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Sample records for activity-dependent bulk endocytosis

  1. VAMP4 Is an Essential Cargo Molecule for Activity-Dependent Bulk Endocytosis.

    PubMed

    Nicholson-Fish, Jessica C; Kokotos, Alexandros C; Gillingwater, Thomas H; Smillie, Karen J; Cousin, Michael A

    2015-12-02

    The accurate formation of synaptic vesicles (SVs) and incorporation of their protein cargo during endocytosis is critical for the maintenance of neurotransmission. During intense neuronal activity, a transient and acute accumulation of SV cargo occurs at the plasma membrane. Activity-dependent bulk endocytosis (ADBE) is the dominant SV endocytosis mode under these conditions; however, it is currently unknown how ADBE mediates cargo retrieval. We examined the retrieval of different SV cargo molecules during intense stimulation using a series of genetically encoded pH-sensitive reporters in neuronal cultures. The retrieval of only one reporter, VAMP4-pHluorin, was perturbed by inhibiting ADBE. This selective recovery was confirmed by the enrichment of endogenous VAMP4 in purified bulk endosomes formed by ADBE. VAMP4 was also essential for ADBE, with a cytoplasmic di-leucine motif being critical for this role. Therefore, VAMP4 is the first identified ADBE cargo and is essential for this endocytosis mode to proceed.

  2. Calcineurin Selectively Docks with the Dynamin Ixb Splice Variant to Regulate Activity-dependent Bulk Endocytosis

    PubMed Central

    Xue, Jing; Graham, Mark E.; Novelle, Aimee E.; Sue, Nancy; Gray, Noah; McNiven, Mark A.; Smillie, Karen J.; Cousin, Michael A.; Robinson, Phillip J.

    2011-01-01

    Depolarization of nerve terminals stimulates rapid dephosphorylation of two isoforms of dynamin I (dynI), mediated by the calcium-dependent phosphatase calcineurin (CaN). Dephosphorylation at the major phosphorylation sites Ser-774/778 promotes a dynI-syndapin I interaction for a specific mode of synaptic vesicle endocytosis called activity-dependent bulk endocytosis (ADBE). DynI has two main splice variants at its extreme C terminus, long or short (dynIxa and dynIxb) varying only by 20 (xa) or 7 (xb) residues. Recombinant GST fusion proteins of dynIxa and dynIxb proline-rich domains (PRDs) were used to pull down interacting proteins from rat brain nerve terminals. Both bound equally to syndapin, but dynIxb PRD exclusively bound to the catalytic subunit of CaNA, which recruited CaNB. Binding of CaN was increased in the presence of calcium and was accompanied by further recruitment of calmodulin. Point mutations showed that the entire C terminus of dynIxb is a CaN docking site related to a conserved CaN docking motif (PXIXI(T/S)). This sequence is unique to dynIxb among all other dynamin variants or genes. Peptide mimetics of the dynIxb tail blocked CaN binding in vitro and selectively inhibited depolarization-evoked dynI dephosphorylation in nerve terminals but not of other dephosphins. Therefore, docking to dynIxb is required for the regulation of both dynI splice variants, yet it does not regulate the phosphorylation cycle of other dephosphins. The peptide blocked ADBE, but not clathrin-mediated endocytosis of synaptic vesicles. Our results indicate that Ca2+ influx regulates assembly of a fully active CaN-calmodulin complex selectively on the tail of dynIxb and that the complex is recruited to sites of ADBE in nerve terminals. PMID:21730063

  3. Synaptic vesicle exocytosis and increased cytosolic calcium are both necessary but not sufficient for activity-dependent bulk endocytosis.

    PubMed

    Morton, Andrew; Marland, Jamie R K; Cousin, Michael A

    2015-08-01

    Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle (SV) endocytosis mode in central nerve terminals during intense neuronal activity. By definition this mode is triggered by neuronal activity; however, key questions regarding its mechanism of activation remain unaddressed. To determine the basic requirements for ADBE triggering in central nerve terminals, we decoupled SV fusion events from activity-dependent calcium influx using either clostridial neurotoxins or buffering of intracellular calcium. ADBE was monitored both optically and morphologically by observing uptake of the fluid phase markers tetramethylrhodamine-dextran and horse radish peroxidase respectively. Ablation of SV fusion with tetanus toxin resulted in the arrest of ADBE, but had no effect on other calcium-dependent events such as activity-dependent dynamin I dephosphorylation, indicating that SV exocytosis is necessary for triggering. Furthermore, the calcium chelator EGTA abolished ADBE while leaving SV exocytosis intact, demonstrating that ADBE is triggered by intracellular free calcium increases outside the active zone. Activity-dependent dynamin I dephosphorylation was also arrested in EGTA-treated neurons, consistent with its proposed role in triggering ADBE. Thus, SV fusion and increased cytoplasmic free calcium are both necessary but not sufficient individually to trigger ADBE. Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle (SV) endocytosis mode in central nerve terminals during intense neuronal activity. To determine the minimal requirements for ADBE triggering, we decoupled SV fusion events from activity-dependent calcium influx using either clostridial neurotoxins or buffering of intracellular calcium. We found that SV fusion and increased cytoplasmic free calcium are both necessary but not sufficient to trigger ADBE.

  4. Release activity-dependent control of vesicle endocytosis by the synaptic adhesion molecule N-cadherin

    PubMed Central

    van Stegen, Bernd; Dagar, Sushma; Gottmann, Kurt

    2017-01-01

    At synapses in the mammalian brain, continuous information transfer requires the long-term maintenance of homeostatic coupling between exo- and endocytosis of synaptic vesicles. Because classical endocytosis is orders of magnitude slower than the millisecond-range exocytosis of vesicles, high frequency vesicle fusion could potentially compromise structural stability of synapses. However, the molecular mechanisms mediating the tight coupling of exo- and endocytosis are largely unknown. Here, we investigated the role of the transsynaptic adhesion molecules N-cadherin and Neuroligin1 in regulating vesicle exo- and endocytosis by using activity-induced FM4–64 staining and by using synaptophysin-pHluorin fluorescence imaging. The synaptic adhesion molecules N-cadherin and Neuroligin1 had distinct impacts on exo- and endocytosis at mature cortical synapses. Expression of Neuroligin1 enhanced vesicle release in a N-cadherin-dependent way. Most intriguingly, expression of N-cadherin enhanced both vesicle exo- and endocytosis. Further detailed analysis of N-cadherin knockout neurons revealed that the boosting of endocytosis by N-cadherin was largely dependent on preceding high levels of vesicle release activity. In summary, regulation of vesicle endocytosis was mediated at the molecular level by N-cadherin in a release activity-dependent manner. Because of its endocytosis enhancing function, N-cadherin might play an important role in the coupling of vesicle exo- and endocytosis. PMID:28106089

  5. Release activity-dependent control of vesicle endocytosis by the synaptic adhesion molecule N-cadherin.

    PubMed

    van Stegen, Bernd; Dagar, Sushma; Gottmann, Kurt

    2017-01-20

    At synapses in the mammalian brain, continuous information transfer requires the long-term maintenance of homeostatic coupling between exo- and endocytosis of synaptic vesicles. Because classical endocytosis is orders of magnitude slower than the millisecond-range exocytosis of vesicles, high frequency vesicle fusion could potentially compromise structural stability of synapses. However, the molecular mechanisms mediating the tight coupling of exo- and endocytosis are largely unknown. Here, we investigated the role of the transsynaptic adhesion molecules N-cadherin and Neuroligin1 in regulating vesicle exo- and endocytosis by using activity-induced FM4-64 staining and by using synaptophysin-pHluorin fluorescence imaging. The synaptic adhesion molecules N-cadherin and Neuroligin1 had distinct impacts on exo- and endocytosis at mature cortical synapses. Expression of Neuroligin1 enhanced vesicle release in a N-cadherin-dependent way. Most intriguingly, expression of N-cadherin enhanced both vesicle exo- and endocytosis. Further detailed analysis of N-cadherin knockout neurons revealed that the boosting of endocytosis by N-cadherin was largely dependent on preceding high levels of vesicle release activity. In summary, regulation of vesicle endocytosis was mediated at the molecular level by N-cadherin in a release activity-dependent manner. Because of its endocytosis enhancing function, N-cadherin might play an important role in the coupling of vesicle exo- and endocytosis.

  6. Synaptotagmin-11 inhibits clathrin-mediated and bulk endocytosis.

    PubMed

    Wang, Changhe; Wang, Yeshi; Hu, Meiqin; Chai, Zuying; Wu, Qihui; Huang, Rong; Han, Weiping; Zhang, Claire Xi; Zhou, Zhuan

    2016-01-01

    Precise and efficient endocytosis is essential for vesicle recycling during a sustained neurotransmission. The regulation of endocytosis has been extensively studied, but inhibitors have rarely been found. Here, we show that synaptotagmin-11 (Syt11), a non-Ca(2+)-binding Syt implicated in schizophrenia and Parkinson's disease, inhibits clathrin-mediated endocytosis (CME) and bulk endocytosis in dorsal root ganglion neurons. The frequency of both types of endocytic event increases in Syt11 knockdown neurons, while the sizes of endocytosed vesicles and the kinetics of individual bulk endocytotic events remain unaffected. Specifically, clathrin-coated pits and bulk endocytosis-like structures increase on the plasma membrane in Syt11-knockdown neurons. Structural-functional analysis reveals distinct domain requirements for Syt11 function in CME and bulk endocytosis. Importantly, Syt11 also inhibits endocytosis in hippocampal neurons, implying a general role of Syt11 in neurons. Taken together, we propose that Syt11 functions to ensure precision in vesicle retrieval, mainly by limiting the sites of membrane invagination at the early stage of endocytosis.

  7. Adaptor protein complexes 1 and 3 are essential for generation of synaptic vesicles from activity-dependent bulk endosomes.

    PubMed

    Cheung, Giselle; Cousin, Michael A

    2012-04-25

    Activity-dependent bulk endocytosis is the dominant synaptic vesicle retrieval mode during high intensity stimulation in central nerve terminals. A key event in this endocytosis mode is the generation of new vesicles from bulk endosomes, which replenish the reserve vesicle pool. We have identified an essential requirement for both adaptor protein complexes 1 and 3 in this process by employing morphological and optical tracking of bulk endosome-derived synaptic vesicles in rat primary neuronal cultures. We show that brefeldin A inhibits synaptic vesicle generation from bulk endosomes and that both brefeldin A knockdown and shRNA knockdown of either adaptor protein 1 or 3 subunits inhibit reserve pool replenishment from bulk endosomes. Conversely, no plasma membrane function was found for adaptor protein 1 or 3 in either bulk endosome formation or clathrin-mediated endocytosis. Simultaneous knockdown of both adaptor proteins 1 and 3 indicated that they generated the same population of synaptic vesicles. Thus, adaptor protein complexes 1 and 3 play an essential dual role in generation of synaptic vesicles during activity-dependent bulk endocytosis.

  8. Adaptor protein complexes 1 and 3 are essential for generation of synaptic vesicles from activity-dependent bulk endosomes

    PubMed Central

    Cheung, Giselle; Cousin, Michael Alan

    2012-01-01

    Activity-dependent bulk endocytosis is the dominant synaptic vesicle retrieval mode during high intensity stimulation in central nerve terminals. A key event in this endocytosis mode is the generation of new vesicles from bulk endosomes, which replenish the reserve vesicle pool. We have identified an essential requirement for both adaptor protein complexes 1 and 3 in this process by employing morphological and optical tracking of bulk endosome-derived synaptic vesicles in rat primary neuronal cultures. We show that brefeldin A inhibits synaptic vesicle generation from bulk endosomes, and that both brefeldin A and shRNA knockdown of either adaptor protein 1 or 3 subunits inhibit reserve pool replenishment from bulk endosomes. Conversely, no plasma membrane function was found for adaptor proteins 1 or 3 in either bulk endosome formation or clathrin-mediated endocytosis. Simultaneous knockdown of both adaptor protein 1 and 3 indicated that they generated the same population of SVs. Thus adaptor protein complex 1 and 3 play an essential dual role in generation of synaptic vesicles during activity-dependent bulk endocytosis. PMID:22539861

  9. Actin- and Dynamin-Dependent Maturation of Bulk Endocytosis Restores Neurotransmission following Synaptic Depletion

    PubMed Central

    Nguyen, Tam H.; Maucort, Guillaume; Sullivan, Robert K. P.; Schenning, Mitja; Lavidis, Nickolas A.; McCluskey, Adam; Robinson, Phillip J.; Meunier, Frederic A.

    2012-01-01

    Bulk endocytosis contributes to the maintenance of neurotransmission at the amphibian neuromuscular junction by regenerating synaptic vesicles. How nerve terminals internalize adequate portions of the presynaptic membrane when bulk endocytosis is initiated before the end of a sustained stimulation is unknown. A maturation process, occurring at the end of the stimulation, is hypothesised to precisely restore the pools of synaptic vesicles. Using confocal time-lapse microscopy of FM1-43-labeled nerve terminals at the amphibian neuromuscular junction, we confirm that bulk endocytosis is initiated during a sustained tetanic stimulation and reveal that shortly after the end of the stimulation, nerve terminals undergo a maturation process. This includes a transient bulging of the plasma membrane, followed by the development of large intraterminal FM1-43-positive donut-like structures comprising large bulk membrane cisternae surrounded by recycling vesicles. The degree of bulging increased with stimulation frequency and the plasmalemma surface retrieved following the transient bulging correlated with the surface membrane internalized in bulk cisternae and recycling vesicles. Dyngo-4a, a potent dynamin inhibitor, did not block the initiation, but prevented the maturation of bulk endocytosis. In contrast, cytochalasin D, an inhibitor of actin polymerization, hindered both the initiation and maturation processes. Both inhibitors hampered the functional recovery of neurotransmission after synaptic depletion. Our data confirm that initiation of bulk endocytosis occurs during stimulation and demonstrates that a delayed maturation process controlled by actin and dynamin underpins the coupling between exocytosis and bulk endocytosis. PMID:22629340

  10. Small misfolded Tau species are internalized via bulk endocytosis and anterogradely and retrogradely transported in neurons.

    PubMed

    Wu, Jessica W; Herman, Mathieu; Liu, Li; Simoes, Sabrina; Acker, Christopher M; Figueroa, Helen; Steinberg, Joshua I; Margittai, Martin; Kayed, Rakez; Zurzolo, Chiara; Di Paolo, Gilbert; Duff, Karen E

    2013-01-18

    The accumulation of Tau into aggregates is associated with key pathological events in frontotemporal lobe degeneration (FTD-Tau) and Alzheimer disease (AD). Recent data have shown that misfolded Tau can be internalized by cells in vitro (Frost, B., Jacks, R. L., and Diamond, M. I. (2009) J. Biol. Chem. 284, 12845-12852) and propagate pathology in vivo (Clavaguera, F., Bolmont, T., Crowther, R. A., Abramowski, D., Frank, S., Probst, A., Fraser, G., Stalder, A. K., Beibel, M., Staufenbiel, M., Jucker, M., Goedert, M., and Tolnay, M. (2009) Nat. Cell Biol. 11, 909-913; Lasagna-Reeves, C. A., Castillo-Carranza, D. L., Sengupta, U., Guerrero-Munoz, M. J., Kiritoshi, T., Neugebauer, V., Jackson, G. R., and Kayed, R. (2012) Sci. Rep. 2, 700). Here we show that recombinant Tau misfolds into low molecular weight (LMW) aggregates prior to assembly into fibrils, and both extracellular LMW Tau aggregates and short fibrils, but not monomers, long fibrils, nor long filaments purified from brain extract are taken up by neurons. Remarkably, misfolded Tau can be internalized at the somatodendritic compartment, or the axon terminals and it can be transported anterogradely, retrogradely, and can enhance tauopathy in vivo. The internalized Tau aggregates co-localize with dextran, a bulk-endocytosis marker, and with the endolysosomal compartments. Our findings demonstrate that exogenous Tau can be taken up by cells, uptake depends on both the conformation and size of the Tau aggregates and once inside cells, Tau can be transported. These data provide support for observations that tauopathy can spread trans-synaptically in vivo, via cell-to-cell transfer.

  11. Small Misfolded Tau Species Are Internalized via Bulk Endocytosis and Anterogradely and Retrogradely Transported in Neurons*

    PubMed Central

    Wu, Jessica W.; Herman, Mathieu; Liu, Li; Simoes, Sabrina; Acker, Christopher M.; Figueroa, Helen; Steinberg, Joshua I.; Margittai, Martin; Kayed, Rakez; Zurzolo, Chiara; Di Paolo, Gilbert; Duff, Karen E.

    2013-01-01

    The accumulation of Tau into aggregates is associated with key pathological events in frontotemporal lobe degeneration (FTD-Tau) and Alzheimer disease (AD). Recent data have shown that misfolded Tau can be internalized by cells in vitro (Frost, B., Jacks, R. L., and Diamond, M. I. (2009) J. Biol. Chem. 284, 12845–12852) and propagate pathology in vivo (Clavaguera, F., Bolmont, T., Crowther, R. A., Abramowski, D., Frank, S., Probst, A., Fraser, G., Stalder, A. K., Beibel, M., Staufenbiel, M., Jucker, M., Goedert, M., and Tolnay, M. (2009) Nat. Cell Biol. 11, 909–913; Lasagna-Reeves, C. A., Castillo-Carranza, D. L., Sengupta, U., Guerrero-Munoz, M. J., Kiritoshi, T., Neugebauer, V., Jackson, G. R., and Kayed, R. (2012) Sci. Rep. 2, 700). Here we show that recombinant Tau misfolds into low molecular weight (LMW) aggregates prior to assembly into fibrils, and both extracellular LMW Tau aggregates and short fibrils, but not monomers, long fibrils, nor long filaments purified from brain extract are taken up by neurons. Remarkably, misfolded Tau can be internalized at the somatodendritic compartment, or the axon terminals and it can be transported anterogradely, retrogradely, and can enhance tauopathy in vivo. The internalized Tau aggregates co-localize with dextran, a bulk-endocytosis marker, and with the endolysosomal compartments. Our findings demonstrate that exogenous Tau can be taken up by cells, uptake depends on both the conformation and size of the Tau aggregates and once inside cells, Tau can be transported. These data provide support for observations that tauopathy can spread trans-synaptically in vivo, via cell-to-cell transfer. PMID:23188818

  12. Building a better dynasore: the dyngo compounds potently inhibit dynamin and endocytosis.

    PubMed

    McCluskey, Adam; Daniel, James A; Hadzic, Gordana; Chau, Ngoc; Clayton, Emma L; Mariana, Anna; Whiting, Ainslie; Gorgani, Nick N; Lloyd, Jonathan; Quan, Annie; Moshkanbaryans, Lia; Krishnan, Sai; Perera, Swetha; Chircop, Megan; von Kleist, Lisa; McGeachie, Andrew B; Howes, Mark T; Parton, Robert G; Campbell, Michael; Sakoff, Jennette A; Wang, Xuefeng; Sun, Jian-Yuan; Robertson, Mark J; Deane, Fiona M; Nguyen, Tam H; Meunier, Frederic A; Cousin, Michael A; Robinson, Phillip J

    2013-12-01

    Dynamin GTPase activity increases when it oligomerizes either into helices in the presence of lipid templates or into rings in the presence of SH3 domain proteins. Dynasore is a dynamin inhibitor of moderate potency (IC₅₀ ~ 15 μM in vitro). We show that dynasore binds stoichiometrically to detergents used for in vitro drug screening, drastically reducing its potency (IC₅₀ = 479 μM) and research tool utility. We synthesized a focused set of dihydroxyl and trihydroxyl dynasore analogs called the Dyngo™ compounds, five of which had improved potency, reduced detergent binding and reduced cytotoxicity, conferred by changes in the position and/or number of hydroxyl substituents. The Dyngo compound 4a was the most potent compound, exhibiting a 37-fold improvement in potency over dynasore for liposome-stimulated helical dynamin activity. In contrast, while dynasore about equally inhibited dynamin assembled in its helical or ring states, 4a and 6a exhibited >36-fold reduced activity against rings, suggesting that they can discriminate between helical or ring oligomerization states. 4a and 6a inhibited dynamin-dependent endocytosis of transferrin in multiple cell types (IC₅₀ of 5.7 and 5.8 μM, respectively), at least sixfold more potently than dynasore, but had no effect on dynamin-independent endocytosis of cholera toxin. 4a also reduced synaptic vesicle endocytosis and activity-dependent bulk endocytosis in cultured neurons and synaptosomes. Overall, 4a and 6a are improved and versatile helical dynamin and endocytosis inhibitors in terms of potency, non-specific binding and cytotoxicity. The data further suggest that the ring oligomerization state of dynamin is not required for clathrin-mediated endocytosis.

  13. HDL endocytosis and resecretion☆

    PubMed Central

    Röhrl, Clemens; Stangl, Herbert

    2013-01-01

    HDL removes excess cholesterol from peripheral tissues and delivers it to the liver and steroidogenic tissues via selective lipid uptake without catabolism of the HDL particle itself. In addition, endocytosis of HDL holo-particles has been debated for nearly 40 years. However, neither the connection between HDL endocytosis and selective lipid uptake, nor the physiological relevance of HDL uptake has been delineated clearly. This review will focus on HDL endocytosis and resecretion and its relation to cholesterol transfer. We will discuss the role of HDL endocytosis in maintaining cholesterol homeostasis in tissues and cell types involved in atherosclerosis, focusing on liver, macrophages and endothelium. We will critically summarize the current knowledge on the receptors mediating HDL endocytosis including SR-BI, F1-ATPase and CD36 and on intracellular HDL transport routes. Dependent on the tissue, HDL is either resecreted (retro-endocytosis) or degraded after endocytosis. Finally, findings on HDL transcytosis across the endothelial barrier will be summarized. We suggest that HDL endocytosis and resecretion is a rather redundant pathway under physiologic conditions. In case of disturbed lipid metabolism, however, HDL retro-endocytosis represents an alternative pathway that enables tissues to maintain cellular cholesterol homeostasis. PMID:23939397

  14. HDL endocytosis and resecretion.

    PubMed

    Röhrl, Clemens; Stangl, Herbert

    2013-11-01

    HDL removes excess cholesterol from peripheral tissues and delivers it to the liver and steroidogenic tissues via selective lipid uptake without catabolism of the HDL particle itself. In addition, endocytosis of HDL holo-particles has been debated for nearly 40years. However, neither the connection between HDL endocytosis and selective lipid uptake, nor the physiological relevance of HDL uptake has been delineated clearly. This review will focus on HDL endocytosis and resecretion and its relation to cholesterol transfer. We will discuss the role of HDL endocytosis in maintaining cholesterol homeostasis in tissues and cell types involved in atherosclerosis, focusing on liver, macrophages and endothelium. We will critically summarize the current knowledge on the receptors mediating HDL endocytosis including SR-BI, F1-ATPase and CD36 and on intracellular HDL transport routes. Dependent on the tissue, HDL is either resecreted (retro-endocytosis) or degraded after endocytosis. Finally, findings on HDL transcytosis across the endothelial barrier will be summarized. We suggest that HDL endocytosis and resecretion is a rather redundant pathway under physiologic conditions. In case of disturbed lipid metabolism, however, HDL retro-endocytosis represents an alternative pathway that enables tissues to maintain cellular cholesterol homeostasis.

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

    PubMed

    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.

