demonstration of endocytic processes during germination and opens new perspectives about the role of the extracellular matrix and membrane components in seed germination. PMID:22476454

  19. Shaping inhibition: activity dependent structural plasticity of GABAergic synapses

    Pablo Mendez

    2014-10-01

    Full Text Available Inhibitory transmission through the neurotransmitter Ɣ-aminobutyric acid (GABA shapes network activity in the mammalian cerebral cortex by filtering synaptic incoming information and dictating the activity of principal cells. The incredibly diverse population of cortical neurons that use GABA as neurotransmitter shows an equally diverse range of mechanisms that regulate changes in the strength of GABAergic synaptic transmission and allow them to dynamically follow and command the activity of neuronal ensembles. Similarly to glutamatergic synaptic transmission, activity-dependent functional changes in inhibitory neurotransmission are accompanied by alterations in GABAergic synapse structure that range from morphological reorganization of postsynaptic density to de novo formation and elimination of inhibitory contacts. Here we review several aspects of structural plasticity of inhibitory synapses, including its induction by different forms of neuronal activity, behavioral and sensory experience and the molecular mechanisms and signaling pathways involved. We discuss the functional consequences of GABAergic synapse structural plasticity for information processing and memory formation in view of the heterogenous nature of the structural plasticity phenomena affecting inhibitory synapses impinging on somatic and dendritic compartments of cortical and hippocampal neurons.

  20. Solar activity dependence of nightside aurora in winter conditions

    Zhou, Su; Luan, Xiaoli; Dou, Xiankang

    2016-02-01

    The dependence of the nightside (21:00-03:00 MLT; magnetic local time) auroral energy flux on solar activity was quantitatively studied for winter/dark and geomagnetically quiet conditions. Using data combined from Thermosphere, Ionosphere, Mesosphere Energetics and Dynamics/Global Ultraviolet Imager and Defense Meteorological Satellite Program/Special Sensor Ultraviolet Spectrographic Imager observations, we separated the effects of geomagnetic activity from those of solar flux on the nightside auroral precipitation. The results showed that the nightside auroral power was reduced by ~42% in solar maximum (F10.7 = 200 sfu; solar flux unit 1 sfu = 10-22 W m-2 Hz-1) with respect to that under solar minimum (F10.7 = 70 sfu) for the Kp = 1 condition, and this change rate became less (~21%) for the Kp = 3 condition. In addition, the solar cycle dependence of nightside auroral power was similar with that from both the premidnight (21:00-23:00 MLT) and postmidnight (01:00-03:00 MLT) sectors. These results indicated that as the ionospheric ionization increases with the enhanced auroral and geomagnetic activities, the solar activity dependences of nightside auroral power become weaker, at least under geomagnetically quiet conditions.

  1. Spatiotemporal analysis of endocytosis and membrane distribution of fluorescent sterols in living cells

    Wüstner, Daniel; Faergeman, Nils J

    2008-01-01

    regions of the cell surface, and endocytosis contributed by 62% to total sterol uptake in J774 cells. DHE co-localized with fluorescent transferrin (Tf) in vesicles right after onset of endocytosis and in deepened surface patches of energy depleted cells. Surface caveolae labeled with GFP-tagged caveolin...

  2. The TPLATE Adaptor Complex Drives Clathrin-Mediated Endocytosis in Plants

    Gadeyne, A.; Sanchez-Rodriguez, C.; Rubbo, Di S.; Ketelaar, T.

    2014-01-01

    Clathrin-mediated endocytosis is the major mechanism for eukaryotic plasma membrane-based proteome turn-over. In plants, clathrin-mediated endocytosis is essential for physiology and development, but the identification and organization of the machinery operating this process remains largely obscure.

  3. Bile acids reduce endocytosis of high-density lipoprotein (HDL in HepG2 cells.

    Clemens Röhrl

    Full Text Available High-density lipoprotein (HDL transports lipids to hepatic cells and the majority of HDL-associated cholesterol is destined for biliary excretion. Cholesterol is excreted into the bile directly or after conversion to bile acids, which are also present in the plasma as they are effectively reabsorbed through the enterohepatic cycle. Here, we provide evidence that bile acids affect HDL endocytosis. Using fluorescent and radiolabeled HDL, we show that HDL endocytosis was reduced in the presence of high concentrations of taurocholate, a natural non-cell-permeable bile acid, in human hepatic HepG2 and HuH7 cells. In contrast, selective cholesteryl-ester (CE uptake was increased. Taurocholate exerted these effects extracellularly and independently of HDL modification, cell membrane perturbation or blocking of endocytic trafficking. Instead, this reduction of endocytosis and increase in selective uptake was dependent on SR-BI. In addition, cell-permeable bile acids reduced HDL endocytosis by farnesoid X receptor (FXR activation: chenodeoxycholate and the non-steroidal FXR agonist GW4064 reduced HDL endocytosis, whereas selective CE uptake was unaltered. Reduced HDL endocytosis by FXR activation was independent of SR-BI and was likely mediated by impaired expression of the scavenger receptor cluster of differentiation 36 (CD36. Taken together we have shown that bile acids reduce HDL endocytosis by transcriptional and non-transcriptional mechanisms. Further, we suggest that HDL endocytosis and selective lipid uptake are not necessarily tightly linked to each other.

  4. Endocytosis of simian virus 40 into the endoplasmic reticulum

    The endocytosis of SV-40 into CV-1 cells we studied using biochemical and ultrastructural techniques. The half-time of binding of [35S]methionine-radiolabeled SV-40 to CV-1 cells was 25 min. Most of the incoming virus particles remained undegraded for several hours. Electron microscopy showed that some virus entered the endosomal/lysosomal pathway via coated vesicles, while the majority were endocytosed via small uncoated vesicles. After infection at high multiplicity, one third of total cell-associated virus was observed to enter the ER, starting 1-2 h after virus application. The viruses were present in large, tubular, smooth membrane networks generated as extentions of the ER. The results describe a novel and unique membrane transport pathway that allows endocytosed viral particles to be targeted from the plasma membrane to the ER

  5. Clostridial glucosylating toxins enter cells via clathrin-mediated endocytosis.

    Papatheodorou, Panagiotis; Zamboglou, Constantinos; Genisyuerek, Selda; Guttenberg, Gregor; Aktories, Klaus

    2010-01-01

    Clostridium difficile toxin A (TcdA) and toxin B (TcdB), C. sordellii lethal toxin (TcsL) and C. novyi alpha-toxin (TcnA) are important pathogenicity factors, which represent the family of the clostridial glucosylating toxins (CGTs). Toxin A and B are associated with antibiotic-associated diarrhea and pseudomembraneous colitis. Lethal toxin is involved in toxic shock syndrome after abortion and alpha-toxin in gas gangrene development. CGTs enter cells via receptor-mediated endocytosis and require an acidified endosome for translocation of the catalytic domain into the cytosol. Here we studied the endocytic processes that mediate cell internalization of the CGTs. Intoxication of cells was monitored by analyzing cell morphology, status of Rac glucosylation in cell lysates and transepithelial resistance of cell monolayers. We found that the intoxication of cultured cells by CGTs was strongly delayed when cells were preincubated with dynasore, a cell-permeable inhibitor of dynamin, or chlorpromazine, an inhibitor of the clathrin-dependent endocytic pathway. Additional evidence about the role of clathrin in the uptake of the prototypical CGT family member toxin B was achieved by expression of a dominant-negative inhibitor of the clathrin-mediated endocytosis (Eps15 DN) or by siRNA against the clathrin heavy chain. Accordingly, cells that expressed dominant-negative caveolin-1 were not protected from toxin B-induced cell rounding. In addition, lipid rafts impairment by exogenous depletion of sphingomyelin did not decelerate intoxication of HeLa cells by CGTs. Taken together, our data indicate that the endocytic uptake of the CGTs involves a dynamin-dependent process that is mainly governed by clathrin. PMID:20498856

  6. Temperature effect on endocytosis and exocytosis by rabbit alveolar macrophages

    Endocytosis of 125I-mannose-bovine serum albumin (BSA) and exocytosis of 125I-mannose-poly-D-lysine by rabbit alveolar macrophages were examined as a function of temperature. A plot for total ligand uptake (cell-associated ligand plus degraded ligand) versus time shows a single inflection point at 20 degrees C. Ligand degradation does not occur below 20 degrees C. Internalization of surface-bound 125I-mannose-BSA is negligible below 10 degrees C. The rate constant for internalization increases dramatically above 20 degrees C: 0.02 min-1 at 20 degrees C, 0.05 min-1 at 25 degrees C, 0.13 min-1 at 30 degrees C, and 0.29 min-1 at 35 degrees C. 125I-Mannose-N-acetyl-poly-D-lysine preloaded in lysosomes is exocytosed in a temperature and time-dependent fashion. Even at lower temperatures (2-10 degrees C), secretion of 125I-mannose-N-acetyl-poly-D-lysine was detected, indicating that movement of lysosomal content to plasma membrane and beyond cannot be suppressed at these temperatures. Thus, the temperature dependence of exocytosis of an 125I-labeled ligand is quite different from that of endocytosis, suggesting that the two processes are controlled by different mechanisms. Stimulation of secretion of preloaded 125I-mannose-N-acetyl-poly-D-lysine by mannose-BSA was more pronounced at lower temperatures with a sharp inflection point at 10 degrees C. These findings suggest that endosomes containing newly internalized mannose-BSA interact with the exocytosis pathway and enhance secretion of 125I-mannose-N-acetyl-poly-D-lysine from lysosomes

  7. A role for the dynamin-like protein Vps1 during endocytosis in yeast.

    Smaczynska-de Rooij, Iwona I; Allwood, Ellen G; Aghamohammadzadeh, Soheil; Hettema, Ewald H; Goldberg, Martin W; Ayscough, Kathryn R

    2010-10-15

    Dynamins are a conserved family of proteins involved in membrane fusion and fission. Although mammalian dynamins are known to be involved in several membrane-trafficking events, the role of dynamin-1 in endocytosis is the best-characterised role of this protein family. Despite many similarities between endocytosis in yeast and mammalian cells, a comparable role for dynamins in yeast has not previously been demonstrated. The reported lack of involvement of dynamins in yeast endocytosis has raised questions over the general applicability of the current yeast model of endocytosis, and has also precluded studies using well-developed methods in yeast, to further our understanding of the mechanism of dynamin function during endocytosis. Here, we investigate the yeast dynamin-like protein Vps1 and demonstrate a transient burst of localisation to sites of endocytosis. Using live-cell imaging of endocytic reporters in strains lacking vps1, and also electron microscopy and biochemical approaches, we demonstrate a role for Vps1 in facilitating endocytic invagination. Vps1 mutants were generated, and analysis in several assays reveals a role for the C-terminal self-assembly domain in endocytosis but not in other membrane fission events with which Vps1 has previously been associated. PMID:20841380

  8. Clathrin- and dynamin-independent endocytosis of FGFR3--implications for signalling.

    Ellen Margrethe Haugsten

    Full Text Available Endocytosis of tyrosine kinase receptors can influence both the duration and the specificity of the signal emitted. We have investigated the mechanisms of internalization of fibroblast growth factor receptor 3 (FGFR3 and compared it to that of FGFR1 which is internalized predominantly through clathrin-mediated endocytosis. Interestingly, we observed that FGFR3 was internalized at a slower rate than FGFR1 indicating that it may use a different endocytic mechanism than FGFR1. Indeed, after depletion of cells for clathrin, internalization of FGFR3 was only partly inhibited while endocytosis of FGFR1 was almost completely abolished. Similarly, expression of dominant negative mutants of dynamin resulted in partial inhibition of the endocytosis of FGFR3 whereas internalization of FGFR1 was blocked. Interfering with proposed regulators of clathrin-independent endocytosis such as Arf6, flotillin 1 and 2 and Cdc42 did not affect the endocytosis of FGFR1 or FGFR3. Furthermore, depletion of clathrin decreased the degradation of FGFR1 resulting in sustained signalling. In the case of FGFR3, both the degradation and the signalling were only slightly affected by clathrin depletion. The data indicate that clathrin-mediated endocytosis is required for efficient internalization and downregulation of FGFR1 while FGFR3, however, is internalized by both clathrin-dependent and clathrin-independent mechanisms.

  9. Endocytosis of adiponectin receptor 1 through a clathrin-and Rab5-dependent pathway

    Qiurong Ding; Zhenzhen Wang; Yan Chen

    2009-01-01

    In eukaryotic cells, receptor endocytosis is a key event regulating signaling transduction. Adiponectin receptors belong to a new receptor family that is distinct from G-protein-coupled receptors and has critical roles in the pathogen-esis of diabetes and metabolic syndrome. Here, we analyzed the endocytosis of adiponectin and adiponectin receptor 1 (AdipoR1) and found that they are both internalized into transferrin-positive compartments that follow similar traffic routes. Blocking clathrin-mediated endocytosis by expressing Epsl5 mutants or depleting K+ trapped AdipoRl at the plasma membrane, and K+ depletion abolished adiponectin internalization, indicating that the endocytosis of AdipoRl and adiponectin is clathrin-dependent. Depletion of K+ and overexpression of Eps15 mutants enhance adiponectin-stimulated AMP-activated protein kinase phosphorylation, suggesting that the endocytosis of AdipoR1 might down-regulate adiponectin signaling. In addition, AdipoR1 colocalizes with the small GTPase Rab5, and a dominant negative Rab5 abrogates AdipoR1 endocytosis. These data indicate that AdipoRl is internalized through a clathrin- and Rab5-dependent pathway and that endocytosis may play a role in the regulation of adiponectin signaling.

  10. Ultrasound Microbubble Treatment Enhances Clathrin-Mediated Endocytosis and Fluid-Phase Uptake through Distinct Mechanisms

    Fekri, Farnaz; Delos Santos, Ralph Christian; Karshafian, Raffi

    2016-01-01

    Drug delivery to tumors is limited by several factors, including drug permeability of the target cell plasma membrane. Ultrasound in combination with microbubbles (USMB) is a promising strategy to overcome these limitations. USMB treatment elicits enhanced cellular uptake of materials such as drugs, in part as a result of sheer stress and formation of transient membrane pores. Pores formed upon USMB treatment are rapidly resealed, suggesting that other processes such as enhanced endocytosis may contribute to the enhanced material uptake by cells upon USMB treatment. How USMB regulates endocytic processes remains incompletely understood. Cells constitutively utilize several distinct mechanisms of endocytosis, including clathrin-mediated endocytosis (CME) for the internalization of receptor-bound macromolecules such as Transferrin Receptor (TfR), and distinct mechanism(s) that mediate the majority of fluid-phase endocytosis. Tracking the abundance of TfR on the cell surface and the internalization of its ligand transferrin revealed that USMB acutely enhances the rate of CME. Total internal reflection fluorescence microscopy experiments revealed that USMB treatment altered the assembly of clathrin-coated pits, the basic structural units of CME. In addition, the rate of fluid-phase endocytosis was enhanced, but with delayed onset upon USMB treatment relative to the enhancement of CME, suggesting that the two processes are distinctly regulated by USMB. Indeed, vacuolin-1 or desipramine treatment prevented the enhancement of CME but not of fluid phase endocytosis upon USMB, suggesting that lysosome exocytosis and acid sphingomyelinase, respectively, are required for the regulation of CME but not fluid phase endocytosis upon USMB treatment. These results indicate that USMB enhances both CME and fluid phase endocytosis through distinct signaling mechanisms, and suggest that strategies for potentiating the enhancement of endocytosis upon USMB treatment may improve targeted

  11. Ultrasound Microbubble Treatment Enhances Clathrin-Mediated Endocytosis and Fluid-Phase Uptake through Distinct Mechanisms.

    Fekri, Farnaz; Delos Santos, Ralph Christian; Karshafian, Raffi; Antonescu, Costin N

    2016-01-01

    Drug delivery to tumors is limited by several factors, including drug permeability of the target cell plasma membrane. Ultrasound in combination with microbubbles (USMB) is a promising strategy to overcome these limitations. USMB treatment elicits enhanced cellular uptake of materials such as drugs, in part as a result of sheer stress and formation of transient membrane pores. Pores formed upon USMB treatment are rapidly resealed, suggesting that other processes such as enhanced endocytosis may contribute to the enhanced material uptake by cells upon USMB treatment. How USMB regulates endocytic processes remains incompletely understood. Cells constitutively utilize several distinct mechanisms of endocytosis, including clathrin-mediated endocytosis (CME) for the internalization of receptor-bound macromolecules such as Transferrin Receptor (TfR), and distinct mechanism(s) that mediate the majority of fluid-phase endocytosis. Tracking the abundance of TfR on the cell surface and the internalization of its ligand transferrin revealed that USMB acutely enhances the rate of CME. Total internal reflection fluorescence microscopy experiments revealed that USMB treatment altered the assembly of clathrin-coated pits, the basic structural units of CME. In addition, the rate of fluid-phase endocytosis was enhanced, but with delayed onset upon USMB treatment relative to the enhancement of CME, suggesting that the two processes are distinctly regulated by USMB. Indeed, vacuolin-1 or desipramine treatment prevented the enhancement of CME but not of fluid phase endocytosis upon USMB, suggesting that lysosome exocytosis and acid sphingomyelinase, respectively, are required for the regulation of CME but not fluid phase endocytosis upon USMB treatment. These results indicate that USMB enhances both CME and fluid phase endocytosis through distinct signaling mechanisms, and suggest that strategies for potentiating the enhancement of endocytosis upon USMB treatment may improve targeted

  12. [Molecular physiology of receptor mediated endocytosis and its role in overcoming multidrug resistance].

    Severin, E S; Posypanova, G A

    2011-06-01

    Receptor-mediated endocytosis plays important role in the selective uptake of proteins at the plasma membrane of eukaryotic cells. Endocytosis regulates many processes of cell signalling by controlling the number of functional receptors on the cell surface. The article reviews the mechanism of clathrin-dependent endocytosis and the possibility of using this phenomenon for the targeted delivery of drugs. Use of certain proteins as targeting component of drug delivery systems can significantly improve the selectivity of this drug, as well as to overcome the multidrug resistance of cells resulting from the activity of the ABC-transporters. PMID:21874867

  13. A luminescent assay for real-time measurements of receptor endocytosis in living cells.

    Robers, Matthew B; Binkowski, Brock F; Cong, Mei; Zimprich, Chad; Corona, Cesear; McDougall, Mark; Otto, George; Eggers, Christopher T; Hartnett, Jim; Machleidt, Thomas; Fan, Frank; Wood, Keith V

    2015-11-15

    Ligand-mediated endocytosis is a key autoregulatory mechanism governing the duration and intensity of signals emanating from cell surface receptors. Due to the mechanistic complexity of endocytosis and its emerging relevance in disease, simple methods capable of tracking this dynamic process in cells have become increasingly desirable. We have developed a bioluminescent reporter technology for real-time analysis of ligand-mediated receptor endocytosis using genetic fusions of NanoLuc luciferase with various G-protein-coupled receptors (GPCRs). This method is compatible with standard microplate formats, which should decrease work flows for high-throughput screens. This article also describes the application of this technology to endocytosis of epidermal growth factor receptor (EGFR), demonstrating potential applicability of the method beyond GPCRs. PMID:26278171

  14. Sensing the delivery and endocytosis of nanoparticles using magneto-photo-acoustic imaging

    M. Qu

    2015-09-01

    Full Text Available Many biomedical applications necessitate a targeted intracellular delivery of the nanomaterial to specific cells. Therefore, a non-invasive and reliable imaging tool is required to detect both the delivery and cellular endocytosis of the nanoparticles. Herein, we demonstrate that magneto-photo-acoustic (MPA imaging can be used to monitor the delivery and to identify endocytosis of magnetic and optically absorbing nanoparticles. The relationship between photoacoustic (PA and magneto-motive ultrasound (MMUS signals from the in vitro samples were analyzed to identify the delivery and endocytosis of nanoparticles. The results indicated that during the delivery of nanoparticles to the vicinity of the cells, both PA and MMUS signals are almost linearly proportional. However, accumulation of nanoparticles within the cells leads to nonlinear MMUS-PA relationship, due to non-linear MMUS signal amplification. Therefore, through longitudinal MPA imaging, it is possible to monitor the delivery of nanoparticles and identify the endocytosis of the nanoparticles by living cells.

  15. Bradykinin release avoids high molecular weight kininogen endocytosis.

    Igor Z Damasceno

    Full Text Available Human H-kininogen (120 kDa plays a role in many pathophysiological processes and interacts with the cell surface through protein receptors and proteoglycans, which mediate H-kininogen endocytosis. In the present work we demonstrate that H-kininogen containing bradykinin domain is internalized and different endogenous kininogenases are present in CHO-K1 cells. We used CHO-K1 (wild type and CHO-745 (mutant deficient in proteoglycans biosynthesis cell lines. H-kininogen endocytosis was studied using confocal microscopy, and its hydrolysis by cell lysate fraction was determined by immunoblotting. Bradykinin release was also measured by radioimmunoassay. H-kininogen interaction with the cell surface of CHO-745 cells resulted in bradykinin release by serine proteases. In CHO-K1 cells, which produce heparan and chondroitin sulfate proteoglycans, internalization of H-kininogen through its bradykinin domain can occur on lipid raft domains/caveolae. Nevertheless bradykinin-free H-kininogen was not internalized by CHO-K1 cells. The H-kininogen present in acidic endosomal vesicles in CHO-K1 was approximately 10-fold higher than the levels in CHO-745. CHO-K1 lysate fractions were assayed at pH 5.5 and intact H-kininogen was totally hydrolyzed into a 62 kDa fragment. By contrast, at an assay pH 7.4, the remained fragments were 115 kDa, 83 kDa, 62 kDa and 48 kDa in size. The antipain-Sepharose chromatography separated endogenous kininogenases from CHO-K1 lysate fraction. No difference was detected in the assays at pH 5.5 or 7.4, but the proteins in the fraction bound to the resin released bradykinin from H-kininogen. However, the proteins in the unbound fraction cleaved intact H-kininogen at other sites but did not release bradykinin. H-kininogen can interact with extravascular cells, and is internalized dependent on its bradykinin domain and cell surface proteoglycans. After internalization, H-kininogen is proteolytically processed by intracellular

  16. Discovery of New Cargo Proteins that enter Cells through Clathrin-Independent Endocytosis

    Eyster, Craig A.; Higginson, Jason D.; Huebner, Robert; Porat-Shliom, Natalie; Weigert, Roberto; Wu, Wells W.; Shen, Rong-Fong; Donaldson, Julie G.

    2009-01-01

    Clathrin-independent endocytosis (CIE) allows internalization of plasma membrane proteins lacking clathrin-targeting sequences, such as the major histocompatibility complex Class I protein (MHCI), into cells. After internalization, vesicles containing MHCI fuse with transferrin-containing endosomes generated from clathrin-dependent endocytosis. In HeLa cells, MHCI is subsequently routed to late endosomes or recycled back out to the plasma membrane (PM) in distinctive tubular carriers. Arf6 is...

  17. Clathrin-mediated endocytosis at the synaptic terminal: bridging the gap between physiology and molecules

    Royle, Stephen J; Lagnado, Leon

    2010-01-01

    It has long been known that the maintenance of fast communication between neurons requires that presynaptic terminals recycle the small vesicles from which neurotransmitter is released. But the mechanisms that retrieve vesicles from the cell surface are still not understood. Although we have a wealth of information about the molecular details of endocytosis in non-neuronal cells, it is clear that endocytosis at the synapse is faster and regulated in distinct ways. A satisfying understanding o...

  18. Bile Acids Reduce Endocytosis of High-Density Lipoprotein (HDL) in HepG2 Cells

    Clemens Röhrl; Karin Eigner; Stefanie Fruhwürth; Herbert Stangl

    2014-01-01

    High-density lipoprotein (HDL) transports lipids to hepatic cells and the majority of HDL-associated cholesterol is destined for biliary excretion. Cholesterol is excreted into the bile directly or after conversion to bile acids, which are also present in the plasma as they are effectively reabsorbed through the enterohepatic cycle. Here, we provide evidence that bile acids affect HDL endocytosis. Using fluorescent and radiolabeled HDL, we show that HDL endocytosis was reduced in the presence...

  19. Mechanistic analysis of massive endocytosis in relation to functionally defined surface membrane domains

    Hilgemann, Donald W.; Fine, Michael

    2011-01-01

    A large fraction of endocytosis in eukaryotic cells occurs without adaptors or dynamins. Here, we present evidence for the involvement of lipid domains in massive endocytosis (MEND) activated by both large Ca transients and amphipathic compounds in baby hamster kidney and HEK293 cells. First, we demonstrate functional coupling of the two MEND types. Ca transients can strongly facilitate detergent-activated MEND. Conversely, an amphipath with dual alkyl chains, ditridecylphthalate, is without ...

  20. Cytosol- and clathrin-dependent stimulation of endocytosis in vitro by purified adaptors

    1992-01-01

    Using stage-specific assays for receptor-mediated endocytosis of transferrin (Tfn) into perforated A431 cells we show that purified adaptors stimulate coated pit assembly and ligand sequestration into deeply invaginated coated pits. Late events in endocytosis involving membrane fission and coated vesicle budding which lead to the internalization of Tfn are unaffected. AP2, plasma membrane adaptors, are active at physiological concentrations, whereas AP1, Golgi adaptors, are inactive. Adaptor-...

  1. Quantification of endocytosis using a folate functionalized silica hollow nanoshell platform.

    Sandoval, Sergio; Mendez, Natalie; Alfaro, Jesus G; Yang, Jian; Aschemeyer, Sharraya; Liberman, Alex; Trogler, William C; Kummel, Andrew C

    2015-08-01

    A quantification method to measure endocytosis was designed to assess cellular uptake and specificity of a targeting nanoparticle platform. A simple N -hydroxysuccinimide ester conjugation technique to functionalize 100-nm hollow silica nanoshell particles with fluorescent reporter fluorescein isothiocyanate and folate or polyethylene glycol (PEG) was developed. Functionalized nanoshells were characterized using scanning electron microscopy and transmission electron microscopy and the maximum amount of folate functionalized on nanoshell surfaces was quantified with UV-Vis spectroscopy. The extent of endocytosis by HeLa cervical cancer cells and human foreskin fibroblast (HFF-1) cells was investigated in vitro using fluorescence and confocal microscopy. A simple fluorescence ratio analysis was developed to quantify endocytosis versus surface adhesion. Nanoshells functionalized with folate showed enhanced endocytosis by cancer cells when compared to PEG functionalized nanoshells. Fluorescence ratio analyses showed that 95% of folate functionalized silica nanoshells which adhered to cancer cells were endocytosed, while only 27% of PEG functionalized nanoshells adhered to the cell surface and underwent endocytosis when functionalized with 200 and 900  μg , respectively. Additionally, the endocytosis of folate functionalized nanoshells proved to be cancer cell selective while sparing normal cells. The developed fluorescence ratio analysis is a simple and rapid verification/validation method to quantify cellular uptake between datasets by using an internal control for normalization. PMID:26315280

  2. Ouabain uptake by endocytosis in isolated guinea pig atria

    Mammalian cells specifically internalize some molecular species through receptor-mediated endocytosis (RME). The authors have used four different experimental protocols to investigate whether ouabain enters cardiac cells of guinea pig atrium through this pathway. First, by electron microscope morphometry the authors found that ouabain increased endocytic vesicles in atrial cells. Second, by scintillation counting they found that [3H]ouabain uptake by the tissue is decreased by three treatments that decrease RME, i.e., NH4Cl, trifluoperazine, and 16 mM [K+]0. Third, by radioautography at the electron microscope level, they checked that in preceding experiments [3H]ouabain was washed out of plasma membrane after 60-min rinse and interiorized into the cardiac cells. Fourth, isometric tension recordings showed that the positive inotropic effect of ouabain was diminished in the presence of inhibitors, whereas that of a hydrophobic analogue, ouabagenin, was not affected. These results suggest that ouabain enters cardiac cells through RME and also that an intracellular site may, at least in part, be responsible for its inotropic effect

  3. A yeast t-SNARE involved in endocytosis.

    Séron, K; Tieaho, V; Prescianotto-Baschong, C; Aust, T; Blondel, M O; Guillaud, P; Devilliers, G; Rossanese, O W; Glick, B S; Riezman, H; Keränen, S; Haguenauer-Tsapis, R

    1998-10-01

    The ORF YOL018c (TLG2) of Saccharomyces cerevisiae encodes a protein that belongs to the syntaxin protein family. The proteins of this family, t-SNAREs, are present on target organelles and are thought to participate in the specific interaction between vesicles and acceptor membranes in intracellular membrane trafficking. TLG2 is not an essential gene, and its deletion does not cause defects in the secretory pathway. However, its deletion in cells lacking the vacuolar ATPase subunit Vma2p leads to loss of viability, suggesting that Tlg2p is involved in endocytosis. In tlg2Delta cells, internalization was normal for two endocytic markers, the pheromone alpha-factor and the plasma membrane uracil permease. In contrast, degradation of alpha-factor and uracil permease was delayed in tlg2Delta cells. Internalization of positively charged Nanogold shows that the endocytic pathway is perturbed in the mutant, which accumulates Nanogold in primary endocytic vesicles and shows a greatly reduced complement of early endosomes. These results strongly suggest that Tlg2p is a t-SNARE involved in early endosome biogenesis. PMID:9763449

  4. β-Hydroxybutyrate supports synaptic vesicle cycling but reduces endocytosis and exocytosis in rat brain synaptosomes.