  16. Synaptic Vesicle Endocytosis

    PubMed Central

    Saheki, Yasunori; De Camilli, Pietro

    2012-01-01

    Neurons can sustain high rates of synaptic transmission without exhausting their supply of synaptic vesicles. This property relies on a highly efficient local endocytic recycling of synaptic vesicle membranes, which can be reused for hundreds, possibly thousands, of exo-endocytic cycles. Morphological, physiological, molecular, and genetic studies over the last four decades have provided insight into the membrane traffic reactions that govern this recycling and its regulation. These studies have shown that synaptic vesicle endocytosis capitalizes on fundamental and general endocytic mechanisms but also involves neuron-specific adaptations of such mechanisms. Thus, investigations of these processes have advanced not only the field of synaptic transmission but also, more generally, the field of endocytosis. This article summarizes current information on synaptic vesicle endocytosis with an emphasis on the underlying molecular mechanisms and with a special focus on clathrin-mediated endocytosis, the predominant pathway of synaptic vesicle protein internalization. PMID:22763746

  17. Endocytosis of Nanomedicines

    PubMed Central

    Sahay, Gaurav; Alakhova, Daria Y; Kabanov, Alexander V

    2010-01-01

    Novel nanomaterials are being developed to improve diagnosis and therapy of diseases through effective delivery of drugs, biopharmaceutical molecules and imaging agents to target cells in disease sites. Such diagnostic and therapeutic nanomaterials, also termed “nanomedicines”, often require site-specific cellular entry to deliver their payload to subcellular locations hidden beneath cell membranes. Nanomedicines can employ multiple pathways for cellular entry, which are currently insufficiently understood. This review, first, classifies various mechanisms of endocytosis available to nanomedicines including phagocytosis and pinocytosis through clathrin-dependent and clathrin-independent pathways. Second, it describes the current experimental tools to study endocytosis of nanomedicines. Third, it provides specific examples from recent literature and our own work on endocytosis of nanomedicines. Finally, these examples are used to ascertain 1) the role of particle size, shape, material composition, surface chemistry and/or charge for utilization of a selected pathway(s); 2) the effect of cell type on the processing of nanomedicines; 3) the effect of nanomaterial-cell interactions on the processes of endocytosis, the fate of the nanomedicines and the resulting cellular responses. This review will be useful to a diverse audience of students and scientists who are interested in understanding endocytosis of nanomedicines. PMID:20226220

  18. Endocytosis in enterocytes.

    PubMed

    Zimmer, Klaus-Peter; de Laffolie, Jan; Barone, Maria Vittoria; Naim, Hassan Y

    2016-05-01

    Endocytosis is a fundamental cell biological process, which carries out essential functions in a polarized epithelial cell such as enterocytes provided with a huge surface area of the brush border membrane. Major tasks of enterocytes, which are regulated by endocytic signals, are digestion and absorption of nutrients and drugs/pharmacological agents, barrier permeability to microorganism, toxins and antigens, and transcytotic crosstalk between intestinal lumen and lamina propria cells with access to the circulation.Investigations on inflammatory bowel diseases such as food allergy, celiac disease, Crohn's disease, and ulcerative colitis focus on immune processes originating within enterocytes as antigen presenting cells. Thus the initiation of oral tolerance, that is, the binding of food antigens to MHC class II proteins, might be localized within late endosomes of enterocytes. Furthermore, the late endosomal compartment of enterocytes seems to be involved in the processing of luminal antigens during the pathogenesis of celiac disease and inflammatory bowel diseases. Investigations of inherited diseases such as microvillus inclusion disease have revealed a pathogenetic defect in the autophagocytotic and/or recycling pathway of enterocytes.Our progress in the cell and molecular biological understanding of the endocytosis and the methodical opportunities of translational research offer now new therapeutic options for patients suffering from endocytosis-related diseases of enterocytes.

  19. Overlapping Role of Dynamin Isoforms in Synaptic Vesicle Endocytosis

    PubMed Central

    Raimondi, Andrea; Ferguson, Shawn M.; Lou, Xuelin; Armbruster, Moritz; Paradise, Summer; Giovedi, Silvia; Messa, Mirko; Kono, Nao; Takasaki, Junko; Cappello, Valentina; O’Toole, Eileen; Ryan, Timothy A.; De Camilli, Pietro

    2011-01-01

    The existence of neuron specific endocytic protein isoforms raises questions about their importance for specialized neuronal functions. Dynamin, a GTPase implicated in the fission reaction of endocytosis, is encoded by three genes, two of which, dynamin 1 and 3, are highly expressed in neurons. We show that dynamin 3, thought to play a predominantly postsynaptic role, has a major presynaptic function. While lack of dynamin 3 does not produce an overt phenotype in mice, it worsens the dynamin 1 KO phenotype, leading to perinatal lethality and a more severe defect in activity-dependent synaptic vesicle endocytosis. Thus, dynamin 1 and 3, which together account for the overwhelming majority of brain dynamin, cooperate in supporting optimal rates of synaptic vesicle endocytosis. Persistence of synaptic transmission in their absence indicates that if dynamin plays essential functions in neurons, such functions can be achieved by the very low levels of dynamin 2. PMID:21689597

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

    PubMed Central

    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

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

    PubMed

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

    2016-08-18

    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.

  2. Lipid-Mediated Endocytosis

    PubMed Central

    Ewers, Helge; Helenius, Ari

    2011-01-01

    Receptor-mediated endocytosis is used by a number of viruses and toxins to gain entry into cells. Some have evolved to use specific lipids in the plasma membrane as their receptors. They include bacterial toxins such as Shiga and Cholera toxin and viruses such as mouse polyoma virus and simian virus 40. Through multivalent binding to glycosphingolipids, they induce lipid clustering and changes in membrane properties. Internalization occurs by unusual endocytic mechanisms involving lipid rafts, induction of membrane curvature, trans-bilayer coupling, and activation of signaling pathways. Once delivered to early endosomes, they follow diverse intracellular routes to the lumen of the ER, from which they penetrate into the cytosol. The role of the lipid receptors is central in these well-studied processes. PMID:21576253

  3. Cell biology of neuronal endocytosis.

    PubMed

    Parton, R G; Dotti, C G

    1993-09-01

    Endocytosis is the process by which cells take in fluid and components of the plasma membrane. In this way cells obtain nutrients and trophic factors, retrieve membrane proteins for degradation, and sample their environment. In neuronal cells endocytosis is essential for the recycling of membrane after neurotransmitter release and plays a critical role during early developmental stages. Moreover, alterations of the endocytic pathway have been attributed a crucial role in the pathophysiology of certain neurological diseases. Although well characterized at the ultrastructural level, little is known of the dynamics and molecular organization of the neuronal endocytic pathways. In this respect most of our knowledge comes from studies of non-neuronal cells. In this review we will examine the endocytic pathways in neurons from a cell biological viewpoint by making comparisons with non-neuronal cells and in particular with another polarized cell, the epithelial cell.

  4. Endocytosis of Viruses and Bacteria

    PubMed Central

    Cossart, Pascale; Helenius, Ari

    2014-01-01

    Of the many pathogens that infect humans and animals, a large number use cells of the host organism as protected sites for replication. To reach the relevant intracellular compartments, they take advantage of the endocytosis machinery and exploit the network of endocytic organelles for penetration into the cytosol or as sites of replication. In this review, we discuss the endocytic entry processes used by viruses and bacteria and compare the strategies used by these dissimilar classes of pathogens. PMID:25085912

  5. Mint proteins are required for synaptic activity-dependent amyloid precursor protein (APP) trafficking and amyloid β generation.

    PubMed

    Sullivan, Sarah E; Dillon, Gregory M; Sullivan, Josefa M; Ho, Angela

    2014-05-30

    Aberrant amyloid β (Aβ) production plays a causal role in Alzheimer disease pathogenesis. A major cellular pathway for Aβ generation is the activity-dependent endocytosis and proteolytic cleavage of the amyloid precursor protein (APP). However, the molecules controlling activity-dependent APP trafficking in neurons are less defined. Mints are adaptor proteins that directly interact with the endocytic sorting motif of APP and are functionally important in regulating APP endocytosis and Aβ production. We analyzed neuronal cultures from control and Mint knockout neurons that were treated with either glutamate or tetrodotoxin to stimulate an increase or decrease in neuronal activity, respectively. We found that neuronal activation by glutamate increased APP endocytosis, followed by elevated APP insertion into the cell surface, stabilizing APP at the plasma membrane. Conversely, suppression of neuronal activity by tetrodotoxin decreased APP endocytosis and insertion. Interestingly, we found that activity-dependent APP trafficking and Aβ generation were blocked in Mint knockout neurons. We showed that wild-type Mint1 can rescue APP internalization and insertion in Mint knockout neurons. In addition, we found that Mint overexpression increased excitatory synaptic activity and that APP was internalized predominantly to endosomes associated with APP processing. We demonstrated that presenilin 1 (PS1) endocytosis requires interaction with the PDZ domains of Mint1 and that this interaction facilitates activity-dependent colocalization of APP and PS1. These findings demonstrate that Mints are necessary for activity-induced APP and PS1 trafficking and provide insight into the cellular fate of APP in endocytic pathways essential for Aβ production. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Mint Proteins Are Required for Synaptic Activity-dependent Amyloid Precursor Protein (APP) Trafficking and Amyloid β Generation*

    PubMed Central

    Sullivan, Sarah E.; Dillon, Gregory M.; Sullivan, Josefa M.; Ho, Angela

    2014-01-01

    Aberrant amyloid β (Aβ) production plays a causal role in Alzheimer disease pathogenesis. A major cellular pathway for Aβ generation is the activity-dependent endocytosis and proteolytic cleavage of the amyloid precursor protein (APP). However, the molecules controlling activity-dependent APP trafficking in neurons are less defined. Mints are adaptor proteins that directly interact with the endocytic sorting motif of APP and are functionally important in regulating APP endocytosis and Aβ production. We analyzed neuronal cultures from control and Mint knockout neurons that were treated with either glutamate or tetrodotoxin to stimulate an increase or decrease in neuronal activity, respectively. We found that neuronal activation by glutamate increased APP endocytosis, followed by elevated APP insertion into the cell surface, stabilizing APP at the plasma membrane. Conversely, suppression of neuronal activity by tetrodotoxin decreased APP endocytosis and insertion. Interestingly, we found that activity-dependent APP trafficking and Aβ generation were blocked in Mint knockout neurons. We showed that wild-type Mint1 can rescue APP internalization and insertion in Mint knockout neurons. In addition, we found that Mint overexpression increased excitatory synaptic activity and that APP was internalized predominantly to endosomes associated with APP processing. We demonstrated that presenilin 1 (PS1) endocytosis requires interaction with the PDZ domains of Mint1 and that this interaction facilitates activity-dependent colocalization of APP and PS1. These findings demonstrate that Mints are necessary for activity-induced APP and PS1 trafficking and provide insight into the cellular fate of APP in endocytic pathways essential for Aβ production. PMID:24742670

  7. Endocytosis and its regulation in plants.

    PubMed

    Fan, Lusheng; Li, Ruili; Pan, Jianwei; Ding, Zhaojun; Lin, Jinxing

    2015-06-01

    Endocytosis provides a major route of entry for membrane proteins, lipids, and extracellular molecules into the cell. Recent evidence indicates that multiple cellular processes require endocytosis, including nutrient uptake, signaling transduction, and plant-microbe interactions. Also, advanced microscopy, combined with biochemical and genetic approaches, has provided more insights into the molecular machinery and functions of endocytosis in plants. Here we review mechanisms of the clathrin-dependent and membrane microdomain-associated endocytic routes in plant cells. In addition, degradation of endocytosed proteins and endosomal sorting complex required for transport (ESCRT)-mediated vesicle formation at the endosome are discussed. Finally, we summarize the essential roles of various regulators during plant endocytosis.

  8. Evidence for a Clathrin-independent mode of endocytosis at a continuously active sensory synapse

    PubMed Central

    Fuchs, Michaela; Brandstätter, Johann Helmut; Regus-Leidig, Hanna

    2014-01-01

    Synaptic vesicle exocytosis at chemical synapses is followed by compensatory endocytosis. Multiple pathways including Clathrin-mediated retrieval of single vesicles, bulk retrieval of large cisternae, and kiss-and-run retrieval have been reported to contribute to vesicle recycling. Particularly at the continuously active ribbon synapses of retinal photoreceptor and bipolar cells, compensatory endocytosis plays an essential role to provide ongoing vesicle supply. Yet, little is known about the mechanisms that contribute to endocytosis at these highly complex synapses. To identify possible specializations in ribbon synaptic endocytosis during different states of activity, we exposed mice to controlled lighting conditions and compared the distribution of endocytotic proteins at rod and cone photoreceptor, and ON bipolar cell ribbon synapses with light and electron microscopy. In mouse ON bipolar cell terminals, Clathrin-mediated endocytosis seemed to be the dominant mode of endocytosis at all adaptation states analyzed. In contrast, in mouse photoreceptor terminals in addition to Clathrin-coated pits, clusters of membranously connected electron-dense vesicles appeared during prolonged darkness. These clusters labeled for Dynamin3, Endophilin1, and Synaptojanin1, but not for AP180, Clathrin LC, and hsc70. We hypothesize that rod and cone photoreceptors possess an additional Clathrin-independent mode of vesicle retrieval supporting the continuous synaptic vesicle supply during prolonged high activity. PMID:24616664

  9. Endocytosis and Signaling during Development

    PubMed Central

    Bökel, Christian

    2014-01-01

    The development of multicellular organisms relies on an intricate choreography of intercellular communication events that pattern the embryo and coordinate the formation of tissues and organs. It is therefore not surprising that developmental biology, especially using genetic model organisms, has contributed significantly to the discovery and functional dissection of the associated signal-transduction cascades. At the same time, biophysical, biochemical, and cell biological approaches have provided us with insights into the underlying cell biological machinery. Here we focus on how endocytic trafficking of signaling components (e.g., ligands or receptors) controls the generation, propagation, modulation, reception, and interpretation of developmental signals. A comprehensive enumeration of the links between endocytosis and signal transduction would exceed the limits of this review. We will instead use examples from different developmental pathways to conceptually illustrate the various functions provided by endocytic processes during key steps of intercellular signaling. PMID:24591521

  10. Endocytosis and signaling during development.

    PubMed

    Bökel, Christian; Brand, Michael

    2014-03-01

    The development of multicellular organisms relies on an intricate choreography of intercellular communication events that pattern the embryo and coordinate the formation of tissues and organs. It is therefore not surprising that developmental biology, especially using genetic model organisms, has contributed significantly to the discovery and functional dissection of the associated signal-transduction cascades. At the same time, biophysical, biochemical, and cell biological approaches have provided us with insights into the underlying cell biological machinery. Here we focus on how endocytic trafficking of signaling components (e.g., ligands or receptors) controls the generation, propagation, modulation, reception, and interpretation of developmental signals. A comprehensive enumeration of the links between endocytosis and signal transduction would exceed the limits of this review. We will instead use examples from different developmental pathways to conceptually illustrate the various functions provided by endocytic processes during key steps of intercellular signaling.

  11. Mitochondrial Calcium Uptake Modulates Synaptic Vesicle Endocytosis in Central Nerve Terminals.

    PubMed

    Marland, Jamie Roslin Keynes; Hasel, Philip; Bonnycastle, Katherine; Cousin, Michael Alan

    2016-01-29

    Presynaptic calcium influx triggers synaptic vesicle (SV) exocytosis and modulates subsequent SV endocytosis. A number of calcium clearance mechanisms are present in central nerve terminals that regulate intracellular free calcium levels both during and after stimulation. During action potential stimulation, mitochondria rapidly accumulate presynaptic calcium via the mitochondrial calcium uniporter (MCU). The role of mitochondrial calcium uptake in modulating SV recycling has been debated extensively, but a definitive conclusion has not been achieved. To directly address this question, we manipulated the expression of the MCU channel subunit in primary cultures of neurons expressing a genetically encoded reporter of SV turnover. Knockdown of MCU resulted in ablation of activity-dependent mitochondrial calcium uptake but had no effect on the rate or extent of SV exocytosis. In contrast, the rate of SV endocytosis was increased in the absence of mitochondrial calcium uptake and slowed when MCU was overexpressed. MCU knockdown did not perturb activity-dependent increases in presynaptic free calcium, suggesting that SV endocytosis may be controlled by calcium accumulation and efflux from mitochondria in their immediate vicinity.

  12. Activation-dependent plasticity of polarized GPCR distribution on the neuronal surface.

    PubMed

    Simon, Anne C; Loverdo, Claude; Gaffuri, Anne-Lise; Urbanski, Michel; Ladarre, Delphine; Carrel, Damien; Rivals, Isabelle; Leterrier, Christophe; Benichou, Olivier; Dournaud, Pascal; Szabo, Bela; Voituriez, Raphael; Lenkei, Zsolt

    2013-08-01

    Directionality of information flow through neuronal networks is sustained at cellular level by polarized neurons. However, specific targeting or anchoring motifs responsible for polarized distribution on the neuronal surface have only been identified for a few neuronal G-protein-coupled receptors (GPCRs). Here, through mutational and pharmacological modifications of the conformational state of two model GPCRs, the axonal CB1R cannabinoid and the somatodendritic SSTR2 somatostatin receptors, we show important conformation-dependent variations in polarized distribution. The underlying mechanisms include lower efficiency of conformation-dependent GPCR endocytosis in axons, compared with dendrites, particularly at moderate activation levels, as well as endocytosis-dependent transcytotic delivery of GPCRs from the somatodendritic domain to distal axonal portions, shown by using compartmentalized microfluidic devices. Kinetic modeling predicted that GPCR distribution polarity is highly regulated by steady-state endocytosis, which is conformation dependent and is able to regulate the relative amount of GPCRs targeted to axons and that axonally polarized distribution is an intermediary phenotype that appears at moderate basal activation levels. Indeed, we experimentally show that gradual changes in basal activation-dependent endocytosis lead to highly correlated shifts of polarized GPCR distribution on the neuronal surface, which can even result in a fully reversed polarized distribution of naturally somatodendritic or axonal GPCRs. In conclusion, polarized distribution of neuronal GPCRs may have a pharmacologically controllable component, which, in the absence of dominant targeting motifs, could even represent the principal regulator of sub-neuronal distribution. Consequently, chronic modifications of basal GPCR activation by therapeutic or abused drugs may lead to previously unanticipated changes in brain function through perturbation of polarized GPCR distribution on

  13. Lineage-specific proteins essential for endocytosis in trypanosomes.

    PubMed

    Manna, Paul T; Obado, Samson O; Boehm, Cordula; Gadelha, Catarina; Sali, Andrej; Chait, Brian T; Rout, Michael P; Field, Mark C

    2017-04-15

    Clathrin-mediated endocytosis (CME) is the most evolutionarily ancient endocytic mechanism known, and in many lineages the sole mechanism for internalisation. Significantly, in mammalian cells CME is responsible for the vast bulk of endocytic flux and has likely undergone multiple adaptations to accommodate specific requirements by individual species. In African trypanosomes, we previously demonstrated that CME is independent of the AP-2 adaptor protein complex, that orthologues to many of the animal and fungal CME protein cohort are absent, and that a novel, trypanosome-restricted protein cohort interacts with clathrin and drives CME. Here, we used a novel cryomilling affinity isolation strategy to preserve transient low-affinity interactions, giving the most comprehensive trypanosome clathrin interactome to date. We identified the trypanosome AP-1 complex, Trypanosoma brucei (Tb)EpsinR, several endosomal SNAREs plus orthologues of SMAP and the AP-2 associated kinase AAK1 as interacting with clathrin. Novel lineage-specific proteins were identified, which we designate TbCAP80 and TbCAP141. Their depletion produced extensive defects in endocytosis and endomembrane system organisation, revealing a novel molecular pathway subtending an early-branching and highly divergent form of CME, which is conserved and likely functionally important across the kinetoplastid parasites. © 2017. Published by The Company of Biologists Ltd.

  14. Endocytosis of Nanoscale Systems for Cancer Treatments.

    PubMed

    Chen, Kai; Li, Xue; Zhu, Hongyan; Gong, Qiyong; Luo, Kui

    2017-04-28

    Advances of nanoscale systems for cancer treatment have been involved in enabling highly regulated site-specific localization to sub cellular organelles hidden beneath cell membranes. Thus far, the cellular entry of these nanoscale systems has been not fully understood. Endocytosisis a form of active transport in which cell transports elected extracellular molecules (such as proteins, viruses, micro-organisms and nanoscale systems) are allowed into cell interiors by engulfing them in an energy-dependent process. This process appears at the plasma membrane surface and contains internalization of the cell membrane as well as the membrane proteins and lipids of cell. There are multiform pathways of endocytosis for nanoscale systems. Further comprehension for the mechanisms of endocytosis is achieved with a combination of efficient genetic manipulations, cell dynamic imaging, and chemical endocytosis inhibitors. This review provides an account of various endocytic pathways, itemizes current methods to study endocytosis of nanoscale systems, discusses some factors associated with cellular uptake for nanoscale systems and introduces the trafficking behavior for nanoscale systems with active targeting. An insight into the endocytosis mechanism is urgent and significant for developing safe and efficient nanoscale systems for cancer diagnosis and therapy. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  15. Multiple Functions of Sterols in Yeast Endocytosis

    PubMed Central

    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. The lack of phosphorylation is not due to a defect in Ste2p localization or in ligand–receptor interaction. Contrary to most known endocytic factors, sterols seem to function in internalization independently of actin. Furthermore, sterol structures are required at a postinternalization step of endocytosis. ergΔ cells were able to take up the membrane marker FM4-64, but exhibited defects in FM4-64 movement through endosomal compartments to the vacuole. Therefore, there are at least two roles for sterols in endocytosis. Based on sterol analysis, the sterol structural requirements for these two processes were different, suggesting that sterols may have distinct functions at different places in the endocytic pathway. Interestingly, sterol structures unable to support endocytosis allowed transport of the glycosylphosphatidylinositol-anchored protein Gas1p from the endoplasmic reticulum to Golgi compartment. PMID:12181337

  16. Ultrafast endocytosis at mouse hippocampal synapses

    NASA Astrophysics Data System (ADS)

    Watanabe, Shigeki; Rost, Benjamin R.; Camacho-Pérez, Marcial; Davis, M. Wayne; Söhl-Kielczynski, Berit; Rosenmund, Christian; Jorgensen, Erik M.

    2013-12-01

    To sustain neurotransmission, synaptic vesicles and their associated proteins must be recycled locally at synapses. Synaptic vesicles are thought to be regenerated approximately 20s after fusion by the assembly of clathrin scaffolds or in approximately 1s by the reversal of fusion pores via `kiss-and-run' endocytosis. Here we use optogenetics to stimulate cultured hippocampal neurons with a single stimulus, rapidly freeze them after fixed intervals and examine the ultrastructure using electron microscopy--`flash-and-freeze' electron microscopy. Docked vesicles fuse and collapse into the membrane within 30ms of the stimulus. Compensatory endocytosis occurs within 50 to 100ms at sites flanking the active zone. Invagination is blocked by inhibition of actin polymerization, and scission is blocked by inhibiting dynamin. Because intact synaptic vesicles are not recovered, this form of recycling is not compatible with kiss-and-run endocytosis; moreover, it is 200-fold faster than clathrin-mediated endocytosis. It is likely that `ultrafast endocytosis' is specialized to restore the surface area of the membrane rapidly.

  17. Extremely Low Frequency Electromagnetic Fields Facilitate Vesicle Endocytosis by Increasing Presynaptic Calcium Channel Expression at a Central Synapse

    PubMed Central

    Sun, Zhi-cheng; Ge, Jian-long; Guo, Bin; Guo, Jun; Hao, Mei; Wu, Yi-chen; Lin, Yi-an; La, Ting; Yao, Pan-tong; Mei, Yan-ai; Feng, Yi; Xue, Lei

    2016-01-01

    Accumulating evidence suggests significant biological effects caused by extremely low frequency electromagnetic fields (ELF-EMF). Although exo-endocytosis plays crucial physical and biological roles in neuronal communication, studies on how ELF-EMF regulates this process are scarce. By directly measuring calcium currents and membrane capacitance at a large mammalian central nervous synapse, the calyx of Held, we report for the first time that ELF-EMF critically affects synaptic transmission and plasticity. Exposure to ELF-EMF for 8 to 10 days dramatically increases the calcium influx upon stimulation and facilitates all forms of vesicle endocytosis, including slow and rapid endocytosis, endocytosis overshoot and bulk endocytosis, but does not affect the RRP size and exocytosis. Exposure to ELF-EMF also potentiates PTP, a form of short-term plasticity, increasing its peak amplitude without impacting its time course. We further investigated the underlying mechanisms and found that calcium channel expression, including the P/Q, N, and R subtypes, at the presynaptic nerve terminal was enhanced, accounting for the increased calcium influx upon stimulation. Thus, we conclude that exposure to ELF-EMF facilitates vesicle endocytosis and synaptic plasticity in a calcium-dependent manner by increasing calcium channel expression at the nerve terminal. PMID:26887777

  18. Activity dependent CAM cleavage and neurotransmission

    PubMed Central

    Conant, Katherine; Allen, Megan; Lim, Seung T.

    2015-01-01

    Spatially localized proteolysis represents an elegant means by which neuronal activity dependent changes in synaptic structure, and thus experience dependent learning and memory, can be achieved. In vitro and in vivo studies suggest that matrix metalloproteinase and adamalysin activity is concentrated at the cell surface, and emerging evidence suggests that increased peri-synaptic expression, release and/or activation of these proteinases occurs with enhanced excitatory neurotransmission. Synaptically expressed cell adhesion molecules (CAMs) could therefore represent important targets for neuronal activity-dependent proteolysis. Several CAM subtypes are expressed at the synapse, and their cleavage can influence the efficacy of synaptic transmission through a variety of non-mutually exclusive mechanisms. In the following review, we discuss mechanisms that regulate neuronal activity-dependent synaptic CAM shedding, including those that may be calcium dependent. We also highlight CAM targets of activity-dependent proteolysis including neuroligin and intercellular adhesion molecule-5 (ICAM-5). We include discussion focused on potential consequences of synaptic CAM shedding, with an emphasis on interactions between soluble CAM cleavage products and specific pre- and post-synaptic receptors. PMID:26321910

  19. Mitotic inhibition of clathrin-mediated endocytosis

    PubMed Central

    Fielding, Andrew B.; Royle, Stephen J.

    2014-01-01

    Endocytosis and mitosis are fundamental processes in a cell’s life. Nearly fifty years of research suggest that these processes are linked and that endocytosis is shut down as cells undergo the early stages of mitosis. Precisely how this occurs at a molecular level is an open question. In this review, we summarize the early work characterizing the inhibition of clathrin-mediated endocytosis and discuss recent challenges to this established concept. We also set out four proposed mechanisms for the inhibition: mitotic phosphorylation of endocytic proteins, altered membrane tension, moonlighting of endocytic proteins and a mitotic spindle-dependent mechanism. Finally, we speculate the functional consequences of endocytic shutdown during mitosis and where an understanding of the mechanism of inhibition will lead us in the future. PMID:23307073

  20. Ultrafast endocytosis at Caenorhabditis elegans neuromuscular junctions

    PubMed Central

    Watanabe, Shigeki; Liu, Qiang; Davis, M Wayne; Hollopeter, Gunther; Thomas, Nikita; Jorgensen, Nels B; Jorgensen, Erik M

    2013-01-01

    Synaptic vesicles can be released at extremely high rates, which places an extraordinary demand on the recycling machinery. Previous ultrastructural studies of vesicle recycling were conducted in dissected preparations using an intense stimulation to maximize the probability of release. Here, a single light stimulus was applied to motor neurons in intact Caenorhabditis elegans nematodes expressing channelrhodopsin, and the animals rapidly frozen. We found that docked vesicles fuse along a broad active zone in response to a single stimulus, and are replenished with a time constant of about 2 s. Endocytosis occurs within 50 ms adjacent to the dense projection and after 1 s adjacent to adherens junctions. These studies suggest that synaptic vesicle endocytosis may occur on a millisecond time scale following a single physiological stimulus in the intact nervous system and is unlikely to conform to current models of endocytosis. DOI: http://dx.doi.org/10.7554/eLife.00723.001 PMID:24015355

  1. Clathrin-Independent Pathways of Endocytosis

    PubMed Central

    Mayor, Satyajit; Parton, Robert G.; Donaldson, Julie G.

    2014-01-01

    There are many pathways of endocytosis at the cell surface that apparently operate at the same time. With the advent of new molecular genetic and imaging tools, an understanding of the different ways by which a cell may endocytose cargo is increasing by leaps and bounds. In this review we explore pathways of endocytosis that occur in the absence of clathrin. These are referred to as clathrin-independent endocytosis (CIE). Here we primarily focus on those pathways that function at the small scale in which some have distinct coats (caveolae) and others function in the absence of specific coated intermediates. We follow the trafficking itineraries of the material endocytosed by these pathways and finally discuss the functional roles that these pathways play in cell and tissue physiology. It is likely that these pathways will play key roles in the regulation of plasma membrane area and tension and also control the availability of membrane during cell migration. PMID:24890511

  2. An endocytosis pathway initiated through neuropilin-1 and regulated by nutrient availability

    PubMed Central

    Pang, Hong-Bo; Braun, Gary B.; Friman, Tomas; Aza-Blanc, Pedro; Ruidiaz, Manuel E.; Sugahara, Kazuki N.; Teesalu, Tambet; Ruoslahti, Erkki

    2014-01-01

    Neuropilins (NRPs) are trans-membrane receptors involved in axon guidance and vascular development. Many growth factors and other signaling molecules bind to NRPs through a C-terminal, basic sequence motif (C-end Rule or CendR motif). Peptides with this motif (CendR peptides) are taken up into cells by endocytosis. Tumor-homing CendR peptides penetrate through tumor tissue and have shown utility in enhancing drug delivery into tumors. Here we show, using RNAi screening and subsequent validation studies, that NRP1-mediated endocytosis of CendR peptides is distinct from known endocytic pathways. Ultrastructurally, CendR endocytosis resembles macropinocytosis, but is mechanistically different. We also show that nutrient-sensing networks such as mTOR signaling regulate CendR endocytosis and subsequent intercellular transport of CendR cargo, both of which are stimulated by nutrient depletion. As CendR is a bulk transport pathway, our results suggest a role for it in nutrient transport; CendR-enhanced drug delivery then makes use of this natural pathway. PMID:25277522

  3. Endocytosis in Giardia: Evidence of Absence.

    PubMed

    Zamponi, Nahuel; Feliziani, Constanza; Touz, María C

    2016-11-01

    Zumthor et al. recently reported a novel function for clathrin coatomer in Giardia lamblia endocytosis. On the basis of old and new data, we propose an updated model of the participation of clathrin function in this parasite. Copyright © 2016. Published by Elsevier Ltd.

  4. An immersed boundary method for endocytosis

    NASA Astrophysics Data System (ADS)

    Tseng, Yu-Hau; Huang, Huaxiong

    2014-09-01

    Endocytosis is one of the cellular functions for capturing (engulfing) vesicles or microorganisms. Understanding the biophysical mechanisms of this cellular process is essential from a bioengineering point of view since it will provide guidance for developing effective targeted drug delivery therapies. In this paper, we propose an immersed boundary (IB) method that can be used to simulate the dynamical process of this important biological function. In our model, membranes of the vesicle and the cell are treated as Canham-Helfrich Hamiltonian interfaces. The membrane-bound molecules are modeled as insoluble surfactants such that the molecules after binding are regarded as a product of a “chemical” reaction. Our numerical examples show that the immersed boundary method is a useful simulation tool for studying endocytosis, where the roles of interfacial energy, fluid flow and viscous dissipation in the success of the endocytosis process can be investigated in detail. A distinct feature of our IB method is the treatment of the two binding membranes that is different from the merging of fluid-fluid interfaces. Another important feature of our method is the strict conservation of membrane-borne receptors and ligands, which is important for predicting the dynamics of the endocytosis process.

  5. On the Modeling of Endocytosis in Yeast

    PubMed Central

    Zhang, Tao; Sknepnek, Rastko; Bowick, M.J.; Schwarz, J.M.

    2015-01-01

    The cell membrane deforms during endocytosis to surround extracellular material and draw it into the cell. Results of experiments on endocytosis in yeast show general agreement that 1) actin polymerizes into a network of filaments exerting active forces on the membrane to deform it, and 2) the large-scale membrane deformation is tubular in shape. In contrast, there are three competing proposals for precisely how the actin filament network organizes itself to drive the deformation. We use variational approaches and numerical simulations to address this competition by analyzing a meso-scale model of actin-mediated endocytosis in yeast. The meso-scale model breaks up the invagination process into three stages: 1) initiation, where clathrin interacts with the membrane via adaptor proteins; 2) elongation, where the membrane is then further deformed by polymerizing actin filaments; and 3) pinch-off. Our results suggest that the pinch-off mechanism may be assisted by a pearling-like instability. We rule out two of the three competing proposals for the organization of the actin filament network during the elongation stage. These two proposals could be important in the pinch-off stage, however, where additional actin polymerization helps break off the vesicle. Implications and comparisons with earlier modeling of endocytosis in yeast are discussed. PMID:25650919

  6. Multiscale perspectives of virus entry via endocytosis.

    PubMed

    Barrow, Eric; Nicola, Anthony V; Liu, Jin

    2013-06-05

    Most viruses take advantage of endocytic pathways to gain entry into host cells and initiate infections. Understanding of virus entry via endocytosis is critically important for the design of antiviral strategies. Virus entry via endocytosis is a complex process involving hundreds of cellular proteins. The entire process is dictated by events occurring at multiple time and length scales. In this review, we discuss and evaluate the available means to investigate virus endocytic entry, from both experimental and theoretical/numerical modeling fronts, and highlight the importance of multiscale features. The complexity of the process requires investigations at a systems biology level, which involves the combination of different experimental approaches, the collaboration of experimentalists and theorists across different disciplines, and the development of novel multiscale models.

  7. A role of endocytosis in plant cytokinesis.

    PubMed

    Karahara, Ichirou; Staehelin, L Andrew; Mineyuki, Yoshinobu

    2010-01-01

    The preprophase band (PPB) of microtubules (MTs) marks the site of the future division plane irrespective of the orientation of the equatorial plane. Because the PPB MTs disappear during prometaphase, some positional information is thought to remain in the cortical cytoplasm after the disappearance of the PPB MTs. Cytoskeletal proteins are known to be excluded from the PPB site during mitosis. These depleted zones of cytoskeletal proteins are potential candidates for a "negative memory" system. However, how these depleted zones of the cytoskeletal proteins are produced remains unknown. In a recent paper, we have quantified the distribution of clathrin-coated pits and vesicles as well as of secretory structures during PPB formation using a combination of high-pressure freezing and electron tomography techniques. Our results demonstrated that the rate of endocytosis is enhanced in PPB regions. We postulate that the removal of membrane proteins by endocytosis plays a role in the creation of PPB "memory" structures.

  8. Dynamic spectrin/ankyrin-G microdomains promote lateral membrane assembly by opposing endocytosis

    PubMed Central

    Jenkins, Paul M.; He, Meng; Bennett, Vann

    2015-01-01

    Current physical models for plasma membranes emphasize dynamic 10- to 300-nm compartments at thermodynamic equilibrium but subject to thermal fluctuations. However, epithelial lateral membranes contain micrometer-sized domains defined by an underlying membrane skeleton composed of spectrin and its partner ankyrin-G. We demonstrate that these spectrin/ankyrin-G domains exhibit local microtubule-dependent movement on a time scale of minutes and encounter most of the lateral membranes within an hour. Spectrin/ankyrin-G domains exclude clathrin and clathrin-dependent cargo, and inhibit both receptor-mediated and bulk endocytosis. Moreover, inhibition of endocytosis fully restores lateral membrane height in spectrin- or ankyrin-G–depleted cells. These findings support a non-equilibrium cellular-scale model for epithelial lateral membranes, where spectrin/ankyrin-G domains actively patrol the plasma membrane, analogous to “window washers,” and promote columnar morphology by blocking membrane uptake. PMID:26523289

  9. Dynamic spectrin/ankyrin-G microdomains promote lateral membrane assembly by opposing endocytosis.

    PubMed

    Jenkins, Paul M; He, Meng; Bennett, Vann

    2015-09-11

    Current physical models for plasma membranes emphasize dynamic 10- to 300-nm compartments at thermodynamic equilibrium but subject to thermal fluctuations. However, epithelial lateral membranes contain micrometer-sized domains defined by an underlying membrane skeleton composed of spectrin and its partner ankyrin-G. We demonstrate that these spectrin/ankyrin-G domains exhibit local microtubule-dependent movement on a time scale of minutes and encounter most of the lateral membranes within an hour. Spectrin/ankyrin-G domains exclude clathrin and clathrin-dependent cargo, and inhibit both receptor-mediated and bulk endocytosis. Moreover, inhibition of endocytosis fully restores lateral membrane height in spectrin- or ankyrin-G-depleted cells. These findings support a non-equilibrium cellular-scale model for epithelial lateral membranes, where spectrin/ankyrin-G domains actively patrol the plasma membrane, analogous to "window washers," and promote columnar morphology by blocking membrane uptake.

  10. Actin-Regulator Feedback Interactions during Endocytosis

    PubMed Central

    Wang, Xinxin; Galletta, Brian J.; Cooper, John A.; Carlsson, Anders E.

    2016-01-01

    Endocytosis mediated by clathrin, a cellular process by which cells internalize membrane receptors and their extracellular ligands, is an important component of cell signaling regulation. Actin polymerization is involved in endocytosis in varying degrees depending on the cellular context. In yeast, clathrin-mediated endocytosis requires a pulse of polymerized actin and its regulators, which recruit and activate the Arp2/3 complex. In this article, we seek to identify the main protein-protein interactions that 1) cause actin and its regulators to appear in pulses, and 2) determine the effects of key mutations and drug treatments on actin and regulator assembly. We perform a joint modeling/experimental study of actin and regulator dynamics during endocytosis in the budding yeast Saccharomyces cerevisiae. We treat both a stochastic model that grows an explicit three-dimensional actin network, and a simpler two-variable Fitzhugh-Nagumo type model. The models include a negative-feedback interaction of F-actin onto the Arp2/3 regulators. Both models explain the pulse time courses and the effects of interventions on actin polymerization: the surprising increase in the peak F-actin count caused by reduced regulator branching activity, the increase in F-actin resulting from slowing of actin disassembly, and the increased Arp2/3 regulator lifetime resulting from latrunculin treatment. In addition, they predict that decreases in the regulator branching activity lead to increases in accumulation of regulators, and we confirmed this prediction with experiments on yeast harboring mutations in the Arp2/3 regulators, using quantitative fluorescence microscopy. Our experimental measurements suggest that the regulators act quasi-independently, in the sense that accumulation of a particular regulator is most strongly affected by mutations of that regulator, as opposed to the others. PMID:27028652

  11. Actin and Endocytosis in Budding Yeast

    PubMed Central

    Goode, Bruce L.; Eskin, Julian A.; Wendland, Beverly

    2015-01-01

    Endocytosis, the process whereby the plasma membrane invaginates to form vesicles, is essential for bringing many substances into the cell and for membrane turnover. The mechanism driving clathrin-mediated endocytosis (CME) involves > 50 different protein components assembling at a single location on the plasma membrane in a temporally ordered and hierarchal pathway. These proteins perform precisely choreographed steps that promote receptor recognition and clustering, membrane remodeling, and force-generating actin-filament assembly and turnover to drive membrane invagination and vesicle scission. Many critical aspects of the CME mechanism are conserved from yeast to mammals and were first elucidated in yeast, demonstrating that it is a powerful system for studying endocytosis. In this review, we describe our current mechanistic understanding of each step in the process of yeast CME, and the essential roles played by actin polymerization at these sites, while providing a historical perspective of how the landscape has changed since the preceding version of the YeastBook was published 17 years ago (1997). Finally, we discuss the key unresolved issues and where future studies might be headed. PMID:25657349

  12. Endocytosis and Signaling: Cell Logistics Shape the Eukaryotic Cell Plan

    PubMed Central

    Sigismund, Sara; Confalonieri, Stefano; Ciliberto, Andrea; Polo, Simona; Scita, Giorgio; Di Fiore, Pier Paolo

    2017-01-01

    Our understanding of endocytosis has evolved remarkably in little more than a decade. This is the result not only of advances in our knowledge of its molecular and biological workings, but also of a true paradigm shift in our understanding of what really constitutes endocytosis and of its role in homeostasis. Although endocytosis was initially discovered and studied as a relatively simple process to transport molecules across the plasma membrane, it was subsequently found to be inextricably linked with almost all aspects of cellular signaling. This led to the notion that endocytosis is actually the master organizer of cellular signaling, providing the cell with understandable messages that have been resolved in space and time. In essence, endocytosis provides the communications and supply routes (the logistics) of the cell. Although this may seem revolutionary, it is still likely to be only a small part of the entire story. A wealth of new evidence is uncovering the surprisingly pervasive nature of endocytosis in essentially all aspects of cellular regulation. In addition, many newly discovered functions of endocytic proteins are not immediately interpretable within the classical view of endocytosis. A possible framework, to rationalize all this new knowledge, requires us to “upgrade” our vision of endocytosis. By combining the analysis of biochemical, biological, and evolutionary evidence, we propose herein that endocytosis constitutes one of the major enabling conditions that in the history of life permitted the development of a higher level of organization, leading to the actuation of the eukaryotic cell plan. PMID:22298658

  13. Cell mobility after endocytosis of carbon nanotubes

    NASA Astrophysics Data System (ADS)

    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.

  14. Erythrocyte membrane skeleton inhibits nanoparticle endocytosis

    NASA Astrophysics Data System (ADS)

    Gao, Xinli; Yue, Tongtao; Tian, Falin; Liu, Zhiping; Zhang, Xianren

    2017-06-01

    Red blood cells (RBCs), also called erythrocytes, have been experimentally proposed in recent decades as the biological drug delivery systems through entrapping certain drugs by endocytosis. However, the internalization pathway of endocytosis seems to conflict with the robust mechanical properties of RBCs that is induced by the spectrin-actin network of erythrocyte membrane skeleton. In this work, we employed a minimum realistic model and the dissipative particle dynamics method to investigate the influence of the spectrin-actin membrane skeleton on the internalization of nanoparticles (NPs). Our simulations show that the existence of skeleton meshwork indeed induces an inhibiting effect that effectively prevents NPs from internalization. The inhibiting effect is found to depend on the membrane-NP attraction, skeleton tension and relative size of the NP to the membrane skeleton mesh. However, our simulations also demonstrate that there are two possibilities for successful internalization of NPs in the presence of the membrane skeleton. The first case is for NPs that has a much smaller size than the dimension of skeleton meshes, and the other is that the skeleton tension is rather weak so that the formed vesicle can still move inward for NP internalization.

  15. Endocytosis of Integrin-Binding Human Picornaviruses

    PubMed Central

    Merilahti, Pirjo; Koskinen, Satu; Heikkilä, Outi; Karelehto, Eveliina; Susi, Petri

    2012-01-01

    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. PMID:23227048

  16. Shape transitions during clathrin-induced endocytosis

    NASA Astrophysics Data System (ADS)

    Kumar, Gaurav; Sain, Anirban

    2016-12-01

    Endocytosis is among the most common transport mechanisms which cells employ to receive macromolecules, the so-called cargo, from its extra cellular environment. Clathrin-mediated endocytosis (CME), in particular, involves the cytoplasmic protein clathrin which induces formation and internalization of clathrin-coated membrane buds that contain extra-cellular cargo. Decades of experimental work have established that the morphology of the clathrin coat evolves with time and induces its curvature on the membrane bud; but energetics of the process remain unclear. Recent experiments by Avinoam et al. [Science 348, 1369 (2015), 10.1126/science.aaa9555] reported that the area of the clathrin coat remains fixed while its curvature increases with time and also the clathrin molecules in the coat turn over rapidly. We show that these observations challenge existing models of coated membrane bud formation. We analyze their data to bring out certain features consistent with the underlying lattice structure of the coat. We hypothesize that membrane curvature inhibits clathrin deposition and propose a kinetic model that explains the area distribution of clathrin coats. We also show that their data on shape evolution of the coated membrane bud can be approximately understood from simple geometric considerations. However, the energetics of the coat formation which controls the kinetics of the process remains a puzzle.

  17. Yeast Exocytic v-SNAREs Confer Endocytosis

    PubMed Central

    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 deficient in their ability to deliver the soluble dye FM4–64 to the vacuole. Under conditions in which vesicles accumulate, FM4–64 stained primarily the cytoplasm as well as fragmented vacuoles. In addition, α-factor–stimulated endocytosis of the α-factor receptor, Ste2, was fully blocked, as evidenced using a Ste2-green fluorescent protein fusion protein as well as metabolic labeling studies. This suggests a direct role for Snc v-SNAREs in the retrieval of membrane proteins from the cell surface. Moreover, this idea is supported by genetic and physical data that demonstrate functional interactions with t-SNAREs that confer endosomal transport (e.g., Tlg1,2). Notably, Snc1ala43 was found to be nonfunctional in cells lacking Tlg1 or Tlg2. Thus, we propose that synaptobrevin/VAMP family members are engaged in anterograde and retrograde protein sorting steps between the Golgi and the plasma membrane. PMID:11029060

  18. Kinetics of virus entry by endocytosis

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2015-04-01

    Entry of virions into the host cells is either endocytotic or fusogenic. In both cases, it occurs via reversible formation of numerous relatively weak bonds resulting in wrapping of a virion by the host membrane with subsequent membrane rupture or scission. The corresponding kinetic models are customarily focused on the formation of bonds and do not pay attention to the energetics of the whole process, which is crucially dependent, especially in the case of endocytosis, on deformation of actin filaments forming the cytoskeleton of the host cell. The kinetic model of endocytosis, proposed by the author, takes this factor into account and shows that the whole process can be divided into a rapid initial transient stage and a long steady-state stage. The entry occurs during the latter stage and can be described as a first-order reaction. Depending on the details of the dependence of the grand canonical potential on the number of bonds, the entry can be limited either by the interplay of bond formation and membrane rupture (or scission) or by reaching a maximum of this potential.

  19. Polarised clathrin-mediated endocytosis of EGFR during chemotactic invasion.

    PubMed

    Mutch, Laura Jane; Howden, Jake Davey; Jenner, Emma Poppy Louise; Poulter, Natalie Sarah; Rappoport, Joshua Zachary

    2014-06-01

    Directed cell migration is critical for numerous physiological processes including development and wound healing. However chemotaxis is also exploited during cancer progression. Recent reports have suggested links between vesicle trafficking pathways and directed cell migration. Very little is known about the potential roles of endocytosis pathways during metastasis. Therefore we performed a series of studies employing a previously characterised model for chemotactic invasion of cancer cells to assess specific hypotheses potentially linking endocytosis to directed cell migration. Our results demonstrate that clathrin-mediated endocytosis is indispensable for epidermal growth factor (EGF) directed chemotactic invasion of MDA-MB-231 cells. Conversely, caveolar endocytosis is not required in this mode of migration. We further found that chemoattractant receptor (EGFR) trafficking occurs by clathrin-mediated endocytosis and is polarised towards the front of migrating cells. However, we found no role for clathrin-mediated endocytosis in focal adhesion disassembly in this migration model. Thus, this study has characterised the role of endocytosis during chemotactic invasion and has identified functions mechanistically linking clathrin-mediated endocytosis to directed cell motility.

  20. Clathrin-mediated endocytosis is inhibited during mitosis.

    PubMed

    Fielding, Andrew B; Willox, Anna K; Okeke, Emmanuel; Royle, Stephen J

    2012-04-24

    A long-standing paradigm in cell biology is the shutdown of endocytosis during mitosis. There is consensus that transferrin uptake is inhibited after entry into prophase and that it resumes in telophase. A recent study proposed that endocytosis is continuous throughout the cell cycle and that the observed inhibition of transferrin uptake is due to a decrease in available transferrin receptor at the cell surface, and not to a shutdown of endocytosis. This challenge to the established view is gradually becoming accepted. Because of this controversy, we revisited the question of endocytic activity during mitosis. Using an antibody uptake assay and controlling for potential changes in surface receptor density, we demonstrate the strong inhibition of endocytosis in mitosis of CD8 chimeras containing any of the three major internalization motifs for clathrin-mediated endocytosis (YXXΦ, [DE]XXXL[LI], or FXNPXY) or a CD8 protein with the cytoplasmic tail of the cation-independent mannose 6-phosphate receptor. The shutdown is not gradual: We describe a binary switch from endocytosis being "on" in interphase to "off" in mitosis as cells traverse the G(2)/M checkpoint. In addition, we show that the inhibition of transferrin uptake in mitosis occurs despite abundant transferrin receptor at the surface of HeLa cells. Our study finds no support for the recent idea that endocytosis continues during mitosis, and we conclude that endocytosis is temporarily shutdown during early mitosis.

  1. Clathrin-mediated endocytosis is inhibited during mitosis

    PubMed Central

    Fielding, Andrew B.; Willox, Anna K.; Okeke, Emmanuel; Royle, Stephen J.

    2012-01-01

    A long-standing paradigm in cell biology is the shutdown of endocytosis during mitosis. There is consensus that transferrin uptake is inhibited after entry into prophase and that it resumes in telophase. A recent study proposed that endocytosis is continuous throughout the cell cycle and that the observed inhibition of transferrin uptake is due to a decrease in available transferrin receptor at the cell surface, and not to a shutdown of endocytosis. This challenge to the established view is gradually becoming accepted. Because of this controversy, we revisited the question of endocytic activity during mitosis. Using an antibody uptake assay and controlling for potential changes in surface receptor density, we demonstrate the strong inhibition of endocytosis in mitosis of CD8 chimeras containing any of the three major internalization motifs for clathrin-mediated endocytosis (YXXΦ, [DE]XXXL[LI], or FXNPXY) or a CD8 protein with the cytoplasmic tail of the cation-independent mannose 6-phosphate receptor. The shutdown is not gradual: We describe a binary switch from endocytosis being “on” in interphase to “off” in mitosis as cells traverse the G2/M checkpoint. In addition, we show that the inhibition of transferrin uptake in mitosis occurs despite abundant transferrin receptor at the surface of HeLa cells. Our study finds no support for the recent idea that endocytosis continues during mitosis, and we conclude that endocytosis is temporarily shutdown during early mitosis. PMID:22493256

  2. Polarised Clathrin-Mediated Endocytosis of EGFR During Chemotactic Invasion

    PubMed Central

    Mutch, Laura Jane; Howden, Jake Davey; Jenner, Emma Poppy Louise; Poulter, Natalie Sarah; Rappoport, Joshua Zachary

    2014-01-01

    Directed cell migration is critical for numerous physiological processes including development and wound healing. However chemotaxis is also exploited during cancer progression. Recent reports have suggested links between vesicle trafficking pathways and directed cell migration. Very little is known about the potential roles of endocytosis pathways during metastasis. Therefore we performed a series of studies employing a previously characterised model for chemotactic invasion of cancer cells to assess specific hypotheses potentially linking endocytosis to directed cell migration. Our results demonstrate that clathrin-mediated endocytosis is indispensable for epidermal growth factor (EGF) directed chemotactic invasion of MDA-MB-231 cells. Conversely, caveolar endocytosis is not required in this mode of migration. We further found that chemoattractant receptor (EGFR) trafficking occurs by clathrin-mediated endocytosis and is polarised towards the front of migrating cells. However, we found no role for clathrin-mediated endocytosis in focal adhesion disassembly in this migration model. Thus, this study has characterised the role of endocytosis during chemotactic invasion and has identified functions mechanistically linking clathrin-mediated endocytosis to directed cell motility. PMID:24921075

  3. Role for Phospholipase D in Receptor-Mediated Endocytosis

    PubMed Central

    Shen, Yingjie; Xu, Lizhong; Foster, David A.