    Hrynevich, Sviatlana V; Waseem, Tatyana V; Hébert, Audrey; Pellerin, Luc; Fedorovich, Sergei V

    2016-02-01

    The ketogenic diet is used as a prophylactic treatment for different types of brain diseases, such as epilepsy or Alzheimer's disease. In such a diet, carbohydrates are replaced by fats in everyday food, resulting in an elevation of blood-borne ketone bodies levels. Despite clinical applications of this treatment, the molecular mechanisms by which the ketogenic diet exerts its beneficial effects are still uncertain. In this study, we investigated the effect of replacing glucose by the ketone body β-hydroxybutyrate as the main energy substrate on synaptic vesicle recycling in rat brain synaptosomes. First, we observed that exposing presynaptic terminals to nonglycolytic energy substrates instead of glucose did not alter the plasma membrane potential. Next, we found that synaptosomes were able to maintain the synaptic vesicle cycle monitored with the fluorescent dye acridine orange when glucose was replaced by β-hydroxybutyrate. However, in presence of β-hydroxybutyrate, synaptic vesicle recycling was modified with reduced endocytosis. Replacing glucose by pyruvate also led to a reduced endocytosis. Addition of β-hydroxybutyrate to glucose-containing incubation medium was without effect. Reduced endocytosis in presence of β-hydroxybutyrate as sole energy substrate was confirmed using the fluorescent dye FM2-10. Also we found that replacement of glucose by ketone bodies leads to inhibition of exocytosis, monitored by FM2-10. However this reduction was smaller than the effect on endocytosis under the same conditions. Using both acridine orange in synaptosomes and the genetically encoded sensor synaptopHluorin in cortical neurons, we observed that replacing glucose by β-hydroxybutyrate did not modify the pH gradient of synaptic vesicles. In conclusion, the nonglycolytic energy substrates β-hydroxybutyrate and pyruvate are able to support synaptic vesicle recycling. However, they both reduce endocytosis. Reduction of both endocytosis and exocytosis together with

  5. Calcyon is Necessary for Activity Dependent AMPA Receptor Internalization and LTD in CA1 Neurons of Hippocampus

    Davidson, Heather Trantham; Xiao, Jiping; Dai, Rujuan; Bergson, Clare

    2009-01-01

    Calcyon is a single transmembrane endocytic protein that regulates clathrin assembly and clathrin mediated endocytosis in brain. Ultrastructural studies indicate that calcyon localizes to spines, but whether it regulates glutamate neurotransmission is not known. Here, we show that deletion of the calcyon gene in mice inhibits agonist stimulated endocytosis of AMPA receptors, without altering basal surface levels of the GluR1 or GluR2 subunits. Whole cell patch clamp studies of hippocampal neu...

  6. Endocytosis and intracellular protein degradation in cystic fibrosis fibroblasts

    Normal rates of pinocytosis of [3H]sucrose were measured in cystic fibrosis fibroblasts, and were not affected by the addition of cystic fibrosis serum. Bulk protein degradation (a significant proportion of which occurs intralysosomally following autophagy) and its regulation by growth state were apparently identical in normal and cystic fibrosis cultures. (Auth.)

  7. HSV-1 Glycoproteins Are Delivered to Virus Assembly Sites Through Dynamin-Dependent Endocytosis.

    Albecka, Anna; Laine, Romain F; Janssen, Anne F J; Kaminski, Clemens F; Crump, Colin M

    2016-01-01

    Herpes simplex virus-1 (HSV-1) is a large enveloped DNA virus that belongs to the family of Herpesviridae. It has been recently shown that the cytoplasmic membranes that wrap the newly assembled capsids are endocytic compartments derived from the plasma membrane. Here, we show that dynamin-dependent endocytosis plays a major role in this process. Dominant-negative dynamin and clathrin adaptor AP180 significantly decrease virus production. Moreover, inhibitors targeting dynamin and clathrin lead to a decreased transport of glycoproteins to cytoplasmic capsids, confirming that glycoproteins are delivered to assembly sites via endocytosis. We also show that certain combinations of glycoproteins colocalize with each other and with the components of clathrin-dependent and -independent endocytosis pathways. Importantly, we demonstrate that the uptake of neutralizing antibodies that bind to glycoproteins when they become exposed on the cell surface during virus particle assembly leads to the production of non-infectious HSV-1. Our results demonstrate that transport of viral glycoproteins to the plasma membrane prior to endocytosis is the major route by which these proteins are localized to the cytoplasmic virus assembly compartments. This highlights the importance of endocytosis as a major protein-sorting event during HSV-1 envelopment. PMID:26459807

  8. Characterization of endocytosis and exocytosis of cationic nanoparticles in airway epithelium cells

    Dombu, Christophe Youta; Kroubi, Maya; Zibouche, Rima; Matran, Regis; Betbeder, Didier, E-mail: dbetbeder@aol.com [EA 4483, IFR 114, Laboratoire de Physiologie, Faculte de Medecine Pole Recherche, Universite de Lille 2, 1 place de Verdun, 59045 Lille Cedex (France)

    2010-09-03

    A major challenge of drug delivery using colloids via the airway is to understand the mechanism implied in their interactions with epithelial cells. The purpose of this work was to characterize the process of endocytosis and exocytosis of cationic nanoparticles (NPs) made of maltodextrin which were developed as a delivery system for antigens in vaccine applications. Confocal microscopy demonstrated that these NP are rapidly endocytosed after as little as 3 min incubation, and that the endocytosis was also faster than NP binding since most of the NPs were found in the middle of the cells around the nuclei. A saturation limit was observed after a 40 min incubation, probably due to an equilibrium becoming established between endocytosis and exocytosis. Endocytosis was dramatically reduced at 4 deg. C compared with 37 deg. C, or by NaN{sub 3} treatment, both results suggesting an energy dependent process. Protamine pretreatment of the cells inhibited NPs uptake and we found that clathrin pathway is implied in their endocytosis. Cholesterol depletion increased NP uptake by 300% and this phenomenon was explained by the fact that cholesterol depletion totally blocked NP exocytosis. These results suggest that these cationic NPs interact with anionic sites, are quickly endocytosed via the clathrin pathway and that their exocytosis is cholesterol dependent, and are similar to those obtained in other studies with viruses such as influenza.

  9. Endocytosis as a biological response in receptor pharmacology: evaluation by fluorescence microscopy.

    Víctor M Campa

    Full Text Available The activation of G-protein coupled receptors by agonist compounds results in diverse biological responses in cells, such as the endocytosis process consisting in the translocation of receptors from the plasma membrane to the cytoplasm within internalizing vesicles or endosomes. In order to functionally evaluate endocytosis events resulted from pharmacological responses, we have developed an image analysis method -the Q-Endosomes algorithm- that specifically discriminates the fluorescent signal originated at endosomes from that one observed at the plasma membrane in images obtained from living cells by fluorescence microscopy. Mu opioid (MOP receptor tagged at the carboxy-terminus with yellow fluorescent protein (YFP and permanently expressed in HEK293 cells was used as experimental model to validate this methodology. Time-course experiments performed with several agonists resulted in different sigmoid curves depending on the drug used to initiate MOP receptor endocytosis. Thus, endocytosis resulting from the simultaneous activation of co-expressed MOP and serotonin 5-HT2C receptors by morphine plus serotonin was significantly different, in kinetics as well as in maximal response parameters, from the one caused by DAMGO, sufentanyl or methadone. Therefore, this analytical tool permits the pharmacological characterization of receptor endocytosis in living cells with functional and temporal resolution.

  10. On the ultrastructure of Trichomonas vaginalis: cytoskeleton, endocytosis and hydrogenosomes

    SIXTO RAUL COSTAMAGNA

    2001-07-01

    Full Text Available This paper is focused on the study of the ultrastructure of Trichomonas vaginalis in liquid cultures. Its cytoskeleton, the morphology of its hydrogenosomes, and endocytosis phenomena have been observed by scanning electron microscopy (SEM and transmission electron microscopy (TEM. For the present study, the traditional techniques for SEM and TEM have been slightly modified for the processing of this flagellate. Following our experiments, it can be concluded that: 1. The modified techniques are adequate for the ultrastructural study of this protozoon. 2. There are no mitochondria in T. vaginalis. 3. T. vaginalis might use micropinocytosis processes related to coated vesicles as a habitual endo- and exocytosis mechanism, while phagocytosis is observed for major vesicles. 4. As to the cytoskeleton, microtubules are numerous and display different structures, which are analyzed in this paper. 5. Many hydrogenosomes are found in the cytoplasm of T. vaginalis underneath the undulating membrane and along the axostyle, each with electron-dense deposits in the manner of «operculums.»El presente estudio está referido a la ultraestructura de Trichomonas vaginalis, cultivadas en medio líquido. Su citoesqueleto, fenómenos de endocitosis y la morfología de sus hidrogenosomas fueron observados por microscopía electrónica de barrido y de transmisión. Las técnicas clásicas para el procesamiento al SEM y TEM de este flagelado, fueron discreta y sutilmente modificadas por nosotros. Como resultado de nuestras experiencias se concluye que: 1. Las técnicas modificadas empleadas son adecuadas para el estudio morfológico y ultraestructural de este Protozoo. 2. Al TEM su citoplasma no muestra mitocondrias. 3. Utiliza los fenómenos de micropinocitosis asociados con vesículas con cubierta como mecanismo habitual de endo y exocitosis selectiva, mientras que para partículas mayores la fagocitosis es frecuentemente vista. 4. Con referencia al citoesqueleto

  11. Caveolae-mediated endocytosis of biocompatible gold nanoparticles in living Hela cells

    Efficient intracellular delivery of gold nanoparticles (AuNPs) and unraveling the mechanism underlying the intracellular delivery are essential for advancing the applications of AuNPs toward in vivo imaging and therapeutic interventions. We employed fluorescence microscopy to investigate the internalization mechanism of small-size AuNPs by living Hela cells. Herein, we found that the caveolae-mediated endocytosis was the dominant pathway for the intracellular delivery of small-size AuNPs. The intracellular delivery was suppressed when we depleted the cholesterol with methyl-β-cyclodextrin (MβCD); in contrast, the sucrose that disrupts the formation of clathrin-mediated endocytosis did not block the endocytosis of AuNPs. Meanwhile, we examined the intracellular localization of AuNPs in endocytic vesicles by fluorescent colocalization. This work would provide a potential technique to study the intracellular delivery of small-size nanoparticles for biomedical applications. (paper)

  12. Impaired activity-dependent neural circuit assembly and refinement in autism spectrum disorder genetic models

    Caleb Andrew Doll; Kendal eBroadie

    2014-01-01

    Early-use activity during circuit-specific critical periods refines brain circuitry by the coupled processes of eliminating inappropriate synapses and strengthening maintained synapses. We theorize these activity-dependent developmental processes are specifically impaired in autism spectrum disorders (ASDs). ASD genetic models in both mouse and Drosophila have pioneered our insights into normal activity-dependent neural circuit assembly and consolidation, and how these developmental mechanism...

  13. Drosophila king tubby (ktub mediates light-induced rhodopsin endocytosis and retinal degeneration

    Chen Shu-Fen

    2012-12-01

    Full Text Available Background The tubby (tub and tubby-like protein (tulp genes encode a small family of proteins found in many organisms. Previous studies have shown that TUB and TULP genes in mammalian involve in obesity, neural development, and retinal degeneration. The purpose of this study was to investigate the role of Drosophila king tubby (ktub in rhodopsin 1 (Rh1 endocytosis and retinal degeneration upon light stimulation. Results Drosophila ktub mutants were generated using imprecise excision. Wild type and mutant flies were raised in dark or constant light conditions. After a period of light stimulation, retinas were dissected, fixed and stained with anti-Rh1 antibody to reveal Rh1 endocytosis. Confocal and transmission electron microscope were used to examine the retinal degeneration. Immunocytochemical analysis shows that Ktub is expressed in the rhabdomere domain under dark conditions. When flies receive light stimulation, the Ktub translocates from the rhabdomere to the cytoplasm and the nucleus of the photoreceptor cells. Wild type photoreceptors form Rh1-immunopositive large vesicles (RLVs shortly after light stimulation. In light-induced ktub mutants, the majority of Rh1 remains at the rhabdomere, and only a few RLVs appear in the cytoplasm of photoreceptor cells. Mutation of norpA allele causes massive Rh1 endocytosis in light stimulation. In ktub and norpA double mutants, however, Rh1 endocytosis is blocked under light stimulation. This study also shows that ktub and norpA double mutants rescue the light-induced norpA retinal degeneration. Deletion constructs further demonstrate that the Tubby domain of the Ktub protein participates in an important role in Rh1 endocytosis. Conclusions The results in this study delimit the novel function of Ktub in Rh1 endocytosis and retinal degeneration.

  14. Prominin-2 expression increases protrusions, decreases caveolae and inhibits Cdc42 dependent fluid phase endocytosis

    Highlights: •Prominin-2 expression induced protrusions that co-localized with lipid raft markers. •Prominin-2 expression decreased caveolae, caveolar endocytosis and increased pCav1. •Prominin-2 expression inhibited fluid phase endocytosis by inactivation of Cdc42. •These endocytic effects can be reversed by adding exogenous cholesterol. •Caveolin1 knockdown restored fluid phase endocytosis in Prominin2 expressing cells. -- Abstract: Background: Membrane protrusions play important roles in biological processes such as cell adhesion, wound healing, migration, and sensing of the external environment. Cell protrusions are a subtype of membrane microdomains composed of cholesterol and sphingolipids, and can be disrupted by cholesterol depletion. Prominins are pentaspan membrane proteins that bind cholesterol and localize to plasma membrane (PM) protrusions. Prominin-1 is of great interest as a marker for stem and cancer cells, while Prominin-2 (Prom2) is reportedly restricted to epithelial cells. Aim: To characterize the effects of Prom-2 expression on PM microdomain organization. Methods: Prom2-fluorescent protein was transfected in human skin fibroblasts (HSF) and Chinese hamster ovary (CHO) cells for PM raft and endocytic studies. Caveolae at PM were visualized using transmission electron microscopy. Cdc42 activation was measured and caveolin-1 knockdown was performed using siRNAs. Results: Prom2 expression in HSF and CHO cells caused extensive Prom2-positive protrusions that co-localized with lipid raft markers. Prom2 expression significantly decreased caveolae at the PM, reduced caveolar endocytosis and increased caveolin-1 phosphorylation. Prom2 expression also inhibited Cdc42-dependent fluid phase endocytosis via decreased Cdc42 activation. Effects on endocytosis were reversed by addition of cholesterol. Knockdown of caveolin-1 by siRNA restored Cdc42 dependent fluid phase endocytosis in Prom2-expressing cells. Conclusions: Prom2 protrusions primarily

  15. Prominin-2 expression increases protrusions, decreases caveolae and inhibits Cdc42 dependent fluid phase endocytosis

    Singh, Raman Deep, E-mail: Takhter.Ramandeep@mayo.edu; Schroeder, Andreas S.; Scheffer, Luana; Holicky, Eileen L.; Wheatley, Christine L.; Marks, David L., E-mail: Marks.david@mayo.edu; Pagano, Richard E.

    2013-05-10

    Highlights: •Prominin-2 expression induced protrusions that co-localized with lipid raft markers. •Prominin-2 expression decreased caveolae, caveolar endocytosis and increased pCav1. •Prominin-2 expression inhibited fluid phase endocytosis by inactivation of Cdc42. •These endocytic effects can be reversed by adding exogenous cholesterol. •Caveolin1 knockdown restored fluid phase endocytosis in Prominin2 expressing cells. -- Abstract: Background: Membrane protrusions play important roles in biological processes such as cell adhesion, wound healing, migration, and sensing of the external environment. Cell protrusions are a subtype of membrane microdomains composed of cholesterol and sphingolipids, and can be disrupted by cholesterol depletion. Prominins are pentaspan membrane proteins that bind cholesterol and localize to plasma membrane (PM) protrusions. Prominin-1 is of great interest as a marker for stem and cancer cells, while Prominin-2 (Prom2) is reportedly restricted to epithelial cells. Aim: To characterize the effects of Prom-2 expression on PM microdomain organization. Methods: Prom2-fluorescent protein was transfected in human skin fibroblasts (HSF) and Chinese hamster ovary (CHO) cells for PM raft and endocytic studies. Caveolae at PM were visualized using transmission electron microscopy. Cdc42 activation was measured and caveolin-1 knockdown was performed using siRNAs. Results: Prom2 expression in HSF and CHO cells caused extensive Prom2-positive protrusions that co-localized with lipid raft markers. Prom2 expression significantly decreased caveolae at the PM, reduced caveolar endocytosis and increased caveolin-1 phosphorylation. Prom2 expression also inhibited Cdc42-dependent fluid phase endocytosis via decreased Cdc42 activation. Effects on endocytosis were reversed by addition of cholesterol. Knockdown of caveolin-1 by siRNA restored Cdc42 dependent fluid phase endocytosis in Prom2-expressing cells. Conclusions: Prom2 protrusions primarily

  16. Confocal microscopy of FM4-64 as a tool for analysing endocytosis and vesicle trafficking in living fungal hyphae

    Fischer-Parton, S; Parton, R M; Hickey, P C; Dijksterhuis, J; Atkinson, H A; Read, N D

    2000-01-01

    Confocal microscopy of amphiphilic styryl dyes has been used to investigate endocytosis and vesicle trafficking in living fungal hyphae. Hyphae were treated with FM4-64, FM1-43 or TMA-DPH, three of the most commonly used membrane-selective dyes reported as markers of endocytosis. All three dyes were

  17. Trypanosoma cruzi: antigen-receptor mediated endocytosis of antibody

    Judith Abelha

    1981-06-01

    Full Text Available Trypanomastigote forms of Trypanosoma cruzi were derived from tissue culture and incubated with immune and non-immune human sera. All immune sera showed high titers of specific humoral antibodies of the IgM or the IgG type. Agglutination and swelling of parasites were observed after incubation at 37ºC, but many trypomastigotes remained free-swimming in the sera for two to three days. The quantitiy of immune serum capable of lysing a maximum of 10 x 10 [raised to the power of 6] sensitized red cells was not capable of lysing 4 x 10 [raised to the power of 3] tripomastigotes. Typically, the parasites underwent cyclical changes with the formation of clumps of amastigotes and the appearance of epimastigote forms. Multiplication of the parasites was observed in immune sera. Further, the infectivity of the parasites to susceptible mice was not lost. All sera used produced similar general effects on the growth of the parasite. The antibody bound to T. cruzi appeard to enter cells by antigen-receptor mediated endocytosis. The ferritin-conjugated antibody was internalized and delivered to phagolysosomes where they might be completely degraded to amino-acids. This seemed to be a coupled process by which the immunoglobulin is first bound to specific parasite surface receptor and then rapidly endocytosed by the cell.Formas tripomastigotas de Trypanosoma cruzi derivadas de cultura de tecido foram encubadas com soros humanos imunes e não-imunes.Todos os soros humanos usados tinham títulos elevados de anticorpos das classes IgM ou IgG. Aglutinação e entumescimento dos parasitos eram observados apos encubação a 37ºC mas muitos tripomastigotas permaneceram circulando livremente nos soros por dois a três dias. A quantidade de soro imune capaz de lisar um máximo de 10 x 10 [elevado a 6] hemácias sensibilizadas não foi capaz de lisar 4 x 10 [elevado a 3] tripomastigotas. Tipicamente, os parasitos apresentavam alterações cíclicas com formação de

  18. Endophilin mutations block clathrin-mediated endocytosis but not neurotransmitter release

    Verstreken, Patrik; Kjaerulff, Ole; Lloyd, Thomas E;

    2002-01-01

    We have identified mutations in Drosophila endophilin to study its function in vivo. Endophilin is required presynaptically at the neuromuscular junction, and absence of Endophilin dramatically impairs endocytosis in vivo. Mutant larvae that lack Endophilin fail to take up FM1-43 dye in synaptic ...

  19. Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor

    George Kourouniotis

    2016-07-01

    Full Text Available The binding of epidermal growth factor (EGF to EGF receptor (EGFR stimulates cell mitogenesis and survival through various signalling cascades. EGF also stimulates rapid EGFR endocytosis and its eventual degradation in lysosomes. The immediate events induced by ligand binding include receptor dimerization, activation of intrinsic tyrosine kinase and autophosphorylation. However, in spite of intensified efforts, the results regarding the roles of these events in EGFR signalling and internalization is still very controversial. In this study, we constructed a chimeric EGFR by replacing its extracellular domain with leucine zipper (LZ and tagged a green fluorescent protein (GFP at its C-terminus. We showed that the chimeric LZ-EGFR-GFP was constitutively dimerized. The LZ-EGFR-GFP dimer autophosphorylated each of its five well-defined C-terminal tyrosine residues as the ligand-induced EGFR dimer does. Phosphorylated LZ-EGFR-GFP was localized to both the plasma membrane and endosomes, suggesting it is capable of endocytosis. We also showed that LZ-EGFR-GFP activated major signalling proteins including Src homology collagen-like (Shc, extracellular signal-regulated kinase (ERK and Akt. Moreover, LZ-EGFR-GFP was able to stimulate cell proliferation. These results indicate that non-ligand induced dimerization is sufficient to activate EGFR and initiate cell signalling and EGFR endocytosis. We conclude that receptor dimerization is a critical event in EGF-induced cell signalling and EGFR endocytosis.

  20. Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor

    Kourouniotis, George; Wang, Yi; Pennock, Steven; Chen, Xinmei; Wang, Zhixiang

    2016-01-01

    The binding of epidermal growth factor (EGF) to EGF receptor (EGFR) stimulates cell mitogenesis and survival through various signalling cascades. EGF also stimulates rapid EGFR endocytosis and its eventual degradation in lysosomes. The immediate events induced by ligand binding include receptor dimerization, activation of intrinsic tyrosine kinase and autophosphorylation. However, in spite of intensified efforts, the results regarding the roles of these events in EGFR signalling and internalization is still very controversial. In this study, we constructed a chimeric EGFR by replacing its extracellular domain with leucine zipper (LZ) and tagged a green fluorescent protein (GFP) at its C-terminus. We showed that the chimeric LZ-EGFR-GFP was constitutively dimerized. The LZ-EGFR-GFP dimer autophosphorylated each of its five well-defined C-terminal tyrosine residues as the ligand-induced EGFR dimer does. Phosphorylated LZ-EGFR-GFP was localized to both the plasma membrane and endosomes, suggesting it is capable of endocytosis. We also showed that LZ-EGFR-GFP activated major signalling proteins including Src homology collagen-like (Shc), extracellular signal-regulated kinase (ERK) and Akt. Moreover, LZ-EGFR-GFP was able to stimulate cell proliferation. These results indicate that non-ligand induced dimerization is sufficient to activate EGFR and initiate cell signalling and EGFR endocytosis. We conclude that receptor dimerization is a critical event in EGF-induced cell signalling and EGFR endocytosis. PMID:27463710

  1. Silence of synaptotagmin I in INS-1 cells inhibits fast exocytosis and fast endocytosis

    Synaptotagmin I (Syt I) is a Ca2+ sensor for triggering fast synchronized release of neurotransmitters. However, controversy remains whether Syt I is also obligatory for the exocytosis and endocytosis of larger dense core vesicles (LDCVs) in endocrine cells. In this study, we used a short hairpin RNA (shRNA) to silence the expression of Syt I and investigated the roles of Syt I on exocytosis and endocytosis in INS-1 cells. Our results demonstrated that expression of Syt I is remarkably reduced by the Syt I gene targeting shRNA. Using high-time resolution capacitance measurement, we found that the silence of Syt I decreased the calcium sensitivity of fusion of insulin granules and therefore reduced the exocytotic burst triggered by step-like [Ca2+] i elevation. In addition, the occurrence frequency and amplitude of fast endocytosis were remarkably reduced in the silenced cells. We conclude that Syt I not only participates in the Ca2+-sensing of LDCV fusion with plasmalemma, but also plays a crucial role in fast endocytosis in INS-1 cells

  2. NtGNL1a ARF-GEF acts in endocytosis in tobacco cells

    Jelínková, Adriana; Müller, Karel; Pařezová, Markéta; Petrášek, Jan

    2015-01-01

    Roč. 15, NOV 5 (2015), s. 272. ISSN 1471-2229 R&D Projects: GA ČR GPP305/11/P797 Institutional support: RVO:61389030 Keywords : Endocytosis * PIN1 protein trafficking * Inhibitors of endomembrane trafficking Subject RIV: EA - Cell Biology Impact factor: 3.813, year: 2014

  3. Activity-dependent brain-derived neurotrophic factor expression regulates cortistatin-interneurons and sleep behavior

    Martinowich Keri

    2011-03-01

    Full Text Available Abstract Background Sleep homeostasis is characterized by a positive correlation between sleep length and intensity with the duration of the prior waking period. A causal role for brain-derived neurotrophic factor (BDNF in sleep homeostasis has been suggested, but the underlying mechanisms remain unclear. Cortistatin, a neuropeptide expressed primarily in a subset of cortical GABAergic interneurons, is another molecule implicated in sleep homeostasis. Results We confirmed that sleep deprivation leads to an increase in cortical cortistatin mRNA expression. Disruption of activity-dependent BDNF expression in a genetically modified mouse line impairs both baseline levels of cortistatin mRNA as well as its levels following sleep deprivation. Disruption of activity-dependent BDNF also leads to a decrease in sleep time during the active (dark phase. Conclusion Our studies suggest that regulation of cortistatin-expressing interneurons by activity-dependent BDNF expression may contribute to regulation of sleep behavior.