    2001-01-01

    In response to epidermal growth factor (EGF), the EGF receptor is endocytosed and degraded. A substantial lag period exists between endocytosis and degradation, suggesting that endocytosis is more than a simple negative feedback. Phospholipase D (PLD), which has been implicated in vesicle formation in the Golgi, is activated in response to EGF and other growth factors. We report here that EGF receptor endocytosis is dependent upon PLD and the PLD1 regulators, protein kinase C α and RalA. EGF-induced receptor degradation is accelerated by overexpression of either wild-type PLD1 or PLD2 and retarded by overexpression of catalytically inactive mutants of either PLD1 or PLD2. EGF-induced activation of mitogen-activated protein kinase, which is dependent upon receptor endocytosis, is also dependent upon PLD. These data suggest a role for PLD in signaling that facilitates receptor endocytosis. PMID:11134345

  4. Clathrin-mediated endocytosis persists during unperturbed mitosis.

    PubMed

    Tacheva-Grigorova, Silvia K; Santos, António J M; Boucrot, Emmanuel; Kirchhausen, Tom

    2013-08-29

    How does mitosis influence the critical process of endocytosis? Some experiments lead to the conclusion that endocytosis arrests completely during mitosis, whereas others indicate that endocytosis persists. We have resolved this apparent discrepancy by showing how conditions of the experiment influence its outcome. The dynamics of clathrin-coated pit formation and the uptake of transferrin are maintained in naturally dividing cells but are nearly absent in mitotic cells arrested chemically by treatment with nocodazole, S-Trityl-L-cysteine, or RO-3306. Moreover, sequentially incubating cells at 4°C and then shifting them to 37°C or to serum starvation artificially increases the amount of transferrin receptor at the surface of naturally dividing cells, leading to the incorrect conclusion that endocytosis has ceased during mitosis. Thus, our data show that endocytosis is unaffected during all stages of natural cell division. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Endocytosis-like protein uptake in the bacterium Gemmata obscuriglobus.

    PubMed

    Lonhienne, Thierry G A; Sagulenko, Evgeny; Webb, Richard I; Lee, Kuo-Chang; Franke, Josef; Devos, Damien P; Nouwens, Amanda; Carroll, Bernard J; Fuerst, John A

    2010-07-20

    Endocytosis is a process by which extracellular material such as macromolecules can be incorporated into cells via a membrane-trafficking system. Although universal among eukaryotes, endocytosis has not been identified in Bacteria or Archaea. However, intracellular membranes are known to compartmentalize cells of bacteria in the phylum Planctomycetes, suggesting the potential for endocytosis and membrane trafficking in members of this phylum. Here we show that cells of the planctomycete Gemmata obscuriglobus have the ability to uptake proteins present in the external milieu in an energy-dependent process analogous to eukaryotic endocytosis, and that internalized proteins are associated with vesicle membranes. Occurrence of such ability in a bacterium is consistent with autogenous evolution of endocytosis and the endomembrane system in an ancestral noneukaryote cell.

  6. Endocytosis-like protein uptake in the bacterium Gemmata obscuriglobus

    PubMed Central

    Lonhienne, Thierry G. A.; Sagulenko, Evgeny; Webb, Richard I.; Lee, Kuo-Chang; Franke, Josef; Devos, Damien P.; Nouwens, Amanda; Carroll, Bernard J.; Fuerst, John A.

    2010-01-01

    Endocytosis is a process by which extracellular material such as macromolecules can be incorporated into cells via a membrane-trafficking system. Although universal among eukaryotes, endocytosis has not been identified in Bacteria or Archaea. However, intracellular membranes are known to compartmentalize cells of bacteria in the phylum Planctomycetes, suggesting the potential for endocytosis and membrane trafficking in members of this phylum. Here we show that cells of the planctomycete Gemmata obscuriglobus have the ability to uptake proteins present in the external milieu in an energy-dependent process analogous to eukaryotic endocytosis, and that internalized proteins are associated with vesicle membranes. Occurrence of such ability in a bacterium is consistent with autogenous evolution of endocytosis and the endomembrane system in an ancestral noneukaryote cell. PMID:20566852

  7. Receptor-mediated Endocytosis in the Caenorhabditis elegans Oocyte

    PubMed Central

    Grant, Barth; Hirsh, David

    1999-01-01

    The Caenorhabditis elegans oocyte is a highly amenable system for forward and reverse genetic analysis of receptor-mediated endocytosis. We describe the use of transgenic strains expressing a vitellogenin::green fluorescent protein (YP170::GFP) fusion to monitor yolk endocytosis by the C. elegans oocyte in vivo. This YP170::GFP reporter was used to assay the functions of C. elegans predicted proteins homologous to vertebrate endocytosis factors using RNA-mediated interference. We show that the basic components and pathways of endocytic trafficking are conserved between C. elegans and vertebrates, and that this system can be used to test the endocytic functions of any new gene. We also used the YP170::GFP assay to identify rme (receptor-mediated endocytosis) mutants. We describe a new member of the low-density lipoprotein receptor superfamily, RME-2, identified in our screens for endocytosis defective mutants. We show that RME-2 is the C. elegans yolk receptor. PMID:10588660

  8. Bulk undercooling

    NASA Technical Reports Server (NTRS)

    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.

  9. Systematic analysis of endocytosis by cellular perturbations.

    PubMed

    Kühling, Lena; Schelhaas, Mario

    2014-01-01

    Endocytosis is an essential process of eukaryotic cells that facilitates numerous cellular and organismal functions. The formation of vesicles from the plasma membrane serves the internalization of ligands and receptors and leads to their degradation or recycling. A number of distinct mechanisms have been described over the years, several of which are only partially characterized in terms of mechanism and function. These are often referred to as novel endocytic pathways. The pathways differ in their mode of uptake and in their intracellular destination. Here, an overview of the set of cellular proteins that facilitate the different pathways is provided. Further, the approaches to distinguish between the pathways by different modes of perturbation are critically discussed, emphasizing the use of genetic tools such as dominant negative mutant proteins.

  10. Physical Principles of Nanoparticle Cellular Endocytosis.

    PubMed

    Zhang, Sulin; Gao, Huajian; Bao, Gang

    2015-09-22

    This review article focuses on the physiochemical mechanisms underlying nanoparticle uptake into cells. When nanoparticles are in close vicinity to a cell, the interactions between the nanoparticles and the cell membrane generate forces from different origins. This leads to the membrane wrapping of the nanoparticles followed by cellular uptake. This article discusses how the kinetics, energetics, and forces are related to these interactions and dependent on the size, shape, and stiffness of nanoparticles, the biomechanical properties of the cell membrane, as well as the local environment of the cells. The discussed fundamental principles of the physiochemical causes for nanoparticle-cell interaction may guide new studies of nanoparticle endocytosis and lead to better strategies to design nanoparticle-based approaches for biomedical applications.

  11. Multifactorial Regulation of G Protein-Coupled Receptor Endocytosis

    PubMed Central

    Zhang, Xiaohan; Kim, Kyeong-Man

    2017-01-01

    Endocytosis is a process by which cells absorb extracellular materials via the inward budding of vesicles formed from the plasma membrane. Receptor-mediated endocytosis is a highly selective process where receptors with specific binding sites for extracellular molecules internalize via vesicles. G protein-coupled receptors (GPCRs) are the largest single family of plasma-membrane receptors with more than 1000 family members. But the molecular mechanisms involved in the regulation of GPCRs are believed to be highly conserved. For example, receptor phosphorylation in collaboration with β-arrestins plays major roles in desensitization and endocytosis of most GPCRs. Nevertheless, a number of subsequent studies showed that GPCR regulation, such as that by endocytosis, occurs through various pathways with a multitude of cellular components and processes. This review focused on i) functional interactions between homologous and heterologous pathways, ii) methodologies applied for determining receptor endocytosis, iii) experimental tools to determine specific endocytic routes, iv) roles of small guanosine triphosphate-binding proteins in GPCR endocytosis, and v) role of post-translational modification of the receptors in endocytosis. PMID:28035080

  12. Kainate-induced endocytosis in retinal amacrine cells.

    PubMed

    Borsello, Tiziana; Bressoud, Raymond; Mottier, Vincent; González, Nicolas; Gomez, Gabriel; Clarke, Peter G H

    2003-10-13

    Endocytosis is enhanced in some cases of neuronal death. We report for the first time that intraocular injections, in chick embryos, of excitotoxic doses of kainate induce strong endocytosis in retinal amacrine cells destined to die and that even subtoxic doses can induce some degree of endocytosis. That the uptake was due to endocytosis rather than passive diffusion through the plasma membrane was shown ultrastructurally. The endocytosis was demonstrated by using three unrelated tracers--horseradish peroxidase, microperoxidase, and 4.4-kDa fluorescein isothiocyanate (FITC)-labeled dextran--suggesting that it does not depend on the binding of the tracers to a particular receptor. However, it appears to be surprisingly sensitive to the size of the ligand, because a heavier (42-kDa) FITC-dextran was not endocytosed. The induction of endocytosis by kainate can occur even when protein synthesis is blocked. These results indicate that toxic or near-toxic doses of kainate induce endocytosis, raising the question of whether this is mechanistically implicated in causing or preventing excitotoxic neuronal death. Copyright 2003 Wiley-Liss, Inc.

  13. Synthetic antigens reveal dynamics of BCR endocytosis during inhibitory signaling.

    PubMed

    Courtney, Adam H; Bennett, Nitasha R; Zwick, Daniel B; Hudon, Jonathan; Kiessling, Laura L

    2014-01-17

    B cells detect foreign antigens through their B cell antigen receptor (BCR). The BCR, when engaged by antigen, initiates a signaling cascade. Concurrent with signaling is endocytosis of the BCR complex, which acts to downregulate signaling and facilitate uptake of antigen for processing and display on the cell surface. The relationship between signaling and BCR endocytosis is poorly defined. Here, we explore the interplay between BCR endocytosis and antigens that either promote or inhibit B cell activation. Specifically, synthetic antigens were generated that engage the BCR alone or both the BCR and the inhibitory co-receptor CD22. The lectin CD22, a member of the Siglec family, binds sialic acid-containing glycoconjugates found on host tissues, inhibiting BCR signaling to prevent erroneous B cell activation. At low concentrations, antigens that can cocluster the BCR and CD22 promote rapid BCR endocytosis; whereas, slower endocytosis occurs with antigens that bind only the BCR. At higher antigen concentrations, rapid BCR endocytosis occurs upon treatment with either stimulatory or inhibitory antigens. Endocytosis of the BCR, in response to synthetic antigens, results in its entry into early endocytic compartments. Although the CD22-binding antigens fail to activate key regulators of antigen presentation (e.g., Syk), they also promote BCR endocytosis, indicating that inhibitory antigens can be internalized. Together, our observations support a functional role for BCR endocytosis in downregulating BCR signaling. The reduction of cell surface BCR levels in the absence of B cell activation should raise the threshold for BCR subsequent activation. The ability of the activating synthetic antigens to trigger both signaling and entry of the BCR into early endosomes suggests strategies for targeted antigen delivery.

  14. [Lipids in the process of synaptic vesicle exo- and endocytosis].

    PubMed

    Zefirov, A L; Petrov, A M

    2010-08-01

    The phenomenon of synaptic transmission is based on the processes of synaptic vesicle exo- and endocytosis carried out with complex protein-dependent mechanisms. The SNARE-complex forming proteins (synaptobrevin, syntaxin, SNAP-25), synaptotagmin, Munc13, Munc18, NSF, alpha-SNAP are involved in exocytosis, while the synaptic vesicle endocytosis is mediated by another protein (clathrin, AP-2, epsin, endophilin, amphiphysin, dynamin, synaptojanin, Hsc70). In recent years, data on critical role of various lipids in exo- and encocytosis are collected. Most interesting results are received about significance of the cholesterol, phosphoinositides, phosphatidic and polynonsaturated fat acids in the exo-endocytosis cycle. Participation of lipid rafts in synaptic vesicle recycling is discussed. In this article, the data of the last years, including the authors' own data about role of some lipids and lipid-modifying enzimes in processes of exo- and endocytosis are presented.

  15. Synucleins regulate the kinetics of synaptic vesicle endocytosis.

    PubMed

    Vargas, Karina J; Makani, Sachin; Davis, Taylor; Westphal, Christopher H; Castillo, Pablo E; Chandra, Sreeganga S

    2014-07-09

    Genetic and pathological studies link α-synuclein to the etiology of Parkinson's disease (PD), but the normal function of this presynaptic protein remains unknown. α-Synuclein, an acidic lipid binding protein, shares high sequence identity with β- and γ-synuclein. Previous studies have implicated synucleins in synaptic vesicle (SV) trafficking, although the precise site of synuclein action continues to be unclear. Here we show, using optical imaging, electron microscopy, and slice electrophysiology, that synucleins are required for the fast kinetics of SV endocytosis. Slowed endocytosis observed in synuclein null cultures can be rescued by individually expressing mouse α-, β-, or γ-synuclein, indicating they are functionally redundant. Through comparisons to dynamin knock-out synapses and biochemical experiments, we suggest that synucleins act at early steps of SV endocytosis. Our results categorize α-synuclein with other familial PD genes known to regulate SV endocytosis, implicating this pathway in PD.

  16. Activity-dependent regulation of NMDA receptors in substantia nigra dopaminergic neurones

    PubMed Central

    Wild, Angela R; Jones, Susan; Gibb, Alasdair J

    2014-01-01

    N-Methyl-d-aspartate receptors (NMDARs) are Ca2+-permeable glutamate receptors that play a critical role in synaptic plasticity and promoting cell survival. However, overactive NMDARs can trigger cell death signalling pathways and have been implicated in substantia nigra pars compacta (SNc) pathology in Parkinson's disease. Calcium ion influx through NMDARs recruits Ca2+-dependent proteins that can regulate NMDAR activity. The surface density of NMDARs can also be regulated dynamically in response to receptor activity via Ca2+-independent mechanisms. We have investigated the activity-dependent regulation of NMDARs in SNc dopaminergic neurones. Repeated whole-cell agonist applications resulted in a decline in the amplitude of NMDAR currents (current run-down) that was use dependent and not readily reversible. Run-down was reduced by increasing intracellular Ca2+ buffering or by reducing Ca2+ influx but did not appear to be mediated by the same regulatory proteins that cause Ca2+-dependent run-down in hippocampal neurones. The NMDAR current run-down may be mediated in part by a Ca2+-independent mechanism, because intracellular dialysis with a dynamin-inhibitory peptide reduced run-down, suggesting a role for clathrin-mediated endocytosis in the regulation of the surface density of receptors. Synaptic NMDARs were also subject to current run-down during repeated low-frequency synaptic stimulation in a Ca2+-dependent but dynamin-independent manner. Thus, we report, for the first time, regulation of NMDARs in SNc dopaminergic neurones by changes in intracellular Ca2+ at both synaptic and extrasynaptic sites and provide evidence for activity-dependent changes in receptor trafficking. These mechanisms may contribute to intracellular Ca2+ homeostasis in dopaminergic neurones by limiting Ca2+ influx through the NMDAR. PMID:24344168

  17. Calcyon is necessary for activity-dependent AMPA receptor internalization and LTD in CA1 neurons of hippocampus.

    PubMed

    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 the 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 alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), without altering basal surface levels of the GluR1 or GluR2 subunits. Whole-cell patch-clamp studies of hippocampal neurons in culture and CA1 synapses in slices revealed that knockout (KO) of calcyon abolishes long-term synaptic depression (LTD), whereas mini-analysis in slices indicated basal transmission in the hippocampus is unaffected by the deletion. Further, transfection of green fluorescent protein-tagged calcyon rescued the ability of KO cultures to undergo LTD. In contrast, intracellular dialysis of a fusion protein containing the clathrin light-chain-binding domain of calcyon blocked the induction of LTD in wild-type hippocampal slices. Taken together, the present studies involving biochemical, immunological and electrophysiological analyses raise the possibility that calcyon plays a specialized role in regulating activity-dependent removal of synaptic AMPARs.

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

    PubMed Central

    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 neurons in culture and CA1 synapses in slices revealed that knockout of calcyon abolishes long term synaptic depression (LTD) whereas mini-analysis in slices indicated basal transmission in hippocampus is unaffected by the deletion. Further, transfection of GFP-tagged calcyon rescued the ability of knockout cultures to undergo LTD. In contrast, intracellular dialysis of a fusion protein containing the clathrin light chain binding domain of calcyon blocked the induction of LTD in wild type hippocampal slices. Taken together, the present studies involving biochemical, immunological and electrophysiological analyses raise the possibility that calcyon plays a specialized role in regulating activity-dependent removal of synaptic AMPA receptors. PMID:19120439

  19. Mechanics of receptor-mediated endocytosis

    NASA Astrophysics Data System (ADS)

    Gao, Huajian; Shi, Wendong; Freund, Lambert B.

    2005-07-01

    Most viruses and bioparticles endocytosed by cells have characteristic sizes in the range of tens to hundreds of nanometers. The process of viruses entering and leaving animal cells is mediated by the binding interaction between ligand molecules on the viral capid and their receptor molecules on the cell membrane. How does the size of a bioparticle affect receptor-mediated endocytosis? Here, we study how a cell membrane containing diffusive mobile receptors wraps around a ligand-coated cylindrical or spherical particle. It is shown that particles in the size range of tens to hundreds of nanometers can enter or exit cells via wrapping even in the absence of clathrin or caveolin coats, and an optimal particles size exists for the smallest wrapping time. This model can also be extended to include the effect of clathrin coat. The results seem to show broad agreement with experimental observations. Author contributions: H.G. and L.B.F. designed research; H.G., W.S., and L.B.F. performed research; and H.G., W.S., and L.B.F. wrote the paper.Abbreviations: CNT, carbon nanotube; SWNT, single-walled nanotube.

  20. Effects of mutant rat dynamin on endocytosis

    PubMed Central

    1993-01-01

    Dynamin is a 100-kD microtubule-activated GTPase. Recent evidence has revealed a high degree of sequence homology with the product of the Drosophila gene shibire, mutations in which block the recycling of synaptic vesicles and, more generally, the formation of coated and non- coated vesicles at the plasma membrane. We have now transfected cultured mammalian COS-7 cells with both wild-type and mutant dynamin cDNAs. Point mutations in the GTP-binding consensus sequence elements of dynamin equivalent to dominant negative mutations in ras, and an NH2- terminal deletion of the entire GTP-binding domain of dynamin, block transferrin uptake and alter the distribution of clathrin heavy chain and alpha-, but not gamma-, adaptin. COOH-terminal deletions reverse these effects, identifying this portion of dynamin as a site of interaction with other components of the endocytic pathway. Over- expression of neither wild-type nor mutant forms of dynamin affected the distribution of microtubules. These results demonstrate a specific role for dynamin and for GTP in the initial stages of receptor-mediated endocytosis. PMID:8335685

  1. Ankyrin-G Inhibits Endocytosis of Cadherin Dimers*

    PubMed Central

    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

  2. Vesicle Formation and Endocytosis: Function, Machinery, Mechanisms, and Modeling

    PubMed Central

    Parkar, Nihal S.; Akpa, Belinda S.; Nitsche, Ludwig C.; Wedgewood, Lewis E.; Place, Aaron T.; Sverdlov, Maria S.; Chaga, Oleg

    2009-01-01

    Abstract Vesicle formation provides a means of cellular entry for extracellular substances and for recycling of membrane constituents. Mechanisms governing the two primary endocytic pathways (i.e., caveolae- and clathrin-mediated endocytosis, as well as newly emerging vesicular pathways) have become the focus of intense investigation to improve our understanding of nutrient, hormone, and drug delivery, as well as opportunistic invasion of pathogens. In this review of endocytosis, we broadly discuss the structural and signaling proteins that compose the molecular machinery governing endocytic vesicle formation (budding, invagination, and fission from the membrane), with some regard for the specificity observed in certain cell types and species. Important biochemical functions of endocytosis and diseases caused by their disruption also are discussed, along with the structures of key components of endocytic pathways and their known mechanistic contributions. The mechanisms by which principal components of the endocytic machinery are recruited to the plasma membrane, where they interact to induce vesicle formation, are discussed, together with computational approaches used to simulate simplified versions of endocytosis with the hope of clarifying aspects of vesicle formation that may be difficult to determine experimentally. Finally, we pose several unanswered questions intended to stimulate further research interest in the cell biology and modeling of endocytosis. Antioxid. Redox Signal. 11, 1301–1312. PMID:19113823

  3. Intracellular Trafficking Network of Protein Nanocapsules: Endocytosis, Exocytosis and Autophagy

    PubMed Central

    Zhang, Jinxie; Zhang, Xudong; Liu, Gan; Chang, Danfeng; Liang, Xin; Zhu, Xianbing; Tao, Wei; Mei, Lin

    2016-01-01

    The inner membrane vesicle system is a complex transport system that includes endocytosis, exocytosis and autophagy. However, the details of the intracellular trafficking pathway of nanoparticles in cells have been poorly investigated. Here, we investigate in detail the intracellular trafficking pathway of protein nanocapsules using more than 30 Rab proteins as markers of multiple trafficking vesicles in endocytosis, exocytosis and autophagy. We observed that FITC-labeled protein nanoparticles were internalized by the cells mainly through Arf6-dependent endocytosis and Rab34-mediated micropinocytosis. In addition to this classic pathway: early endosome (EEs)/late endosome (LEs) to lysosome, we identified two novel transport pathways: micropinocytosis (Rab34 positive)-LEs (Rab7 positive)-lysosome pathway and EEs-liposome (Rab18 positive)-lysosome pathway. Moreover, the cells use slow endocytosis recycling pathway (Rab11 and Rab35 positive vesicles) and GLUT4 exocytosis vesicles (Rab8 and Rab10 positive) transport the protein nanocapsules out of the cells. In addition, protein nanoparticles are observed in autophagosomes, which receive protein nanocapsules through multiple endocytosis vesicles. Using autophagy inhibitor to block these transport pathways could prevent the degradation of nanoparticles through lysosomes. Using Rab proteins as vesicle markers to investigation the detail intracellular trafficking of the protein nanocapsules, will provide new targets to interfere the cellular behaver of the nanoparticles, and improve the therapeutic effect of nanomedicine. PMID:27698943

  4. Ankyrin-G Inhibits Endocytosis of Cadherin Dimers.

    PubMed

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

    2016-01-08

    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.

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

    PubMed Central

    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 dim2-1, revealed defects in the endocytic pathway. Like other yeast endocytosis mutants, the temperature-sensitive dim mutant were unable to endocytose FM4-64 or radiolabeled alpha-factor as efficiently as wild-type cells. In addition, double mutants with either dim1-delta or dim2-1 and the endocytosis mutants end4-1 or act1-1 displayed synthetic growth defects, indicating that the DIM gene products function in a common or parallel endocytic pathway. Complementation cloning of the DIM genes revealed identity of DIM1 to SHE4 and DIM2 to PAN1. Pan1p shares homology with the mammalian clathrin adaptor-associated protein, eps15. Both proteins contain multiple EH (eps15 homology) domains, a motif proposed to mediate protein-protein interactions. Phalloidin labeling of filamentous actin revealed profound defects in the actin cytoskeleton in both dim mutants. EM analysis revealed that the dim mutants accumulate vesicles and tubulo-vesicular structures reminiscent of mammalian early endosomes. In addition, the accumulation of novel plasma membrane invaginations where endocytosis is likely to occur were visualized in the mutants by electron microscopy using cationized ferritin as a marker for the endocytic pathway. This new screening strategy demonstrates a role for She4p and Pan1p in endocytosis, and provides a new general method for the identification of additional endocytosis mutants. PMID:8978817

  6. Dextrans as markers for endocytosis in innervated and denervated skeletal muscle.

    PubMed

    Elmquist, S; Libelius, R; Lawoko, G; Tågerud, S

    1992-08-01

    Fluorescence-labeled dextrans were evaluated as markers for endocytosis in skeletal muscle. Fluorescein isothiocyanate (FITC)-labeled dextrans (average molecular weight 3900 to 71200) showed a higher uptake in denervated than in innervated muscle both in vitro and in vivo. The in vitro uptake of FITC-dextran (35.600) increased linearly with time at 37 degrees C, and was almost completely inhibited by low temperature (4 degrees C). The uptake was not a pure bulk uptake, because a saturable component was evident from the concentration dependence and from competition experiments with unlabeled dextran. The uptake of FITC-labeled or rhodamine B isothiocyanate (RITC)-labeled dextrans in denervated muscle occurred mainly in small segments of the fibers centered around the denervated endplate region. However, not all denervated fibers showed such segments. Periodic acid Schiff's base staining for carbohydrates stained dextrans in denervated muscle fibers. Some staining, probably of lysosomes, was also observed in denervated muscle not exposed to dextran.

  7. Quantitative analysis of endocytosis with cytoplasmic pHluorin chimeras.

    PubMed

    Prosser, Derek C; Whitworth, Karen; Wendland, Beverly

    2010-09-01

    The pH-sensitive green fluorescent protein (GFP) variant pHluorin is typically fused to the extracellular domain of transmembrane proteins to monitor endocytosis. Here, we have turned pHluorin inside-out, and show that cytoplasmic fusions of pHluorin are effective quantitative reporters for endocytosis and multivesicular body (MVB) sorting. In yeast in particular, fusion of GFP and its variants on the extracellular side of transmembrane proteins can result in perturbed trafficking. In contrast, cytoplasmic fusions are well tolerated, allowing for the quantitative assessment of trafficking of virtually any transmembrane protein. Quenching of degradation-resistant pHluorin in the acidic vacuole permits quantification of extravacuolar cargo proteins at steady-state levels and is compatible with kinetic analysis of endocytosis in live cells.

  8. Endocytosis and exocytosis of nanoparticles in mammalian cells

    PubMed Central

    Oh, Nuri; Park, Ji-Ho

    2014-01-01

    Engineered nanoparticles that can be injected into the human body hold tremendous potential to detect and treat complex diseases. Understanding of the endocytosis and exocytosis mechanisms of nanoparticles is essential for safe and efficient therapeutic application. In particular, exocytosis is of significance in the removal of nanoparticles with drugs and contrast agents from the body, while endocytosis is of great importance for the targeting of nanoparticles in disease sites. Here, we review the recent research on the endocytosis and exocytosis of functionalized nanoparticles based on various sizes, shapes, and surface chemistries. We believe that this review contributes to the design of safe nanoparticles that can efficiently enter and leave human cells and tissues. PMID:24872703

  9. Multiscale Modeling of Virus Entry via Receptor-Mediated Endocytosis

    NASA Astrophysics Data System (ADS)

    Liu, Jin

    2012-11-01

    Virus infections are ubiquitous and remain major threats to human health worldwide. Viruses are intracellular parasites and must enter host cells to initiate infection. Receptor-mediated endocytosis is the most common entry pathway taken by viruses, the whole process is highly complex and dictated by various events, such as virus motions, membrane deformations, receptor diffusion and ligand-receptor reactions, occurring at multiple length and time scales. We develop a multiscale model for virus entry through receptor-mediated endocytosis. The binding of virus to cell surface is based on a mesoscale three dimensional stochastic adhesion model, the internalization (endocytosis) of virus and cellular membrane deformation is based on the discretization of Helfrich Hamiltonian in a curvilinear space using Monte Carlo method. The multiscale model is based on the combination of these two models. We will implement this model to study the herpes simplex virus entry into B78 cells and compare the model predictions with experimental measurements.

  10. Synapse Maturation by Activity-Dependent Ectodomain Shedding of SIRPα

    PubMed Central

    Toth, Anna B.; Terauchi, Akiko; Zhang, Lily Y.; Johnson-Venkatesh, Erin M.; Larsen, David J.; Sutton, Michael A.; Umemori, Hisashi

    2013-01-01

    Formation of appropriate synaptic connections is critical for proper functioning of the brain. After initial synaptic differentiation, active synapses are stabilized by neural activity-dependent signals to establish functional synaptic connections. However, the molecular mechanisms underlying activity-dependent synapse maturation remain to be elucidated. Here we show that activity-dependent ectodomain shedding of SIRPα mediates presynaptic maturation. Two target-derived molecules, FGF22 and SIRPα, sequentially organize the glutamatergic presynaptic terminals during the initial synaptic differentiation and synapse maturation stages, respectively, in the mouse hippocampus. SIRPα drives presynaptic maturation in an activity-dependent fashion. Remarkably, neural activity cleaves the extracellular domain of SIRPα, and the shed ectodomain, in turn, promotes the maturation of the presynaptic terminal. This process involves CaM kinase, matrix metalloproteinases, and the presynaptic receptor CD47. Finally, SIRPα-dependent synapse maturation has significant impacts on synaptic function and plasticity. Thus, ectodomain shedding of SIRPα is an activity-dependent trans-synaptic mechanism for the maturation of functional synapses. PMID:24036914

  11. Clathrin-independent Endocytosis: A cargo-centric view

    PubMed Central

    Maldonado-Báez, Lymarie; Williamson, Chad; Donaldson, Julie G.

    2014-01-01

    Clathrin-independent endocytosis occurs in all cells and interest in this mode of cellular entry has grown. Although this form of endocytosis was first described for entry of bacterial toxins, here we focus our attention on the endogenous cell surface “cargo” proteins that enter cells by this mechanism. The cargo proteins entering by this mechanism are varied and include nutrient transporters, ion channels, cell adhesion molecules and proteins associated with the immune system. Despite the apparent lack of selection at the cell surface, we provide some examples of specific sorting of these cargo proteins after entry, leading to distinct itineraries and cellular fates. PMID:23954817

  12. The immunological synapse: a focal point for endocytosis and exocytosis

    PubMed Central

    Tsun, Andy; Stinchcombe, Jane C.

    2010-01-01

    There are many different cells in the immune system. To mount an effective immune response, they need to communicate with each other. One way in which this is done is by the formation of immunological synapses between cells. Recent developments show that the immune synapse serves as a focal point for exocytosis and endocytosis, directed by centrosomal docking at the plasma membrane. In this respect, formation of the immunological synapse bears striking similarities to cilia formation and cytokinesis. These intriguing observations suggest that the centrosome may play a conserved role in designating a specialized area of membrane for localized endocytosis and exocytosis. PMID:20439993

  13. Cdk5 and the mystery of synaptic vesicle endocytosis.

    PubMed

    Nguyen, Chan; Bibb, James A

    2003-11-24

    Regulation of endocytosis by protein phosphorylation and dephosphorylation is critical to synaptic vesicle recycling. Two groups have now identified the neuronal kinase Cdk5 (cyclin-dependent kinase 5) as an important regulator of this process. Robinson and coworkers recently demonstrated that Cdk5 is necessary for synaptic vesicle endocytosis (SVE) (Tan et al., 2003), whereas a new report in this issue claims that Cdk5 negatively regulates SVE (Tomizawa et al., 2003). Careful examination of the data reveals a model that helps resolve the apparently contradictory nature of these reports.

  14. Fluid-phase endocytosis in yeasts other than Saccharomyces cerevisiae.

    PubMed

    Fernandez, N; Puente, P; Leal, F

    1990-05-01

    A FITC-dextran internalization assay with Saccharomyces cerevisiae as positive control was used to determine whether fluid-phase endocytosis is a general characteristic of yeasts. Schizosaccharomyces pombe, Pichia polymorpha, Kluyveromyces phaseolosporus, Yarrowia lipolytica and Candida albicans were clearly positive, whereas results obtained with Debaryomyces marama were inconclusive. In all cases internalized FITC-dextran was found to be localized in the vacuoles and the process was always time- and temperature-dependent. Lower eucaryotes, particularly yeasts, appear to have the ability to incorporate substances from the extracellular medium through fluid-phase endocytosis.

  15. Protein Kinase C-dependent Ubiquitination and Clathrin-mediated Endocytosis of the Cationic Amino Acid Transporter CAT-1*

    PubMed Central

    Vina-Vilaseca, Arnau; Bender-Sigel, Julia; Sorkina, Tatiana; Closs, Ellen Ildicho; Sorkin, Alexander

    2011-01-01

    Cationic amino acid transporter 1 (CAT-1) is responsible for the bulk of the uptake of cationic amino acids in most mammalian cells. Activation of protein kinase C (PKC) leads to down-regulation of the cell surface CAT-1. To examine the mechanisms of PKC-induced down-regulation of CAT-1, a functional mutant of CAT-1 (CAT-1-HA-GFP) was generated in which a hemagglutinin antigen (HA) epitope tag was introduced into the second extracellular loop and GFP was attached to the carboxyl terminus. CAT-1-HA-GFP was stably expressed in porcine aorthic endothelial and human epithelial kidney (HEK) 293 cells. Using the HA antibody internalization assay we have demonstrated that PKC-dependent endocytosis was strongly inhibited by siRNA depletion of clathrin heavy chain, indicating that CAT-1-HA-GFP internalization requires clathrin-coated pits. Internalized CAT-1-HA-GFP was accumulated in early, recycling, and late endosomes. PKC activation also resulted in ubiquitination of CAT-1. CAT-1 ubiquitination and endocytosis in phorbol ester-stimulated porcine aorthic endothelial and HEK293 cells were inhibited by siRNA knockdown of NEDD4-2 and NEDD4-1 E3 ubiquitin ligases, respectively. In contrast, ubiquitination and endocytosis of the dopamine transporter was dependent on NEDD4-2 in all cell types tested. Altogether, our data suggest that ubiquitination mediated by NEDD4-2 or NEDD4-1 leading to clathrin-mediated endocytosis is the common mode of regulation of various transporter proteins by PKC. PMID:21212261

  16. Regulation of spine and synapse formation by activity-dependent intracellular signaling pathways

    PubMed Central

    Saneyoshi, Takeo; Fortin, Dale A; Soderling, Thomas R

    2010-01-01

    Formation of the human brain during embryonic and postnatal development is an extraordinarily complex process resulting at maturity in billions of neurons with trillions of specialized connections called synapses. These synapses, composed of a varicosity or bouton from a presynaptic neuron that communicates with a dendritic spine of the postsynaptic neuron, comprise the neural network that is essential for complex behavioral phenomena and cognition. Inappropriate synapse formation or structure is thought to underlie several developmental neuropathologies. Even in the mature CNS, alterations in synapse structure and function continues to be a very dynamic process that is foundational to learning and memory as well as other adaptive abilities of the brain. This synaptic plasticity in mature neurons, which is often triggered by certain patterns of neural activity, is again multifaceted and involves post-translational modifications (e.g. phosphorylation) and subcellular relocalization or trafficking (endocytosis/exocytosis) of existing synaptic proteins, initiation of protein synthesis from existing mRNAs localized in dendrites or spines, and triggering of new gene transcription in the nucleus. These various cellular processes support varying temporal components of synaptic plasticity that begin within 1–2 min but can persist for hours to days. This review will give a critical assessment of activity-dependent molecular modulations of synapses reported over the past couple years. Owing to space limitations, it will focus on mammalian excitatory (i.e. glutamatergic) synapses and will not consider several activity-independent signaling pathways (e.g. ephrinB receptor) that also modulate spine and synapse formation [1,2]. PMID:19896363

  17. Revisiting the endocytosis of the m2 muscarinic acetylcholine receptor.

    PubMed

    Ockenga, Wymke; Tikkanen, Ritva

    2015-05-12

    The agonist-induced endocytosis of the muscarinic acetylcholine receptor M2 is different from that of the other members of the muscarinic receptor family. The uptake of the M2 receptor involves the adapter proteins of the β-arrestin family and the small GTPase ADP-ribosylation factor 6. However, it has remained inconclusive if M2 endocytosis is dependent on clathrin or the large GTPase dynamin. We here show by means of knocking down the clathrin heavy chain that M2 uptake upon agonist stimulation requires clathrin. The expression of various dominant-negative dynamin-2 mutants and the use of chemical inhibitors of dynamin function revealed that dynamin expression and membrane localization as such appear to be necessary for M2 endocytosis, whereas dynamin GTPase activity is not required for this process. Based on the data from the present and from previous studies, we propose that M2 endocytosis takes place by means of an atypical clathrin-mediated pathway that may involve a specific subset of clathrin-coated pits/vesicles.

  18. Revisiting the Endocytosis of the M2 Muscarinic Acetylcholine Receptor

    PubMed Central

    Ockenga, Wymke; Tikkanen, Ritva

    2015-01-01

    The agonist-induced endocytosis of the muscarinic acetylcholine receptor M2 is different from that of the other members of the muscarinic receptor family. The uptake of the M2 receptor involves the adapter proteins of the β-arrestin family and the small GTPase ADP-ribosylation factor 6. However, it has remained inconclusive if M2 endocytosis is dependent on clathrin or the large GTPase dynamin. We here show by means of knocking down the clathrin heavy chain that M2 uptake upon agonist stimulation requires clathrin. The expression of various dominant-negative dynamin-2 mutants and the use of chemical inhibitors of dynamin function revealed that dynamin expression and membrane localization as such appear to be necessary for M2 endocytosis, whereas dynamin GTPase activity is not required for this process. Based on the data from the present and from previous studies, we propose that M2 endocytosis takes place by means of an atypical clathrin-mediated pathway that may involve a specific subset of clathrin-coated pits/vesicles. PMID:25985102

  19. Bladder Uptake of Liposomes after Intravesical Administration Occurs by Endocytosis

    PubMed Central

    Rajaganapathy, Bharathi Raja; Chancellor, Michael B.; Nirmal, Jayabalan; Dang, Loan; Tyagi, Pradeep

    2015-01-01

    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. PMID:25811468

  20. Cell adhesion defines the topology of endocytosis and signaling

    PubMed Central

    Grossier, Jean-Philippe; Xouri, Georgia; Goud, Bruno; Schauer, Kristine

    2014-01-01

    Preferred sites of endocytosis have been observed in various cell types, but whether they occur randomly or are linked to cellular cues is debated. Here, we quantified the sites of endocytosis of transferrin (Tfn) and epidermal growth factor (EGF) in cells whose adhesion geometry was defined by micropatterns. 3D probabilistic density maps revealed that Tfn was enriched in adhesive sites during uptake, whereas EGF endocytosis was restricted to the dorsal cellular surface. This spatial separation was not due to distributions of corresponding receptors but was regulated by uptake mechanisms. Asymmetric uptake of Tfn resulted from the enrichment of clathrin and adaptor protein 2 at adhesive areas. Asymmetry in EGF uptake was strongly dependent on the actin cytoskeleton and led to asymmetry in EGF receptor activation. Mild alteration of actin dynamics abolished asymmetry in EGF uptake and decreased EGF-induced downstream signaling, suggesting that cellular adhesion cues influence signal propagation. We propose that restriction of endocytosis at distinct sites allows cells to sense their environment in an “outside-in” mechanism. PMID:24366944

  1. Signalling through phospholipase C interferes with clathrin-mediated endocytosis.

    PubMed

    Carvou, Nicolas; Norden, Anthony G W; Unwin, Robert J; Cockcroft, Shamshad

    2007-01-01

    We investigated if phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2) hydrolysis by phospholipase C activation through cell surface receptors would interfere with clathrin-mediated endocytosis as recruitment of clathrin assembly proteins is PtdIns(4,5)P2-dependent. In the WKPT renal epithelial cell line, endocytosed insulin and beta2-glycoprotein I (beta2gpI) were observed in separate compartments, although endocytosis of both ligands was clathrin-dependent as demonstrated by expression of the clathrin-binding C-terminal domain of AP180 (AP180-C). The two uptake mechanisms were different as only insulin uptake was reduced when the mu2-subunit of the adaptor complex AP-2 was silenced by RNA interference. ATP receptors are expressed at the apical surface of renal cells and, thus, we examined the effect of extracellular ATP on insulin and beta2gpI uptake. ATP stimulated phospholipase C activity, and also suppressed uptake of insulin, but not beta2gpI. This effect was reversed by the PLC inhibitor U-73122. In polarized cell cultures, insulin uptake was apical, whereas beta2gpI uptake was through the basolateral membrane, thus providing an explanation for selective inhibition of insulin endocytosis by ATP. Taken together, these results demonstrate that stimulation of apical G-protein-coupled P2Y receptors, which are coupled to phospholipase C activation diminishes clathrin-mediated endocytosis without interfering with basolateral endocytic mechanisms.

  2. Entry of aminoglycosides into renal tubular epithelial cells via endocytosis-dependent and endocytosis-independent pathways.

    PubMed

    Nagai, Junya; Takano, Mikihisa

    2014-08-15

    Aminoglycoside antibiotics such as gentamicin and amikacin are well recognized as a clinically important antibiotic class because of their reliable efficacy and low cost. However, the clinical use of aminoglycosides is limited by their nephrotoxicity and ototoxicity. Nephrotoxicity is induced mainly due to high accumulation of the antibiotics in renal proximal tubular cells. Therefore, a lot of studies on characterization of the renal transport system for aminoglycosides so far reported involved various in-vivo and in-vitro techniques. Early studies revealed that aminoglycosides are taken up through adsorptive endocytosis in renal epithelial cells. Subsequently, it was found that megalin, a multiligand endocytic receptor abundantly expressed on the apical side of renal proximal tubular cells, can bind aminoglycosides and that megalin-mediated endocytosis plays a crucial role in renal accumulation of aminoglycosides. Therefore, megalin has been suggested to be a promising molecular target for the prevention of aminoglycoside-induced nephrotoxicity. On the other hand, recently, some reports have indicated that aminoglycosides are transported via a pathway that does not require endocytosis, such as non-selective cation channel-mediated entry, in cultured renal tubular cells as well as cochlear outer hair cells. In this commentary article, we review the cellular transport of aminoglycosides in renal epithelial cells, focusing on endocytosis-dependent and -independent pathways.

  3. Activity-Regulated Cytoskeleton-Associated Protein Controls AMPAR Endocytosis through a Direct Interaction with Clathrin-Adaptor Protein 2123

    PubMed Central

    Wall, Mark J.; P. de Almeida, Luciana; Wauters, Sandrine C.; Januário, Yunan C.; Müller, Jürgen

    2016-01-01

    Abstract The activity-regulated cytoskeleton-associated (Arc) protein controls synaptic strength by facilitating AMPA receptor (AMPAR) endocytosis. Here we demonstrate that Arc targets AMPAR to be internalized through a direct interaction with the clathrin-adaptor protein 2 (AP-2). We show that Arc overexpression in dissociated hippocampal neurons obtained from C57BL/6 mouse reduces the density of AMPAR GluA1 subunits at the cell surface and reduces the amplitude and rectification of AMPAR-mediated miniature-EPSCs (mEPSCs). Mutations of Arc, that prevent the AP-2 interaction reduce Arc-mediated endocytosis of GluA1 and abolish the reduction in AMPAR-mediated mEPSC amplitude and rectification. Depletion of the AP-2 subunit µ2 blocks the Arc-mediated reduction in mEPSC amplitude, an effect that is restored by reintroducing µ2. The Arc–AP-2 interaction plays an important role in homeostatic synaptic scaling as the Arc-dependent decrease in mEPSC amplitude, induced by a chronic increase in neuronal activity, is inhibited by AP-2 depletion. These data provide a mechanism to explain how activity-dependent expression of Arc decisively controls the fate of AMPAR at the cell surface and modulates synaptic strength, via the direct interaction with the endocytic clathrin adaptor AP-2. PMID:27257628

  4. The yin and yang of calcium effects on synaptic vesicle endocytosis.

    PubMed

    Wu, Xin-Sheng; Wu, Ling-Gang

    2014-02-12

    A large number of studies suggest that calcium triggers and accelerates vesicle endocytosis at many synapses and non-neuronal secretory cells. However, many studies show that prolonging the duration of the stimulation train, which induces more calcium influx, slows down endocytosis; and several studies suggest that instead of triggering endocytosis, calcium actually inhibits endocytosis. Here we addressed this apparent conflict at a large nerve terminal, the calyx of Held in rat brainstem, in which recent studies suggest that transient calcium increase up to tens of micromolar concentration at the micro/nano domain triggers endocytosis. By dialyzing 0-1 μM calcium into the calyx via a whole-cell pipette, we found that slow endocytosis was inhibited by calcium dialysis in a concentration-dependent manner. Thus, prolonged, small, and global calcium increase inhibits endocytosis, whereas transient and large calcium increase at the micro/nano domain triggers endocytosis and facilitates endocytosis. This yin and yang effect of calcium may reconcile apparent conflicts regarding whether calcium accelerates or inhibits endocytosis. Whether endocytosis is fast or slow depends on the net outcome between the yin and yang effect of calcium.

  5. Myosin light chain kinase accelerates vesicle endocytosis at the calyx of Held synapse.

    PubMed

    Yue, Hai-Yuan; Xu, Jianhua

    2014-01-01

    Neuronal activity triggers endocytosis at synaptic terminals to retrieve efficiently the exocytosed vesicle membrane, ensuring the membrane homeostasis of active zones and the continuous supply of releasable vesicles. The kinetics of endocytosis depends on Ca(2+) and calmodulin which, as a versatile signal pathway, can activate a broad spectrum of downstream targets, including myosin light chain kinase (MLCK). MLCK is known to regulate vesicle trafficking and synaptic transmission, but whether this kinase regulates vesicle endocytosis at synapses remains elusive. We investigated this issue at the rat calyx of Held synapse, where previous studies using whole-cell membrane capacitance measurement have characterized two common forms of Ca(2+)/calmodulin-dependent endocytosis, i.e., slow clathrin-dependent endocytosis and rapid endocytosis. Acute inhibition of MLCK with pharmacological agents was found to slow down the kinetics of both slow and rapid forms of endocytosis at calyces. Similar impairment of endocytosis occurred when blocking myosin II, a motor protein that can be phosphorylated upon MLCK activation. The inhibition of endocytosis was not accompanied by a change in Ca(2+) channel current. Combined inhibition of MLCK and calmodulin did not induce synergistic inhibition of endocytosis. Together, our results suggest that activation of MLCK accelerates both slow and rapid forms of vesicle endocytosis at nerve terminals, likely by functioning downstream of Ca(2+)/calmodulin.

  6. Activity-dependent dendritic release of BDNF and biological consequences

    PubMed Central

    Kuczewski, Nicola; Porcher, Christophe; Lessmann, Volkmar; Medina, Igor; Gaiarsa, Jean-Luc

    2009-01-01

    Network construction and reorganization is modulated by the level and pattern of synaptic activity generated in the nervous system. During the past decades, neurotrophins, and in particular brain-derived neurotrophic factor (BDNF), have emerged as attractive candidates for linking synaptic activity and brain plasticity. Thus, neurotrophin expression and secretion are under the control of activity-dependent mechanisms and, besides their classical role in supporting neuronal survival neurotrophins, modulate nearly all key steps of network construction from neuronal migration to experience-dependent refinement of local connections. In this paper, we provide an overview of recent findings showing that BDNF can serve as a target-derived messenger for activity-dependent synaptic plasticity and development at the single cell level. PMID:19156544

  7. Activity-dependent plasticity of hippocampal place maps

    PubMed Central

    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

  8. USP17 is required for clathrin mediated endocytosis of epidermal growth factor receptor

    PubMed Central

    Jaworski, Jakub; de la Vega, Michelle; Fletcher, Sarah J.; McFarlane, Cheryl; Greene, Michelle K.; Smyth, Andrew W.; Van Schaeybroeck, Sandra; Johnston, James A.; Scott, Christopher J.; Rappoport, Joshua Z.; Burrows, James F.

    2014-01-01

    Previously we have shown that expression of the deubiquitinating enzyme USP17 is required for cell proliferation and motility. More recently we reported that USP17 deubiquitinates RCE1 isoform 2 and thus regulates the processing of ‘CaaX’ motif proteins. Here we now show that USP17 expression is induced by epidermal growth factor and that USP17 expression is required for clathrin mediated endocytosis of epidermal growth factor receptor. In addition, we show that USP17 is required for the endocytosis of transferrin, an archetypal substrate for clathrin mediated endocytosis, and that USP17 depletion impedes plasma membrane recruitment of the machinery required for clathrin mediated endocytosis. Thus, our data reveal that USP17 is necessary for epidermal growth factor receptor and transferrin endocytosis via clathrin coated pits, indicate this is mediated via the regulation of the recruitment of the components of the endocytosis machinery and suggest USP17 may play a general role in receptor endocytosis. PMID:25026282

  9. Endoplasmic reticulum stress contributes to acetylcholine receptor degradation by promoting endocytosis in skeletal muscle cells.

    PubMed

    Du, Ailian; Huang, Shiqian; Zhao, Xiaonan; Zhang, Yun; Zhu, Lixun; Ding, Ji; Xu, Congfeng

    2016-01-15

    After binding by acetylcholine released from a motor neuron, a nicotinic acetylcholine receptor at the neuromuscular junction produces a localized end-plate potential, which leads to muscle contraction. Improper turnover and renewal of acetylcholine receptors contributes to the pathogenesis of myasthenia gravis. In the present study, we demonstrate that endoplasmic reticulum (ER) stress contributes to acetylcholine receptor degradation in C2C12 myocytes. We further show that ER stress promotes acetylcholine receptor endocytosis and lysosomal degradation, which was dampened by blocking endocytosis or treating with lysosome inhibitor. Knockdown of ER stress proteins inhibited acetylcholine receptor endocytosis and degradation, while rescue assay restored its endocytosis and degradation, confirming the effects of ER stress on promoting endocytosis-mediated degradation of junction acetylcholine receptors. Thus, our studies identify ER stress as a factor promoting acetylcholine receptor degradation through accelerating endocytosis in muscle cells. Blocking ER stress and/or endocytosis might provide a novel therapeutic approach for myasthenia gravis.