  4. Definition of a Bidirectional Activity-Dependent Pathway Involving BDNF and Narp

    Abigail Mariga

    2015-12-01

    Full Text Available One of the cardinal features of neural development and adult plasticity is the contribution of activity-dependent signaling pathways. However, the interrelationships between different activity-dependent genes are not well understood. The immediate early gene neuronal-activity-regulated pentraxin (NPTX2 or Narp encodes a protein that has been associated with excitatory synaptogenesis, AMPA receptor aggregation, and the onset of critical periods. Here, we show that Narp is a direct transcriptional target of brain-derived neurotrophic factor (BDNF, another highly regulated activity-dependent gene involved in synaptic plasticity. Unexpectedly, Narp is bidirectionally regulated by BDNF. Acute BDNF withdrawal results in downregulation of Narp, whereas transcription of Narp is greatly enhanced by BDNF. Furthermore, our results show that BDNF directly regulates Narp to mediate glutamatergic transmission and mossy fiber plasticity. Hence, Narp serves as a significant epistatic target of BDNF to regulate synaptic plasticity during periods of dynamic activity.

  5. Listeria monocytogenes internalin B activates junctional endocytosis to accelerate intestinal invasion.

    Mickey Pentecost

    2010-05-01

    Full Text Available Listeria monocytogenes (Lm uses InlA to invade the tips of the intestinal villi, a location at which cell extrusion generates a transient defect in epithelial polarity that exposes the receptor for InlA, E-cadherin, on the cell surface. As the dying cell is removed from the epithelium, the surrounding cells reorganize to form a multicellular junction (MCJ that Lm exploits to find its basolateral receptor and invade. By examining individual infected villi using 3D-confocal imaging, we uncovered a novel role for the second major invasin, InlB, during invasion of the intestine. We infected mice intragastrically with isogenic strains of Lm that express or lack InlB and that have a modified InlA capable of binding murine E-cadherin and found that Lm lacking InlB invade the same number of villi but have decreased numbers of bacteria within each infected villus tip. We studied the mechanism of InlB action at the MCJs of polarized MDCK monolayers and find that InlB does not act as an adhesin, but instead accelerates bacterial internalization after attachment. InlB locally activates its receptor, c-Met, and increases endocytosis of junctional components, including E-cadherin. We show that MCJs are naturally more endocytic than other sites of the apical membrane, that endocytosis and Lm invasion of MCJs depends on functional dynamin, and that c-Met activation by soluble InlB or hepatocyte growth factor (HGF increases MCJ endocytosis. Also, in vivo, InlB applied through the intestinal lumen increases endocytosis at the villus tips. Our findings demonstrate a two-step mechanism of synergy between Lm's invasins: InlA provides the specificity of Lm adhesion to MCJs at the villus tips and InlB locally activates c-Met to accelerate junctional endocytosis and bacterial invasion of the intestine.

  6. Effect of sulfur dioxide on pulmonary macrophage endocytosis at rest and during exercise

    Inhaled SO2 may cause damage by injuring upper airways. To what extent can SO2 also alter pulmonary macrophage function in the parenchyma and what is the impact of exercise? We studied the effect of SO2 on pulmonary macrophage endocytosis in resting and in exercising animals by measuring the rates of macrophage endocytosis in situ for 1 h of a test particle of insoluble radioactive colloidal gold (198Au), 1, 24, or 48 h after inhalation exposure to SO2. Resting hamsters exposed for 4 h to 50 ppm SO2 had no significant reduction in macrophage endocytosis compared with air-breathing control hamsters. However, if hamsters were exposed to the same concentration of SO2 while continuously running (40 min at 0.9 km/h), macrophage endocytosis was significantly reduced 1 h after exposure even though the exposure time was only one-sixth as long. Twenty-four hours later, the percentage of gold ingested by pulmonary macrophages remained significantly depressed. By 48 h, the rate had returned to control values. Exercise alone did not affect endocytosis. Hamsters exposed to 50 ppm SO2, with or without exercise, also showed significant reductions in the number of lavaged macrophages. This decrease was greatest and most persistent in the SO2 plus exercise group. These data indicate that even when animals are exposed to water-soluble gases, which are normally removed by the upper airways, exercise can potentiate damage to more peripheral components of the pulmonary defense system such as the macrophage

  7. Phosphorylation decreases ubiquitylation of the thiazide-sensitive cotransporter NCC and subsequent clathrin-mediated endocytosis.

    Rosenbaek, Lena L; Kortenoeven, Marleen L A; Aroankins, Takwa S; Fenton, Robert A

    2014-05-01

    The thiazide-sensitive sodium chloride cotransporter, NCC, is the major NaCl transport protein in the distal convoluted tubule (DCT). The transport activity of NCC can be regulated by phosphorylation, but knowledge of modulation of NCC trafficking by phosphorylation is limited. In this study, we generated novel tetracycline-inducible Madin-Darby canine kidney type I (MDCKI) cell lines expressing NCC to examine the role of NCC phosphorylation and ubiquitylation on NCC endocytosis. In MDCKI-NCC cells, NCC was highly glycosylated at molecular weights consistent with NCC monomers and dimers. NCC constitutively cycles to the apical plasma membrane of MDCKI-NCC cells, with 20-30% of the membrane pool of NCC internalized within 30 min. The use of dynasore, PitStop2, methyl-β-cyclodextrin, nystatin, and filipin (specific inhibitors of either clathrin-dependent or -independent endocytosis) demonstrated that NCC is internalized via a clathrin-mediated pathway. Reduction of endocytosis resulted in greater levels of NCC in the plasma membrane. Immunogold electron microscopy confirmed the association of NCC with the clathrin-mediated internalization pathway in rat DCT cells. Compared with controls, inducing phosphorylation of NCC via low chloride treatment or mimicking phosphorylation by replacing Thr-53, Thr-58, and Ser-71 residues with Asp resulted in increased membrane abundance and reduced rates of NCC internalization. NCC ubiquitylation was lowest in the conditions with greatest NCC phosphorylation, thus providing a mechanism for the reduced endocytosis. In conclusion, our data support a model where NCC is constitutively cycled to the plasma membrane, and upon stimulation, it can be phosphorylated to both increase NCC activity and decrease NCC endocytosis, together increasing NaCl transport in the DCT. PMID:24668812

  8. Impaired activity-dependent neural circuit assembly and refinement in autism spectrum disorder genetic models

    Caleb Andrew Doll

    2014-02-01

    Full Text Available Early-use activity during circuit-specific critical periods refines brain circuitry by the coupled processes of eliminating inappropriate synapses and strengthening maintained synapses. We theorize these activity-dependent developmental processes are specifically impaired in autism spectrum disorders (ASDs. ASD genetic models in both mouse and Drosophila have pioneered our insights into normal activity-dependent neural circuit assembly and consolidation, and how these developmental mechanisms go awry in specific genetic conditions. The monogenic Fragile X syndrome (FXS, a common cause of heritable ASD and intellectual disability, has been particularly well linked to defects in activity-dependent critical period processes. The Fragile X Mental Retardation Protein (FMRP is positively activity-regulated in expression and function, in turn regulates excitability and activity in a negative feedback loop, and appears to be required for the activity-dependent remodeling of synaptic connectivity during early-use critical periods. The Drosophila FXS model has been shown to functionally conserve the roles of human FMRP in synaptogenesis, and has been centrally important in generating our current mechanistic understanding of the FXS disease state. Recent advances in Drosophila optogenetics, transgenic calcium reporters, highly-targeted transgenic drivers for individually-identified neurons, and a vastly improved connectome of the brain are now being combined to provide unparalleled opportunities to both manipulate and monitor activity-dependent processes during critical period brain development in defined neural circuits. The field is now poised to exploit this new Drosophila transgenic toolbox for the systematic dissection of activity-dependent mechanisms in normal versus ASD brain development, particularly utilizing the well-established Drosophila FXS disease model.

  9. Bulk chemicals from biomass

    Haveren, van J.; Scott, E.L.; Sanders, J.P.M.

    2008-01-01

    Given the current robust forces driving sustainable production, and available biomass conversion technologies, biomass-based routes are expected to make a significant impact on the production of bulk chemicals within 10 years, and a huge impact within 20-30 years. In the Port of Rotterdam there is a

  10. Ferromagnetic bulk glassy alloys

    This paper deals with the review on the formation, thermal stability and magnetic properties of the Fe-based bulk glassy alloys in as-cast bulk and melt-spun ribbon forms. A large supercooled liquid region over 50 K before crystallization was obtained in Fe-(Al, Ga)-(P, C, B, Si), Fe-(Cr, Mo, Nb)-(Al, Ga)-(P, C, B) and (Fe, Co, Ni)-Zr-M-B (M=Ti, Hf, V, Nb, Ta, Cr, Mo and W) systems and bulk glassy alloys were produced in a thickness range below 2 mm for the Fe-(Al, Ga)-(P, C, B, Si) system and 6 mm for the Fe-Co-(Zr, Nb, Ta)-(Mo, W)-B system by copper-mold casting. The ring-shaped glassy Fe-(Al, Ga)-(P, C, B, Si) alloys exhibit much better soft magnetic properties as compared with the ring-shaped alloy made from the melt-spun ribbon because of the formation of the unique domain structure. The good combination of high glass-forming ability and good soft magnetic properties indicates the possibility of future development as a new bulk glassy magnetic material

  11. Heat transport in bulk/nanoporous/bulk silicon devices

    Criado-Sancho, M. [Departamento de Ciencias y Técnicas Físicoquimicas, Facultad de Ciencias, UNED, Senda del Rey 9, 20040 Madrid (Spain); Jou, D., E-mail: David.Jou@uab.cat [Departament de Física, Universitat Autònoma de Barcelona, 08193 Bellaterra, Catalonia (Spain); Institut d' Estudis Catalans, Carme 47, 08001 Barcelona, Catalonia (Spain)

    2013-02-04

    We study heat transport in bulk/nanoporous/bulk silicon devices; we show that, despite bulk/nanoporous devices may act as thermal rectifiers, the non-linear aspects of their joint thermal conductance are not strong enough to lead to a negative differential thermal resistance, necessary to allow bulk/nanoporous/bulk Si devices to act as thermal transistors. Furthermore, we explicitly study the effective thermal conductivity of the mentioned devices for several temperatures, geometries, porosities, and pore size.

  12. Activity-dependent branching ratios in stocks, solar x-ray flux, and the Bak-Tang-Wiesenfeld sandpile model

    Martin, Elliot; Shreim, Amer; Paczuski, Maya

    2010-01-01

    We define an activity-dependent branching ratio that allows comparison of different time series Xt . The branching ratio bx is defined as bx=E[ξx/x] . The random variable ξx is the value of the next signal given that the previous one is equal to x , so ξx={Xt+1∣Xt=x} . If bx>1 , the process is on average supercritical when the signal is equal to x , while if bxefficient market hypothesis.” For stock volumes, solar x-ray flux intensities, and the Bak-Tang-Wiesenfeld (BTW) sandpile model, bx is supercritical for small values of activity and subcritical for the largest ones, indicating a tendency to return to a typical value. For stock volumes this tendency has an approximate power-law behavior. For solar x-ray flux and the BTW model, there is a broad regime of activity where bx≃1 , which we interpret as an indicator of critical behavior. This is true despite different underlying probability distributions for Xt and for ξx . For the BTW model the distribution of ξx is Gaussian, for x sufficiently larger than 1, and its variance grows linearly with x . Hence, the activity in the BTW model obeys a central limit theorem when sampling over past histories. The broad region of activity where bx is close to one disappears once bulk dissipation is introduced in the BTW model—supporting our hypothesis that it is an indicator of criticality.

  13. DREAM controls the on/off switch of specific activity-dependent transcription pathways.

    Mellström, Britt; Sahún, Ignasi; Ruiz-Nuño, Ana; Murtra, Patricia; Gomez-Villafuertes, Rosa; Savignac, Magali; Oliveros, Juan C; Gonzalez, Paz; Kastanauskaite, Asta; Knafo, Shira; Zhuo, Min; Higuera-Matas, Alejandro; Errington, Michael L; Maldonado, Rafael; DeFelipe, Javier; Jefferys, John G R; Bliss, Tim V P; Dierssen, Mara; Naranjo, Jose R

    2014-03-01

    Changes in nuclear Ca(2+) homeostasis activate specific gene expression programs and are central to the acquisition and storage of information in the brain. DREAM (downstream regulatory element antagonist modulator), also known as calsenilin/KChIP-3 (K(+) channel interacting protein 3), is a Ca(2+)-binding protein that binds DNA and represses transcription in a Ca(2+)-dependent manner. To study the function of DREAM in the brain, we used transgenic mice expressing a Ca(2+)-insensitive/CREB-independent dominant active mutant DREAM (daDREAM). Using genome-wide analysis, we show that DREAM regulates the expression of specific activity-dependent transcription factors in the hippocampus, including Npas4, Nr4a1, Mef2c, JunB, and c-Fos. Furthermore, DREAM regulates its own expression, establishing an autoinhibitory feedback loop to terminate activity-dependent transcription. Ablation of DREAM does not modify activity-dependent transcription because of gene compensation by the other KChIP family members. The expression of daDREAM in the forebrain resulted in a complex phenotype characterized by loss of recurrent inhibition and enhanced long-term potentiation (LTP) in the dentate gyrus and impaired learning and memory. Our results indicate that DREAM is a major master switch transcription factor that regulates the on/off status of specific activity-dependent gene expression programs that control synaptic plasticity, learning, and memory. PMID:24366545

  14. Bulk materials handling review

    NONE

    2007-02-15

    The paper provides details of some of the most important coal handling projects and technologies worldwide. It describes development by Aubema Crushing Technology GmbH, Bedeschi, Cimbria Moduflex, DBT, Dynamic Air Conveying Systems, E & F Services, InBulk Technologies, Nord-Sen Metal Industries Ltd., Pebco Inc, Primasonics International Ltd., R.J.S. Silo Clean (International) Ltd., Takraf GmbH, and The ACT Group. 17 photos.

  15. pHuji, a pH-sensitive red fluorescent protein for imaging of exo- and endocytosis

    Shen, Yi; Rosendale, Morgane; Robert E Campbell; Perrais, David

    2014-01-01

    Fluorescent proteins with pH-sensitive fluorescence are valuable tools for the imaging of exocytosis and endocytosis. The Aequorea green fluorescent protein mutant superecliptic pHluorin (SEP) is particularly well suited to these applications. Here we describe pHuji, a red fluorescent protein with a pH sensitivity that approaches that of SEP, making it amenable for detection of single exocytosis and endocytosis events. To demonstrate the utility of the pHuji plus SEP pair, we perform simultan...

  16. Binding of heparin and of the small proteoglycan decorin to the same endocytosis receptor proteins leads to different metabolic consequences

    1991-01-01

    Decorin, a small interstitial dermatan sulfate proteoglycan, is turned over in cultured cells of mesenchymal origin by receptor-mediated endocytosis followed by intralysosomal degradation. Two endosomal proteins of 51 and 26 kD have been implicated in the endocytotic process because of their interaction with decorin core protein. However, heparin and protein-free dermatan sulfate were able to inhibit endocytosis of decorin in a concentration-dependent manner. After Western blotting of endosom...

  17. Clathrin is Important for Normal Actin Dynamics and Progression of Sla2p-Containing Patches During Endocytosis in Yeast

    Newpher, Thomas M.; Lemmon, Sandra K.

    2006-01-01

    Clathrin is a major vesicle coat protein involved in receptor-mediated endocytosis. In yeast and higher eukaryotes, clathrin is recruited to the plasma membrane during the early stage of endocytosis along with clathrin-associated adaptors. As coated pits undergo maturation, a burst of actin polymerization accompanies and helps drive vesicle internalization. Here, we investigate the dynamics of clathrin relative to the early endocytic patch protein Sla2p. We find that clathrin is recruited to ...

  18. Systemic blockade of the Hyaluronan Receptor for Endocytosis (HARE) prevents lymph node metastasis of prostate cancer

    Simpson, Melanie A.; Weigel, Janet A.; Weigel, Paul H.

    2012-01-01

    Tumor progression and metastasis are promoted by the remodeling of organized tissue architecture and engagement of molecular interactions that support tumor cell passage through endothelial barriers. Prostate tumor cells that secrete and turn over excessive quantities of pericellular hyaluronan (HA) exhibit accelerated growth kinetics and spontaneous lymph node metastasis in mice. The HA Receptor for Endocytosis (HARE) is an endocytic clearance receptor for HA in the liver that is also highly...

  19. Rosiglitazone Balances Insulin-Induced Exo- And Endocytosis In Single 3t3-L1 Adipocytes

    Velebit, Jelena; Chowdhury, Helena H.; Kreft, Marko; Zorec, Robert

    2011-01-01

    Abstract Rosiglitazone (Rosi) improves insulin sensitivity and increases the translocation of glucose transporter 4 (GLUT4) to the plasma membrane (PM). This involves the fusion of membrane-bound compartments with the plasma membrane, thus increasing the plasma membrane area. However, recent work has shown that in Rosi-pretreated 3T3-L1 adipocytes membrane area did not increase following insulin application, suggesting that the rates of exo- and endocytosis are balanced. Here we ex...

  20. Hepcidin-induced endocytosis of ferroportin is dependent on ferroportin ubiquitination

    Qiao, Bo; Sugianto, Priscilla; Fung, Eileen; del-Castillo-Rueda, Alejandro; Moran-Jimenez, Maria-Josefa; Ganz, Tomas; Nemeth, Elizabeta

    2012-01-01

    Ferroportin exports iron into plasma from absorptive enterocytes, erythrophagocytosing macrophages, and hepatic stores. The hormone hepcidin controls cellular iron export and plasma iron concentrations by binding to ferroportin and causing its internalization and degradation. We explored the mechanism of hepcidin-induced endocytosis of ferroportin, the key molecular event in systemic iron homeostasis. Hepcidin binding caused rapid ubiquitination of ferroportin in cell lines overexpressing fer...

  1. Identification and Selected Reaction Monitoring (SRM) Quantification of Endocytosis Factors Associated with Numb*

    Krieger, Jonathan R; Taylor, Paul; Gajadhar, Aaron S.; Guha, Abhijit; Moran, Michael F.; McGlade, C. Jane

    2012-01-01

    Numb is an endocytic adaptor protein that regulates the endocytosis and trafficking of transmembrane receptors including Notch, E-cadherin, and integrins. Vertebrate Numb is alternatively spliced at exons 3 and 9 to give rise to four protein isoforms. Expression of these isoforms varies at different developmental stages, and although the function of Numb isoforms containing exon 3 has been studied, the role of exon 9 inclusion has not been shown. Here we use affinity purification and tandem m...

  2. Analysis of occludin trafficking, demonstrating continuous endocytosis, degradation, recycling and biosynthetic secretory trafficking.

    Sarah J Fletcher

    Full Text Available Tight junctions (TJs link adjacent cells and are critical for maintenance of apical-basolateral polarity in epithelial monolayers. The TJ protein occludin functions in disparate processes, including wound healing and Hepatitis C Virus infection. Little is known about steady-state occludin trafficking into and out of the plasma membrane. Therefore, we determined the mechanisms responsible for occludin turnover in confluent Madin-Darby canine kidney (MDCK epithelial monolayers. Using various biotin-based trafficking assays we observed continuous and rapid endocytosis of plasma membrane localised occludin (the majority internalised within 30 minutes. By 120 minutes a significant reduction in internalised occludin was observed. Inhibition of lysosomal function attenuated the reduction in occludin signal post-endocytosis and promoted co-localisation with the late endocytic system. Using a similar method we demonstrated that ∼20% of internalised occludin was transported back to the cell surface. Consistent with these findings, significant co-localisation between internalised occludin and recycling endosomal compartments was observed. We then quantified the extent to which occludin synthesis and transport to the plasma membrane contributes to plasma membrane occludin homeostasis, identifying inhibition of protein synthesis led to decreased plasma membrane localised occludin. Significant co-localisation between occludin and the biosynthetic secretory pathway was demonstrated. Thus, under steady-state conditions occludin undergoes turnover via a continuous cycle of endocytosis, recycling and degradation, with degradation compensated for by biosynthetic exocytic trafficking. We developed a mathematical model to describe the endocytosis, recycling and degradation of occludin, utilising experimental data to provide quantitative estimates for the rates of these processes.

  3. Effects of receptor-mediated endocytosis and tubular protein composition on volume retention in experimental glomerulonephritis

    Kastner, Christian; Pohl, Marcus; Sendeski, Mauricio;

    2009-01-01

    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...

  4. Capacitance measurements. An analysis of the phase detector technique used to study exocytosis and endocytosis.

    Joshi, C.; Fernandez, J M

    1988-01-01

    We have studied the admittance of patch-clamped mast cells during exocytosis and found that they are adequately described by a four parameter equivalent circuit. On the basis of these measurements, we show that, contrary to current belief, when using a phase sensitive detector, small capacitance changes due to exocytosis or endocytosis should be studied by measuring current 90 degrees out of phase, relative to the component that corresponds to changes in series resistance. We have extended th...

  5. Cyclin dependent kinase 5 regulates endocytosis in nerve terminals via dynamin I phosphorylation

    Full text: Synaptic vesicle endocytosis (SVE) in nerve terminals is essential for normal synaptic transmission and for memory retrieval. Dynamin I is a 96kDa nerve terminal phosphoprotein necessary for synaptic vesicle endocytosis in the nerve terminal. Dynamin I is dephosphorylated and rephosphorylated in a cyclical fashion with nerve terminal depolarisation and repolarisation. A number of kinases phosphorylate dynamin I in vitro including PKC, MAP kinase and cdc2. PKC phosphorylates dynamin in the proline rich domain on Ser 795 and is also thought to be the in vivo kinase for dynamin I. Another candidate is the neuron specific kinase cdk5, crucial for CNS development. The aim of this study is to identify the kinase which phosphorylates dynamin I in intact nerve terminals. Here we show that cyclin-dependent kinase 5 (cdk5) phosphorylates dynamin I in the proline-rich tail on Ser-774 or Ser-778. The phosphorylation of these sites but not Ser-795 also occurred in intact nerve terminals suggesting that cdk5 is the physiologically relevant enzyme for dynamin I. Synaptosomes prepared from rat brains (after cervical dislocations) and labelled with 32 Pi, were incubated with 100 M roscovitine (a selective inhibitor of cdks), 10 M Ro 31-8220 (a selective PKC inhibitor) and 100 M PD 98059 (a MEK kinase inhibitor). Dynamin rephosphorylation during repolarisation was reduced in synaptosomes treated with roscovitine and Ro 38-8220 but not in synaptosomes treated with PD 98059. Fluorimetric experiments on intact synaptosomes utilising FM-210 (a fluorescent dye) indicate that endocytosis was reduced in synaptosomes treated with 100 M roscovitine. Our results suggest that dynamin phosphorylation in intact nerve terminals may not be regulated by PKC or MAP kinase and that dynamin phosphorylation by cdk5 may regulate endocytosis. Copyright (2002) Australian Neuroscience Society

  6. Human vascular endothelial cells transport foreign exosomes from cow's milk by endocytosis.

    Kusuma, Rio Jati; Manca, Sonia; Friemel, Taylor; Sukreet, Sonal; Nguyen, Christopher; Zempleni, Janos

    2016-05-15

    Encapsulation of microRNAs in exosomes confers protection against degradation and a vehicle for shuttling of microRNAs between cells and tissues, and cellular uptake by endocytosis. Exosomes can be found in foods including milk. Humans absorb cow's milk exosomes and deliver the microRNA cargo to peripheral tissues, consistent with gene regulation by dietary nucleic acids across species boundaries. Here, we tested the hypothesis that human vascular endothelial cells transport milk exosomes by endocytosis, constituting a step crucial for the delivery of dietary exosomes and their cargo to peripheral tissues. We tested this hypothesis by using human umbilical vein endothelial cells and fluorophore-labeled exosomes isolated from cow's milk. Exosome uptake followed Michaelis-Menten kinetics (Vmax = 0.057 ± 0.004 ng exosome protein × 40,000 cells/h; Km = 17.97 ± 3.84 μg exosomal protein/200 μl media) and decreased by 80% when the incubation temperature was lowered from 37°C to 4°C. When exosome surface proteins were removed by treatment with proteinase K, or transport was measured in the presence of the carbohydrate competitor d-galactose or measured in the presence of excess unlabeled exosomes, transport rates decreased by 45% to 80% compared with controls. Treatment with an inhibitor of endocytosis, cytochalasin D, caused a 50% decrease in transport. When fluorophore-labeled exosomes were administered retro-orbitally, exosomes accumulated in liver, spleen, and lungs in mice. We conclude that human vascular endothelial cells transport bovine exosomes by endocytosis and propose that this is an important step in the delivery of dietary exosomes and their cargo to peripheral tissues. PMID:26984735

  7. Contributions of herpes simplex virus type 1 envelope proteins to entry by endocytosis

    Herpes simplex virus (HSV) proteins specifically required for endocytic entry but not direct penetration have not been identified. HSVs deleted of gE, gG, gI, gJ, gM, UL45, or Us9 entered cells via either pH-dependent or pH-independent endocytosis and were inactivated by mildly acidic pH. Thus, the ...

  8. Substrate-Induced Ubiquitylation and Endocytosis of Yeast Amino Acid Permeases

    Ghaddar, Kassem; Merhi, Ahmad; Saliba, Elie; Krammer, Eva-Maria; Prévost, Martine; André, Bruno

    2014-01-01

    Many plasma membrane transporters are downregulated by ubiquitylation, endocytosis, and delivery to the lysosome in response to various stimuli. We report here that two amino acid transporters of Saccharomyces cerevisiae, the general amino acid permease (Gap1) and the arginine-specific permease (Can1), undergo ubiquitin-dependent downregulation in response to their substrates and that this downregulation is not due to intracellular accumulation of the transported amino acids but to transport ...

  9. Enhanced endocytosis of nano-curcumin in nasopharyngeal cancer cells: An atomic force microscopy study

    Prasanth, R.; Nair, Greshma; Girish, C. M.

    2011-10-01

    Recent studies in drug development have shown that curcumin can be a good competent due to its improved anticancer, antioxidant, anti-proliferative, and anti-inflammatory activities. A detailed real time characterization of drug (curcumin)-cell interaction is carried out in human nasopharyngeal cancer cells using atomic force microscopy. Nanocurcumin shows an enhanced uptake over micron sized drugs attributed to the receptor mediated route. Cell membrane stiffness plays a critical role in the drug endocytosis in nasopharyngeal cancer cells.

  10. Human immunodeficiency virus infection of T cells and monocytes proceeds via receptor-mediated endocytosis

    1988-01-01

    The rates of internalization and uncoating of 32P-labelled human immunodeficiency virus (HIV) in the human T lymphoid cell line CEM are consonant with a receptor-mediated endocytosis mechanism of entry. This interpretation was affirmed by electron microscopic observation of virions within endosomes. Virus binding and infectivity were inhibited to the same extent by pretreatment with OKT4A antibody, therefore, the CD4 receptor-dependent pathway of internalization appears to be the infectious r...

  11. Parameter estimation with bio-inspired meta-heuristic optimization : modeling the dynamics of endocytosis.

    Tashkova Katerina; Korošec Peter; Šilc Jurij; Todorovski Ljupčo; Džeroski Sašo

    2011-01-01

    Abstract Background We address the task of parameter estimation in models of the dynamics of biological systems based on ordinary differential equations (ODEs) from measured data, where the models are typically non-linear and have many parameters, the measurements are imperfect due to noise, and the studied system can often be only partially observed. A representative task is to estimate the parameters in a model of the dynamics of endocytosis, i.e., endosome maturation, reflected in a cut-ou...

  12. HIV-1 Vpu promotes release and prevents endocytosis of nascent retrovirus particles from the plasma membrane.

    2006-05-01

    Full Text Available The human immunodeficiency virus (HIV type-1 viral protein U (Vpu protein enhances the release of diverse retroviruses from human, but not monkey, cells and is thought to do so by ablating a dominant restriction to particle release. Here, we determined how Vpu expression affects the subcellular distribution of HIV-1 and murine leukemia virus (MLV Gag proteins in human cells where Vpu is, or is not, required for efficient particle release. In HeLa cells, where Vpu enhances HIV-1 and MLV release approximately 10-fold, concentrations of HIV-1 Gag and MLV Gag fused to cyan fluorescent protein (CFP were initially detected at the plasma membrane, but then accumulated over time in early and late endosomes. Endosomal accumulation of Gag-CFP was prevented by Vpu expression and, importantly, inhibition of plasma membrane to early endosome transport by dominant negative mutants of Rab5a, dynamin, and EPS-15. Additionally, accumulation of both HIV and MLV Gag in endosomes required a functional late-budding domain. In human HOS cells, where HIV-1 and MLV release was efficient even in the absence of Vpu, Gag proteins were localized predominantly at the plasma membrane, irrespective of Vpu expression or manipulation of endocytic transport. While these data indicated that Vpu inhibits nascent virion endocytosis, Vpu did not affect transferrin endocytosis. Moreover, inhibition of endocytosis did not restore Vpu-defective HIV-1 release in HeLa cells, but instead resulted in accumulation of mature virions that could be released from the cell surface by protease treatment. Thus, these findings suggest that a specific activity that is present in HeLa cells, but not in HOS cells, and is counteracted by Vpu, traps assembled retrovirus particles at the cell surface. This entrapment leads to subsequent endocytosis by a Rab5a- and clathrin-dependent mechanism and intracellular sequestration of virions in endosomes.

  13. Endocytosis of activated receptors and clathrin-coated pit formation: deciphering the chicken or egg relationship

    1996-01-01

    The fundamental mechanisms by which receptors once targeted for endocytosis are found in coated pits is an important yet unresolved question. Specifically, are activated receptors simply trapped on encountering preexisting coated pits, subsequently being rapidly internalized? Or do the receptors themselves, by active recruitment, gather soluble coat and cytosolic components and initiate the rapid assembly of new coated pits that then mediate their internalization? To explore this question, we...

  14. Multiple GTP-binding proteins participate in clathrin-coated vesicle- mediated endocytosis

    1993-01-01

    We have examined the effects of various agonists and antagonists of GTP- binding proteins on receptor-mediated endocytosis in vitro. Stage- specific assays which distinguish coated pit assembly, invagination, and coat vesicle budding have been used to demonstrate requirements for GTP-binding protein(s) in each of these events. Coated pit invagination and coated vesicle budding are both stimulated by addition of GTP and inhibited by GDP beta S. Although coated pit invagination is resistant to ...

  15. Responses of plant calmodulin to endocytosis induced by rare earth elements.

    Wang, Lihong; Cheng, Mengzhu; Chu, Yunxia; Li, Xiaodong; Chen, David D Y; Huang, Xiaohua; Zhou, Qing

    2016-07-01

    The wide application of rare earth elements (REEs) have led to their diffusion and accumulation in the environment. The activation of endocytosis is the primary response of plant cells to REEs. Calmodulin (CaM), as an important substance in calcium (Ca) signaling systems, regulating almost all of the physiological activities in plants, such as cellular metabolism, cell growth and division. However, the response of CaM to endocytosis activated by REEs remains unknown. By using immunofluorescence labeling and a confocal laser scanning microscope, we found that trivalent lanthanum [La(III)], an REE ion, affected the expression of CaM in endocytosis. Using circular dichroism, X-ray photoelectron spectroscopy and computer simulations, we demonstrated that a low concentration of La(III) could interact with extracellular CaM by electrostatic attraction and was then bound to two Ca-binding sites of CaM, making the molecular structure more compact and orderly, whereas a high concentration of La(III) could be coordinated with cytoplasmic CaM or bound to other Ca-binding sites, making the molecular structure more loose and disorderly. Our results provide a reference for revealing the action mechanisms of REEs in plant cells. PMID:27081794

  16. Hyaluronic acid binding, endocytosis and degradation by sinusoidal liver endothelial cells

    The binding, endocytosis, and degradation of 125I-hyaluronic acid (125I-HA) by liver endothelial cells (LEC) was studied under several conditions. The dissociation of receptor-bound 125I-HA was rapid, with a half time of ∼31 min and a Koff of 6.3 x 10-4/sec. A large reversible increase in 125I-HA binding to LEC at pH 5.0 was due to an increase in the observed affinity of the binding interaction. Pronase digestion suggested the protein nature of the receptor and the intracellular location of the digitonin exposed binding activity. Binding and endocytosis occur in the presence of 10 mM EGTA indicating that divalent cations are not required for receptor function. To study the degradation of 125I-HA by LEC, a cetylpyridinium chloride (CPC) precipitation assay was characterized. The minimum HA length required for precipitation was elucidated. The fate of the LEC HA receptor after endocytosis was examined

  17. Hyaluronic acid binding, endocytosis and degradation by sinusoidal liver endothelial cells

    McGary, C.T.

    1988-01-01

    The binding, endocytosis, and degradation of {sup 125}I-hyaluronic acid ({sup 125}I-HA) by liver endothelial cells (LEC) was studied under several conditions. The dissociation of receptor-bound {sup 125}I-HA was rapid, with a half time of {approx}31 min and a K{sub off} of 6.3 {times} 10{sup {minus}4}/sec. A large reversible increase in {sup 125}I-HA binding to LEC at pH 5.0 was due to an increase in the observed affinity of the binding interaction. Pronase digestion suggested the protein nature of the receptor and the intracellular location of the digitonin exposed binding activity. Binding and endocytosis occur in the presence of 10 mM EGTA indicating that divalent cations are not required for receptor function. To study the degradation of {sup 125}I-HA by LEC, a cetylpyridinium chloride (CPC) precipitation assay was characterized. The minimum HA length required for precipitation was elucidated. The fate of the LEC HA receptor after endocytosis was examined.

  18. The translocation of fullerenic nanoparticles into lysosome via the pathway of clathrin-mediated endocytosis

    Li Wei; Chen Chunying; Ye Chang; Zhao Yuliang; Chen Zhen; Meng Huan; Gao Yuxi; Yuan Hui; Xing Genmei; Zhao Feng; Chai Zhifang [Laboratory for Bio-Environmental Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Nanotechnology of China and Institute of High Energy Physics, Chinese Academy of Science, Yuquan Road 19B, Beijing 100049 (China); Wei Taotao; Zhang Xujia; Yang Fuyu [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101 (China); Lao Fang; Han Dong [National Center for Nanoscience and Technology of China, No 2, Ist North Street Zhongguancun, Beijing 100080 (China); Tang Xianhua; Zhang Yingge [Chinese Academy of Military Medical Sciences, Beijing 100039 (China)], E-mail: chenchy@nanoctr.cn, E-mail: weitt@moon.ibp.ac.cn, E-mail: zhaoyuliang@ihep.ac.cn

    2008-04-09

    Manufactured fullerene nanoparticles easily enter into cells and hence have been rapidly developed for biomedical uses. However, it is generally unknown which route the nanoparticles undergo when crossing cell membranes and where they localize to the intracellular compartments. Herein we have used both microscopic imaging and biological techniques to explore the processes of [C{sub 60}(C(COOH){sub 2}){sub 2}]{sub n} nanoparticles across cellular membranes and their intracellular translocation in 3T3 L1 and RH-35 living cells. The fullerene nanoparticles are quickly internalized by the cells and then routed to the cytoplasm with punctate localization. Upon entering the cell, they are synchronized to lysosome-like vesicles. The [C{sub 60}(C(COOH){sub 2}){sub 2}]{sub n} nanoparticles entering cells are mainly via endocytosis with time-, temperature- and energy-dependent manners. The cellular uptake of [C{sub 60}(C(COOH){sub 2}){sub 2}]{sub n} nanoparticles was found to be clathrin-mediated but not caveolae-mediated endocytosis. The endocytosis mechanism and the subcellular target location provide key information for the better understanding and predicting of the biomedical function of fullerene nanoparticles inside cells.

  19. The translocation of fullerenic nanoparticles into lysosome via the pathway of clathrin-mediated endocytosis

    Li, Wei; Chen, Chunying; Ye, Chang; Wei, Taotao; Zhao, Yuliang; Lao, Fang; Chen, Zhen; Meng, Huan; Gao, Yuxi; Yuan, Hui; Xing, Genmei; Zhao, Feng; Chai, Zhifang; Zhang, Xujia; Yang, Fuyu; Han, Dong; Tang, Xianhua; Zhang, Yingge

    2008-04-01

    Manufactured fullerene nanoparticles easily enter into cells and hence have been rapidly developed for biomedical uses. However, it is generally unknown which route the nanoparticles undergo when crossing cell membranes and where they localize to the intracellular compartments. Herein we have used both microscopic imaging and biological techniques to explore the processes of [C60(C(COOH)2)2]n nanoparticles across cellular membranes and their intracellular translocation in 3T3 L1 and RH-35 living cells. The fullerene nanoparticles are quickly internalized by the cells and then routed to the cytoplasm with punctate localization. Upon entering the cell, they are synchronized to lysosome-like vesicles. The [C60(C(COOH)2)2]n nanoparticles entering cells are mainly via endocytosis with time-, temperature- and energy-dependent manners. The cellular uptake of [C60(C(COOH)2)2]n nanoparticles was found to be clathrin-mediated but not caveolae-mediated endocytosis. The endocytosis mechanism and the subcellular target location provide key information for the better understanding and predicting of the biomedical function of fullerene nanoparticles inside cells.

  20. Expression of activity-dependent neuroprotective protein in the brain of adult rats

    Gennet, N.; Herden, C.; Bubb, V J; Quinn, J P; Kipar, A.

    2008-01-01

    Activity-dependent neuroprotective protein (ADNP) is a VIP-regulated gene, which is essential for brain development. A synthetic peptide (NAP) derived from the ADNP sequence is highly neuroprotective, therefore it has been hypothesised that ADNP has a similar role. ADNP contains classical transcription factor motifs and nuclear localisation domains, but it has also been reported to be secreted and to co-localise with microtubules, indicating that ADNP may have multiple...

  1. Robustness and Enhancement of Neural Synchronization by Activity-Dependent Coupling

    Zhigulin, Valentin P.; Mikhail I. Rabinovich; Huerta, Ramon; Abarbanel, Henry D. I.

    2002-01-01

    We study the synchronization of two model neurons coupled through a synapse having an activity-dependent strength. Our synapse follows the rules of Spike-Timing Dependent Plasticity (STDP). We show that this plasticity of the coupling between neurons produces enlarged frequency locking zones and results in synchronization that is more rapid and much more robust against noise than classical synchronization arising from connections with constant strength. We also present a simple discrete map m...

  2. The Development and Activity-Dependent Expression of Aggrecan in the Cat Visual Cortex

    Kind, P. C.; Sengpiel, F.; Beaver, C. J.; Crocker-Buque, A.; Kelly, G. M.; R. T. Matthews; Mitchell, D E

    2012-01-01

    The Cat-301 monoclonal antibody identifies aggrecan, a chondroitin sulfate proteoglycan in the cat visual cortex and dorsal lateral geniculate nucleus (dLGN). During development, aggrecan expression increases in the dLGN with a time course that matches the decline in plasticity. Moreover, examination of tissue from selectively visually deprived cats shows that expression is activity dependent, suggesting a role for aggrecan in the termination of the sensitive period. Here, we demonstrate for ...

  3. Robustness and Enhancement of Neural Synchronization by Activity-Dependent Coupling

    Zhigulin, V P; Huerta, R; Abarbanel, Henry D I; Zhigulin, Valentin P.; Rabinovich, Mikhail I.; Huerta, Ramon; Abarbanel, Henry D I

    2003-01-01

    We study the synchronization of two model neurons coupled through a synapse having an activity-dependent strength. Our synapse follows the rules of Spike-Timing Dependent Plasticity (STDP). We show that this plasticity of the coupling between neurons produces enlarged frequency locking zones and results in synchronization that is more rapid and much more robust against noise than classical synchronization arising from connections with constant strength. We also present a simple discrete map model that demonstrates the generality of the phenomenon.

  4. Activity-dependent targeting of TRPV1 with a pore-permeating capsaicin analog

    Hui LI; Wang, Shu; Chuang, Alexander Y.; Cohen, Bruce E.; Chuang, Huai-hu

    2011-01-01

    The capsaicin receptor TRPV1 is the principal transduction channel for nociception. Excessive TRPV1 activation causes pathological pain. Ideal pain mangement requires selective inhibition of hyperactive pain-sensing neurons, but sparing normal nociception. We sought to determine whether it is possible to use activity-dependent TRPV1 agonists to identify nerves with excessive TRPV1 activity, as well as exploit the TRPV1 pore to deliver charged anesthetics for neuronal silencing. We synthesized...

  5. The activity-dependent transcription factor NPAS4 regulates domain-specific inhibition

    Bloodgood, Brenda L.; Sharma, Nikhil; Browne, Heidi Adlman; Trepman, Alissa Z.; Greenberg, Michael E.

    2013-01-01

    A heterogeneous population of inhibitory neurons controls the flow of information through a neural circuit1–3. Inhibitory synapses that form on pyramidal neuron dendrites modulate the summation of excitatory synaptic potentials4–6 and prevent the generation of dendritic calcium spikes7,8. Precisely timed somatic inhibition limits both the number of action potentials and the time window during which firing can occur8,9. The activity-dependent transcription factor NPAS4 regulates inhibitory syn...

  6. Wormholes in Bulk Viscous Cosmology

    Jamil, Mubasher

    2008-01-01

    We investigate the effects of the accretion of phantom energy with non-zero bulk viscosity onto a Morris-Thorne wormhole. We have found that if the bulk viscosity is large then the mass of wormhole increases rapidly as compared to small or zero bulk viscosity.

  7. Explosive bulk charge

    Miller, Jacob Lee

    2015-04-21

    An explosive bulk charge, including: a first contact surface configured to be selectively disposed substantially adjacent to a structure or material; a second end surface configured to selectively receive a detonator; and a curvilinear side surface joining the first contact surface and the second end surface. The first contact surface, the second end surface, and the curvilinear side surface form a bi-truncated hemispherical structure. The first contact surface, the second end surface, and the curvilinear side surface are formed from an explosive material. Optionally, the first contact surface and the second end surface each have a substantially circular shape. Optionally, the first contact surface and the second end surface consist of planar structures that are aligned substantially parallel or slightly tilted with respect to one another. The curvilinear side surface has one of a smooth curved geometry, an elliptical geometry, and a parabolic geometry.

  8. Creating bulk nanocrystalline metal.

    Fredenburg, D. Anthony (Georgia Institute of Technology, Atlanta, GA); Saldana, Christopher J. (Purdue University, West Lafayette, IN); Gill, David D.; Hall, Aaron Christopher; Roemer, Timothy John (Ktech Corporation, Albuquerque, NM); Vogler, Tracy John; Yang, Pin

    2008-10-01

    Nanocrystalline and nanostructured materials offer unique microstructure-dependent properties that are superior to coarse-grained materials. These materials have been shown to have very high hardness, strength, and wear resistance. However, most current methods of producing nanostructured materials in weapons-relevant materials create powdered metal that must be consolidated into bulk form to be useful. Conventional consolidation methods are not appropriate due to the need to maintain the nanocrystalline structure. This research investigated new ways of creating nanocrystalline material, new methods of consolidating nanocrystalline material, and an analysis of these different methods of creation and consolidation to evaluate their applicability to mesoscale weapons applications where part features are often under 100 {micro}m wide and the material's microstructure must be very small to give homogeneous properties across the feature.

  9. The Incredible Bulk

    Fukushima, Keita; Kumar, Jason; Sandick, Pearl; Yamamoto, Takahiro

    2014-01-01

    Recent experimental results from the LHC have placed strong constraints on the masses of colored superpartners. The MSSM parameter space is also constrained by the measurement of the Higgs boson mass, and the requirement that the relic density of lightest neutralinos be consistent with observations. Although large regions of the MSSM parameter space can be excluded by these combined bounds, leptophilic versions of the MSSM can survive these constraints. In this paper we consider a scenario in which the requirements of minimal flavor violation, vanishing $CP$-violation, and mass universality are relaxed, specifically focusing on scenarios with light sleptons. We find a large region of parameter space, analogous to the original bulk region, for which the lightest neutralino is a thermal relic with an abundance consistent with that of dark matter. We find that these leptophilic models are constrained by measurements of the magnetic and electric dipole moments of the electron and muon, and that these models have ...

  10. Bulk muscles, loose cables.

    Liyanage, Chamari R D G; Kodali, Venkata

    2014-01-01

    The accessibility and usage of body building supplements is on the rise with stronger internet marketing strategies by the industry. The dangers posed by the ingredients in them are underestimated. A healthy young man came to the emergency room with palpitations and feeling unwell. Initial history and clinical examination were non-contributory to find the cause. ECG showed atrial fibrillation. A detailed history for any over the counter or herbal medicine use confirmed that he was taking supplements to bulk muscle. One of the components in these supplements is yohimbine; the onset of symptoms coincided with the ingestion of this product and the patient is symptom free after stopping it. This report highlights the dangers to the public of consuming over the counter products with unknown ingredients and the consequential detrimental impact on health. PMID:25326558

  11. Early survival factor deprivation in the olfactory epithelium enhances activity-dependent survival

    Adrien eFrançois

    2013-12-01

    Full Text Available The neuronal olfactory epithelium undergoes permanent renewal because of environmental aggression. This renewal is partly regulated by factors modulating the level of neuronal apoptosis. Among them, we had previously characterized endothelin as neuroprotective. In this study, we explored the effect of cell survival factor deprivation in the olfactory epithelium by intranasal delivery of endothelin receptors antagonists to rat pups. This treatment induced an overall increase of apoptosis in the olfactory epithelium. The responses to odorants recorded by electroolfactogram were decreased in treated animal, a result consistent with a loss of olfactory sensory neurons (OSNs. However, the treated animal performed better in an olfactory orientation test based on maternal odor compared to non-treated littermates. This improved performance could be due to activity-dependent neuronal survival of OSNs in the context of increased apoptosis level. In order to demonstrate it, we odorized pups with octanal, a known ligand for the rI7 olfactory receptor (Olr226. We quantified the number of OSN expressing rI7 by RT-qPCR and whole mount in situ hybridization. While this number was reduced by the survival factor removal treatment, this reduction was abolished by the presence of its ligand. This improved survival was optimal for low concentration of odorant and was specific for rI7-expressing OSNs. Meanwhile, the number of rI7-expressing OSNs was not affected by the odorization in non-treated littermates; showing that the activity-dependant survival of OSNs did not affect the OSN population during the 10 days of odorization in control conditions. Overall, our study shows that when apoptosis is promoted in the olfactory mucosa, the activity-dependent neuronal plasticity allows faster tuning of the olfactory sensory neuron population towards detection of environmental odorants.

  12. Endocytosis of a functionally enhanced GFP-tagged transferrin receptor in CHO cells.

    Qi He

    Full Text Available The endocytosis of transferrin receptor (TfR has served as a model to study the receptor-targeted cargo delivery system for cancer therapy for many years. To accurately evaluate and optically measure this TfR targeting delivery in vitro, a CHO cell line with enhanced green fluorescent protein (EGFP-tagged human TfR was established. A chimera of the hTfR and EGFP was engineered by fusing EGFP to the amino terminus of hTfR. Data were provided to demonstrate that hTfR-EGFP chimera was predominantly localized on the plasma membrane with some intracellular fluorescent structures on CHO cells and the EGFP moiety did not affect the endocytosis property of hTfR. Receptor internalization occurred similarly to that of HepG2 cells expressing wild-type hTfR. The internalization percentage of this chimeric receptor was about 81 ± 3% of wild type. Time-dependent co-localization of hTfR-EGFP and PE-conjugated anti-hTfR mAb in living cells demonstrated the trafficking of mAb-receptor complexes through the endosomes followed by segregation of part of the mAb and receptor at the late stages of endocytosis. The CHO-hTfR cells preferentially took up anti-hTfR mAb conjugated nanoparticles. This CHO-hTfR cell line makes it feasible for accurate evaluation and visualization of intracellular trafficking of therapeutic agents conjugated with transferrin or Abs targeting the hTfRs.

  13. Akt Links Insulin Signaling to Albumin Endocytosis in Proximal Tubule Epithelial Cells.

    Coffey, Sam; Costacou, Tina; Orchard, Trevor; Erkan, Elif

    2015-01-01

    Diabetes mellitus (DM) has become an epidemic, causing a significant decline in quality of life of individuals due to its multisystem involvement. Kidney is an important target organ in DM accounting for the majority of patients requiring renal replacement therapy at dialysis units. Microalbuminuria (MA) has been a valuable tool to predict end-organ damage in DM but its low sensitivity has driven research efforts to seek other alternatives. Albumin is taken up by albumin receptors, megalin and cubilin in the proximal tubule epithelial cells. We demonstrated that insulin at physiological concentrations induce albumin endocytosis through activation of protein kinase B (Akt) in proximal tubule epithelial cells. Inhibition of Akt by a phosphorylation deficient construct abrogated insulin induced albumin endocytosis suggesting a role for Akt in insulin-induced albumin endocytosis. Furthermore we demonstrated a novel interaction between Akt substrate 160kDa (AS160) and cytoplasmic tail of megalin. Mice with type 1 DM (T1D) displayed decreased Akt, megalin, cubilin and AS160 expression in their kidneys in association with urinary cubilin shedding preceding significant MA. Patients with T1D who have developed MA in the EDC (The Pittsburgh Epidemiology of Diabetes Complications) study demonstrated urinary cubilin shedding prior to development of MA. We hypothesize that perturbed insulin-Akt cascade in DM leads to alterations in trafficking of megalin and cubilin, which results in urinary cubilin shedding as a prelude to MA in early diabetic nephropathy. We propose that utilization of urinary cubilin shedding, as a urinary biomarker, will allow us to detect and intervene in diabetic nephropathy (DN) at an earlier stage. PMID:26465605

  14. Akt Links Insulin Signaling to Albumin Endocytosis in Proximal Tubule Epithelial Cells.

    Sam Coffey

    Full Text Available Diabetes mellitus (DM has become an epidemic, causing a significant decline in quality of life of individuals due to its multisystem involvement. Kidney is an important target organ in DM accounting for the majority of patients requiring renal replacement therapy at dialysis units. Microalbuminuria (MA has been a valuable tool to predict end-organ damage in DM but its low sensitivity has driven research efforts to seek other alternatives. Albumin is taken up by albumin receptors, megalin and cubilin in the proximal tubule epithelial cells. We demonstrated that insulin at physiological concentrations induce albumin endocytosis through activation of protein kinase B (Akt in proximal tubule epithelial cells. Inhibition of Akt by a phosphorylation deficient construct abrogated insulin induced albumin endocytosis suggesting a role for Akt in insulin-induced albumin endocytosis. Furthermore we demonstrated a novel interaction between Akt substrate 160kDa (AS160 and cytoplasmic tail of megalin. Mice with type 1 DM (T1D displayed decreased Akt, megalin, cubilin and AS160 expression in their kidneys in association with urinary cubilin shedding preceding significant MA. Patients with T1D who have developed MA in the EDC (The Pittsburgh Epidemiology of Diabetes Complications study demonstrated urinary cubilin shedding prior to development of MA. We hypothesize that perturbed insulin-Akt cascade in DM leads to alterations in trafficking of megalin and cubilin, which results in urinary cubilin shedding as a prelude to MA in early diabetic nephropathy. We propose that utilization of urinary cubilin shedding, as a urinary biomarker, will allow us to detect and intervene in diabetic nephropathy (DN at an earlier stage.

  15. Inhibition of HIV-1 endocytosis allows lipid mixing at the plasma membrane, but not complete fusion

    de la Vega Michelle

    2011-12-01

    Full Text Available Abstract Background We recently provided evidence that HIV-1 enters HeLa-derived TZM-bl and lymphoid CEMss cells by fusing with endosomes, whereas its fusion with the plasma membrane does not proceed beyond the lipid mixing step. The mechanism of restriction of HIV-1 fusion at the cell surface and/or the factors that aid the virus entry from endosomes remain unclear. Results We examined HIV-1 fusion with a panel of target cells lines and with primary CD4+ T cells. Kinetic measurements of fusion combined with time-resolved imaging of single viruses further reinforced the notion that HIV-1 enters the cells via endocytosis and fusion with endosomes. Furthermore, we attempted to deliberately redirect virus fusion to the plasma membrane, using two experimental strategies. First, the fusion reaction was synchronized by pre-incubating the viruses with cells at reduced temperature to allow CD4 and coreceptors engagement, but not the virus uptake or fusion. Subsequent shift to a physiological temperature triggered accelerated virus uptake followed by entry from endosomes, but did not permit fusion at the cell surface. Second, blocking HIV-1 endocytosis by a small-molecule dynamin inhibitor, dynasore, resulted in transfer of viral lipids to the plasma membrane without any detectable release of the viral content into the cytosol. We also found that a higher concentration of dynasore is required to block the HIV-endosome fusion compared to virus internalization. Conclusions Our results further support the notion that HIV-1 enters disparate cell types through fusion with endosomes. The block of HIV-1 fusion with the plasma membrane at a post-lipid mixing stage shows that this membrane is not conducive to fusion pore formation and/or enlargement. The ability of dynasore to interfere with the virus-endosome fusion suggests that dynamin could be involved in two distinct steps of HIV-1 entry - endocytosis and fusion within intracellular compartments.

  16. Inflation from bulk viscosity

    Bamba, Kazuharu

    2015-01-01

    We explore the perfect fluid description of the inflationary universe. In particular, we investigate a fluid model with the bulk-viscosity term. We find that the three observables of inflationary cosmology: the spectral index of the curvature perturbations, the tensor-to-scalar ratio of the density perturbations, and the running of the spectral index, can be consistent with the recent Planck results. We also reconstruct the explicit equation of state (EoS) of the viscous fluid from the spectral index of the curvature perturbations compatible with the Planck analysis. In the reconstructed models of the viscous fluid, the tensor-to-scalar ratio of the density perturbations can satisfy the constraints obtained from the Planck satellite. The running of the spectral index can explain the Planck data. In addition, it is demonstrated that in the reconstructed models of the viscous fluid, the graceful exit from inflation can be realized. Furthermore, we show that the singular inflation can occur in the viscous fluid ...

  17. Bulk-Fill Resin Composites

    Benetti, Ana Raquel; Havndrup-Pedersen, Cæcilie; Honoré, Daniel;

    2015-01-01

    restorative procedure. The aim of this study, therefore, was to compare the depth of cure, polymerization contraction, and gap formation in bulk-fill resin composites with those of a conventional resin composite. To achieve this, the depth of cure was assessed in accordance with the International Organization...... for Standardization 4049 standard, and the polymerization contraction was determined using the bonded-disc method. The gap formation was measured at the dentin margin of Class II cavities. Five bulk-fill resin composites were investigated: two high-viscosity (Tetric EvoCeram Bulk Fill, SonicFill) and...... three low-viscosity (x-tra base, Venus Bulk Fill, SDR) materials. Compared with the conventional resin composite, the high-viscosity bulk-fill materials exhibited only a small increase (but significant for Tetric EvoCeram Bulk Fill) in depth of cure and polymerization contraction, whereas the low...