  10. Dysferlin is essential for endocytosis in the sea star oocyte.

    PubMed

    Oulhen, Nathalie; Onorato, Thomas M; Ramos, Isabela; Wessel, Gary M

    2014-04-01

    Dysferlin is a calcium-binding transmembrane protein involved in membrane fusion and membrane repair. In humans, mutations in the dysferlin gene are associated with muscular dystrophy. In this study, we isolated plasma membrane-enriched fractions from full-grown immature oocytes of the sea star, and identified dysferlin by mass spectrometry analysis. The full-length dysferlin sequence is highly conserved between human and the sea star. We learned that in the sea star Patiria miniata, dysferlin RNA and protein are expressed from oogenesis to gastrulation. Interestingly, the protein is highly enriched in the plasma membrane of oocytes. Injection of a morpholino against dysferlin leads to a decrease of endocytosis in oocytes, and to a developmental arrest during gastrulation. These results suggest that dysferlin is critical for normal endocytosis during oogenesis and for embryogenesis in the sea star and that this animal may be a useful model for studying the relationship of dysferlin structure as it relates to its function.

  11. Rare earth elements activate endocytosis in plant cells

    PubMed Central

    Wang, Lihong; Li, Jigang; Zhou, Qing; Yang, Guangmei; Ding, Xiao Lan; Li, Xiaodong; Cai, Chen Xin; Zhang, Zhao; Wei, Hai Yan; Lu, Tian Hong; Deng, Xing Wang; Huang, Xiao Hua

    2014-01-01

    It has long been observed that rare earth elements (REEs) regulate multiple facets of plant growth and development. However, the underlying mechanisms remain largely unclear. Here, using electron microscopic autoradiography, we show the life cycle of a light REE (lanthanum) and a heavy REE (terbium) in horseradish leaf cells. Our data indicate that REEs were first anchored on the plasma membrane in the form of nanoscale particles, and then entered the cells by endocytosis. Consistently, REEs activated endocytosis in plant cells, which may be the cellular basis of REE actions in plants. Moreover, we discovered that a portion of REEs was successively released into the cytoplasm, self-assembled to form nanoscale clusters, and finally deposited in horseradish leaf cells. Taken together, our data reveal the life cycle of REEs and their cellular behaviors in plant cells, which shed light on the cellular mechanisms of REE actions in living organisms. PMID:25114214

  12. Anchored but not internalized: shape dependent endocytosis of nanodiamond

    NASA Astrophysics Data System (ADS)

    Zhang, Bokai; Feng, Xi; Yin, Hang; Ge, Zhenpeng; Wang, Yanhuan; Chu, Zhiqin; Raabova, Helena; Vavra, Jan; Cigler, Petr; Liu, Renbao; Wang, Yi; Li, Quan

    2017-04-01

    Nanoparticle-cell interactions begin with the cellular uptake of the nanoparticles, a process that eventually determines their cellular fate. In the present work, we show that the morphological features of nanodiamonds (NDs) affect both the anchoring and internalization stages of their endocytosis. While a prickly ND (with sharp edges/corners) has no trouble of anchoring onto the plasma membrane, it suffers from difficult internalization afterwards. In comparison, the internalization of a round ND (obtained by selective etching of the prickly ND) is not limited by its lower anchoring amount and presents a much higher endocytosis amount. Molecular dynamics simulation and continuum modelling results suggest that the observed difference in the anchoring of round and prickly NDs likely results from the reduced contact surface area with the cell membrane of the former, while the energy penalty associated with membrane curvature generation, which is lower for a round ND, may explain its higher probability of the subsequent internalization.

  13. Receptor palmitoylation and ubiquitination regulate anthrax toxin endocytosis

    PubMed Central

    Abrami, Laurence; Leppla, Stephen H.; van der Goot, F. Gisou

    2006-01-01

    The anthrax toxin is composed of three independent polypeptide chains. Successful intoxication only occurs when heptamerization of the receptor-binding polypeptide, the protective antigen (PA), allows binding of the two enzymatic subunits before endocytosis. We show that this tailored behavior is caused by two counteracting posttranslational modifications in the cytoplasmic tail of PA receptors. The receptor is palmitoylated, and this unexpectedly prevents its association with lipid rafts and, thus, its premature ubiquitination. This second modification, which is mediated by the E3 ubiquitin ligase Cbl, only occurs in rafts and is required for rapid endocytosis of the receptor. As a consequence, cells expressing palmitoylation-defective mutant receptors are less sensitive to anthrax toxin because of a lower number of surface receptors as well as premature internalization of PA without a requirement for heptamerization. PMID:16401723

  14. Receptor palmitoylation and ubiquitination regulate anthrax toxin endocytosis.

    PubMed

    Abrami, Laurence; Leppla, Stephen H; van der Goot, F Gisou

    2006-01-16

    The anthrax toxin is composed of three independent polypeptide chains. Successful intoxication only occurs when heptamerization of the receptor-binding polypeptide, the protective antigen (PA), allows binding of the two enzymatic subunits before endocytosis. We show that this tailored behavior is caused by two counteracting posttranslational modifications in the cytoplasmic tail of PA receptors. The receptor is palmitoylated, and this unexpectedly prevents its association with lipid rafts and, thus, its premature ubiquitination. This second modification, which is mediated by the E3 ubiquitin ligase Cbl, only occurs in rafts and is required for rapid endocytosis of the receptor. As a consequence, cells expressing palmitoylation-defective mutant receptors are less sensitive to anthrax toxin because of a lower number of surface receptors as well as premature internalization of PA without a requirement for heptamerization.

  15. Signaling-mediated control of ubiquitin ligases in endocytosis.

    PubMed

    Polo, Simona

    2012-03-15

    Ubiquitin-dependent regulation of endocytosis plays an important part in the control of signal transduction, and a critical issue in the understanding of signal transduction therefore relates to regulation of ubiquitination in the endocytic pathway. We discuss here what is known of the mechanisms by which signaling controls the activity of the ubiquitin ligases that specifically recognize the targets of ubiquitination on the endocytic pathway, and suggest alternative mechanisms that deserve experimental investigation.

  16. Characterization of Ligand-Induced Endocytosis of EGF-Receptors

    DTIC Science & Technology

    1997-12-01

    Dili domain of Epsl5. They showed that a GST-protein containing the entire COOH-terminal domain of Epsl5 could efficiently precipitate AP- 2 complexes...from cytosol. A construct lacking amino acids 661-739, GST-DfflAl, could also precipitate AP- 2 complexes albiet less efficiently . In contrast...purified AP- 2 complexes restored highly efficient endocytosis. Furhter confirmation of this hypothesis was obtained in another set of experiments

  17. Sorting Nexin 9 facilitates podocin endocytosis in the injured podocyte

    PubMed Central

    Sasaki, Yu; Hidaka, Teruo; Ueno, Takashi; Akiba-Takagi, Miyuki; Trejo, Juan Alejandro Oliva; Seki, Takuto; Nagai-Hosoe, Yoshiko; Tanaka, Eriko; Horikoshi, Satoshi; Tomino, Yasuhiko; Suzuki, Yusuke; Asanuma, Katsuhiko

    2017-01-01

    The irreversibility of glomerulosclerotic changes depends on the degree of podocyte injury. We have previously demonstrated the endocytic translocation of podocin to the subcellular area in severely injured podocytes and found that this process is the primary disease trigger. Here we identified the protein sorting nexin 9 (SNX9) as a novel facilitator of podocin endocytosis in a yeast two-hybrid analysis. SNX9 is involved in clathrin-mediated endocytosis, actin rearrangement and vesicle transport regulation. Our results revealed and confirmed that SNX9 interacts with podocin exclusively through the Bin–Amphiphysin–Rvs (BAR) domain of SNX9. Immunofluorescence staining revealed the expression of SNX9 in response to podocyte adriamycin-induced injury both in vitro and in vivo. Finally, an analysis of human glomerular disease biopsy samples demonstrated strong SNX9 expression and co-localization with podocin in samples representative of severe podocyte injury, such as IgA nephropathy with poor prognosis, membranous nephropathy and focal segmental glomerulosclerosis. In conclusion, we identified SNX9 as a facilitator of podocin endocytosis in severe podocyte injury and demonstrated the expression of SNX9 in the podocytes of both nephropathy model mice and human patients with irreversible glomerular disease. PMID:28266622

  18. Actin growth profile in clathrin-mediated endocytosis

    NASA Astrophysics Data System (ADS)

    Tweten, D. J.; Bayly, P. V.; Carlsson, A. E.

    2017-05-01

    Clathrin-mediated endocytosis in yeast is driven by a protein patch containing close to 100 different types of proteins. Among the proteins are 5000 -10 000 copies of polymerized actin, and successful endocytosis requires growth of the actin network. Since it is not known exactly how actin network growth drives endocytosis, we calculate the spatial distribution of actin growth required to generate the force that drives the process. First, we establish the force distribution that must be supplied by actin growth, by combining membrane-bending profiles obtained via electron microscopy with established theories of membrane mechanics. Next, we determine the profile of actin growth, using a continuum mechanics approach and an iterative procedure starting with an actin growth profile obtained from a linear analysis. The profile has fairly constant growth outside a central hole of radius 45-50 nm, but very little growth in this hole. This growth profile can reproduce the required forces if the actin shear modulus exceeds 80 kPa, and the growing filaments can exert very large polymerization forces. The growth profile prediction could be tested via electron-microscopy or super-resolution experiments in which the turgor pressure is suddenly turned off.

  19. Sorting Nexin 9 facilitates podocin endocytosis in the injured podocyte.

    PubMed

    Sasaki, Yu; Hidaka, Teruo; Ueno, Takashi; Akiba-Takagi, Miyuki; Trejo, Juan Alejandro Oliva; Seki, Takuto; Nagai-Hosoe, Yoshiko; Tanaka, Eriko; Horikoshi, Satoshi; Tomino, Yasuhiko; Suzuki, Yusuke; Asanuma, Katsuhiko

    2017-03-07

    The irreversibility of glomerulosclerotic changes depends on the degree of podocyte injury. We have previously demonstrated the endocytic translocation of podocin to the subcellular area in severely injured podocytes and found that this process is the primary disease trigger. Here we identified the protein sorting nexin 9 (SNX9) as a novel facilitator of podocin endocytosis in a yeast two-hybrid analysis. SNX9 is involved in clathrin-mediated endocytosis, actin rearrangement and vesicle transport regulation. Our results revealed and confirmed that SNX9 interacts with podocin exclusively through the Bin-Amphiphysin-Rvs (BAR) domain of SNX9. Immunofluorescence staining revealed the expression of SNX9 in response to podocyte adriamycin-induced injury both in vitro and in vivo. Finally, an analysis of human glomerular disease biopsy samples demonstrated strong SNX9 expression and co-localization with podocin in samples representative of severe podocyte injury, such as IgA nephropathy with poor prognosis, membranous nephropathy and focal segmental glomerulosclerosis. In conclusion, we identified SNX9 as a facilitator of podocin endocytosis in severe podocyte injury and demonstrated the expression of SNX9 in the podocytes of both nephropathy model mice and human patients with irreversible glomerular disease.

  20. Visualization of Receptor-mediated Endocytosis in Yeast

    PubMed Central

    Mulholland, Jon; Konopka, James; Singer-Kruger, Birgit; Zerial, Marino; Botstein, David

    1999-01-01

    We studied the ligand-induced endocytosis of the yeast α-factor receptor Ste2p by immuno-electron microscopy. We observed and quantitated time-dependent loss of Ste2p from the plasma membrane of cells exposed to α-factor. This ligand-induced internalization of Ste2p was blocked in the well-characterized endocytosis-deficient mutant sac6Δ. We provide evidence that implicates furrow-like invaginations of the plasma membrane as the site of receptor internalization. These invaginations are distinct from the finger-like plasma membrane invaginations within actin cortical patches. Consistent with this, we show that Ste2p is not located within the cortical actin patch before and during receptor-mediated endocytosis. In wild-type cells exposed to α-factor we also observed and quantitated a time-dependent accumulation of Ste2p in intracellular, membrane-bound compartments. These compartments have a characteristic electron density but variable shape and size and are often located adjacent to the vacuole. In immuno-electron microscopy experiments these compartments labeled with antibodies directed against the rab5 homologue Ypt51p (Vps21p), the resident vacuolar protease carboxypeptidase Y, and the vacuolar H+-ATPase Vph1p. Using a new double-labeling technique we have colocalized antibodies against Ste2p and carboxypeptidase Y to this compartment, thereby identifying these compartments as prevacuolar late endosomes. PMID:10069819

  1. Architectural remodeling of the tonoplast during fluid-phase endocytosis

    PubMed Central

    Etxeberria, Ed; Gonzalez, Pedro; Pozueta-Romero, Javier

    2013-01-01

    During fluid phase endocytosis (FPE) in plant storage cells, the vacuole receives a considerable amount of membrane and fluid contents. If allowed to accumulate over a period of time, the enlarging tonoplast and increase in fluids would invariably disrupt the structural equilibrium of the mature cells. Therefore, a membrane retrieval process must exist that will guarantee membrane homeostasis in light of tonoplast expansion by membrane addition during FPE. We examined the morphological changes to the vacuolar structure during endocytosis in red beet hypocotyl tissue using scanning laser confocal microscopy and immunohistochemistry. The heavily pigmented storage vacuole allowed us to visualize all architectural transformations during treatment. When red beet tissue was incubated in 200 mM sucrose, a portion of the sucrose accumulated entered the cell by means of FPE. The accumulation process was accompanied by the development of vacuole-derived vesicles which transiently counterbalanced the addition of surplus endocytic membrane during rapid rates of endocytosis. Topographic fluorescent confocal micrographs showed an ensuing reduction in the size of the vacuole-derived vesicles and further suggest their reincorporation into the vacuole to maintain vacuolar unity and solute concentration. PMID:23656870

  2. Endocytosis in the plant-pathogenic fungus Ustilago maydis.

    PubMed

    Fuchs, U; Steinberg, G

    2005-10-01

    Filamentous fungi are an important group of tip-growing organisms, which include numerous plant pathogens such as Magnaporthe grisea and Ustilago maydis. Despite their ecological and economical relevance, we are just beginning to unravel the importance of endocytosis in filamentous fungi. Most evidence for endocytosis in filamentous fungi is based on the use of endocytic tracer dyes that are taken up into the cell and delivered to the vacuole. Moreover, genomewide screening for candidate genes in Neurospora crassa and U. maydis confirmed the presence of most components of the endocytic machinery, indicating that endocytosis participates in filamentous growth. Indeed, it was shown that in U. maydis early endosomes cluster at sites of growth, where they support morphogenesis and polar growth, most likely via endosome-based membrane recycling. In humans, such recycling processes to the plasma membrane involve small GTPases such as Rab4. A homologue of this protein is encoded in the genome of U. maydis but is absent from the yeast Saccharomyces cerevisiae, suggesting that Rab4-mediated recycling is important for filamentous growth. Furthermore, human Rab4 regulates traffic of early endosomes along microtubules, and a similar microtubule-based transport is described for U. maydis. These observations suggest that Rab4-like GTPases might regulate endosome- and microtubule-based recycling during tip growth of filamentous fungi.

  3. Spatiotemporal control of endocytosis by phosphatidylinositol-3,4-bisphosphate.

    PubMed

    Posor, York; Eichhorn-Gruenig, Marielle; Puchkov, Dmytro; Schöneberg, Johannes; Ullrich, Alexander; Lampe, André; Müller, Rainer; Zarbakhsh, Sirus; Gulluni, Federico; Hirsch, Emilio; Krauss, Michael; Schultz, Carsten; Schmoranzer, Jan; Noé, Frank; Haucke, Volker

    2013-07-11

    Phosphoinositides serve crucial roles in cell physiology, ranging from cell signalling to membrane traffic. Among the seven eukaryotic phosphoinositides the best studied species is phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), which is concentrated at the plasma membrane where, among other functions, it is required for the nucleation of endocytic clathrin-coated pits. No phosphatidylinositol other than PI(4,5)P2 has been implicated in clathrin-mediated endocytosis, whereas the subsequent endosomal stages of the endocytic pathway are dominated by phosphatidylinositol-3-phosphates(PI(3)P). How phosphatidylinositol conversion from PI(4,5)P2-positive endocytic intermediates to PI(3)P-containing endosomes is achieved is unclear. Here we show that formation of phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2) by class II phosphatidylinositol-3-kinase C2α (PI(3)K C2α) spatiotemporally controls clathrin-mediated endocytosis. Depletion of PI(3,4)P2 or PI(3)K C2α impairs the maturation of late-stage clathrin-coated pits before fission. Timed formation of PI(3,4)P2 by PI(3)K C2α is required for selective enrichment of the BAR domain protein SNX9 at late-stage endocytic intermediates. These findings provide a mechanistic framework for the role of PI(3,4)P2 in endocytosis and unravel a novel discrete function of PI(3,4)P2 in a central cell physiological process.

  4. Clathrin-mediated endocytosis of gold nanoparticles in vitro.

    PubMed

    Ng, Cheng Teng; Tang, Florence Mei Ai; Li, Jasmine Jia'en; Ong, Cynthia; Yung, Lanry Lin Yue; Bay, Boon Huat

    2015-02-01

    Gold nanoparticles (AuNPs) have potential biomedical and scientific applications. In this study, we evaluated the uptake and internalization of FBS-coated 20 nm AuNPs into lung fibroblasts and liver cells by different microscopy techniques. AuNP aggregates were observed inside MRC5 lung fibroblasts and Chang liver cells under light microscopy, especially after enhancement with automegallography. Clusters of AuNPs were observed to be adsorbed on the cell surface by scanning electron microscopy. Ultrathin sections showed that AuNPs were mainly enclosed within cytoplasmic vesicles when viewed under transmission electron microscopy. We also investigated the mechanism of uptake for AuNPs, using endocytosis inhibitors and quantification of Au with inductively coupled plasma mass spectrometry. Cells treated with concanavalin A and chlorpromazine showed significant decrease of Au uptake in MRC5 lung fibroblasts and Chang liver cells, respectively, implying that the uptake of AuNPs was facilitated by clathrin-mediated endocytosis. It would therefore appear that uptake of 20 nm AuNPs in both cell types with different tissues of origin, was dependent upon clathrin-mediated endocytosis.

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

    SciTech Connect

    Americo, Tatiana A.; Chiarini, Luciana B.; Linden, Rafael . E-mail: rlinden@biof.ufrj.br

    2007-06-29

    The co-chaperone hop/STI-1 is a ligand of the cell surface prion protein (PrP{sup C}), and their interaction leads to signaling and biological effects. Among these, hop/STI-1 induces proliferation of A172 glioblastoma cells, dependent on both PrP{sup C} 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 PrP{sup C} 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.

  6. Translocation and Endocytosis for Cell-penetrating Peptide Internalization

    PubMed Central

    Jiao, Chen-Yu; Delaroche, Diane; Burlina, Fabienne; Alves, Isabel D.; Chassaing, Gérard; Sagan, Sandrine

    2009-01-01

    Cell-penetrating peptides (CPPs) share the property of cellular internalization. The question of how these peptides reach the cytoplasm of cells is still widely debated. Herein, we have used a mass spectrometry-based method that enables quantification of internalized and membrane-bound peptides. Internalization of the most used CPP was studied at 37 °C (endocytosis and translocation) and 4 °C (translocation) in wild type and proteoglycan-deficient Chinese hamster ovary cells. Both translocation and endocytosis are internalization pathways used by CPP. The choice of one pathway versus the other depends on the peptide sequence (not the number of positive changes), the extracellular peptide concentration, and the membrane components. There is no relationship between the high affinity of these peptides for the cell membrane and their internalization efficacy. Translocation occurs at low extracellular peptide concentration, whereas endocytosis, a saturable and cooperative phenomenon, is activated at higher concentrations. Translocation operates in a narrow time window, which implies a specific lipid/peptide co-import in cells. PMID:19833724

  7. Endocytosis of cholera toxin by human enterocytes is developmentally regulated.

    PubMed

    Lu, Lei; Khan, Sameer; Lencer, Wayne; Walker, W Allan

    2005-08-01

    Many secretory diarrheas including cholera are more prevalent and fulminant in young infants than in older children and adults. Cholera toxin (CT) elicits a cAMP-dependent chloride secretory response in intestinal epithelia, which accounts for the fundamental pathogenesis of this toxigenic diarrhea. We have previously reported that the action of this bacterial enterotoxin is excessive in immature enterocytes and under developmental regulation. In this study, we tested the hypothesis that enhanced endocytosis by immature human enterocytes may, in part, account for the excessive secretory response to CT noted in the immature intestine and that enterocyte endocytosis of CT is developmentally regulated. To test this hypothesis, we used specific inhibitors to define endocytic pathways in mature and immature cell lines. We showed that internalization of CT in adult enterocytes is less and occurs via the caveolae/raft-mediated pathway in contrast to an enhanced immature human enterocyte CT uptake that occurs via a clathrin pathway. We also present evidence that this clathrin pathway is developmentally regulated as demonstrated by its response to corticosteroids, a known maturation factor that causes a decreased CT endocytosis by this pathway.

  8. Dimerization drives EGFR endocytosis through two sets of compatible endocytic codes.

    PubMed

    Wang, Qian; Chen, Xinmei; Wang, Zhixiang

    2015-03-01

    We have shown previously that epidermal growth factor (EGF) receptor (EGFR) endocytosis is controlled by EGFR dimerization. However, it is not clear how the dimerization drives receptor internalization. We propose that EGFR endocytosis is driven by dimerization, bringing two sets of endocytic codes, one contained in each receptor monomer, in close proximity. Here, we tested this hypothesis by generating specific homo- or hetero-dimers of various receptors and their mutants. We show that ErbB2 and ErbB3 homodimers are endocytosis deficient owing to the lack of endocytic codes. Interestingly, EGFR-ErbB2 or EGFR-ErbB3 heterodimers are also endocytosis deficient. Moreover, the heterodimer of EGFR and the endocytosis-deficient mutant EGFRΔ1005-1017 is also impaired in endocytosis. These results indicate that two sets of endocytic codes are required for receptor endocytosis. We found that an EGFR-PDGFRβ heterodimer is endocytosis deficient, although both EGFR and PDGFRβ homodimers are endocytosis-competent, indicating that two compatible sets of endocytic codes are required. Finally, we found that to mediate the endocytosis of the receptor dimer, the two sets of compatible endocytic codes, one contained in each receptor molecule, have to be spatially coordinated.

  9. Cholesterol regulates multiple forms of vesicle endocytosis at a mammalian central synapse.

    PubMed

    Yue, Hai-Yuan; Xu, Jianhua

    2015-07-01

    Endocytosis in synapses sustains neurotransmission by recycling vesicle membrane and maintaining the homeostasis of synaptic membrane. A role of membrane cholesterol in synaptic endocytosis remains controversial because of conflicting observations, technical limitations in previous studies, and potential interference from non-specific effects after cholesterol manipulation. Furthermore, it remains unclear whether cholesterol participates in distinct forms of endocytosis that function under different activity levels. In this study, applying the whole-cell membrane capacitance measurement to monitor endocytosis in real time at the rat calyx of Held terminals, we found that disrupting cholesterol with dialysis of cholesterol oxidase or methyl-β-cyclodextrin impaired three different forms of endocytosis, including slow endocytosis, rapid endocytosis, and endocytosis of the retrievable membrane that exists at the surface before stimulation. The effects were observed when disruption of cholesterol was mild enough not to change Ca(2+) channel current or vesicle exocytosis, indicative of stringent cholesterol requirement in synaptic endocytosis. Extracting cholesterol with high concentrations of methyl-β-cyclodextrin reduced exocytosis, mainly by decreasing the readily releasable pool and the vesicle replenishment after readily releasable pool depletion. Our study suggests that cholesterol is an important, universal regulator in multiple forms of vesicle endocytosis at mammalian central synapses.

  10. Reversible, activity-dependent targeting of profilin to neuronal nuclei

    SciTech Connect

    Birbach, Andreas . E-mail: andreas.birbach@lbicr.lbg.ac.at; Verkuyl, J. Martin; Matus, Andrew . E-mail: aim@fmi.ch

    2006-07-15

    The actin cytoskeleton in pyramidal neurons plays a major role in activity-dependent processes underlying neuronal plasticity. The small actin-binding protein profilin shows NMDA receptor-dependent accumulation in dendritic spines, which is correlated with suppression of actin dynamics and long-term stabilization of synaptic morphology. Here we show that following NMDA receptor activation profilin also accumulates in the nucleus of hippocampal neurons via a process involving rearrangement of the actin cytoskeleton. This simultaneous targeting to dendritic spines and the cell nucleus suggests a novel mechanism of neuronal plasticity in which profilin both tags activated synapses and influences nuclear events.