  18. Fc receptor endocytosis is controlled by a cytoplasmic domain determinant that actively prevents coated pit localization

    1992-01-01

    Macrophages and B-lymphocytes express two major isoforms of Fc receptor (FcRII-B2 and FcRII-B1) that exhibit distinct capacities for endocytosis. This difference in function reflects the presence of an in- frame insertion of 47 amino acids in the cytoplasmic domain of the lymphocyte isoform (FcRII-B1) due to alternative mRNA splicing. By expressing wild type and mutant FcRII cDNAs in fibroblasts, we have now examined the mechanism by which the insertion acts to prevent coated pit localization...

  19. Role of coated vesicles, microfilaments, and calmodulin in receptor- mediated endocytosis by cultured B lymphoblastoid cells

    1980-01-01

    Cell surface receptor IgM molecules of cultured human lymlphoblastoid cells (WiL2) patch and redistribute into a cap over the Golgi region of the cell after treatment with multivalent anti-IgM antibodies. During and after the redistribution, ligand-receptor clusters are endocytosed into coated pits and coated vesicles. Morphometric analysis of the distribution of ferritin-labeled ligand at EM resolution reveals the following sequence of events in the endocytosis of cell surface IgM: (a) bindi...

  20. Role of endocytosis and cathepsin-mediated activation in Nipah virus entry

    The recent discovery that the Nipah virus (NiV) fusion protein (F) is activated by endosomal cathepsin L raised the question if NiV utilize pH- and protease-dependent mechanisms of entry. We show here that the NiV receptor ephrin B2, virus-like particles and infectious NiV are internalized from the cell surface. However, endocytosis, acidic pH and cathepsin-mediated cleavage are not necessary for the initiation of infection of new host cells. Our data clearly demonstrate that proteolytic activation of the NiV F protein is required before incorporation into budding virions but not after virus entry

  1. Equine arteritis virus is delivered to an acidic compartment of host cells via clathrin-dependent endocytosis

    Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae. Infection by EAV requires the release of the viral genome by fusion with the respective target membrane of the host cell. We have investigated the entry pathway of EAV into Baby Hamster Kindey cells (BHK). Infection of cells assessed by the plaque reduction assay was strongly inhibited by substances which interfere with clathrin-dependent endocytosis and by lysosomotropic compounds. Furthermore, infection of BHK cells was suppressed when clathrin-dependent endocytosis was inhibited by expression of antisense RNA of the clathrin-heavy chain before infection. These results strongly suggest that EAV is taken up via clathrin-dependent endocytosis and is delivered to acidic endosomal compartments

  2. Uptake of fluorescent nano beads into BY2-cells involves clathrin-dependent and clathrin-independent endocytosis.

    Bandmann, Vera; Müller, Jasmin Daniela; Köhler, Tim; Homann, Ulrike

    2012-10-19

    To follow endocytosis in BY-2 cells we made use of fluorescent nano beads. Beads with 20nm in diameter were internalised rapidly and accumulated partially in compartments also labelled by the endocytic marker FM4-64. Studies in BY-2 cells and protoplasts revealed that larger beads (100nm) were excluded from uptake into turgescent and plasmolysed cells while protoplasts were able to internalise beads with a diameter of up to 1000nm. Endocytosis of beads was only partially inhibited by the clathrin-specific inhibitor Ikarugamycin and strongly blocked by wortmannin. These results imply that uptake of beads involves clathrin-dependent and clathrin-independent endocytic mechanisms and supports the hypothesis that clathrin-independent endocytosis plays a general role in plants. PMID:23046971

  3. Activity-dependent regulation of calcium and ribosomes in the chick cochlear nucleus.

    Call, C L; Hyson, R L

    2016-03-01

    Cochlea removal results in the death of 20-30% of neurons in the chick cochlear nucleus, nucleus magnocellularis (NM). Two potentially cytotoxic events, a dramatic rise in intracellular calcium concentration ([Ca(2+)]i) and a decline in the integrity of ribosomes are observed within 1h of deafferentation. Glutamatergic input from the auditory nerve has been shown to preserve NM neuron health by activating metabotropic glutamate receptors (mGluRs), maintaining both normal [Ca(2+)]i and ribosomal integrity. One interpretation of these results is that a common mGluR-activated signaling cascade is required for the maintenance of both [Ca(2+)]i and ribosomal integrity. This could happen if both responses are influenced directly by a common messenger, or if the loss of mGluR activation causes changes in one component that secondarily causes changes in the other. The present studies tested this common-mediator hypothesis in slice preparations by examining activity-dependent regulation of [Ca(2+)]i and ribosomes in the same tissue after selectively blocking group I mGluRs (1-Aminoindan-1,5-dicarboxylic acid (AIDA)) or group II mGluRs (LY 341495) during unilateral auditory nerve stimulation. Changes in [Ca(2+)]i of NM neurons were measured using fura-2 ratiometric calcium imaging and the tissue was subsequently processed for Y10B immunoreactivity (Y10B-ir), an antibody that recognizes a ribosomal epitope. The group I mGluR antagonist blocked the activity-dependent regulation of both [Ca(2+)]i and Y10B-ir, but the group II antagonist blocked only the activity-dependent regulation of Y10B-ir. That is, even when group II receptors were blocked, stimulation continued to maintain low [Ca(2+)]i, but it did not maintain Y10B-ir. These results suggest a dissociation in how calcium and ribosomes are regulated in NM neurons and that ribosomes can be regulated through a mechanism that is independent of calcium regulation. PMID:26739326

  4. The clathrin-binding motif and the J-domain of Drosophila Auxilin are essential for facilitating Notch ligand endocytosis

    Chang Henry C

    2008-05-01

    Full Text Available Abstract Background Ligand endocytosis plays a critical role in regulating the activity of the Notch pathway. The Drosophila homolog of auxilin (dAux, a J-domain-containing protein best known for its role in the disassembly of clathrin coats from clathrin-coated vesicles, has recently been implicated in Notch signaling, although its exact mechanism remains poorly understood. Results To understand the role of auxilin in Notch ligand endocytosis, we have analyzed several point mutations affecting specific domains of dAux. In agreement with previous work, analysis using these stronger dAux alleles shows that dAux is required for several Notch-dependent processes, and its function during Notch signaling is required in the signaling cells. In support of the genetic evidences, the level of Delta appears elevated in dAux deficient cells, suggesting that the endocytosis of Notch ligand is disrupted. Deletion analysis shows that the clathrin-binding motif and the J-domain, when over-expressed, are sufficient for rescuing dAux phenotypes, implying that the recruitment of Hsc70 to clathrin is a critical role for dAux. However, surface labeling experiment shows that, in dAux mutant cells, Delta accumulates at the cell surface. In dAux mutant cells, clathrin appears to form large aggregates, although Delta is not enriched in these aberrant clathrin-positive structures. Conclusion Our data suggest that dAux mutations inhibit Notch ligand internalization at an early step during clathrin-mediated endocytosis, before the disassembly of clathrin-coated vesicles. Further, the inhibition of ligand endocytosis in dAux mutant cells possibly occurs due to depletion of cytosolic pools of clathrin via the formation of clathrin aggregates. Together, our observations argue that ligand endocytosis is critical for Notch signaling and auxilin participates in Notch signaling by facilitating ligand internalization.

  5. Size-dependent endocytosis of gold nanoparticles studied by three-dimensional mapping of plasmonic scattering images

    Lee Chia-Wei

    2010-12-01

    Full Text Available Abstract Background Understanding the endocytosis process of gold nanoparticles (AuNPs is important for the drug delivery and photodynamic therapy applications. The endocytosis in living cells is usually studied by fluorescent microscopy. The fluorescent labeling suffers from photobleaching. Besides, quantitative estimation of the cellular uptake is not easy. In this paper, the size-dependent endocytosis of AuNPs was investigated by using plasmonic scattering images without any labeling. Results The scattering images of AuNPs and the vesicles were mapped by using an optical sectioning microscopy with dark-field illumination. AuNPs have large optical scatterings at 550-600 nm wavelengths due to localized surface plasmon resonances. Using an enhanced contrast between yellow and blue CCD images, AuNPs can be well distinguished from cellular organelles. The tracking of AuNPs coated with aptamers for surface mucin glycoprotein shows that AuNPs attached to extracellular matrix and moved towards center of the cell. Most 75-nm-AuNPs moved to the top of cells, while many 45-nm-AuNPs entered cells through endocytosis and accumulated in endocytic vesicles. The amounts of cellular uptake decreased with the increase of particle size. Conclusions We quantitatively studied the endocytosis of AuNPs with different sizes in various cancer cells. The plasmonic scattering images confirm the size-dependent endocytosis of AuNPs. The 45-nm-AuNP is better for drug delivery due to its higher uptake rate. On the other hand, large AuNPs are immobilized on the cell membrane. They can be used to reconstruct the cell morphology.

  6. Calcium channel types contributing to chromaffin cell excitability, exocytosis and endocytosis.

    Mahapatra, S; Calorio, C; Vandael, D H F; Marcantoni, A; Carabelli, V; Carbone, E

    2012-01-01

    Voltage gated Ca(2+) channels are effective voltage sensors of plasma membrane which convert cell depolarizations into Ca(2+) signaling. The chromaffin cells of the adrenal medulla utilize a large number of Ca(2+) channel types to drive the Ca(2+)-dependent release of catecholamines into blood circulation, during normal or stress-induced conditions. Some of the Ca(2+) channels expressed in chromaffin cells (L, N, P/Q, R and T), however, do not control only vesicle fusion and catecholamine release. They also subserve a variety of key activities which are vital for the physiological and pathological functioning of the cell, like: (i) shaping the action potentials of electrical oscillations driven either spontaneously or by ACh stimulation, (ii) controlling the action potential frequency of tonic or bursts firing, (iii) regulating the compensatory and excess endocytosis following robust exocytosis and (iv) driving the remodeling of Ca(2+) signaling which occurs during stressors stimulation. Here, we will briefly review the well-established properties of voltage-gated Ca(2+) channels accumulated over the past three decades focusing on the most recent discoveries on the role that L- (Cav1.2, Cav1.3) and T-type (Cav3.2) channels play in the control of excitability, exocytosis and endocytosis of chromaffin cells in normal and stress-mimicking conditions. PMID:22317919

  7. Kinetics of cellular uptake of viruses and nanoparticles via clathrin-mediated endocytosis

    Banerjee, Anand; Berezhkovskii, Alexander; Nossal, Ralph

    2016-02-01

    Several viruses exploit clathrin-mediated endocytosis to gain entry into host cells. This process is also used extensively in biomedical applications to deliver nanoparticles (NPs) to diseased cells. The internalization of these nano-objects is controlled by the assembly of a clathrin-containing protein coat on the cytoplasmic side of the plasma membrane, which drives the invagination of the membrane and the formation of a cargo-containing endocytic vesicle. Current theoretical models of receptor-mediated endocytosis of viruses and NPs do not explicitly take coat assembly into consideration. In this paper we study cellular uptake of viruses and NPs with a focus on coat assembly. We characterize the internalization process by the mean time between the binding of a particle to the membrane and its entry into the cell. Using a coarse-grained model which maps the stochastic dynamics of coat formation onto a one-dimensional random walk, we derive an analytical formula for this quantity. A study of the dependence of the mean internalization time on NP size shows that there is an upper bound above which this time becomes extremely large, and an optimal size at which it attains a minimum. Our estimates of these sizes compare well with experimental data. We also study the sensitivity of the obtained results on coat parameters to identify factors which significantly affect the internalization kinetics.

  8. Amorphous nanosilica induce endocytosis-dependent ROS generation and DNA damage in human keratinocytes

    Hirai Toshiro

    2011-01-01

    Full Text Available Abstract Background Clarifying the physicochemical properties of nanomaterials is crucial for hazard assessment and the safe application of these substances. With this in mind, we analyzed the relationship between particle size and the in vitro effect of amorphous nanosilica (nSP. Specifically, we evaluated the relationship between particle size of nSP and the in vitro biological effects using human keratinocyte cells (HaCaT. Results Our results indicate that exposure to nSP of 70 nm diameter (nSP70 induced an elevated level of reactive oxygen species (ROS, leading to DNA damage. A markedly reduced response was observed using submicron-sized silica particles of 300 and 1000 nm diameter. In addition, cytochalasin D-treatment reduced nSP70-mediated ROS generation and DNA damage, suggesting that endocytosis is involved in nSP70-mediated cellular effects. Conclusions Thus, particle size affects amorphous silica-induced ROS generation and DNA damage of HaCaT cells. We believe clarification of the endocytosis pathway of nSP will provide useful information for hazard assessment as well as the design of safer forms of nSPs.

  9. Effect of endocytosis inhibitors on Coxiella burnetii interaction with host cells

    The obligate intracellular rickettsia Coxiella burnetii has previously been reported to reach the intra-vacuolar compartment of host cells by phagocytosis. With the aim to further examine the mechanisms of C. burnetii internalisation, macrophage monolayers were treated with well characterised inhibitors of endocytosis. The treatment with two general inhibitors, colchicine and methylamine, resulted in a pronounced dose-dependent decrease of radiolabelled phase II rickettsiae retained from the intracellular fraction. A third inhibitor used, amiloride, has been reported to reduce effectively clathrin-independent pinocytic pathways. The internalisation of C. burnetii was shown to be substantially reduced also by amiloride and the effect was dependent on its concentration. The passive role of C. burnetii in the internalisation was verified by using heat-killed C. burnetii. Host cells treated with either of the three inhibitors (amiloride, colchicine and methylamine) showed a similar reduction of intracellular C. burnetii after exposure to killed as weal as live organisms. The data presented indicate that different endocytic mechanisms, pinocytosis as well as phagocytosis, may mediate the uptake of C. burnetii by a host cell. Key words: Coxiella burnetii; internalisation; endocytosis (authors)

  10. Diacylglycerol Guides the Hopping of Clathrin-Coated Pits along Microtubules for Exo-Endocytosis Coupling.

    Yuan, Tianyi; Liu, Lin; Zhang, Yongdeng; Wei, Lisi; Zhao, Shiqun; Zheng, Xiaolu; Huang, Xiaoshuai; Boulanger, Jerome; Gueudry, Charles; Lu, Jingze; Xie, Lihan; Du, Wen; Zong, Weijian; Yang, Lu; Salamero, Jean; Liu, Yanmei; Chen, Liangyi

    2015-10-12

    Many receptor-mediated endocytic processes are mediated by constitutive budding of clathrin-coated pits (CCPs) at spatially randomized sites before slowly pinching off from the plasma membrane (60-100 s). In contrast, clathrin-mediated endocytosis (CME) coupled with regulated exocytosis in excitable cells occurs at peri-exocytic sites shortly after vesicle fusion (∼10 s). The molecular mechanism underlying this spatiotemporal coupling remains elusive. We show that coupled endocytosis makes use of pre-formed CCPs, which hop to nascent fusion sites nearby following vesicle exocytosis. A dynamic cortical microtubular network, anchored at the cell surface by the cytoplasmic linker-associated protein on microtubules and the LL5β/ELKS complex on the plasma membrane, provides the track for CCP hopping. Local diacylglycerol gradients generated upon exocytosis guide the direction of hopping. Overall, the CCP-cytoskeleton-lipid interaction demonstrated here mediates exocytosis-coupled fast recycling of both plasma membrane and vesicular proteins, and it is required for the sustained exocytosis during repetitive stimulations. PMID:26439397

  11. Seamless tube shape is constrained by endocytosis-dependent regulation of active Moesin.

    Schottenfeld-Roames, Jodi; Rosa, Jeffrey B; Ghabrial, Amin S

    2014-08-01

    Most tubes have seams (intercellular or autocellular junctions that seal membranes together into a tube), but "seamless" tubes also exist. In Drosophila, stellate-shaped tracheal terminal cells make seamless tubes, with single branches running through each of dozens of cellular extensions. We find that mutations in braided impair terminal cell branching and cause formation of seamless tube cysts. We show that braided encodes Syntaxin7 and that cysts also form in cells deficient for other genes required either for membrane scission (shibire) or for early endosome formation (Rab5, Vps45, and Rabenosyn-5). These data define a requirement for early endocytosis in shaping seamless tube lumens. Importantly, apical proteins Crumbs and phospho-Moesin accumulate to aberrantly high levels in braided terminal cells. Overexpression of either Crumbs or phosphomimetic Moesin induced lumenal cysts and decreased terminal branching. Conversely, the braided seamless tube cyst phenotype was suppressed by mutations in crumbs or Moesin. Indeed, mutations in Moesin dominantly suppressed seamless tube cyst formation and restored terminal branching. We propose that early endocytosis maintains normal steady-state levels of Crumbs, which recruits apical phosphorylated (active) Moe, which in turn regulates seamless tube shape through modulation of cortical actin filaments. PMID:25065756

  12. The effect of vanadate on receptor-mediated endocytosis of asialoorosomucoid in rat liver parenchymal cells

    Vanadate is a phosphate analogue that inhibits enzymes involved in phosphate release and transfer reactions. Since such reactions may play important roles in endocytosis, we studied the effects of vanadate on various steps in receptor-mediated endocytosis of asialoorosomucoid labeled with 125I-tyramine-cellobiose (125I-TC-AOM). The labeled degradation products formed from 125I-TC-AOM are trapped in the lysosomes and may therefore serve as lysosomal markers in subcellular fractionation studies. Vanadate reduced the amount of active surface asialoglycoprotein receptors approximately 70%, but had no effect on the rate of internalization and retroendocytosis of ligand. The amount of surface asialoglycoprotein receptors can be reduced by lowering the incubation temperature gradually from 37 to 15 degrees C; vanadate affected only the temperature--sensitive receptors. Vanadate inhibited degradation of 125I-TC-AOM 70-80%. Degradation was much more sensitive to vanadate than binding; half-maximal effects were seen at approximately 1 mM vanadate for binding and approximately 0.1 mM vanadate for degradation. By subcellular fractionation in sucrose and Nycodenz gradients, it was shown that vanadate completely prevented the transfer of 125I-TC-AOM from endosomes to lysosomes. Therefore, the inhibition of degradation by vanadate was indirect; in the presence of vanadate, ligand did not gain access to the lysosomes. The limited degradation in the presence of vanadate took place in a prelysosomal compartment. Vanadate did not affect cell viability and ATP content

  13. Single wall carbon nanotubes enter cells by endocytosis and not membrane penetration

    Lösche Mathias

    2011-09-01

    Full Text Available Abstract Background Carbon nanotubes are increasingly being tested for use in cellular applications. Determining the mode of entry is essential to control and regulate specific interactions with cells, to understand toxicological effects of nanotubes, and to develop nanotube-based cellular technologies. We investigated cellular uptake of Pluronic copolymer-stabilized, purified ~145 nm long single wall carbon nanotubes (SWCNTs through a series of complementary cellular, cell-mimetic, and in vitro model membrane experiments. Results SWCNTs localized within fluorescently labeled endosomes, and confocal Raman spectroscopy showed a dramatic reduction in SWCNT uptake into cells at 4°C compared with 37°C. These data suggest energy-dependent endocytosis, as shown previously. We also examined the possibility for non-specific physical penetration of SWCNTs through the plasma membrane. Electrochemical impedance spectroscopy and Langmuir monolayer film balance measurements showed that Pluronic-stabilized SWCNTs associated with membranes but did not possess sufficient insertion energy to penetrate through the membrane. SWCNTs associated with vesicles made from plasma membranes but did not rupture the vesicles. Conclusions These measurements, combined, demonstrate that Pluronic-stabilized SWCNTs only enter cells via energy-dependent endocytosis, and association of SWCNTs to membrane likely increases uptake.

  14. Increased expression of endocytosis-Related proteins in rat hippocampus following 10-day electroconvulsive seizure treatment.

    Enomoto, Shingo; Shimizu, Kunio; Nibuya, Masashi; Toda, Hiroyuki; Yoshino, Aihide; Suzuki, Eiji; Kondo, Takashi; Fukuda, Hiroshi

    2016-06-15

    Although electroconvulsive therapy (ECT) is clinically used for severe depression and drug-resistant Parkinson's disease, its exact biological background and mechanism have not yet been fully elucidated. Two potential explanations have been presented so far to explain the increased neuroplastic and resilient profiles of multiple ECT administrations. One is the alteration of central neurotransmitter receptor densities and the other is the expressional upregulation of brain derived neurotrophic factor in various brain regions with enhanced hippocampal neurogenesis and mossy fiber sprouting. In the present report, western blot analyses revealed significantly upregulated expression of various endocytosis-related proteins following 10-day electroconvulsive seizure (ECS) treatment in rat hippocampal homogenates and hippocampal lipid raft fractions extracted using an ultracentrifugation procedure. Upregulated proteins included endocytosis-related scaffolding proteins (caveolin-1, flotillin-1, and heavy and light chains of clathrin) and small GTPases (Rab5, Rab7, Rab11, and Rab4) specifically expressed on various types of endosomes. Two scaffolding proteins, caveolin-1 and flotillin-1, were also increased in the lipid raft fraction. Together with our previous finding of increased autophagy-related proteins in the hippocampal region, the present results suggest membrane trafficking machinery is enhanced following 10-day ECS treatment. We consider that the membrane trafficking machinery that transports functional proteins in the neuronal cells and from or into the synaptic membranes is one of the new candidates supporting the cellular and behavioral neuroplastic profiles of ECS treatments in animal experiments and ECT administrations in clinical settings. PMID:27177725

  15. Mitochondrial uncouplers inhibit clathrin-mediated endocytosis largely through cytoplasmic acidification

    Dejonghe, Wim; Kuenen, Sabine; Mylle, Evelien; Vasileva, Mina; Keech, Olivier; Viotti, Corrado; Swerts, Jef; Fendrych, Matyáš; Ortiz-Morea, Fausto Andres; Mishev, Kiril; Delang, Simon; Scholl, Stefan; Zarza, Xavier; Heilmann, Mareike; Kourelis, Jiorgos; Kasprowicz, Jaroslaw; Nguyen, Le Son Long; Drozdzecki, Andrzej; Van Houtte, Isabelle; Szatmári, Anna-Mária; Majda, Mateusz; Baisa, Gary; Bednarek, Sebastian York; Robert, Stéphanie; Audenaert, Dominique; Testerink, Christa; Munnik, Teun; Van Damme, Daniël; Heilmann, Ingo; Schumacher, Karin; Winne, Johan; Friml, Jiří; Verstreken, Patrik; Russinova, Eugenia

    2016-01-01

    ATP production requires the establishment of an electrochemical proton gradient across the inner mitochondrial membrane. Mitochondrial uncouplers dissipate this proton gradient and disrupt numerous cellular processes, including vesicular trafficking, mainly through energy depletion. Here we show that Endosidin9 (ES9), a novel mitochondrial uncoupler, is a potent inhibitor of clathrin-mediated endocytosis (CME) in different systems and that ES9 induces inhibition of CME not because of its effect on cellular ATP, but rather due to its protonophore activity that leads to cytoplasm acidification. We show that the known tyrosine kinase inhibitor tyrphostinA23, which is routinely used to block CME, displays similar properties, thus questioning its use as a specific inhibitor of cargo recognition by the AP-2 adaptor complex via tyrosine motif-based endocytosis signals. Furthermore, we show that cytoplasm acidification dramatically affects the dynamics and recruitment of clathrin and associated adaptors, and leads to reduction of phosphatidylinositol 4,5-biphosphate from the plasma membrane. PMID:27271794

  16. CD4- and dynamin-dependent endocytosis of HIV-1 into plasmacytoid dendritic cells

    Pritschet, Kathrin; Donhauser, Norbert; Schuster, Philipp; Ries, Moritz; Haupt, Sabrina; Kittan, Nicolai A.; Korn, Klaus [Institute of Clinical and Molecular Virology, National Reference Centre for Retroviruses, Friedrich-Alexander-Universitaet Erlangen-Nuernberg, 91054 Erlangen (Germany); Poehlmann, Stefan [Institute of Virology, Hannover Medical School, 30625 Hannover (Germany); Holland, Gudrun; Bannert, Norbert [Robert Koch-Institute, Center for Biological Security 4, 13353 Berlin (Germany); Bogner, Elke [Institute of Virology, Charite University Hospital, 10117 Berlin (Germany); Schmidt, Barbara, E-mail: baschmid@viro.med.uni-erlangen.de [Institute of Clinical and Molecular Virology, National Reference Centre for Retroviruses, Friedrich-Alexander-Universitaet Erlangen-Nuernberg, 91054 Erlangen (Germany)

    2012-02-20

    Chronic immune activation, triggered by plasmacytoid dendritic cell (PDC) interferon (IFN)-alpha production, plays an important role in HIV-1 pathogenesis. As the entry of HIV-1 seems to be important for the activation of PDC, we directly characterized the viral entry into these cells using immuno-electron microscopy, cellular fractionation, confocal imaging, and functional experiments. After attachment to PDC, viruses were taken up in an energy-dependent manner. The virions were located in compartments positive for caveolin; early endosomal antigen 1; Rab GTPases 5, 7 and 9; lysosomal-associated membrane protein 1. PDC harbored more virus in endocytic vesicles than CD4+ T cells (p < 0.05). Blocking CD4 inhibited the uptake of virions into cytosolic and endosomal compartments. Dynasore, an inhibitor of dynamin-dependent endocytosis, not the fusion inhibitor T-20, reduced the HIV-1 induced IFN-alpha production. Altogether, our morphological and functional data support the role of endocytosis for the entry and IFN-alpha induction of HIV-1 in PDC.

  17. CD4- and dynamin-dependent endocytosis of HIV-1 into plasmacytoid dendritic cells

    Chronic immune activation, triggered by plasmacytoid dendritic cell (PDC) interferon (IFN)-alpha production, plays an important role in HIV-1 pathogenesis. As the entry of HIV-1 seems to be important for the activation of PDC, we directly characterized the viral entry into these cells using immuno-electron microscopy, cellular fractionation, confocal imaging, and functional experiments. After attachment to PDC, viruses were taken up in an energy-dependent manner. The virions were located in compartments positive for caveolin; early endosomal antigen 1; Rab GTPases 5, 7 and 9; lysosomal-associated membrane protein 1. PDC harbored more virus in endocytic vesicles than CD4+ T cells (p < 0.05). Blocking CD4 inhibited the uptake of virions into cytosolic and endosomal compartments. Dynasore, an inhibitor of dynamin-dependent endocytosis, not the fusion inhibitor T-20, reduced the HIV-1 induced IFN-alpha production. Altogether, our morphological and functional data support the role of endocytosis for the entry and IFN-alpha induction of HIV-1 in PDC.

  18. New Regulators of Clathrin-Mediated Endocytosis Identified in Saccharomyces cerevisiae by Systematic Quantitative Fluorescence Microscopy.

    Farrell, Kristen B; Grossman, Caitlin; Di Pietro, Santiago M

    2015-11-01

    Despite the importance of clathrin-mediated endocytosis (CME) for cell biology, it is unclear if all components of the machinery have been discovered and many regulatory aspects remain poorly understood. Here, using Saccharomyces cerevisiae and a fluorescence microscopy screening approach we identify previously unknown regulatory factors of the endocytic machinery. We further studied the top scoring protein identified in the screen, Ubx3, a member of the conserved ubiquitin regulatory X (UBX) protein family. In vivo and in vitro approaches demonstrate that Ubx3 is a new coat component. Ubx3-GFP has typical endocytic coat protein dynamics with a patch lifetime of 45 ± 3 sec. Ubx3 contains a W-box that mediates physical interaction with clathrin and Ubx3-GFP patch lifetime depends on clathrin. Deletion of the UBX3 gene caused defects in the uptake of Lucifer Yellow and the methionine transporter Mup1 demonstrating that Ubx3 is needed for efficient endocytosis. Further, the UBX domain is required both for localization and function of Ubx3 at endocytic sites. Mechanistically, Ubx3 regulates dynamics and patch lifetime of the early arriving protein Ede1 but not later arriving coat proteins or actin assembly. Conversely, Ede1 regulates the patch lifetime of Ubx3. Ubx3 likely regulates CME via the AAA-ATPase Cdc48, a ubiquitin-editing complex. Our results uncovered new components of the CME machinery that regulate this fundamental process. PMID:26362318

  19. Osmotic induction of fluid-phase endocytosis in onion epidermal cells.

    Oparka, K J; Prior, D A; Harris, N

    1990-03-01

    A transient plasmolysis/deplasmolysis (plasmolytic cycle) of onion epidermal cells has been shown to induce the formation of fluid-phase endocytic vesicles. Plasmolysis in the presence of the membrane-impermeant fluorescent probes Lucifer Yellow CH (LYCH) and Cascade Blue hydrazide resulted in the uptake of these probes by fluid-phase endocytosis. Following deplasmolysis, many of the dye-containing vesicles left their parietal positions within the cell and underwent vigorous streaming in the cytoplasm. Vesicles were observed to move within transvacuolar strands and their movements were recorded over several hours by video-microscopy. Within 2 h of deplasmolysis several of the larger endocytic vesicles had clustered around the nuclear membrane, apparently lodged in the narrow zone of cytoplams surrounding the nucleus. In further experiments LYCH was endocytically loaded into the cells during the first plasmolytic cycle and Cascade Blue subsequently loaded during a second plasmolytic cycle. This resulted in the introduction of two populations of endocytic vesicles into the cells, each containing a different probe. Both sets of vesicles underwent cytoplasmic streaming. The data are discussed in the light of previous observations of fluid-phase endocytosis in plant cells. PMID:24202101

  20. The ulcerative colitis marker protein WAFL interacts with accessory proteins in endocytosis

    You Fu Pan, Ing-Marie Viklund, Heng Hang Tsai, Sven Pettersson, Ichiro N. Maruyama

    2010-01-01

    Full Text Available Ulcerative colitis (UC is one of the major forms of inflammatory bowel disease with unknown cause. A molecular marker, WAFL, has recently been found to be up-regulated in the inflamed colonic mucosa of UC patients. Towards understanding biological function of WAFL, we analyzed proteins interacting with WAFL in HEK-293 cells by immunoprecipitation and mass spectrometry. Among four proteins found to specifically interact with WAFL, both KIAA0196 and KIAA1033 bind to α-appendage of the adaptor protein complex 2 (AP2, which acts as an interaction hub for accessory proteins in endocytosis mediated by clathrin-coated vesicle (CCV. The specific interaction between WAFL and KIAA0196 was also confirmed in human colorectal carcinoma HCT-116 cells by co-immunoprecipitation with specific antibodies. Meta-analyses of the databases of expressed genes suggest that the three genes are co-expressed in many tissues and cell types, and that their molecular function may be classified in the category of 'membrane traffic protein'. Therefore, these results suggest that WAFL may play an important role in endocytosis and subsequent membrane trafficking by interacting with AP2 through KIAA0196 and KIAA1033.

  1. Mining the bulk positron lifetime

    Aourag, H.; Guittom, A. [Centre de Recherche Nucleaire d' Alger (CRNA), Alger Gare - Algiers (Algeria)

    2009-02-15

    We introduce a new approach to investigate the bulk positron lifetimes of new systems based on data-mining techniques. Through data mining of bulk positron lifetimes, we demonstrate the ability to predict the positron lifetimes of new semiconductors on the basis of available semiconductor data already studied. Informatics techniques have been applied to bulk positron lifetimes for different tetrahedrally bounded semiconductors in order to discover computational design rules. (copyright 2009 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  2. Mining the bulk positron lifetime

    We introduce a new approach to investigate the bulk positron lifetimes of new systems based on data-mining techniques. Through data mining of bulk positron lifetimes, we demonstrate the ability to predict the positron lifetimes of new semiconductors on the basis of available semiconductor data already studied. Informatics techniques have been applied to bulk positron lifetimes for different tetrahedrally bounded semiconductors in order to discover computational design rules. (copyright 2009 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  3. Advances in bulk port development

    Soros, P. (Soros Associates Consulting Engineers, New York, NY (USA))

    1991-03-01

    The article features several recently developed bulk ports which illustrate aspects of new technology or concepts in maritime transport. Low handling capacity bulk terminals at Ponta da Madeira, Brazil and Kooragang Island, Australia and the low-cost bulk port at Port of Corpus Christi, Texas are described. Operations at the ports of Pecket and Tocopilla in Chile, which had special technical problems, are mentioned. Coal terminals at Port Kembla, Australia and St. Johns River in Florid Jacksonville, Florida are featured as examples of terminals which had to be designed to meet high environmental standards. 13 refs., 2 figs., 14 photos.

  4. The carboxyl terminus of human cytomegalovirus-encoded 7 transmembrane receptor US28 camouflages agonism by mediating constitutive endocytosis

    Waldhoer, Maria; Casarosa, Paola; Rosenkilde, Mette M;

    2003-01-01

    US28 is one of four 7 transmembrane (7TM) chemokine receptors encoded by human cytomegalovirus and has been shown to both signal and endocytose in a ligand-independent, constitutively active manner. Here we show that the constitutive activity and constitutive endocytosis properties of US28 are se...... a 7TM receptor can camouflage the agonist properties of a ligand....

  5. Sphingosine 1-phosphate-induced motility and endocytosis of dendritic cells is regulated by SWAP-70 through RhoA.

    Ocaña-Morgner, Carlos; Reichardt, Peter; Chopin, Michaël; Braungart, Sarah; Wahren, Christine; Gunzer, Matthias; Jessberger, Rolf

    2011-05-01

    The phospholipid mediator sphingosine 1-phosphate (S1P) enhances motility and endocytosis of mature dendritic cells (DCs). We show that in vitro migration of Swap-70(-/-) bone marrow-derived DCs (BMDCs) in response to S1P and S1P-induced upregulation of endocytosis are significantly reduced. S1P-stimulated movement of Swap-70(-/-) BMDCs, specifically retraction of their trailing edge, in a collagen three-dimensional environment is impaired. These in vitro observations correlate with delayed entry into lymphatic vessels and migration to lymph nodes of skin DCs in Swap-70(-/-) mice. Expression of S1P receptors (S1P(1-3)) by wild-type and Swap-70(-/-) BMDCs is similar, but Swap-70(-/-) BMDCs fail to activate RhoA and to localize Rac1 and RhoA into areas of actin polymerization after S1P stimulus. The Rho-activating G protein Gα(i) interacts with SWAP-70, which also supports the localization of Gα(13) to membrane rafts in BMDCs. LPS-matured Swap-70(-/-) BMDCs contain significantly more active RhoA than wild-type DCs. Preinhibition of Rho activation restored migration to S1P, S1P-induced upregulation of endocytosis in mature Swap-70(-/-) BMDCs, and localization of Gα(13) to membrane rafts. These data demonstrate SWAP-70 as a novel regulator of S1P signaling necessary for DC motility and endocytosis. PMID:21421853

  6. The polyene antimycotics nystatin and filipin disrupt the plasma membrane, whereas natamycin inhibits endocytosis in germinating conidia of Penicillium discolor

    van Leeuwen, M.R.; Golovina, E.A.; Dijksterhuis, J.

    2009-01-01

    AIMS: To investigate the differences in membrane permeability and the effect on endocytosis of the polyene antimycotics nystatin, filipin and natamycin on germinating fungal conidia. METHODS AND RESULTS: The model system was Penicillium discolor, a food spoilage fungus. Filipin resulted in permeabil

  7. The polyene antimycotics nystatin and filipin disrupt the plasma membrane, whereas natamycin inhibits endocytosis in germinating conidia of Penicillium discolor

    Leeuwen, van M.R.; Golovina, E.A.; Dijksterhuis, J.

    2009-01-01

    To investigate the differences in membrane permeability and the effect on endocytosis of the polyene antimycotics nystatin, filipin and natamycin on germinating fungal conidia. Methods and Results: The model system was Penicillium discolor, a food spoilage fungus. Filipin resulted in permeabilizatio

  8. Light-regulated stapled peptides to inhibit protein-protein interactions involved in clathrin-mediated endocytosis.

    Nevola, Laura; Martín-Quirós, Andrés; Eckelt, Kay; Camarero, Núria; Tosi, Sébastien; Llobet, Artur; Giralt, Ernest; Gorostiza, Pau

    2013-07-22

    Control of membrane traffic: Photoswitchable inhibitors of protein-protein interactions were applied to photoregulate clathrin-mediated endocytosis (CME) in living cells. Traffic light (TL) peptides acting as "stop" and "go" signals for membrane traffic can be used to dissect the role of CME in receptor internalization and in cell growth, division, and differentiation. PMID:23775788

  9. MPTP-meditated hippocampal dopamine deprivation modulates synaptic transmission and activity-dependent synaptic plasticity

    Parkinson's disease (PD)-like symptoms including learning deficits are inducible by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Therefore, it is possible that MPTP may disturb hippocampal memory processing by modulation of dopamine (DA)- and activity-dependent synaptic plasticity. We demonstrate here that intraperitoneal (i.p.) MPTP injection reduces the number of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra (SN) within 7 days. Subsequently, the TH expression level in SN and hippocampus and the amount of DA and its metabolite DOPAC in striatum and hippocampus decrease. DA depletion does not alter basal synaptic transmission and changes pair-pulse facilitation (PPF) of field excitatory postsynaptic potentials (fEPSPs) only at the 30 ms inter-pulse interval. In addition, the induction of long-term potentiation (LTP) is impaired whereas the duration of long-term depression (LTD) becomes prolonged. Since both LTP and LTD depend critically on activation of NMDA and DA receptors, we also tested the effect of DA depletion on NMDA receptor-mediated synaptic transmission. Seven days after MPTP injection, the NMDA receptor-mediated fEPSPs are decreased by about 23%. Blocking the NMDA receptor-mediated fEPSP does not mimic the MPTP-LTP. Only co-application of D1/D5 and NMDA receptor antagonists during tetanization resembled the time course of fEPSP potentiation as observed 7 days after i.p. MPTP injection. Together, our data demonstrate that MPTP-induced degeneration of DA neurons and the subsequent hippocampal DA depletion alter NMDA receptor-mediated synaptic transmission and activity-dependent synaptic plasticity. - Highlights: → I.p. MPTP-injection mediates death of dopaminergic neurons. → I.p. MPTP-injection depletes DA and DOPAC in striatum and hippocampus. → I.p. MPTP-injection does not alter basal synaptic transmission. → Reduction of LTP and enhancement of LTD after i.p. MPTP-injection. → Attenuation of NMDA-receptors mediated

  10. Bulk charges in eleven dimensions

    Hawking, Stephen William

    1998-01-01

    Eleven dimensional supergravity has electric type currents arising from the Chern-Simon and anomaly terms in the action. However the bulk charge integrates to zero for asymptotically flat solutions with topological trivial spatial sections. We show that by relaxing the boundary conditions to generalisations of the ALE and ALF boundary conditions in four dimensions one can obtain static solutions with a bulk charge preserving between 1/16 and 1/4 of the supersymmetries. One can introduce membranes with the same sign of charge into these backgrounds. This raises the possibility that these generalized membranes might decay quantum mechanically to leave just a bulk distribution of charge. Alternatively and more probably, a bulk distribution of charge can decay into a collection of singlely charged membranes. Dimensional reductions of these solutions lead to novel representations of extreme black holes in four dimensions with up to four charges. We discuss how the eleven-dimensional Kaluza-Klein monopole wrapped a...

  11. Bulk Nuclear Properties from Reactions

    Danielewicz, P.

    2002-01-01

    Extraction of bulk nuclear properties by comparing reaction observables to results from semiclassical transport-model simulations is discussed. Specific properties include the nuclear viscosity, incompressibility and constraints on the nuclear pressure at supranormal densities.

  12. Three-dimensional imaging of single nanotube molecule endocytosis on plasmonic substrates

    Hong, Guosong; Robinson, Joshua T; Wang, Hailiang; Zhang, Bo; Dai, Hongjie

    2012-01-01

    Investigating the cellular internalization pathways of single molecules or single nano-objects is important to understanding cell-matter interactions and to applications in drug delivery and discovery. Imaging and tracking the motion of single molecules on cell plasma membrane require high spatial resolution in three dimensions (3D). Fluorescence imaging along the axial dimension with nanometer resolution has been highly challenging but critical to revealing displacements in trans-membrane events. Here, utilizing a plasmonic ruler based on the sensitive distance dependence of near-infrared fluorescence enhancement (NIR-FE) of carbon nanotubes on a gold plasmonic substrate, we probe ~10 nm scale trans-membrane displacements through changes in nanotube fluorescence intensity, enabling observations of single nanotube endocytosis in 3D. Cellular uptake and trans-membrane displacements show clear dependences to temperature and clathrin assembly on cell membrane, suggesting that the cellular entry mechanism for a n...

  13. Epidermal growth factor receptor and cancer: control of oncogenic signalling by endocytosis

    Grandal, Michael Vibo; Madshus, I.H.

    2008-01-01

    prevents its down-regulation, underscoring the importance of the cellular background for EGFR effects. Signalling from ErbB proteins can either be terminated by dissociation of ligand resulting in dephosphorylation, or blunted by degradation of the receptors. Although proteasomal targeting of ErbB proteins...... has been described, lysosomal degradation upon ligand-induced endocytosis seems to play the major role in EGFR down-regulation. Preclinical and clinical data have demonstrated that EGFR is a central player in cancer, especially in carcinomas, some brain tumours and in non-small cell lung cancer. Such...... studies have further validated EGFR as an important molecular target in cancer treatment. This review focuses on mechanisms involved in ligand-induced EGFR activation and endocytic down-regulation. A better understanding of EGFR biology should allow development of more tumour-selective therapeutic...

  14. Membrane tension is a key determinant of bud morphology in clathrin-mediated endocytosis

    Hassinger, Julian E; Drubin, David G; Rangamani, Padmini

    2016-01-01

    In clathrin-mediated endocytosis (CME), clathrin and various adaptor proteins coat a patch of the plasma membrane, which is reshaped to form a budded vesicle. Experimental studies have demonstrated that elevated membrane tension can inhibit bud formation by a clathrin coat. In this study, we investigate the impact of membrane tension on the mechanics of membrane budding by simulating clathrin coats that either grow in area or progressively induce greater curvature. At low membrane tension, progressively increasing the area of a curvature-generating coat causes the membrane to smoothly evolve from a flat to budded morphology, whereas the membrane remains essentially flat at high membrane tensions. Interestingly, at physiologically relevant, intermediate membrane tensions, the shape evolution of the membrane undergoes a snapthrough instability in which increasing coat area causes the membrane to "snap" from an open, U-shaped bud to a closed, $\\Omega$-shaped bud. This instability is accompanied by a large energy...

  15. Non-26S Proteasome Proteolytic Role of Ubiquitin in Plant Endocytosis and Endosomal Trafficking

    Miaomiao Tian; Qi Xie

    2013-01-01

    The 76 amino acid protein ubiquitin (Ub) is highly conserved in all eukaryotic species.It plays important roles in many cellular processes by covalently attaching to the target proteins.The best known function of Ub is marking substrate proteins for degradation by the 26S proteasome.In fact,other consequences of ubiquitination have been discovered in yeast and mammals,such as membrane trafficking,DNA repair,chromatin modification,and protein kinase activation.The common mechanism underlying these processes is that Ub serves as a signal to sort proteins to the vacuoles or lysosomes for degradation as opposed to 26S proteasome-dependent degradation.To date,several reports have indicated that a similar function of Ub also exists in plants.This review focuses on a summary and analysis of the recent research progress on Ub acting as a signal to mediate endocytosis and endosomal trafficking in plants.

  16. Endocytosis of pro-inflammatory cytokine receptors and its relevance for signal transduction.

    Hermanns, Heike M; Wohlfahrt, Julia; Mais, Christine; Hergovits, Sabine; Jahn, Daniel; Geier, Andreas

    2016-08-01

    The pro-inflammatory cytokines tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6) are key players of the innate and adaptive immunity. Their activity needs to be tightly controlled to allow the initiation of an appropriate immune response as defense mechanism against pathogens or tissue injury. Excessive or sustained signaling of either of these cytokines leads to severe diseases, including rheumatoid arthritis, inflammatory bowel diseases (Crohn's disease, ulcerative colitis), steatohepatitis, periodic fevers and even cancer. Studies carried out in the last 30 years have emphasized that an elaborate control system for each of these cytokines exists. Here, we summarize what is currently known about the involvement of receptor endocytosis in the regulation of these pro-inflammatory cytokines' signaling cascades. Particularly in the last few years it was shown that this cellular process is far more than a mere feedback mechanism to clear cytokines from the circulation and to shut off their signal transduction. PMID:27071147

  17. Intestinal alkaline phosphatase: selective endocytosis from the enterocyte brush border during fat absorption

    Hansen, Gert Helge; Niels-Christiansen, Lise-Lotte; Immerdal, Lissi;

    2007-01-01

    explants. By immunofluorescence microscopy, fat absorption caused a translocation of IAP from the enterocyte brush border to the interior of the cell, whereas other brush-border enzymes were unaffected. By electron microscopy, the translocation occurred by a rapid (5 min) induction of endocytosis via...... clathrin-coated pits. By 60 min, IAP was seen in subapical endosomes and along membranes surrounding fat droplets. IAP is a well-known lipid raft-associated protein, and fat absorption was accompanied by a marked change in the density and morphology of the detergent-resistant membranes harboring IAP. A...... IAP and may contribute to the appearance of the enzyme in serum and surfactant-like particles....

  18. Analysis of synaptic vesicle endocytosis in synaptosomes by high-content screening.

    Daniel, James A; Malladi, Chandra S; Kettle, Emma; McCluskey, Adam; Robinson, Phillip J

    2012-08-01

    Small molecules modulating synaptic vesicle endocytosis (SVE) may ultimately be useful for diseases where pathological neurotransmission is implicated. Only a small number of specific SVE modulators have been identified to date. Slow progress is due to the laborious nature of traditional approaches to study SVE, in which nerve terminals are identified and studied in cultured neurons, typically yielding data from 10-20 synapses per experiment. We provide a protocol for a quantitative, high-throughput method for studying SVE in thousands of nerve terminals. Rat forebrain synaptosomes are attached to 96-well microplates and depolarized; SVE is then quantified by uptake of the dye FM4-64, which is imaged by high-content screening. Synaptosomes that have been frozen and stored can be used in place of fresh synaptosomes, reducing the experimental time and animal numbers required. With a supply of frozen synaptosomes, the assay can be performed within a day, including data analysis. PMID:22767087

  19. Expression of activity-dependent neuroprotective protein in the brain of adult rats.

    Gennet, N; Herden, C; Bubb, V J; Quinn, J P; Kipar, A

    2008-03-01

    Activity-dependent neuroprotective protein (ADNP) is a VIP-regulated gene, which is essential for brain development. A synthetic peptide (NAP) derived from the ADNP sequence is highly neuroprotective, therefore it has been hypothesised that ADNP has a similar role. ADNP contains classical transcription factor motifs and nuclear localisation domains, but it has also been reported to be secreted and to co-localise with microtubules, indicating that ADNP may have multiple functions. We investigated the pattern of ADNP expression by immunohistology in normal rat brain, in order to generate a framework for future studies examining changes in ADNP expression in response to noxious stimuli or in models of disease. We found widespread ADNP-like immunoreactivity in neurons throughout the rat brain, with the highest expression in the cerebellum, and strong expression in the thalamus, mesencephalon, pons and medulla oblongata. ADNP-like immunoreactivity was mainly observed in the cytoplasm of neurons, and fibre tracts were often strongly positive as well. In addition, positive neuronal nuclei were occasionally observed. ADNP-like immunoreactivity was lost in degenerating "dark" neurons, whereas it appeared to locate to the nucleus in some of the morphologically unaltered adjacent cells. Occasional astrocyte and microglial cells were also positive. We suggest that the widespread expression of ADNP may correlate with the wide-ranging protective effects of NAP, and that the cytoplasmic and axonal localisation of ADNP-like immunoreactivity suggests additional, non-transcriptional functions of ADNP. PMID:18072088

  20. Activity-dependent BDNF release via endocytic pathways is regulated by synaptotagmin-6 and complexin.

    Wong, Yu-Hui; Lee, Chia-Ming; Xie, Wenjun; Cui, Bianxiao; Poo, Mu-ming

    2015-08-11

    Brain-derived neurotrophic factor (BDNF) is known to modulate synapse development and plasticity, but the source of synaptic BDNF and molecular mechanisms regulating BDNF release remain unclear. Using exogenous BDNF tagged with quantum dots (BDNF-QDs), we found that endocytosed BDNF-QDs were preferentially localized to postsynaptic sites in the dendrite of cultured hippocampal neurons. Repetitive neuronal spiking induced the release of BDNF-QDs at these sites, and this process required activation of glutamate receptors. Down-regulating complexin 1/2 (Cpx1/2) expression eliminated activity-induced BDNF-QD secretion, although the overall activity-independent secretion was elevated. Among eight synaptotagmin (Syt) isoforms examined, down-regulation of only Syt6 impaired activity-induced BDNF-QD secretion. In contrast, activity-induced release of endogenously synthesized BDNF did not depend on Syt6. Thus, neuronal activity could trigger the release of endosomal BDNF from postsynaptic dendrites in a Cpx- and Syt6-dependent manner, and endosomes containing BDNF may serve as a source of BDNF for activity-dependent synaptic modulation. PMID:26216953

  1. Activity-dependent synaptic plasticity modulates the critical phase of brain development.

    Chaudhury, Sraboni; Sharma, Vikram; Kumar, Vivek; Nag, Tapas C; Wadhwa, Shashi

    2016-04-01

    Plasticity or neuronal plasticity is a unique and adaptive feature of nervous system which allows neurons to reorganize their interactions in response to an intrinsic or extrinsic stimulation and shapes the formation and maintenance of a functional neuronal circuit. Synaptic plasticity is the most important form of neural plasticity and plays critical role during the development allowing the formation of precise neural connectivity via the process of pruning. In the sensory systems-auditory and visual, this process is heavily dependent on the external cues perceived during the development. Environmental enrichment paradigms in an activity-dependent manner result in early maturation of the synapses and more efficient trans-synaptic signaling or communication flow. This has been extensively observed in the avian auditory system. On the other hand, stimuli results in negative effect can cause alterations in the synaptic connectivity and strength resulting in various developmental brain disorders including autism, fragile X syndrome and rett syndrome. In this review we discuss the role of different forms of activity (spontaneous or environmental) during the development of the nervous system in modifying synaptic plasticity necessary for shaping the adult brain. Also, we try to explore various factors (molecular, genetic and epigenetic) involved in altering the synaptic plasticity in positive and negative way. PMID:26515724

  2. Cell Biological Mechanisms of Activity-Dependent Synapse to Nucleus Translocation of CRTC1 in Neurons

    Toh Hean eCh'ng

    2015-09-01

    Full Text Available Previous studies have revealed a critical role for CREB-regulated transcriptional coactivator (CRTC1 in regulating neuronal gene expression during learning and memory. CRTC1 localizes to synapses but undergoes activity-dependent nuclear translocation to regulate the transcription of CREB target genes. Here we investigate the long-distance retrograde transport of CRTC1 in hippocampal neurons. We show that local elevations in calcium, triggered by activation of synaptic glutamate receptors and L-type voltage-gated calcium channels, initiate active, dynein-mediated retrograde transport of CRTC1 along microtubules. We identify a nuclear localization signal within CRTC1, and characterize three conserved serine residues whose dephosphorylation is required for nuclear import. Domain analysis reveals that the amino-terminal third of CRTC1 contains all of the signals required for regulated nucleocytoplasmic trafficking. We fuse this region to Dendra2 to generate a reporter construct and perform live-cell imaging coupled with local uncaging of glutamate and photoconversion to characterize the dynamics of stimulus-induced retrograde transport and nuclear accumulation.

  3. Activity-dependent synaptic plasticity of a chalcogenide electronic synapse for neuromorphic systems.

    Li, Yi; Zhong, Yingpeng; Zhang, Jinjian; Xu, Lei; Wang, Qing; Sun, Huajun; Tong, Hao; Cheng, Xiaoming; Miao, Xiangshui

    2014-01-01

    Nanoscale inorganic electronic synapses or synaptic devices, which are capable of emulating the functions of biological synapses of brain neuronal systems, are regarded as the basic building blocks for beyond-Von Neumann computing architecture, combining information storage and processing. Here, we demonstrate a Ag/AgInSbTe/Ag structure for chalcogenide memristor-based electronic synapses. The memristive characteristics with reproducible gradual resistance tuning are utilised to mimic the activity-dependent synaptic plasticity that serves as the basis of memory and learning. Bidirectional long-term Hebbian plasticity modulation is implemented by the coactivity of pre- and postsynaptic spikes, and the sign and degree are affected by assorted factors including the temporal difference, spike rate and voltage. Moreover, synaptic saturation is observed to be an adjustment of Hebbian rules to stabilise the growth of synaptic weights. Our results may contribute to the development of highly functional plastic electronic synapses and the further construction of next-generation parallel neuromorphic computing architecture. PMID:24809396

  4. Developmental and activity-dependent plasticity of filiform hair receptors in the locust

    HaraldWolf

    2013-08-01

    Full Text Available A group of wind sensitive filiform hair receptors on the locust thorax and head makes contact onto a pair of identified interneuron, A4I1. The hair receptors’ central nervous projections exhibit pronounced structural dynamics during nymphal development, for example, by gradually eliminating their ipsilateral dendritic field while maintaining the contralateral one. These changes are dependent not only on hormones controlling development but on neuronal activity as well. The hair-to-interneuron system has remarkably high gain (close to 1 and makes contact to flight steering muscles. During stationary flight in front of a wind tunnel, interneuron A4I1 is active in the wing beat rhythm, and in addition it responds strongly to stimulation of sensory hairs in its receptive field. A role of the hair-to-interneuron in flight steering is thus suggested. This system appears suitable for further study of developmental and activity-dependent plasticity in a sensorimotor context with known connectivity patterns.

  5. Long lasting protein synthesis- and activity-dependent spine shrinkage and elimination after synaptic depression.

    Yazmín Ramiro-Cortés

    Full Text Available Neuronal circuits modify their response to synaptic inputs in an experience-dependent fashion. Increases in synaptic weights are accompanied by structural modifications, and activity dependent, long lasting growth of dendritic spines requires new protein synthesis. When multiple spines are potentiated within a dendritic domain, they show dynamic structural plasticity changes, indicating that spines can undergo bidirectional physical modifications. However, it is unclear whether protein synthesis dependent synaptic depression leads to long lasting structural changes. Here, we investigate the structural correlates of protein synthesis dependent long-term depression (LTD mediated by metabotropic glutamate receptors (mGluRs through two-photon imaging of dendritic spines on hippocampal pyramidal neurons. We find that induction of mGluR-LTD leads to robust and long lasting spine shrinkage and elimination that lasts for up to 24 hours. These effects depend on signaling through group I mGluRs, require protein synthesis, and activity. These data reveal a mechanism for long lasting remodeling of synaptic inputs, and offer potential insights into mental retardation.