  11. Isolated rat hepatocytes acquire iron from lactoferrin by endocytosis.

    PubMed

    McAbee, D D

    1995-10-15

    The iron-binding protein lactoferrin (Lf) present in blood is metabolized by the liver. Isolated rat hepatocytes vigorously endocytose bovine Lf via recycling Ca2(+)-dependent binding sites, but the uptake of iron from Lf by hepatocytes has not been examined. In this study, isolated rat hepatocytes were incubated with radiolabelled bovine Lf (125I-Lf, 59Fe-Lf or 125I-59Fe-Lf) at 37 degrees C, then washed at 4 degrees C in the presence of dextran sulphate with either Ca2+ or EGTA to distinguish between total bound and internal radioactivity respectively. Cells internalized 125I-Lf protein and Lf-bound 59Fe at maximal endocytic rates of 1700 and 480 mol.cell-1.s-1 respectively. When Lf was normalized for 59Fe content, these endocytic rates were equivalent and reflected an uptake potential of at least 3400 mol of iron.cell-1.s-1. Cells prebound with 125I-59Fe-Lf to Ca2+(-)dependent sites at 4 degrees C internalized more than 80% of both 125I-Lf protein and Lf-bound 59Fe approx. 6 min after warming to 37 degrees C at similar rates (125I-Lf: k(in) = 0.276 min-1, 59Fe: k(in) = 0.303 min-1). Within 4 h at 37 degrees C, cells had released 25% or less internalized Lf protein in the form of acid-soluble 125I-by-products but retained all the Lf-delivered 59Fe. Hyperosmotic disruption of clathrin-dependent endocytosis blocked the uptake of 125I-Lf and Lf-bound 59Fe. Incubation of cells with 125I-59Fe-Lf and a 100 molar excess of diferric transferrin reduced slightly the endocytosis of 125I-Lf protein and 59Fe accumulation. Treatment of cells with the ferric chelator desferrioxamine did not alter uptake of 125I-Lf protein or Lf-bound 59Fe, but the ferrous chelator bathophenanthroline disulphonate slightly elevated endocytosis of 125I-Lf protein and Lf-bound 59Fe. These findings indicate that Lf does not release its bound iron before endocytosis. It was concluded from this study that hepatocytes take up iron from Lf at high rates by a process that requires endocytosis of Lf

  12. Rotary bulk solids divider

    DOEpatents

    Maronde, Carl P.; Killmeyer, Jr., Richard P.

    1992-01-01

    An apparatus for the disbursement of a bulk solid sample comprising, a gravity hopper having a top open end and a bottom discharge end, a feeder positioned beneath the gravity hopper so as to receive a bulk solid sample flowing from the bottom discharge end, and a conveyor receiving the bulk solid sample from the feeder and rotating on an axis that allows the bulk solid sample to disperse the sample to a collection station.

  13. ROTARY BULK SOLIDS DIVIDER

    DOEpatents

    Maronde, Carl P.; Killmeyer JR., Richard P.

    1992-03-03

    An apparatus for the disbursement of a bulk solid sample comprising, a gravity hopper having a top open end and a bottom discharge end, a feeder positioned beneath the gravity hopper so as to receive a bulk solid sample flowing from the bottom discharge end, and a conveyor receiving the bulk solid sample from the feeder and rotating on an axis that allows the bulk solid sample to disperse the sample to a collection station.

  14. Role of BDNF epigenetics in activity-dependent neuronal plasticity.

    PubMed

    Karpova, Nina N

    2014-01-01

    Brain-derived neurotrophic factor (BDNF) is a key mediator of the activity-dependent processes in the brain that have a major impact on neuronal development and plasticity. Impaired control of neuronal activity-induced BDNF expression mediates the pathogenesis of various neurological and psychiatric disorders. Different environmental stimuli, such as the use of pharmacological compounds, physical and learning exercises or stress exposure, lead to activation of specific neuronal networks. These processes entail tight temporal and spatial transcriptional control of numerous BDNF splice variants through epigenetic mechanisms. The present review highlights recent findings on the dynamic and long-term epigenetic programming of BDNF gene expression by the DNA methylation, histone-modifying and microRNA machineries. The review also summarizes the current knowledge on the activity-dependent BDNF mRNA trafficking critical for rapid local regulation of BDNF levels and synaptic plasticity. Current data open novel directions for discovery of new promising therapeutic targets for treatment of neuropsychiatric disorders. This article is part of the Special Issue entitled 'BDNF Regulation of Synaptic Structure, Function, and Plasticity'. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Activity-dependent inhibitory synapse remodeling through gephyrin phosphorylation.

    PubMed

    Flores, Carmen E; Nikonenko, Irina; Mendez, Pablo; Fritschy, Jean-Marc; Tyagarajan, Shiva K; Muller, Dominique

    2015-01-06

    Maintaining a proper balance between excitation and inhibition is essential for the functioning of neuronal networks. However, little is known about the mechanisms through which excitatory activity can affect inhibitory synapse plasticity. Here we used tagged gephyrin, one of the main scaffolding proteins of the postsynaptic density at GABAergic synapses, to monitor the activity-dependent adaptation of perisomatic inhibitory synapses over prolonged periods of time in hippocampal slice cultures. We find that learning-related activity patterns known to induce N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation and transient optogenetic activation of single neurons induce within hours a robust increase in the formation and size of gephyrin-tagged clusters at inhibitory synapses identified by correlated confocal electron microscopy. This inhibitory morphological plasticity was associated with an increase in spontaneous inhibitory activity but did not require activation of GABAA receptors. Importantly, this activity-dependent inhibitory plasticity was prevented by pharmacological blockade of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), it was associated with an increased phosphorylation of gephyrin on a site targeted by CaMKII, and could be prevented or mimicked by gephyrin phospho-mutants for this site. These results reveal a homeostatic mechanism through which activity regulates the dynamics and function of perisomatic inhibitory synapses, and they identify a CaMKII-dependent phosphorylation site on gephyrin as critically important for this process.

  16. Nitric oxide mediates local activity-dependent excitatory synapse development.

    PubMed

    Nikonenko, Irina; Nikonenko, Alexander; Mendez, Pablo; Michurina, Tatyana V; Enikolopov, Grigori; Muller, Dominique

    2013-10-29

    Learning related paradigms play an important role in shaping the development and specificity of synaptic networks, notably by regulating mechanisms of spine growth and pruning. The molecular events underlying these synaptic rearrangements remain poorly understood. Here we identify NO signaling as a key mediator of activity-dependent excitatory synapse development. We find that chronic blockade of NO production in vitro and in vivo interferes with the development of hippocampal and cortical excitatory spine synapses. The effect results from a selective loss of activity-mediated spine growth mechanisms and is associated with morphological and functional alterations of remaining synapses. These effects of NO are mediated by a cGMP cascade and can be reproduced or prevented by postsynaptic expression of vasodilator-stimulated phosphoprotein phospho-mimetic or phospho-resistant mutants. In vivo analyses show that absence of NO prevents the increase in excitatory synapse density induced by environmental enrichment and interferes with the formation of local clusters of excitatory synapses. We conclude that NO plays an important role in regulating the development of excitatory synapses by promoting local activity-dependent spine-growth mechanisms.

  17. A network model for activity-dependent sleep regulation.

    PubMed

    Roy, Sandip; Krueger, James M; Rector, David M; Wan, Yan

    2008-08-07

    We develop and characterize a dynamical network model for activity-dependent sleep regulation. Specifically, in accordance with the activity-dependent theory for sleep, we view organism sleep as emerging from the local sleep states of functional units known as cortical columns; these local sleep states evolve through integration of local activity inputs, loose couplings with neighboring cortical columns, and global regulation (e.g. by the circadian clock). We model these cortical columns as coupled or networked activity-integrators that transition between sleep and waking states based on thresholds on the total activity. The model dynamics for three canonical experiments (which we have studied both through simulation and system-theoretic analysis) match with experimentally observed characteristics of the cortical-column network. Most notably, assuming connectedness of the network graph, our model predicts the recovery of the columns to a synchronized state upon temporary overstimulation of a single column and/or randomization of the initial sleep and activity-integration states. In analogy with other models for networked oscillators, our model also predicts the possibility for such phenomena as mode-locking.

  18. Fast Endocytosis Is Inhibited by GABA-Mediated Chloride Influx at a Presynaptic Terminal

    PubMed Central

    Hull, Court; von Gersdorff, Henrique

    2013-01-01

    Summary Although multiple kinetic components of synaptic vesicle endocytosis have been identified, it has remained unclear whether neurons can differentially modulate these components. Using membrane capacitance measurements from isolated goldfish bipolar cell terminals, we found that the kinetics of endocytosis in retinal slices (single exponential decay; τ > 10 s) were significantly slower than those in acutely dissociated terminals (double exponential decay; τfast ≈ 1–2 s; τslow > 10 s). Surprisingly, GABAA and/or GABAC receptor antagonists restored the fast component of endocytosis to terminals in retinal slices. Blocking GABAergic feedback from reciprocal synapses or removing external Cl− ions also allowed for fast endocytosis. Elevating internal Cl− via the patch pipette invariably slowed endocytosis, even in terminals dialyzed with increased Ca2+ buffer. These results suggest a new role for GABA and Cl− ions in blocking the trigger for fast endocytosis at this ribbon-type synapse. PMID:15504327

  19. Calcineurin is universally involved in vesicle endocytosis at neuronal and nonneuronal secretory cells.

    PubMed

    Wu, Xin-Sheng; Zhang, Zhen; Zhao, Wei-Dong; Wang, Dongsheng; Luo, Fujun; Wu, Ling-Gang

    2014-05-22

    Calcium influx triggers and accelerates endocytosis in nerve terminals and nonneuronal secretory cells. Whether calcium/calmodulin-activated calcineurin, which dephosphorylates endocytic proteins, mediates this process is highly controversial for different cell types, developmental stages, and endocytic forms. Using three preparations that previously produced discrepant results (i.e., large calyx-type synapses, conventional cerebellar synapses, and neuroendocrine chromaffin cells containing large dense-core vesicles), we found that calcineurin gene knockout consistently slowed down endocytosis, regardless of cell type, developmental stage, or endocytic form (rapid or slow). In contrast, calcineurin and calmodulin blockers slowed down endocytosis at a relatively small calcium influx, but did not inhibit endocytosis at a large calcium influx, resulting in false-negative results. These results suggest that calcineurin is universally involved in endocytosis. They may also help explain the discrepancies among previous pharmacological studies. We therefore suggest that calcineurin should be included as a key player in mediating calcium-triggered and -accelerated vesicle endocytosis.

  20. Rapid kinetics of endocytosis at rod photoreceptor synapses depends upon endocytic load and calcium.

    PubMed

    Cork, Karlene M; Thoreson, Wallace B

    2014-05-01

    Release from rods is triggered by the opening of L-type Ca2+ channels that lie beneath synaptic ribbons. After exocytosis, vesicles are retrieved by compensatory endocytosis. Previous work showed that endocytosis is dynamin-dependent in rods but dynamin-independent in cones. We hypothesized that fast endocytosis in rods may also differ from cones in its dependence upon the amount of Ca2+ influx and/or endocytic load. We measured exocytosis and endocytosis from membrane capacitance (C m) changes evoked by depolarizing steps in voltage clamped rods from tiger salamander retinal slices. Similar to cones, the time constant for endocytosis in rods was quite fast, averaging <200 ms. We manipulated Ca2+ influx and the amount of vesicle release by altering the duration and voltage of depolarizing steps. Unlike cones, endocytosis kinetics in rods slowed after increasing Ca2+ channel activation with longer step durations or more strongly depolarized voltage steps. Endocytosis kinetics also slowed as Ca2+ buffering was decreased by replacing BAPTA (10 or 1 mM) with the slower Ca2+ buffer EGTA (5 or 0.5 mM) in the pipette solution. These data provide further evidence that endocytosis mechanisms differ in rods and cones and suggest that endocytosis in rods is regulated by both endocytic load and local Ca2+ levels.

  1. Rapid kinetics of endocytosis at rod photoreceptor synapses depends upon endocytic load and calcium

    PubMed Central

    CORK, KARLENE M.; THORESON, WALLACE B.

    2015-01-01

    Release from rods is triggered by the opening of L-type Ca2+ channels that lie beneath synaptic ribbons. After exocytosis, vesicles are retrieved by compensatory endocytosis. Previous work showed that endocytosis is dynamin-dependent in rods but dynamin-independent in cones. We hypothesized that fast endocytosis in rods may also differ from cones in its dependence upon the amount of Ca2+ influx and/or endocytic load. We measured exocytosis and endocytosis from membrane capacitance (Cm) changes evoked by depolarizing steps in voltage clamped rods from tiger salamander retinal slices. Similar to cones, the time constant for endocytosis in rods was quite fast, averaging <200 ms. We manipulated Ca2+ influx and the amount of vesicle release by altering the duration and voltage of depolarizing steps. Unlike cones, endocytosis kinetics in rods slowed after increasing Ca2+ channel activation with longer step durations or more strongly depolarized voltage steps. Endocytosis kinetics also slowed as Ca2+ buffering was decreased by replacing BAPTA (10 or 1 mM) with the slower Ca2+ buffer EGTA (5 or 0.5 mM) in the pipette solution. These data provide further evidence that endocytosis mechanisms differ in rods and cones and suggest that endocytosis in rods is regulated by both endocytic load and local Ca2+ levels. PMID:24735554

  2. Activity-Dependent Neuronal Model on Complex Networks

    PubMed Central

    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

  3. Bilateral Activity-Dependent Interactions in the Developing Corticospinal System

    PubMed Central

    Friel, Kathleen M.; Martin, John H.

    2009-01-01

    Activity-dependent competition between the corticospinal (CS) systems in each hemisphere drives postnatal development of motor skills and stable CS tract connections with contralateral spinal motor circuits. Unilateral restriction of motor cortex (M1) activity during an early postnatal critical period impairs contralateral visually guided movements later in development and in maturity. Silenced M1 develops aberrant connections with the contralateral spinal cord whereas the initially active M1, in the other hemisphere, develops bilateral connections. In this study, we determined whether the aberrant pattern of CS tract terminations and motor impairments produced by early postnatal M1 activity restriction could be abrogated by reducing activity-dependent synaptic competition from the initially active M1 later in development. We first inactivated M1 unilaterally between postnatal weeks 5–7. We next inactivated M1 on the other side from weeks 7–11 (alternate inactivation), to reduce the competitive advantage that this side may have over the initially inactivated side. Alternate inactivation redirected aberrant contralateral CS tract terminations from the initially silenced M1 to their normal spinal territories and reduced the density of aberrant ipsilateral terminations from the initially active side. Normal movement endpoint control during visually guided locomotion was fully restored. This reorganization of CS terminals reveals an unsuspected late plasticity after the critical period for establishing the pattern of CS terminations in the spinal cord. Our findings show that robust bilateral interactions between the developing CS systems on each side are important for achieving balance between contralateral and ipsilateral CS tract connections and visuomotor control. PMID:17928450

  4. ATP depletion inhibits the endocytosis of ClC-2.

    PubMed

    Dhani, Sonja U; Kim Chiaw, Patrick; Huan, Ling-Jun; Bear, Christine E

    2008-01-01

    The chloride channel, ClC-2 is expressed ubiquitously and participates in multiple physiological processes. In particular, ClC-2 has been implicated in the regulation of neuronal chloride ion homeostasis and mutations in ClC-2 are associated with idiopathic generalized epilepsy. Despite the physiological and pathophysiological significance of this channel, its regulation remains incompletely understood. The functional expression of ClC-2 at the cell surface has been shown to be enhanced by depletion of cellular ATP, implicating its possible role in cellular energy sensing. In the present study, biochemical assays of cell surface expression suggest that this gain of function reflects, in part, an increase in channel number due to the reduction in ClC-2 internalization by endocytosis. Cell surface expression of the disease-causing mutant: G715E, thought to lack wild-type nucleotide binding affinity, is similarly affected, suggesting that ATP-depletion modifies the function of proteins in the endocytic pathway rather than ClC-2 directly. Using a combination of immunofluorescence and biochemical studies, we confirmed that ClC-2 is internalized via dynamin-dependent endocytosis and that the change in surface expression evoked by ATP depletion is partially mimicked by inhibition of dynamin function using a dynamin dominant-negative mutant (DynK44A). Furthermore, trafficking via the early endosomal compartment occurs in part through rab5-associated vesicles and recycling of ClC-2 to the cell surface occurs through a rab11 dependent pathway. In summary, we have determined that the internalization of ClC-2 by endocytosis is inhibited by metabolic stress, highlighting the importance for understanding the molecular mechanisms mediating the endosomal trafficking of this channel. (c) 2007 Wiley-Liss, Inc.

  5. TRIM72 modulates caveolar endocytosis in repair of lung cells.

    PubMed

    Nagre, Nagaraja; Wang, Shaohua; Kellett, Thomas; Kanagasabai, Ragu; Deng, Jing; Nishi, Miyuki; Shilo, Konstantin; Oeckler, Richard A; Yalowich, Jack C; Takeshima, Hiroshi; Christman, John; Hubmayr, Rolf D; Zhao, Xiaoli

    2016-03-01

    Alveolar epithelial and endothelial cell injury is a major feature of the acute respiratory distress syndrome, in particular when in conjunction with ventilation therapies. Previously we showed [Kim SC, Kellett T, Wang S, Nishi M, Nagre N, Zhou B, Flodby P, Shilo K, Ghadiali SN, Takeshima H, Hubmayr RD, Zhao X. Am J Physiol Lung Cell Mol Physiol 307: L449-L459, 2014.] that tripartite motif protein 72 (TRIM72) is essential for amending alveolar epithelial cell injury. Here, we posit that TRIM72 improves cellular integrity through its interaction with caveolin 1 (Cav1). Our data show that, in primary type I alveolar epithelial cells, lack of TRIM72 led to significant reduction of Cav1 at the plasma membrane, accompanied by marked attenuation of caveolar endocytosis. Meanwhile, lentivirus-mediated overexpression of TRIM72 selectively increases caveolar endocytosis in rat lung epithelial cells, suggesting a functional association between these two. Further coimmunoprecipitation assays show that deletion of either functional domain of TRIM72, i.e., RING, B-box, coiled-coil, or PRY-SPRY, abolishes the physical interaction between TRIM72 and Cav1, suggesting that all theoretical domains of TRIM72 are required to forge a strong interaction between these two molecules. Moreover, in vivo studies showed that injurious ventilation-induced lung cell death was significantly increased in knockout (KO) TRIM72(KO) and Cav1(KO) lungs compared with wild-type controls and was particularly pronounced in double KO mutants. Apoptosis was accompanied by accentuation of gross lung injury manifestations in the TRIM72(KO) and Cav1(KO) mice. Our data show that TRIM72 directly and indirectly modulates caveolar endocytosis, an essential process involved in repair of lung epithelial cells through removal of plasma membrane wounds. Given TRIM72's role in endomembrane trafficking and cell repair, we consider this molecule an attractive therapeutic target for patients with injured lungs.

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

    PubMed

    Lin, Yi-Wen; Lin, Hsiang-Yin; Tsou, Yueh-Liang; Chitra, Ebenezer; Hsiao, Kuang-Nan; Shao, Hsiao-Yun; Liu, Chia-Chyi; Sia, Charles; Chong, Pele; Chow, Yen-Hung

    2012-01-01

    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.

  7. Cargo recognition during clathrin-mediated endocytosis: a team effort.

    PubMed

    Sorkin, Alexander

    2004-08-01

    Transmembrane proteins destined to endosomes are selectively accumulated in clathrin-coated pits at the plasma membrane and rapidly internalized in clathrin-coated vesicles. The recognition of specific sequence motifs in transmembrane cargo by coated-pit proteins confers specificity on the endocytic process. Interaction of membrane cargo with the clathrin adaptor protein complex AP-2 is the major mechanism of cargo sorting into coated pits in mammalian cells. Recent studies have revealed a variety of alternative mechanisms of cargo recruitment involving additional adaptor proteins. These alternative mechanisms appear to be particularly important during clathrin-mediated endocytosis of signaling receptors.

  8. Systems survey of endocytosis by multiparametric image analysis.

    PubMed

    Collinet, Claudio; Stöter, Martin; Bradshaw, Charles R; Samusik, Nikolay; Rink, Jochen C; Kenski, Denise; Habermann, Bianca; Buchholz, Frank; Henschel, Robert; Mueller, Matthias S; Nagel, Wolfgang E; Fava, Eugenio; Kalaidzidis, Yannis; Zerial, Marino

    2010-03-11

    Endocytosis is a complex process fulfilling many cellular and developmental functions. Understanding how it is regulated and integrated with other cellular processes requires a comprehensive analysis of its molecular constituents and general design principles. Here, we developed a new strategy to phenotypically profile the human genome with respect to transferrin (TF) and epidermal growth factor (EGF) endocytosis by combining RNA interference, automated high-resolution confocal microscopy, quantitative multiparametric image analysis and high-performance computing. We identified several novel components of endocytic trafficking, including genes implicated in human diseases. We found that signalling pathways such as Wnt, integrin/cell adhesion, transforming growth factor (TGF)-beta and Notch regulate the endocytic system, and identified new genes involved in cargo sorting to a subset of signalling endosomes. A systems analysis by Bayesian networks further showed that the number, size, concentration of cargo and intracellular position of endosomes are not determined randomly but are subject to specific regulation, thus uncovering novel properties of the endocytic system.

  9. Hepatitis C virus entry depends on clathrin-mediated endocytosis.

    PubMed

    Blanchard, Emmanuelle; Belouzard, Sandrine; Goueslain, Lucie; Wakita, Takaji; Dubuisson, Jean; Wychowski, Czeslaw; Rouillé, Yves

    2006-07-01

    Due to difficulties in cell culture propagation, the mechanisms of hepatitis C virus (HCV) entry are poorly understood. Here, postbinding cellular mechanisms of HCV entry were studied using both retroviral particles pseudotyped with HCV envelope glycoproteins (HCVpp) and the HCV clone JFH-1 propagated in cell culture (HCVcc). HCVpp entry was measured by quantitative real-time PCR after 3 h of contact with target cells, and HCVcc infection was quantified by immunoblot analysis and immunofluorescence detection of HCV proteins expressed in infected cells. The functional role of clathrin-mediated endocytosis in HCV entry was assessed by small interfering RNA-mediated clathrin heavy chain depletion and with chlorpromazine, an inhibitor of clathrin-coated pit formation at the plasma membrane. In both conditions, HCVpp entry and HCVcc infection were inhibited. HCVcc infection was also inhibited by pretreating target cells with bafilomycin A1 or chloroquine, two drugs known to interfere with endosome acidification. These data indicate that HCV enters target cells by clathrin-mediated endocytosis, followed by a fusion step from within an acidic endosomal compartment.

  10. Ebola Virus Uses Clathrin Mediated Endocytosis as an Entry Pathway

    PubMed Central

    Bhattacharyya, Suchita; Warfield, Kelly L.; Ruthel, Gordon; Bavari, Sina; Aman, M. Javad; Hope, Thomas J.