  6. CD4-independent human immunodeficiency virus infection involves participation of endocytosis and cathepsin B.

    Hiroaki Yoshii

    Full Text Available During a comparison of the infectivity of mNDK, a CD4-independent human immunodeficiency virus type 1 (HIV-1 strain, to various cell lines, we found that HeLa cells were much less susceptible than 293T and TE671 cells. Hybridoma cells between HeLa and 293T cells were as susceptible as 293T cells, suggesting that cellular factors enhance the mNDK infection in 293T cells. By screening a cDNA expression library in HeLa cells, cystatin C was isolated as an enhancer of the mNDK infection. Because cathepsin B protease, a natural ligand of cystatin C, was upregulated in HeLa cells, we speculated that the high levels of cathepsin B activities were inhibitory to the CD4-independent infection and that cystatin C enhanced the infection by impairing the excessive cathepsin B activity. Consistent with this idea, pretreatment of HeLa cells with 125 µM of CA-074Me, a cathepsin B inhibitor, resulted in an 8-fold enhancement of the mNDK infectivity. Because cathepsin B is activated by low pH in acidic endosomes, we further examined the potential roles of endosomes in the CD4-independent infection. Suppression of endosome acidification or endocytosis by inhibitors or by an Eps15 dominant negative mutant reduced the infectivity of mNDK in which CD4-dependent infections were not significantly impaired. Taken together, these results suggest that endocytosis, endosomal acidification, and cathepsin B activity are involved in the CD4-independent entry of HIV-1.

  7. C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

    FrançoisMarceau

    2013-09-01

    Full Text Available The C-C chemokine receptor-7 (CCR7 is a G protein coupled receptor that has a role in leukocyte homing, but that is also expressed in aggressive tumor cells. Preclinical research supports that CCR7 is a valid target in oncology. In view of the increasing availability of therapeutic monoclonal antibodies that carry cytotoxic cargoes, we studied the feasibility of forcing intact cells to internalize known monoclonal antibodies by exploiting the cycle of endocytosis and recycling triggered by the CCR7 agonist CCL19. Firstly, an anti-CCR7 antibody (CD197; clone 150503 labeled surface recombinant CCR7 expressed in intact HEK 293a cells and the fluorescent antibody was internalized following CCL19 treatment. Secondly, a recombinant myc-tagged CCL19 construction was exploited along the anti-myc monoclonal antibody 4A6. The myc-tagged ligand was produced as a conditioned medium of transfected HEK 293a cells that contained the equivalent of 430 ng/ml of immunoreactive CCL19 (average value, ELISA determination. CCL19-myc, but not authentic CCL19, carried the fluorophore-labeled antibody 4A6 into other recipient cells that expressed recombinant CCR7 (microscopy, cytofluorometry. The immune complexes were apparent in endosomal structures, colocalized well with the small GTPase Rab5 and progressed toward Rab7-positive endosomes. A dominant negative form of Rab5 (GDP-locked inhibited this endocytosis. Further, endosomes in CCL19-myc- or CCL19-stimulated cells were positive for β-arrestin2, but rarely for β-arrestin1. Following treatment with CCL19-myc and the 4A6 antibody, the melanoma cell line A375 that expresses endogenous CCR7 was specifically stained using a secondary peroxidase-conjugated antibody. Agonist-stimulated CCR7 can transport antibody-based cargoes, with possible therapeutic applications in oncology.

  8. Tissue-type plasminogen activator induces synaptic vesicle endocytosis in cerebral cortical neurons.

    Yepes, M; Wu, F; Torre, E; Cuellar-Giraldo, D; Jia, D; Cheng, L

    2016-04-01

    The release of the serine proteinase tissue-type plasminogen activator (tPA) from the presynaptic terminal of cerebral cortical neurons plays a central role in the development of synaptic plasticity, adaptation to metabolic stress and neuronal survival. Our earlier studies indicate that by inducing the recruitment of the cytoskeletal protein βII-spectrin and voltage-gated calcium channels to the active zone, tPA promotes Ca(2+)-dependent translocation of synaptic vesicles (SVs) to the synaptic release site where they release their load of neurotransmitters into the synaptic cleft. Here we used a combination of in vivo and in vitro experiments to investigate whether this effect leads to depletion of SVs in the presynaptic terminal. Our data indicate that tPA promotes SV endocytosis via a mechanism that does not require the conversion of plasminogen into plasmin. Instead, we show that tPA induces calcineurin-mediated dynamin I dephosphorylation, which is followed by dynamin I-induced recruitment of the actin-binding protein profilin II to the presynaptic membrane, and profilin II-induced F-actin formation. We report that this tPA-induced sequence of events leads to the association of newly formed SVs with F-actin clusters in the endocytic zone. In summary, the data presented here indicate that following the exocytotic release of neurotransmitters tPA activates the mechanism whereby SVs are retrieved from the presynaptic membrane and endocytosed to replenish the pool of vesicles available for a new cycle of exocytosis. Together, these results indicate that in murine cerebral cortical neurons tPA plays a central role coupling SVs exocytosis and endocytosis. PMID:26820595

  9. Mechanisms involved in calcium oxalate endocytosis by Madin-Darby canine kidney cells

    A.H. Campos

    2000-01-01

    Full Text Available Calcium oxalate (CaOx crystals adhere to and are internalized by tubular renal cells and it seems that this interaction is related (positively or negatively to the appearance of urinary calculi. The present study analyzes a series of mechanisms possibly involved in CaOx uptake by Madin-Darby canine kidney (MDCK cells. CaOx crystals were added to MDCK cell cultures and endocytosis was evaluated by polarized light microscopy. This process was inhibited by an increase in intracellular calcium by means of ionomycin (100 nM; N = 6; 43.9% inhibition; P<0.001 or thapsigargin (1 µM; N = 6; 33.3% inhibition; P<0.005 administration, and via blockade of cytoskeleton assembly by the addition of colchicine (10 µM; N = 8; 46.1% inhibition; P<0.001 or cytochalasin B (10 µM; N = 8; 34.2% inhibition; P<0.001. Furthermore, CaOx uptake was reduced when the activity of protein kinase C was inhibited by staurosporine (10 nM; N = 6; 44% inhibition; P<0.01, or that of cyclo-oxygenase by indomethacin (3 µM; N = 12; 17.2% inhibition; P<0.05; however, the uptake was unaffected by modulation of potassium channel activity with glibenclamide (3 µM; N = 6, tetraethylammonium (1 mM; N = 6 or cromakalim (1 µM; N = 6. Taken together, these data indicate that the process of CaOx internalization by renal tubular cells is similar to the endocytosis reported for other systems. These findings may be relevant to cellular phenomena involved in early stages of the formation of renal stones.

  10. Cellular mechanisms of activity-dependent BDNF expression in primary sensory neurons.

    Vermehren-Schmaedick, A; Khanjian, R A; Balkowiec, A

    2015-12-01

    Brain-derived neurotrophic factor (BDNF) is abundantly expressed by both developing and adult rat visceral sensory neurons from the nodose ganglion (NG) in vivo and in vitro. We have previously shown that BDNF is released from neonatal NG neurons by activity and regulates dendritic development in their postsynaptic targets in the brainstem. The current study was carried out to examine the cellular and molecular mechanisms of activity-dependent BDNF expression in neonatal rat NG neurons, using our established in vitro model of neuronal activation by electrical field stimulation with patterns that mimic neuronal activity in vivo. We show that BDNF mRNA (transcript 4) increases over threefold in response to a 4-h tonic or bursting pattern delivered at the frequency of 6 Hz, which corresponds to the normal heart rate of a newborn rat. No significant increase in BDNF expression was observed following stimulation at 1 Hz. The latter effect suggests a frequency-dependent mechanism of regulated BDNF expression. In addition to BDNF transcript 4, which is known to be regulated by activity, transcript 1 also showed significant upregulation. The increases in BDNF mRNA were followed by BDNF protein upregulation of a similar magnitude after 24h of stimulation at 6 Hz. Electrical stimulation-evoked BDNF expression was inhibited by pretreating neurons with the blocker of voltage-gated sodium channels tetrodotoxin and by removing extracellular calcium. Moreover, our data show that repetitive stimulation-evoked BDNF expression requires calcium influx through N-, but not L-type, channels. Together, our study reveals novel mechanisms through which electrical activity stimulates de novo synthesis of BDNF in sensory neurons, and points to the role of N-type calcium channels in regulating BDNF expression in sensory neurons in response to repetitive stimulation. PMID:26459016

  11. Activity-dependent neuroprotective protein (ADNP) exhibits striking sexual dichotomy impacting on autistic and Alzheimer's pathologies.

    Malishkevich, A; Amram, N; Hacohen-Kleiman, G; Magen, I; Giladi, E; Gozes, I

    2015-01-01

    Activity-dependent neuroprotective protein (ADNP) is a most frequent autism spectrum disorder (ASD)-associated gene and the only protein significantly decreasing in the serum of Alzheimer's disease (AD) patients. Is ADNP associated with ASD being more prevalent in boys and AD more prevalent in women? Our results revealed sex-related learning/memory differences in mice, reflecting hippocampal expression changes in ADNP and ADNP-controlled AD/ASD risk genes. Hippocampal ADNP transcript content was doubled in male vs female mice, with females showing equal expression to ADNP haploinsufficient (ADNP(+/)(-)) males and no significant genotype-associated reduction. Increased male ADNP expression was replicated in human postmortem hippocampal samples. The hippocampal transcript for apolipoprotein E (the major risk gene for AD) was doubled in female mice compared with males, and further doubled in the ADNP(+/-) females, contrasting a decrease in ADNP(+/-) males. Previously, overexpression of the eukaryotic translation initiation factor 4E (eIF4E) led to ASD-like phenotype in mice. Here, we identified binding sites on ADNP for eIF4E and co-immunoprecipitation. Furthermore, hippocampal eIF4E expression was specifically increased in young ADNP(+/-) male mice. Behaviorally, ADNP(+/-) male mice exhibited deficiencies in object recognition and social memory compared with ADNP(+/+) mice, while ADNP(+/-) females were partially spared. Contrasting males, which preferred novel over familiar mice, ADNP(+/+) females showed no preference to novel mice and ADNP(+/-) females did not prefer mice over object. ADNP expression, positioned as a master regulator of key ASD and AD risk genes, introduces a novel concept of hippocampal gene-regulated sexual dimorphism and an ADNP(+/-) animal model for translational psychiatry. PMID:25646590

  12. Looking for a bulk point

    Maldacena, Juan; Zhiboedov, Alexander

    2015-01-01

    We consider Lorentzian correlators of local operators. In perturbation theory, singularities occur when we can draw a position-space Landau diagram with null lines. In theories with gravity duals, we can also draw Landau diagrams in the bulk. We argue that certain singularities can arise only from bulk diagrams, not from boundary diagrams. As has been previously observed, these singularities are a clear diagnostic of bulk locality. We analyze some properties of these perturbative singularities and discuss their relation to the OPE and the dimensions of double-trace operators. In the exact nonperturbative theory, we expect no singularity at these locations. We prove this statement in 1+1 dimensions by CFT methods.

  13. Gamma probe dry bulk densities

    The gamma density probe is a useful instrument for measuring water content in small volumes of soil. Essentially, the gamma probe measures the density of the soil and water between a source and a detector. To transpose the gamma densities into water content, the dry bulk density of the soil is needed. A nondestructive method for estimating dry bulk densities for use with the gamma probe is proposed. The procedure is based on the assumption that water content values in a field dry condition were more stable than the dry bulk density values and could be transferred from one point to another. The procedure was successfully used on three areas in Reynolds Creek Experimental Watershed in southwest Idaho. (U.S.)

  14. Bulk Viscosity of Interacting Hadrons

    Wiranata, A.; M. Prakash

    2009-01-01

    We show that first approximations to the bulk viscosity $\\eta_v$ are expressible in terms of factors that depend on the sound speed $v_s$, the enthalpy, and the interaction (elastic and inelastic) cross section. The explicit dependence of $\\eta_v$ on the factor $(\\frac 13 - v_s^2)$ is demonstrated in the Chapman-Enskog approximation as well as the variational and relaxation time approaches. The interesting feature of bulk viscosity is that the dominant contributions at a given temperature ari...

  15. Bulk Viscosity of Interacting Hadrons

    Wiranata, A

    2009-01-01

    We show that first approximations to the bulk viscosity $\\eta_v$ are expressible in terms of factors that depend on the sound speed $v_s$, the enthalpy, and the interaction (elastic and inelastic) cross section. The explicit dependence of $\\eta_v$ on the factor $(\\frac 13 - v_s^2)$ is demonstrated in the Chapman-Enskog approximation as well as the variational and relaxation time approaches. The interesting feature of bulk viscosity is that the dominant contributions at a given temperature arise from particles which are neither extremely nonrelativistic nor extremely relativistic. Numerical results for a model binary mixture are reported.

  16. Geomagnetic and solar activity dependence of ionospheric upflowing O+: FAST observations

    Zhao, K.; Jiang, Y.; Chen, K. W.; Huang, L. F.

    2016-09-01

    This paper investigates the dependence of the occurrence frequency of ionospheric upflowing oxygen (O+) ions on the sunspot cycle and geomagnetic activity. We examine the upflows response to the geomagnetic disturbances as well as the influence of the ion energy factor in controlling the magnitude of the occurrence frequency and the net energy flux. We discuss the spatial distribution of the upflow occurrence frequency and construct a regression model as a function of the magnetic latitude. The results show an overall enhancement of the upflow occurrence frequency during magnetically disturbed periods and indicate that the high-occurrence area spreads out from the source regions during magnetically quiet periods. The high-occurrence areas are located at 70° magnetic latitude (mLat) in the dayside auroral oval zone and between 76-80° mLat in the dayside polar cusp region. In the nightside auroral oval zone, these areas are near 60° mLat, penetrating further equatorward to 55° mLat during magnetically disturbed periods. High energy (≥1 keV) upflowing ions are common in the nightside auroral oval zone while low energy (<1 keV) upflowing ions are found escaping from the high latitude dayside cusp region. A Gaussian function is shown to be a good fit to the occurrence frequency over the magnetic latitude. For high energy upflowing O+ ions, the occurrence frequency exhibits a single peak located at about 60° mLat in the nightside auroral oval zone while for low energy upflowing O+ ions, it exhibits two peaks, one near 60° mLat in the auroral oval zone and the other near 78° mLat in the cusp region. We study the solar activity dependence by analyzing the relationship between the upflow occurrence frequency and the sunspot number (RZ). The statistical result shows that the frequency decreases with declining solar activity level, from ˜30 % at solar maximum to ˜5 % at solar minimum. In addition, the correlation coefficient between the occurrence frequency and RZ

  17. Activity-dependent increases in local oxygen consumption correlate with postsynaptic currents in the mouse cerebellum in vivo

    Mathiesen, Claus; Caesar, Kirsten; Thomsen, Kirsten Engelund;

    2011-01-01

    metabolism via mitochondrial signaling, but whether this also occurs in the intact brain is unknown. Here we applied a pharmacological approach to dissect the effects of ionic currents and cytosolic Ca(2+) rises of neuronal origin on activity-dependent rises in CMRO(2). We used two-photon microscopy and...

  18. Activity-dependent increases in local oxygen consumption correlate with postsynaptic currents in the mouse cerebellum in vivo

    Mathiesen, Claus; Caesar, Kirsten; Thomsen, Kirsten Engelund;

    2011-01-01

    Evoked neural activity correlates strongly with rises in cerebral metabolic rate of oxygen (CMRO(2)) and cerebral blood flow (CBF). Activity-dependent rises in CMRO(2) fluctuate with ATP turnover due to ion pumping. In vitro studies suggest that increases in cytosolic Ca(2+) stimulate oxidative...

  19. Activity-dependent increases in local oxygen consumption correlate with post-synaptic currents in the mouse cerebellum in vivo

    Mathiesen, Claus; Caesar, Kirsten; Thomsen, Kirsten Joan;

    2011-01-01

    Evoked neural activity correlates strongly with rises in cerebral metabolic rate of oxygen (CMRO2) and cerebral blood flow. Activity-dependent rises in CMRO2 fluctuate with ATP turnover due to ion pumping. In vitro studies suggest that increases in cytosolic Ca2+ stimulate oxidative metabolism via...

  20. Bulk charges in eleven dimensions

    Hawking, S. W.; Taylor-Robinson, M. M.

    1998-07-01

    Eleven dimensional supergravity has electric type currents arising from the Chern-Simon and anomaly terms in the action. However the bulk charge integrates to zero for asymptotically flat solutions with topological trivial spatial sections. We show that by relaxing the boundary conditions to generalisations of the ALE and ALF boundary conditions in four dimensions one can obtain static solutions with a bulk charge. Solutions involving anomaly terms preserve between 1/16 and 1/4 of the supersymmetries but Chern-Simons fluxes generally break all of the remaining supersymmetry. One can introduce membranes with the same sign of charge into these backgrounds. This raises the possibility that these generalized membranes might decay quantum mechanically to leave just a bulk distribution of charge. Alternatively and more probably, a bulk distribution of charge can decay into a collection of singly charged membranes. Dimensional reductions of these solutions lead to novel representations of extreme black holes in four dimensions with up to four charges. We discuss how the eleven-dimensional Kaluza-Klein monopole wrapped around a space with non-zero first Pontryagin class picks up an electric charge proportional to the Pontryagin number.

  1. Bulk viscosity and deflationary universes

    Lima, J A S; Waga, I

    2007-01-01

    We analyze the conditions that make possible the description of entropy generation in the new inflationary model by means of a nearequilibrium process. We show that there are situations in which the bulk viscosity cannot describe particle production during the coherent field oscillations phase.

  2. Longitudinal bulk acoustic mass sensor

    Hales, Jan Harry; Teva, Jordi; Boisen, Anja;

    2009-01-01

    A polycrystalline silicon longitudinal bulk acoustic cantilever is fabricated and operated in air at 51 MHz. A mass sensitivity of 100 Hz/fg (1 fg=10(-15) g) is obtained from the preliminary experiments where a minute mass is deposited on the device by means of focused ion beam. The total noise i...

  3. Biodistribution and endocytosis of ICAM-1-targeting antibodies versus nanocarriers in the gastrointestinal tract in mice

    Mane V

    2012-08-01

    Full Text Available Viraj Mane,1 Silvia Muro1, 21Institute for Bioscience and Biotechnology Research, 2Fischell Department of Bioengineering, University of Maryland, College Park, MD, USAAbstract: Drug delivery to the gastrointestinal (GI tract is key for improving treatment of GI maladies, developing oral vaccines, and facilitating drug transport into circulation. However, delivery of formulations to the GI tract is hindered by pH changes, degradative enzymes, mucus, and peristalsis, leading to poor GI retention. Targeting may prolong residence of therapeutics in the GI tract and enhance their interaction with this tissue, improving such aspects. We evaluated nanocarrier (NC and ligand-mediated targeting in the GI tract following gastric gavage in mice. We compared GI biodistribution, degradation, and endocytosis between control antibodies and antibodies targeting the cell surface determinant intercellular adhesion molecule 1 (ICAM-1, expressed on GI epithelium and other cell types. These antibodies were administered either as free entities or coated onto polymer NCs. Fluorescence and radioisotope tracing showed proximal accumulation, with preferential retention in the stomach, jejunum, and ileum; and minimal presence in the duodenum, cecum, and colon by 1 hour after administration. Upstream (gastric retention was enhanced in NC formulations, with decreased downstream (jejunal accumulation. Of the total dose delivered to the GI tract, ~60% was susceptible to enzymatic (but not pH-mediated degradation, verified both in vitro and in vivo. Attenuation of peristalsis by sedation increased upstream retention (stomach, duodenum, and jejunum. Conversely, alkaline NaHCO3, which enhances GI transit by decreasing mucosal viscosity, favored downstream (ileal passage. This suggests passive transit through the GI tract, governed by mucoadhesion and peristalsis. In contrast, both free anti-ICAM and anti-ICAM NCs demonstrated significantly enhanced upstream (stomach and duodenum

  4. Parameter estimation with bio-inspired meta-heuristic optimization: modeling the dynamics of endocytosis

    Tashkova Katerina

    2011-10-01

    Full Text Available Abstract Background We address the task of parameter estimation in models of the dynamics of biological systems based on ordinary differential equations (ODEs from measured data, where the models are typically non-linear and have many parameters, the measurements are imperfect due to noise, and the studied system can often be only partially observed. A representative task is to estimate the parameters in a model of the dynamics of endocytosis, i.e., endosome maturation, reflected in a cut-out switch transition between the Rab5 and Rab7 domain protein concentrations, from experimental measurements of these concentrations. The general parameter estimation task and the specific instance considered here are challenging optimization problems, calling for the use of advanced meta-heuristic optimization methods, such as evolutionary or swarm-based methods. Results We apply three global-search meta-heuristic algorithms for numerical optimization, i.e., differential ant-stigmergy algorithm (DASA, particle-swarm optimization (PSO, and differential evolution (DE, as well as a local-search derivative-based algorithm 717 (A717 to the task of estimating parameters in ODEs. We evaluate their performance on the considered representative task along a number of metrics, including the quality of reconstructing the system output and the complete dynamics, as well as the speed of convergence, both on real-experimental data and on artificial pseudo-experimental data with varying amounts of noise. We compare the four optimization methods under a range of observation scenarios, where data of different completeness and accuracy of interpretation are given as input. Conclusions Overall, the global meta-heuristic methods (DASA, PSO, and DE clearly and significantly outperform the local derivative-based method (A717. Among the three meta-heuristics, differential evolution (DE performs best in terms of the objective function, i.e., reconstructing the output, and in terms of

  5. Polymerisationseigenschaften von Bulk-Fill Kompositen

    Maier, Eva

    2015-01-01

    Hintergrund und Ziele: Untersuchung der Polymerisationseigenschaften von Bulk-Fill Kompositen bzgl. Konversionsrate (degree of conversion = DC), Vickers-Härte (HV), Polymerisationsschrumpfungsstress (PSS) und Polymerisationsvolumenschrumpfung (PVS) im Vergleich zu konventionellen Kompositen. Material und Methode: Untersucht wurden die Bulk-Fill Komposite Filtek Bulk Fill Flowable (FBF, 3M ESPE, Seefeld), Surefil Smart Dentin Replacement (SDR, Dentsply, Konstanz), Tetric EvoCeram Bulk Fill...

  6. Massive endocytosis driven by lipidic forces originating in the outer plasmalemmal monolayer: a new approach to membrane recycling and lipid domains

    Fine, Michael; Llaguno, Marc C.; Lariccia, Vincenzo; Lin, Mei-Jung; Yaradanakul, Alp; Hilgemann, Donald W.

    2011-01-01

    The roles that lipids play in endocytosis are the subject of debate. Using electrical and imaging methods, we describe massive endocytosis (MEND) in baby hamster kidney (BHK) and HEK293 cells when the outer plasma membrane monolayer is perturbed by the nonionic detergents, Triton X-100 (TX100) and NP-40. Some alkane detergents, the amphipathic drugs, edelfosine and tamoxifen, and the phospholipase inhibitor, U73122, are also effective. Uptake of the membrane tracer, FM 4–64, into vesicles and...

  7. Membrane glycoprotein M6A promotes μ-opioid receptor endocytosis and facilitates receptor sorting into the recycling pathway

    Ying-Jian Liang; Dai-Fei Wu; Ralf Stumm; Volker H(o)llt; Thomas Koch

    2008-01-01

    The interaction of μ-opioid receptor (MOPr) with the neuronal membrane glycoprotein M6a is known to facilitate MOPr endocytosis in human embryonic kidney 293 (HEK293) cells. To further study the role of M6a in the post-endocytotic sorting of MOPr, we investigated the agonist-induced co-internalization of MOPr and M6a and protein targeting after internalization in HEK293 cells that co-expressed HA-tagged MOPr and Myc-tagged M6a. We found that M6a, MOPr, and Rab 11, a marker for recycling endosomes, co-localized in endocytotic vesicles, indicating that MOPr and M6a are primarily targeted to recycling endosomes after endocytosis. Furthermore, co-expression of M6a augmented the post-endocytotic sorting of δ-opioid receptors into the recycling pathway, indicating that M6a might have a more general role in opioid receptor post-ndocytotic sorting. The enhanced post-endocytotic sorting of MOPr into the recycling pathway was accompanied by a decrease in agonist-induced receptor down-regulation of M6a in co-expressing cells. We tested the physiological relevance of these findings in primary cultures of cortical neurons and found that co-expression of M6a markedly increased the translocation of MOPrs from the plasma membrane to intracellular vesicles at steady state and significantly enhanced both constitutive and agonist-induced receptor endocytosis. In conclusion, our results strongly indicate that M6a modulates MOPr endocytosis and post-endocytotic sorting and has an important role in receptor regulation.

  8. Preferred sites of exocytosis and endocytosis colocalize during high but not lower frequency stimulation in mouse motor nerve terminals

    Gaffield, Michael A.; Tabares, Lucia; Betz, William J.

    2009-01-01

    The spatial relationship of exocytosis and endocytosis in motor nerve terminals has been explored, with varied results, mostly in fixed preparations and without direct information on the utilization of each exocytic site. We sought to determine these spatial properties in real time using synaptopHluorin (spH) and FM 4-64. Earlier we showed that nerve stimulation elicits the appearance of spH fluorescence hot spots, which mark preferred sites of exocytosis. Here we show that nerve stimulation ...

  9. The overexpressed human 46-kDa mannose 6-phosphate receptor mediates endocytosis and sorting of β-glucuronidase

    The authors studied the function of the human small (46-kDa) mannose 6-phosphate receptor (SMPR) in transfected mouse L cells that do not express the larger insulin-like growth factor II/mannose 6-phosphate receptor. Cells overexpressing human SMPR were studied for enzyme binding to cell surface receptors, for binding to intracellular receptors in permeabilized cells, and for receptor-mediated endocytosis of recombinant human β-glucuronidase. Specific binding to human SMPR in permeabilized cells showed a pH optimum between pH 6.0 and pH 6.5. Binding was significant in the present of EDTA but was enhanced by added divalent cations. Up to 2.3% of the total functional receptor could be detected on the cell surface by enzyme binding. They present experiments showing that at very high levels of overexpression, and at pH 6.5, human SMPR mediated the endocytosis of β-glucuronidase. At pH 7.5, the rate of endocytosis was only 14% the rate seen at pH 6.5. Cells overexpressing human SMPR also showed reduced secretion of newly synthesized β-glucuronidase when compared to cells transfected with vector only, suggesting that overexpressed human SMPR can participate in sorting of newly synthesized β-glucuronidase and partially correct the sorting defect in mouse L cells that do not express the insulin-like growth factor II/mannose 6-phosphate receptor

  10. Influenza A virus H5N1 entry into host cells is through clathrin-dependent endocytosis

    2009-01-01

    Influenza A virus H5N1 presents a major threat to human health. The entry of influenza virus into host cells is believed to be mediated by hemagglutinin (HA), a virus surface glycoprotein that can bind terminal sialic acid residues on host cell glycoproteins and glycolipids. In this study, we elucidated the pathways through which H5N1 enters human lung carcinoma cell line A549. We first proved that H5N1 can enter A549 cells via endocytosis, as lysosomotropic agents, such as bafilomycin A1 and chloroquine, can rescue H5N1-induced A549 cell death. By using specific inhibitors, and siRNAs that target the clathrin pathway, we further found that H5N1 could enter A549 cells via clathrin-mediated endocytosis, while inhibitors targeting caveolae-mediated endocytosis could not inhibit H5N1 cell entry. These findings expand our understanding of H5N1 pathogenesis and provide new information for anti-viral drug research.

  11. Post-fusion structural changes and their roles in exocytosis and endocytosis of dense-core vesicles

    Chiang, Hsueh-Cheng; Shin, Wonchul; Zhao, Wei-Dong; Hamid, Edaeni; Sheng, Jiansong; Baydyuk, Maryna; Wen, Peter J.; Jin, Albert; Momboisse, Fanny; Wu, Ling-Gang

    2014-02-01

    Vesicle fusion with the plasma membrane generates an Ω-shaped membrane profile. Its pore is thought to dilate until flattening (full-collapse), followed by classical endocytosis to retrieve vesicles. Alternatively, the pore may close (kiss-and-run), but the triggering mechanisms and its endocytic roles remain poorly understood. Here, using confocal and stimulated emission depletion microscopy imaging of dense-core vesicles, we find that fusion-generated Ω-profiles may enlarge or shrink while maintaining vesicular membrane proteins. Closure of fusion-generated Ω-profiles, which produces various sizes of vesicles, is the dominant mechanism mediating rapid and slow endocytosis within ~1-30 s. Strong calcium influx triggers dynamin-mediated closure. Weak calcium influx does not promote closure, but facilitates the merging of Ω-profiles with the plasma membrane via shrinking rather than full-collapse. These results establish a model, termed Ω-exo-endocytosis, in which the fusion-generated Ω-profile may shrink to merge with the plasma membrane, change in size or change in size then close in response to calcium, which is the main mechanism to retrieve dense-core vesicles.

  12. Influenza A virus H5N1 entry into host cells is through clathrin-dependent endocytosis

    WANG HongLiang; JIANG ChengYu

    2009-01-01

    Influenza A virus H5N1 presents a major threat to human health. The entry of influenza virus into host cells is believed to be mediated by hemagglutinin (HA), a virus surface glycoprotein that can bind ter-minal sialic acid residues on host cell glycoproteins and glycolipids. In this study, we elucidated the pathways through which H5N1 enters human lung carcinoma cell line A549. We first proved that H5N1 can enter A549 cells via endocytosis, as lysosomotropic agents, such as bafilomycin A1 and chloro. quine, can rescue H5Nl-induced A549 cell death. By using specific inhibitors, and siRNAs that target the clathrin pathway, we further found that H5N1 could enter A549 cells via clathrin-mediated endocy-tosis, while inhibitors targeting caveolae-mediated endocytosis could not inhibit H5N1 cell entry. These findings expand our understanding of H5N1 pathogenesis and provide new information for anti-viral drug research.

  13. Evolutionary Changes on the Way to Clathrin-Mediated Endocytosis in Animals.

    Dergai, Mykola; Iershov, Anton; Novokhatska, Olga; Pankivskyi, Serhii; Rynditch, Alla

    2016-01-01

    Endocytic pathways constitute an evolutionarily ancient system that significantly contributed to the eukaryotic cell architecture and to the diversity of cell type-specific functions and signaling cascades, in particular of metazoans. Here we used comparative proteomic studies to analyze the universal internalization route in eukaryotes, clathrin-mediated endocytosis (CME), to address the issues of how this system evolved and what are its specific features. Among 35 proteins crucially required for animal CME, we identified a subset of 22 proteins common to major eukaryotic branches and 13 gradually acquired during evolution. Based on exploration of structure-function relationship between conserved homologs in sister, distantly related and early diverged branches, we identified novel features acquired during evolution of endocytic proteins on the way to animals: Elaborated way of cargo recruitment by multiple sorting proteins, structural changes in the core endocytic complex AP2, the emergence of the Fer/Cip4 homology domain-only protein/epidermal growth factor receptor substrate 15/intersectin functional complex as an additional interaction hub and activator of AP2, as well as changes in late endocytic stages due to recruitment of dynamin/sorting nexin 9 complex and involvement of the actin polymerization machinery. The evolutionary reconstruction showed the basis of the CME process and its subsequent step-by-step development. Documented changes imply more precise regulation of the pathway, as well as CME specialization for the uptake of specific cargoes and cell type-specific functions. PMID:26872775

  14. Membrane Tension Inhibits Deformation by Coat Proteins in Clathrin-Mediated Endocytosis

    Hassinger, Julian; Drubin, David; Oster, George; Rangamani, Padmini

    2016-02-01

    In clathrin-mediated endocytosis (CME), clathrin and various adaptor proteins coat a patch of the plasma membrane, which is reshaped to form a budded vesicle. Experimental studies have demonstrated that elevated membrane tension can inhibit bud formation by a clathrin coat. In this study, we investigate the impact of membrane tension on the mechanics of membrane budding by simulating clathrin coats that either grow in area or progressively induce greater curvature. At low membrane tension, progressively increasing the area of a curvature-generating coat causes the membrane to smoothly evolve from a flat to budded morphology, whereas the membrane remains essentially flat at high membrane tensions. Interestingly, at physiologically relevant, intermediate membrane tensions, the shape evolution of the membrane undergoes a snapthrough instability in which increasing coat area causes the membrane to "snap" from an open, U-shaped bud to a closed, $\\Omega$-shaped bud. This instability is accompanied by a large energy barrier, which could cause a developing endocytic pit to stall if the binding energy of additional coat is insufficient to overcome this barrier. Similar results were found for a coat of constant area in which the spontaneous curvature progressively increases. Additionally, a pulling force on the bud, simulating a force from actin polymerization, is sufficient to drive a transition from an open to closed bud, overcoming the energy barrier opposing this transition.

  15. Inactivation of Tor proteins affects the dynamics of endocytic proteins in early stage of endocytosis

    Brandon Tenay; Evin Kimberlin; Michelle Williams; Juliette Denise; Joshua Fakilahyel; Kyoungtae Kim

    2013-06-01

    Tor2 is an activator of the Rom2/Rho1 pathway that regulates -factor internalization. Since the recruitment of endocytic proteins such as actin-binding proteins and the amphiphysins precedes the internalization of -factor, we hypothesized that loss of Tor function leads to an alteration in the dynamics of the endocytic proteins. We report here that endocytic proteins, Abp1 and Rvs167, are less recruited to endocytic sites not only in tor2 but also tor1 mutants. Furthermore, we found that the endocytic proteins Rvs167 and Sjl2 are completely mistargeted to the cytoplasm in tor1tor2ts double mutant cells. We also demonstrate here that the efficiency of endocytic internalization or scission in all tor mutants was drastically decreased. In agreement with the Sjl2 mislocalization, we found that in tor1tor2ts double mutant cells, as well as other tor mutant cells, the overall PIP2 level was dramatically increased. Finally, the cell wall chitin content in tor2ts and tor1tor2ts mutant cells was also significantly increased. Taken together, both functional Tor proteins, Tor1 and Tor2, are essentially required for proper endocytic protein dynamics at the early stage of endocytosis.

  16. Elasticity of nanoparticles influences their blood circulation, phagocytosis, endocytosis, and targeting.

    Anselmo, Aaron C; Zhang, Mengwen; Kumar, Sunny; Vogus, Douglas R; Menegatti, Stefano; Helgeson, Matthew E; Mitragotri, Samir

    2015-03-24

    The impact of physical and chemical modifications of nanoparticles on their biological function has been systemically investigated and exploited to improve their circulation and targeting. However, the impact of nanoparticles' flexibility (i.e., elastic modulus) on their function has been explored to a far lesser extent, and the potential benefits of tuning nanoparticle elasticity are not clear. Here, we describe a method to synthesize polyethylene glycol (PEG)-based hydrogel nanoparticles of uniform size (200 nm) with elastic moduli ranging from 0.255 to 3000 kPa. These particles are used to investigate the role of particle elasticity on key functions including blood circulation time, biodistribution, antibody-mediated targeting, endocytosis, and phagocytosis. Our results demonstrate that softer nanoparticles (10 kPa) offer enhanced circulation and subsequently enhanced targeting compared to harder nanoparticles (3000 kPa) in vivo. Furthermore, in vitro experiments show that softer nanoparticles exhibit significantly reduced cellular uptake in immune cells (J774 macrophages), endothelial cells (bEnd.3), and cancer cells (4T1). Tuning nanoparticle elasticity potentially offers a method to improve the biological fate of nanoparticles by offering enhanced circulation, reduced immune system uptake, and improved targeting. PMID:25715979

  17. Egg drop syndrome virus enters duck embryonic fibroblast cells via clathrin-mediated endocytosis.

    Huang, Jingjing; Tan, Dan; Wang, Yang; Liu, Caihong; Xu, Jiamin; Wang, Jingyu

    2015-12-01

    Previous studies of egg drop syndrome virus (EDSV) is restricted to serological surveys, disease diagnostics, and complete viral genome analysis. Consequently, the infection characteristics and entry routes of EDSV are poorly understood. Therefore, we aimed to explore the entry pathway of EDSV into duck embryonic fibroblast (DEF) cells as well as the infection characteristics and proliferation of EDSV in primary DEF and primary chicken embryo liver (CEL) cells. Transmission electron microscopy revealed that the virus triggered DEF cell membrane invagination as early as 10 min post-infection and that integrated endocytic vesicles formed at 20 min post-infection. The virus yield in EDSV-infected DEF cells treated with chlorpromazine (CPZ), sucrose, methyl-β-cyclodextrin (MβCD), or NH4Cl was measured by quantitative real-time PCR. Compared with the mock treatment, CPZ and sucrose greatly inhibited the production of viral progeny in a dose-dependent manner, while MβCD treatment did not result in a significant difference. Furthermore, NH4Cl had a strong inhibitory effect on the production of EDSV progeny. In addition, indirect immunofluorescence demonstrated that virus particles clustered on the surface of DEF cells treated with CPZ or sucrose. These results indicate that EDSV enters DEF cells through clathrin-mediated endocytosis followed by a pH-dependent step, which is similar to the mechanism of entry of human adenovirus types 2 and 5. PMID:26200954

  18. West Nile virus infection causes endocytosis of a specific subset of tight junction membrane proteins.

    Zaikun Xu

    Full Text Available West Nile virus (WNV is a blood-borne pathogen that causes systemic infections and serious neurological disease in human and animals. The most common route of infection is mosquito bites and therefore, the virus must cross a number of polarized cell layers to gain access to organ tissue and the central nervous system. Resistance to trans-cellular movement of macromolecules between epithelial and endothelial cells is mediated by tight junction complexes. While a number of recent studies have documented that WNV infection negatively impacts the barrier function of tight junctions, the intracellular mechanism by which this occurs is poorly understood. In the present study, we report that endocytosis of a subset of tight junction membrane proteins including claudin-1 and JAM-1 occurs in WNV infected epithelial and endothelial cells. This process, which ultimately results in lysosomal degradation of the proteins, is dependent on the GTPase dynamin and microtubule-based transport. Finally, infection of polarized cells with the related flavivirus, Dengue virus-2, did not result in significant loss of tight junction membrane proteins. These results suggest that neurotropic flaviviruses such as WNV modulate the host cell environment differently than hemorrhagic flaviviruses and thus may have implications for understanding the molecular basis for neuroinvasion.

  19. UBE3A Regulates Synaptic Plasticity and Learning and Memory by Controlling SK2 Channel Endocytosis.

    Sun, Jiandong; Zhu, Guoqi; Liu, Yan; Standley, Steve; Ji, Angela; Tunuguntla, Rashmi; Wang, Yubin; Claus, Chad; Luo, Yun; Baudry, Michel; Bi, Xiaoning

    2015-07-21

    Gated solely by activity-induced changes in intracellular calcium, small-conductance potassium channels (SKs) are critical for a variety of functions in the CNS, from learning and memory to rhythmic activity and sleep. While there is a wealth of information on SK2 gating, kinetics, and Ca(2+) sensitivity, little is known regarding the regulation of SK2 subcellular localization. We report here that synaptic SK2 levels are regulated by the E3 ubiquitin ligase UBE3A, whose deficiency results in Angelman syndrome and overexpression in increased risk of autistic spectrum disorder. UBE3A directly ubiquitinates SK2 in the C-terminal domain, which facilitates endocytosis. In UBE3A-deficient mice, increased postsynaptic SK2 levels result in decreased NMDA receptor activation, thereby impairing hippocampal long-term synaptic plasticity. Impairments in both synaptic plasticity and fear conditioning memory in UBE3A-deficient mice are significantly ameliorated by blocking SK2. These results elucidate a mechanism by which UBE3A directly influences cognitive function. PMID:26166566

  20. Enhancement of saporin cytotoxicity by Gypsophila saponins--more than stimulation of endocytosis.

    Weng, A; Bachran, C; Fuchs, H; Krause, E; Stephanowitz, H; Melzig, M F

    2009-10-30

    Saporin is a type I ribosome-inactivating protein with N-glycosidase activity. It removes adenine residues from the 28S ribosomal RNA resulting in inhibition of protein synthesis. Recently we have shown that saporin exerts no cytotoxicity on seven human cell lines. However, the combination of saporin with a special mixture of Gypsophila saponins (Soapwort saponins) from Gypsophila paniculata L. (baby's breath) rendered saporin to a potent cytotoxin comparable to viscumin, a highly toxic type II ribosome-inactivating protein. In this study we investigated whether the enhancement of the saporin-cytotoxicity by Gypsophila saponins is mediated by a saponin-triggered modulation of endocytosis, exocytosis or impaired degradation processes of his-tagged saporin ((his)saporin) in ECV-304 cells. For this purpose (his)saporin was labelled with tritium and cytotoxicity of the toxin alone and in combination with Gypsophila saponins was scrutinized. The transport and degradation processes of (his)saporin were not different in Gypsophila saponin-treated and control cells. However, after ultracentrifugation of a post-nuclear supernatant the amount of cytosolic (his)saporin was significantly higher in saponin-treated cells than in cells, which were only incubated with (his)saporin. This indicates a saponin mediated endosomal escape of saporin. PMID:19615984

  1. Catabolism of circulating enzymes: plasma clearance, endocytosis, and breakdown of lactate dehydrogenase-1 in rabbits

    Smit, M.J.; Beekhuis, H.; Duursma, A.M.; Bouma, J.M.; Gruber, M.

    1988-12-01

    Lactate dehydrogenase-1, intravenously injected into rabbits, was cleared with first-order kinetics (half-life 27 min), until at least 80% of the injected activity had disappeared from plasma. Radioactivity from injected SVI-labeled enzyme disappeared at this same rate. Trichloroacetic-acid-soluble breakdown products started to appear in the circulation shortly after injection of the labeled enzyme. Body scans of the rabbits for 80 min after injection of T I-labeled enzyme revealed rapid accumulation of label in the liver, peaking 10-20 min after injection. Subsequently, activity in the liver declined and radioactivity (probably labeled breakdown products of low molecular mass) steadily accumulated in the bladder. Tissue fractionation of liver, 19 min after injection of labeled enzyme, indicated that the radioactivity was present both in endosomes and in lysosomes, suggesting uptake by endocytosis, followed by breakdown in the lysosomes. Measurements of radioactivity in liver and plasma suggest that the liver is responsible for the breakdown of at least 75% of the injected enzyme. Radioautography of tissue sections of liver and spleen showed accumulated radioactivity in sinusoidal liver cells and red pulpa, respectively. These results are very similar to those for lactate dehydrogenase-5, creatine kinase MM, and several other enzymes that we have previously studied in rats.

  2. Catabolism of circulating enzymes: plasma clearance, endocytosis, and breakdown of lactate dehydrogenase-1 in rabbits.

    Smit, M J; Beekhuis, H; Duursma, A M; Bouma, J M; Gruber, M

    1988-12-01

    Lactate dehydrogenase-1 (EC 1.1.1.27), intravenously injected into rabbits, was cleared with first-order kinetics (half-life 27 min), until at least 80% of the injected activity had disappeared from plasma. Radioactivity from injected 125I-labeled enzyme disappeared at this same rate. Trichloroacetic-acid-soluble breakdown products started to appear in the circulation shortly after injection of the labeled enzyme. Body scans of the rabbits for 80 min after injection of 131I-labeled enzyme revealed rapid accumulation of label in the liver, peaking 10-20 min after injection. Subsequently, activity in the liver declined and radioactivity (probably labeled breakdown products of low molecular mass) steadily accumulated in the bladder. Tissue fractionation of liver, 19 min after injection of labeled enzyme, indicated that the radioactivity was present both in endosomes and in lysosomes, suggesting uptake by endocytosis, followed by breakdown in the lysosomes. Measurements of radioactivity in liver and plasma suggest that the liver is responsible for the breakdown of at least 75% of the injected enzyme. Radioautography of tissue sections of liver and spleen showed accumulated radioactivity in sinusoidal liver cells and red pulpa, respectively. These results are very similar to those for lactate dehydrogenase-5, creatine kinase MM, and several other enzymes that we have previously studied in rats. PMID:3197286

  3. Catabolism of circulating enzymes: plasma clearance, endocytosis, and breakdown of lactate dehydrogenase-1 in rabbits

    Lactate dehydrogenase-1, intravenously injected into rabbits, was cleared with first-order kinetics (half-life 27 min), until at least 80% of the injected activity had disappeared from plasma. Radioactivity from injected 125I-labeled enzyme disappeared at this same rate. Trichloroacetic-acid-soluble breakdown products started to appear in the circulation shortly after injection of the labeled enzyme. Body scans of the rabbits for 80 min after injection of 131I-labeled enzyme revealed rapid accumulation of label in the liver, peaking 10-20 min after injection. Subsequently, activity in the liver declined and radioactivity (probably labeled breakdown products of low molecular mass) steadily accumulated in the bladder. Tissue fractionation of liver, 19 min after injection of labeled enzyme, indicated that the radioactivity was present both in endosomes and in lysosomes, suggesting uptake by endocytosis, followed by breakdown in the lysosomes. Measurements of radioactivity in liver and plasma suggest that the liver is responsible for the breakdown of at least 75% of the injected enzyme. Radioautography of tissue sections of liver and spleen showed accumulated radioactivity in sinusoidal liver cells and red pulpa, respectively. These results are very similar to those for lactate dehydrogenase-5, creatine kinase MM, and several other enzymes that we have previously studied in rats

  4. Receptor-mediated endocytosis of lysozyme in renal proximal tubules of the frog Rana temporaria

    E.V. Seliverstova

    2015-04-01

    Full Text Available The mechanism of protein reabsorption in the kidney of lower vertebrates remains insufficiently investigated in spite of raising interest to the amphibian and fish kidneys as a useful model for physiological and pathophysiological examinations. In the present study, we examined the renal tubular uptake and the internalization rote of lysozyme after its intravenous injection in the wintering frog Rana temporaria using immunohisto- and immunocytochemistry and specific markers for some endocytic compartments. The distinct expression of megalin and cubilin in the proximal tubule cells of lysozyme-injected frogs was revealed whereas kidney tissue of control animals showed no positive immunoreactivity. Lysozyme was detected in the apical endocytic compartment of the tubular cells and colocalized with clathrin 10 min after injection. After 20 min, lysozyme was located in the subapical compartment negative to clathrin (endosomes, and intracellular trafficking of lysozyme was coincided with the distribution of megalin and cubilin. However, internalized protein was retained in the endosomes and did not reach lysosomes within 30 min after treatment that may indicate the inhibition of intracellular trafficking in hibernating frogs. For the first time, we provided the evidence that lysozyme is filtered through the glomeruli and absorbed by receptor-mediated clathrin-dependent endocytosis in the frog proximal tubule cells. Thus, the protein uptake in the amphibian mesonephros is mediated by megalin and cubilin that confirms a critical role of endocytic receptors in the renal reabsorption of proteins in amphibians as in mammals.

  5. Yeast dynamin Vps1 and amphiphysin Rvs167 function together during endocytosis.

    Smaczynska-de Rooij, Iwona I; Allwood, Ellen G; Mishra, Ritu; Booth, Wesley I; Aghamohammadzadeh, Soheil; Goldberg, Martin W; Ayscough, Kathryn R

    2012-02-01

    Dynamins are a conserved family of proteins involved in many membrane fusion and fission events. Previously, the dynamin-related protein Vps1 was shown to localize to endocytic sites, and yeast carrying deletions for genes encoding both the BAR domain protein Rvs167 and Vps1 had a more severe endocytic scission defect than either deletion alone. Vps1 and Rvs167 localize to endocytic sites at the onset of invagination and disassemble concomitant with inward vesicle movement. Rvs167-GFP localization is reduced in cells lacking vps1 suggesting that Vps1 influences Rvs167 association with the endocytic complex. Unlike classical dynamins, Vps1 does not have a proline-arginine domain that could interact with SH3 domain-containing proteins. Thus, while Rvs167 has an SH3 domain, it is not clear how an interaction would be mediated. Here, we demonstrate an interaction between Rvs167 SH3 domain and the single type I SH3-binding motif in Vps1. Mutant Vps1 that cannot bind Rvs167 rescues all membrane fusion/fission functions associated with Vps1 except for endocytic function, demonstrating the specificity and mechanistic importance of the interaction. In vitro, an Rvs161/Rvs167 heterodimer can disassemble Vps1 oligomers. Overall, the data support the idea that Vps1 and the amphiphysins function together to mediate scission during endocytosis in yeast. PMID:22082017

  6. Perspectives on kiss-and-run: role in exocytosis, endocytosis, and neurotransmission.

    Alabi, AbdulRasheed A; Tsien, Richard W

    2013-01-01

    Regulated exocytosis and endocytosis are critical to the function of many intercellular networks, particularly the complex neural circuits underlying mammalian behavior. Kiss-and-run (KR) is an unconventional fusion between secretory vesicles and a target membrane that releases intravesicular content through a transient, nanometer-sized fusion pore. The fusing vesicle retains its gross shape, precluding full integration into the planar membrane, and enough molecular components for rapid retrieval, reacidification, and reuse. KR makes judicious use of finite presynaptic resources, and mounting evidence suggests that it influences synaptic information transfer. Here we detail emerging perspectives on KR and its role in neurotransmission. We additionally formulate a restraining force hypothesis as a plausible mechanistic basis for KR and its physiological modulation in small nerve terminals. Clarification of the mechanism and function of KR has bearing on understanding the kinetic transitions underlying SNARE-mediated fusion, interactions between vesicles and their local environment, and the influence of release dynamics on neural information processing. PMID:23245563

  7. Endocytosis of the major yolk proteins of the silkmoth, Hyalophora cecropia: Uptake kinetics and interactions

    The oocytes of Lepidopteran insects take up several yolk proteins in defined proportions even though their relative availability in the hemolymph changes during the several days required to complete yolk formation in all the eggs. There are three hemolymph yolk precursors, vitellogenin, microvitellogenin and lipophorin; one precursor, paravitellogenin is produced in the ovary. The control mechanism for their proportional endocytosis is not known. In this thesis, the author describe the purification of all four proteins and the radiolabeling of the hemolymph precursors. The radiolabeled proteins were tested with an in vitro incubation system to assess the biological activity of the proteins and the reliability of the incubation methods. All of the labeled probes were transferred from the incubation medium to yolk spheres within the oocyte in a saturable, energy-dependent, and stage-specific manner. The rates of uptake were similar to the estimated rates of uptake in situ. The concentration dependence of in vitro uptake was investigated and found to be consistent with in situ concentrations and the composition of yolk in mature eggs. Two precursors, vitellogenin and lipophorin, competed for uptake indicating that they share a common binding site while the third, microvitellin, did not compete with the others. Though vitellogenin and lipophorin competed for uptake, only vitellogenin displayed the unique ability to increase the uptake rate of microvitellin and fluid in vitro

  8. Down syndrome fibroblast model of Alzheimer-related endosome pathology: accelerated endocytosis promotes late endocytic defects.

    Cataldo, Anne M; Mathews, Paul M; Boiteau, Anne Boyer; Hassinger, Linda C; Peterhoff, Corrinne M; Jiang, Ying; Mullaney, Kerry; Neve, Rachael L; Gruenberg, Jean; Nixon, Ralph A

    2008-08-01

    Endocytic dysfunction is an early pathological change in Alzheimer's disease (AD) and Down's syndrome (DS). Using primary fibroblasts from DS individuals, we explored the interactions among endocytic compartments that are altered in AD and assessed their functional consequences in AD pathogenesis. We found that, like neurons in both AD and DS brains, DS fibroblasts exhibit increased endocytic uptake, fusion, and recycling, and trafficking of lysosomal hydrolases to rab5-positive early endosomes. Moreover, late endosomes identified using antibodies to rab7 and lysobisphosphatidic acid increased in number and appeared as enlarged, perinuclear vacuoles, resembling those in neurons of both AD and DS brains. In control fibroblasts, similar enlargement of rab5-, rab7-, and lysobisphosphatidic acid-positive endosomes was induced when endocytosis and endosomal fusion were increased by expression of either a rab5 or an active rab5 mutant, suggesting that persistent endocytic activation results in late endocytic dysfunction. Conversely, expression of a rab5 mutant that inhibits endocytic uptake reversed early and late endosomal abnormalities in DS fibroblasts. Our results indicate that DS fibroblasts recapitulate the neuronal endocytic dysfunction of AD and DS, suggesting that increased trafficking from early endosomes can account, in part, for downstream endocytic perturbations that occur in neurons in both AD and DS brains. PMID:18535180

  9. RAB-5- and DYNAMIN-1-Mediated Endocytosis of EFF-1 Fusogen Controls Cell-Cell Fusion

    Ksenia Smurova

    2016-02-01

    Full Text Available Cell-cell fusion plays essential roles during fertilization and organogenesis. Previous studies in C. elegans led to the identification of the eukaryotic fusion protein (EFF-1 fusogen, which has structural homology to class II viral fusogens. Transcriptional repression of EFF-1 ensures correct fusion fates, and overexpression of EFF-1 results in embryonic lethality. EFF-1 must be expressed on the surface of both fusing cells; however, little is known regarding how cells regulate EFF-1 surface exposure. Here, we report that EFF-1 is actively removed from the plasma membrane of epidermal cells by dynamin- and RAB-5-dependent endocytosis and accumulates in early endosomes. EFF-1 was transiently localized to apical domains of fusion-competent cells. Effective cell-cell fusion occurred only between pairs of cell membranes in which EFF-1 localized. Downregulation of dynamin or RAB-5 caused EFF-1 mislocalization to all apical membrane domains and excessive fusion. Thus, internalization of EFF-1 is a safety mechanism preventing excessive cell fusion.

  10. Coulombic Fluids Bulk and Interfaces

    Freyland, Werner

    2011-01-01

    Ionic liquids have attracted considerable interest in recent years. In this book the bulk and interfacial physico-chemical characteristics of various fluid systems dominated by Coulomb interactions are treated which includes molten salts, ionic liquids as well as metal-molten salt mixtures and expanded fluid metals. Of particular interest is the comparison of the different systems. Topics in the bulk phase concern the microscopic structure, the phase behaviour and critical phenomena, and the metal-nonmetal transition. Interfacial phenomena include wetting transitions, electrowetting, surface freezing, and the electrified ionic liquid/ electrode interface. With regard to the latter 2D and 3D electrochemical phase formation of metals and semi-conductors on the nanometer scale is described for a number of selected examples. The basic concepts and various experimental methods are introduced making the book suitable for both graduate students and researchers interested in Coulombic fluids.