    2011-01-01

    Ebola virus (EBOV) infects several cell types and while viral entry is known to be pH dependent; the exact entry pathway(s) remains unknown. To gain insights into EBOV entry, the role of several inhibitors of clathrin-mediated endocytosis in blocking infection mediated by HIV pseudotyped with the EBOV envelope glycoprotein (EbGP) was examined. Wild type HIV and envelope-minus HIV pseudotyped with Vesicular Stomatitis Virus glycoprotein (VSVg) were used as controls to assess cell viability after inhibiting clathrin pathway. Inhibition of clathrin pathway using dominant-negative Eps15; siRNA-mediated knockdown of clathrin heavy chain; chlorpromazine and sucrose blocked EbGP pseudotyped HIV infection. Also, both chlorpromazine and Bafilomycin A1 inhibited entry of infectious EBOV. Sensitivity of EbGP pseudotyped HIV as well as infectious EBOV to inhibitors of clathrin suggests that EBOV uses clathrin-mediated endocytosis as an entry pathway. Furthermore, since chlorpromazine inhibits EBOV infection, novel therapeutic modalities could be designed based on this lead compound. PMID:20202662

  11. Effects of Endosomal Photodamage on Membrane Recycling and Endocytosis

    PubMed Central

    Kessel, David; Santiago, Ann Marie; Andrzejak, Michelle

    2011-01-01

    The flux of receptor-independent endocytosis can be estimated by addition of wortmannin to cell cultures. Membrane influx is unaffected but traffic out of late endosomes is impaired, resulting in a substantial enlargement of these organelles. Using the 1c1c7 murine hepatoma, we investigated the effect of endosomal photodamage on this endocytic pathway. We previously reported that photodamage catalyzed by the lysosomal photosensitizer NPe6 prevented wortmannin-induced endosomal swelling, indicating an earlier block in the process. In this study, we show that endosomal photodamage, initiated by photodamage from an asymmetrically-substituted porphine or a phthalocyanine, also prevents wortmannin-induced endosomal swelling, even when the PDT dose is insufficient to cause endosomal disruption. As the PDT dose is increased, endosomal breakage occurs, as does apoptosis and cell death. Very high PDT doses result in necrosis. We propose that photodamage to endosomes results in alterations in the endosomal structure such that influx of new material is inhibited and receptor-independent endocytosis is prevented. In an additional series of studies, we found that the swollen late endosomes induced by wortmannin are unable to retain previously accumulated fluorescent probes or photosensitizers. PMID:21208213

  12. Membrane Mechanics of Endocytosis in Cells with Turgor

    PubMed Central

    Dmitrieff, Serge; Nédélec, François

    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 deformations, 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 constriction by actin may not be required, while its pulling role is essential. Finally, the theory shows that anisotropic curvature effectors stabilize membrane invaginations, and the loss of crescent-shaped BAR domain proteins such as Rvs167 could therefore trigger membrane scission. PMID:26517669

  13. Shank2 redistributes with NaPilla during regulated endocytosis

    PubMed Central

    Dobrinskikh, Evgenia; Giral, Hector; Caldas, Yupanqui A.; Levi, Moshe

    2010-01-01

    Serum phosphate levels are acutely impacted by the abundance of sodium-phosphate cotransporter IIa (NaPiIIa) in the apical membrane of renal proximal tubule cells. PSD-95/Disks Large/Zonula Occludens (PDZ) domain-containing proteins bind NaPiIIa and likely contribute to the delivery, retention, recovery, and trafficking of NaPiIIa. Shank2 is a distinctive PDZ domain protein that binds NaPiIIa. Its role in regulating NaPiIIa activity, distribution, and abundance is unknown. In the present in vivo study, rats were maintained on a low-phosphate diet, and then plasma phosphate levels were acutely elevated by high-phosphate feeding to induce the recovery, endocytosis, and degradation of NaPiIIa. Western blot analysis of renal cortical tissue from rats given high-phosphate feed showed NaPiIIa and Shank2 underwent degradation. Quantitative immunofluorescence analyses, including microvillar versus intracellular intensity ratios and intensity correlation quotients, showed that Shank2 redistributed with NaPiIIa during the time course of NaPiIIa endocytosis. Furthermore, NaPiIIa and Shank2 trafficked through distinct endosomal compartments (clathrin, early endosomes, lysosomes) with the same temporal pattern. These in vivo findings indicate that Shank2 is positioned to coordinate the regulated endocytic retrieval and downregulation of NaPiIIa in rat renal proximal tubule cells. PMID:20810910

  14. Membrane Mechanics of Endocytosis in Cells with Turgor.

    PubMed

    Dmitrieff, Serge; Nédélec, François

    2015-10-01

    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 deformations, 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 constriction by actin may not be required, while its pulling role is essential. Finally, the theory shows that anisotropic curvature effectors stabilize membrane invaginations, and the loss of crescent-shaped BAR domain proteins such as Rvs167 could therefore trigger membrane scission.

  15. Lysosomal trafficking of TGFBIp via caveolae-mediated endocytosis.

    PubMed

    Choi, Seung-Il; Maeng, Yong-Sun; Kim, Tae-Im; Lee, Yangsin; Kim, Yong-Sun; Kim, Eung Kweon

    2015-01-01

    Transforming growth factor-beta-induced protein (TGFBIp) is ubiquitously expressed in the extracellular matrix (ECM) of various tissues and cell lines. Progressive accumulation of mutant TGFBIp is directly involved in the pathogenesis of TGFBI-linked corneal dystrophy. Recent studies reported that mutant TGFBIp accumulates in cells; however, the trafficking of TGFBIp is poorly understood. Therefore, we investigated TGFBIp trafficking to determine the route of its internalization and secretion and to elucidate its roles in the pathogenesis of granular corneal dystrophy type 2 (GCD2). Our data indicate that newly synthesized TGFBIp was secreted via the endoplasmic reticulum/Golgi-dependent secretory pathway, and this secretion was delayed in the corneal fibroblasts of patients with GCD2. We also found that TGFBIp was internalized by caveolae-mediated endocytosis, and the internalized TGFBIp accumulated after treatment with bafilomycin A1, an inhibitor of lysosomal degradation. In addition, the proteasome inhibitor MG132 inhibits the endocytosis of TGFBIp. Co-immunoprecipitation revealed that TGFBIp interacted with integrin αVβ3. Moreover, treatment with arginine-glycine-aspartic acid (RGD) tripeptide suppressed the internalization of TGFBIp. These insights on TGFBIp trafficking could lead to the identification of novel targets and the development of new therapies for TGFBI-linked corneal dystrophy.

  16. Spatial encoding of cyclic AMP signalling specificity by GPCR endocytosis

    PubMed Central

    Tsvetanova, Nikoleta G.; von Zastrow, Mark

    2014-01-01

    G protein-coupled receptors (GPCRs) are well known to signal via cyclic AMP (cAMP) production at the plasma membrane, but it is now clear that various GPCRs also signal after internalization. Apart from its temporal impact through prolonging the cellular response, does the endosome-initiated signal encode any discrete spatial information? Using the beta2-adrenoceptor (β2-AR) as a model, we show that endocytosis is required for the full repertoire of downstream cAMP-dependent transcriptional control. Next, we describe an orthogonal optogenetic approach to definitively establish that the location of cAMP production is indeed the critical variable determining the transcriptional response. Finally, our results suggest that this spatial encoding scheme helps cells functionally discriminate chemically distinct β2-AR ligands according to differences in their ability to promote receptor endocytosis. These findings reveal a discrete principle for achieving cellular signalling specificity, based on endosome-mediated spatial encoding of intracellular second messenger production and ‘location aware’ downstream transcriptional control. PMID:25362359

  17. Transferrin: Endocytosis and Cell Signaling in Parasitic Protozoa

    PubMed Central

    Serrano-Luna, Jesús

    2015-01-01

    Iron is the fourth most abundant element on Earth and the most abundant metal in the human body. This element is crucial for life because almost all organisms need iron for several biological activities. This is the case with pathogenic organisms, which are at the vanguard in the battle with the human host for iron. The latest regulates Fe concentration through several iron-containing proteins, such as transferrin. The transferrin receptor transports iron to each cell that needs it and maintains it away from pathogens. Parasites have developed several strategies to obtain iron as the expression of specific transferrin receptors localized on plasma membrane, internalized through endocytosis. Signal transduction pathways related to the activation of the receptor have functional importance in proliferation. The study of transferrin receptors and other proteins with action in the signaling networks is important because these proteins could be used as therapeutic targets due to their specificity or to differences with the human counterpart. In this work, we describe proteins that participate in signal transduction processes, especially those that involve transferrin endocytosis, and we compare these processes with those found in T. brucei, T. cruzi, Leishmania spp., and E. histolytica parasites. PMID:26090431

  18. Ricin A chain reaches the endoplasmic reticulum after endocytosis

    SciTech Connect

    Liu Qiong; Zhan Jinbiao . E-mail: jzhan2k@zju.edu.cn; Chen Xinhong; Zheng Shu

    2006-05-12

    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.

  19. Dynamin, a GTPase involved in the initial stages of endocytosis.

    PubMed

    Vallee, R B; Herskovits, J S; Aghajanian, J G; Burgess, C C; Shpetner, H S

    1993-01-01

    Dynamin is a high molecular mass (100 kDa) GTPase which binds to and co-purifies with microtubules. Molecular cloning of rat brain dynamin has revealed the three well-established consensus sequence elements for GTP binding within the N-terminal third of the protein, as well as sequence similarity within this region to the interferon-inducible antiviral Mx proteins, the product of the yeast membrane sorting gene VPS1, and the product of the yeast mitochondrial replication gene MGM1. More extensive sequence similarity between rat dynamin and the product of the Drosophila gene shibire, which is involved in endocytosis, has also been found. In in vitro assays microtubules strongly stimulate the dynamin GTPase. This effect can be reversed by removal of the dynamin C-terminus using papain, which abolishes microtubule binding. Overexpression of mutant forms of dynamin in vivo using Cos-7 cells inhibits transferrin uptake and alters the distribution of clathrin and of alpha-adaptin, but not gamma-adaptin. Deletion of the C-terminus of mutant forms of dynamin abolishes these effects. Together these results suggest a critical role for dynamin in the early stages of endocytosis. It is uncertain whether microtubules interact with dynamin in vivo or whether the in vitro effects of microtubules mimic the effects of other regulatory elements in vivo.

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

    PubMed Central

    Flores, Carmen E.; Méndez, Pablo

    2014-01-01

    Inhibitory transmission through the neurotransmitter γ-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. PMID:25386117

  1. Occipital TMS has an activity-dependent suppressive effect

    PubMed Central

    Perini, Francesca; Cattaneo, Luigi; Carrasco, Marisa; Schwarzbach, Jens V.

    2012-01-01

    The effects of transcranial magnetic stimulation (TMS) vary depending on the brain state at the stimulation moment. Four mechanisms have been proposed to underlie these effects: (i) virtual lesion–TMS suppresses neural signals; (ii) preferential activation of less active neurons–TMS drives up activity in the stimulated area, but active neurons are saturating, (iii) noise generation–TMS adds random neuronal activity and its effect interacts with stimulus-intensity; (iv) noise generation–TMS adds random neuronal activity and its effect depends on TMS-intensity. Here we explore these hypotheses by investigating the effects of TMS on early visual cortex on the contrast response function while varying adaptation state of the observers. We tested human participants in an orientation discrimination task, in which performance is contingent upon contrast sensitivity. Before each trial, neuronal activation of visual cortex was altered through contrast adaptation to two flickering gratings. In a factorial design, with or without adaptation, a single TMS pulse was delivered simultaneously with targets of varying contrast. Adaptation decreased contrast sensitivity. The effect of TMS on performance was state-dependent: TMS decreased contrast sensitivity in the absence of adaptation but increased it after adaptation. None of the proposed mechanisms can account for the results in their entirety, in particular, for the facilitatory effect at intermediate to high contrasts after adaptation. We propose an alternative hypothesis: TMS effects are activity-dependent, so that TMS suppresses the most active neurons and thereby changes the balance between excitation and inhibition. PMID:22956826

  2. Solar activity dependence of nightside aurora in winter conditions

    NASA Astrophysics Data System (ADS)

    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.

  3. MT1-MMP regulates the turnover and endocytosis of extracellular matrix fibronectin.

    PubMed

    Shi, Feng; Sottile, Jane

    2011-12-01

    The extracellular matrix (ECM) is dynamically remodeled by cells during development, normal tissue homeostasis and in a variety of disease processes. We previously showed that fibronectin is an important regulator of ECM remodeling. The deposition and/or polymerization of fibronectin into the ECM controls the deposition and stability of other ECM molecules. In addition, agents that inhibit fibronectin polymerization promote the turnover of fibronectin fibrils and enhance ECM fibronectin endocytosis and intracellular degradation. Endocytosis of ECM fibronectin is regulated by β1 integrins, including α5β1 integrin. We have examined the role of extracellular proteases in regulating ECM fibronectin turnover. Our data show that membrane type matrix metalloproteinase 1 (MT1-MMP; also known as MMP14) is a crucial regulator of fibronectin turnover. Cells lacking MT1-MMP show reduced turnover and endocytosis of ECM fibronectin. MT1-MMP regulates ECM fibronectin remodeling by promoting extracellular cleavage of fibronectin and by regulating α5β1-integrin endocytosis. Our data also show that fibronectin polymerization stabilizes fibronectin fibrils and inhibits ECM fibronectin endocytosis by inhibiting α5β1-integrin endocytosis. These data are the first to show that an ECM protein and its modifying enzyme can regulate integrin endocytosis. These data also show that integrin trafficking plays a major role in modulating ECM fibronectin remodeling. The dual dependence of ECM fibronectin turnover on extracellular proteolysis and endocytosis highlights the complex regulatory mechanisms that control ECM remodeling to ensure maintenance of proper tissue function.

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

    PubMed

    Röhrl, Clemens; Eigner, Karin; Fruhwürth, Stefanie; Stangl, Herbert

    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 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.

  5. Notch Ligand Endocytosis Generates Mechanical Pulling Force Dependent on Dynamin, Epsins and Actin

    PubMed Central

    Meloty-Kapella, Laurence; Shergill, Bhupinder; Kuon, Jane; Botvinick, Elliot; Weinmaster, Gerry

    2012-01-01

    SUMMARY Notch signaling induced by cell surface ligands is critical to development and maintenance of many eukaryotic organisms. Notch and its ligands are integral membrane proteins that facilitate direct cell-cell interactions to activate Notch proteolysis and release the intracellular domain that directs Notch-specific cellular responses. Genetic studies suggest Notch ligands require endocytosis, ubiquitylation and epsin endocytic adaptors to activate signaling, yet the exact role ligand endocytosis serves remains unresolved. Here we characterize a molecularly distinct mode of clathrin-mediated endocytosis requiring ligand ubiquitylation, epsins and actin for ligand cells to activate signaling in Notch cells. Using a cell-bead optical tweezers system, we obtained evidence for cell-mediated mechanical force dependent on this distinct mode of ligand endocytosis. We propose mechanical pulling force produced by endocytosis of Notch-bound ligand drives conformational changes in Notch that permit activating proteolysis. PMID:22658936

  6. Adapting for endocytosis: roles for endocytic sorting adaptors in directing neural development

    PubMed Central

    Yap, Chan Choo; Winckler, Bettina

    2015-01-01

    Proper cortical development depends on the orchestrated actions of a multitude of guidance receptors and adhesion molecules and their downstream signaling. The levels of these receptors on the surface and their precise locations can greatly affect guidance outcomes. Trafficking of receptors to a particular surface locale and removal by endocytosis thus feed crucially into the final guidance outcomes. In addition, endocytosis of receptors can affect downstream signaling (both quantitatively and qualitatively) and regulated endocytosis of guidance receptors is thus an important component of ensuring proper neural development. We will discuss the cell biology of regulated endocytosis and the impact on neural development. We focus our discussion on endocytic accessory proteins (EAPs) (such as numb and disabled) and how they regulate endocytosis and subsequent post-endocytic trafficking of their cognate receptors (such as Notch, TrkB, β-APP, VLDLR, and ApoER2). PMID:25904845

  7. Visualizing the endocytosis of phenylephrine in living cells by quantum dot-based tracking.

    PubMed

    Ma, Jing; Wu, Lina; Hou, Zhun; Song, Yao; Wang, Lei; Jiang, Wei

    2014-08-01

    To study the intracellular receptor-drug transportation, a fluorescent probe consisting of phenylephrine-polyethylene glycol-quantum dots conjugate was employed to track endocytosis process of phenylephrine in living cells. This type of movement was studied by continuously filming fluorescent images in the same cell. We also calculated the movement parameters, and divided the endocytosis process into 6 stages. Furthermore, the movement parameters of this probe in different organelles were determined by co-localization of the probe fluorescent images and different cellular organelles. After comparing the parameters in cellular organelles with these in 6 stages, the whole endocytosis pathway was demonstrated. These results verified that this probe successfully tracked the whole intracellular dynamic endocytosis process of phenylephrine. Our method realized the visual tracking the whole receptor-mediated endocytosis, which is a new approach on investigating the molecular mechanisms and kinetic properties of intracellular receptor-drug transportation.

  8. Adapting for endocytosis: roles for endocytic sorting adaptors in directing neural development.

    PubMed

    Yap, Chan Choo; Winckler, Bettina

    2015-01-01

    Proper cortical development depends on the orchestrated actions of a multitude of guidance receptors and adhesion molecules and their downstream signaling. The levels of these receptors on the surface and their precise locations can greatly affect guidance outcomes. Trafficking of receptors to a particular surface locale and removal by endocytosis thus feed crucially into the final guidance outcomes. In addition, endocytosis of receptors can affect downstream signaling (both quantitatively and qualitatively) and regulated endocytosis of guidance receptors is thus an important component of ensuring proper neural development. We will discuss the cell biology of regulated endocytosis and the impact on neural development. We focus our discussion on endocytic accessory proteins (EAPs) (such as numb and disabled) and how they regulate endocytosis and subsequent post-endocytic trafficking of their cognate receptors (such as Notch, TrkB, β-APP, VLDLR, and ApoER2).

  9. Bulk Nanostructured Materials

    NASA Astrophysics Data System (ADS)

    Koch, C. C.; Langdon, T. G.; Lavernia, E. J.

    2017-09-01

    This paper will address three topics of importance to bulk nanostructured materials. Bulk nanostructured materials are defined as bulk solids with nanoscale or partly nanoscale microstructures. This category of nanostructured materials has historical roots going back many decades but has relatively recent focus due to new discoveries of unique properties of some nanoscale materials. Bulk nanostructured materials are prepared by a variety of severe plastic deformation methods, and these will be reviewed. Powder processing to prepare bulk nanostructured materials requires that the powders be consolidated by typical combinations of pressure and temperature, the latter leading to coarsening of the microstructure. The thermal stability of nanostructured materials will also be discussed. An example of bringing nanostructured materials to applications as structural materials will be described in terms of the cryomilling of powders and their consolidation.

  10. Dynamic caveolae exclude bulk membrane proteins and are required for sorting of excess glycosphingolipids

    PubMed Central

    Shvets, Elena; Bitsikas, Vassilis; Howard, Gillian; Hansen, Carsten Gram; Nichols, Benjamin J.

    2015-01-01

    Caveolae have long been implicated in endocytosis. Recent data question this link, and in the absence of specific cargoes the potential cellular function of caveolar endocytosis remains unclear. Here we develop new tools, including doubly genome-edited cell lines, to assay the subcellular dynamics of caveolae using tagged proteins expressed at endogenous levels. We find that around 5% of the cellular pool of caveolae is present on dynamic endosomes, and is delivered to endosomes in a clathrin-independent manner. Furthermore, we show that caveolae are indeed likely to bud directly from the plasma membrane. Using a genetically encoded tag for electron microscopy and ratiometric light microscopy, we go on to show that bulk membrane proteins are depleted within caveolae. Although caveolae are likely to account for only a small proportion of total endocytosis, cells lacking caveolae show fundamentally altered patterns of membrane traffic when loaded with excess glycosphingolipid. Altogether, these observations support the hypothesis that caveolar endocytosis is specialized for transport of membrane lipid. PMID:25897946

  11. The sequence NPFXD defines a new class of endocytosis signal in Saccharomyces cerevisiae

    PubMed Central

    1996-01-01

    The yeast membrane protein Kex2p uses a tyrosine-containing motif within the cytoplasmic domain for localization to a late Golgi compartment. Because Golgi membrane proteins mislocalized to the plasma membrane in yeast can undergo endocytosis, we examined whether the Golgi localization sequence or other sequences in the Kex2p cytoplasmic domain mediate endocytosis. To assess endocytic function, the Kex2p cytoplasmic domain was fused to an endocytosis-defective form of the alpha-factor receptor. Ste2p. Like intact Ste2p, the chimeric protein, Stex22p, undergoes rapid endocytosis that is dependent on clathrin and End3p. Uptake of Stex22p does not require the Kex2p Golgi localization motif. Instead, the sequence NPFSD, located 37 amino acids from the COOH terminus, is essential for Stex22p endocytosis. Internalization was abolished when the N, P, or F residues were converted to alanine and severely impaired upon conversion of D to A. NPFSD restored uptake when added to the COOH terminus of an endocytosis-defective Ste2p chimera lacking lysine-based endocytosis signals present in wild-type Ste2p. An NPF sequence is present in the cytoplasmic domain of the a- factor receptor, Ste3p. Mutation of this sequence prevented pheromone- stimulated endocytosis of a truncated form of Ste3p. Our results identify NPFSD as a clathrin-dependent endocytosis signal that is distinct from the aromatic amino acid-containing Golgi localization motif and lysine-based, ubiquitin-dependent endocytosis signals in yeast. PMID:8991091

  12. Solar activity dependence of the topside ionosphere at low latitudes

    NASA Astrophysics Data System (ADS)

    Chen, Yiding; Liu, Libo; Wan, Weixing; Yue, Xinan; Su, Shin-Yi

    2009-08-01

    We investigated the solar activity dependence of the topside ionosphere with ROCSAT-1 observations. The distribution of the plasma density at 600 km altitude shows features with considerable local time, season, and solar activity differences. In the daytime, plasma density peaks around the dip equator. This peak is more distinct in equinoxes and weaker in May-July, and it enhances with solar activity in all seasons. The seasonal behavior of this peak is primarily controlled by the seasonal variations of neutral density and E × B vertical drift. The enhancement of the peak with solar activity is related to the effect of E × B vertical drift. Around sunset, double peaks are found in the latitudinal distribution of plasma density in solar maximum equinoxes and December solstice, which are mainly attributed to the effects of strong prereversal enhancement (PRE) vertical drift. Moreover, the plasma density at 600 km altitude strongly depends on the solar proxy P = (F 107 + F 107A)/2. At higher altitudes, e.g., 800 km, the amplification trend prevails in the solar activity variations of plasma density. In contrast, the plasma density at 600 km altitude presents three kinds of patterns (linear, amplification, and saturation), which has not been reported. Saturation effect is found at equinox sunset around the dip equator. This saturation effect is attributed to the increase in the PRE vertical drift with solar activity. Solar activity effects of ROCSAT-1 plasma density are argued to be the combined effects induced by the changes in the peak height, the scale height, and the peak electron density, respectively. Among these factors, the rise of the F 2 peak is more important for the equatorial plasma density at 600 km altitude.

  13. Retinal Wave Behavior through Activity-Dependent Refractory Periods

    PubMed Central

    Godfrey, Keith B; Swindale, Nicholas V

    2007-01-01

    In the developing mammalian visual system, spontaneous retinal ganglion cell (RGC) activity contributes to and drives several aspects of visual system organization. This spontaneous activity takes the form of spreading patches of synchronized bursting that slowly advance across portions of the retina. These patches are non-repeating and tile the retina in minutes. Several transmitter systems are known to be involved, but the basic mechanism underlying wave production is still not well-understood. We present a model for retinal waves that focuses on acetylcholine mediated waves but whose principles are adaptable to other developmental stages. Its assumptions are that a) spontaneous depolarizations of amacrine cells drive wave activity; b) amacrine cells are locally connected, and c) cells receiving more input during their depolarization are subsequently less responsive and have longer periods between spontaneous depolarizations. The resulting model produces waves with non-repeating borders and randomly distributed initiation points. The wave generation mechanism appears to be chaotic and does not require neural noise to produce this wave behavior. Variations in parameter settings allow the model to produce waves that are similar in size, frequency, and velocity to those observed in several species. Our results suggest that retinal wave behavior results from activity-dependent refractory periods and that the average velocity of retinal waves depends on the duration a cell is excitatory: longer periods of excitation result in slower waves. In contrast to previous studies, we find that a single layer of cells is sufficient for wave generation. The principles described here are very general and may be adaptable to the description of spontaneous wave activity in other areas of the nervous system. PMID:18052546

  14. Large area bulk superconductors

    DOEpatents

    Miller, Dean J.; Field, Michael B.

    2002-01-01

    A bulk superconductor having a thickness of not less than about 100 microns is carried by a polycrystalline textured substrate having misorientation angles at the surface thereof not greater than about 15.degree.; the bulk superconductor may have a thickness of not less than about 100 microns and a surface area of not less than about 50 cm.sup.2. The textured substrate may have a thickness not less than about 10 microns and misorientation angles at the surface thereof not greater than about 15.degree.. Also disclosed is a process of manufacturing the bulk superconductor and the polycrystalline biaxially textured substrate material.

  15. Endocytosis and Recycling of Tight Junction Proteins in Inflammation

    PubMed Central

    Utech, Markus; Mennigen, Rudolf; Bruewer, Matthias

    2010-01-01

    A critical function of the epithelial lining is to form a barrier that separates luminal contents from the underlying interstitium. This barrier function is primarily regulated by the apical junctional complex (AJC) consisting of tight junctions (TJs) and adherens junctions (AJs) and is compromised under inflammatory conditions. In intestinal epithelial cells, proinflammatory cytokines, for example, interferon-gamma (IFN-γ), induce internalization of TJ proteins by endocytosis. Endocytosed TJ proteins are passed into early and recycling endosomes, suggesting the involvement of recycling of internalized TJ proteins. This review summarizes mechanisms by which TJ proteins under inflammatory conditions are internalized in intestinal epithelial cells and point out comparable mechanism in nonintestinal epithelial cells. PMID:20011071

  16. Imaging and Modeling the Dynamics of Clathrin-Mediated Endocytosis

    PubMed Central

    Mettlen, Marcel

    2014-01-01

    Clathrin-mediated endocytosis (CME) plays a central role in cellular homeostasis and is mediated by clathrin-coated pits (CCPs). Live-cell imaging has revealed a remarkable heterogeneity in CCP assembly kinetics, which can be used as an intrinsic source of mechanistic information on CCP regulation but also poses several major problems for unbiased analysis of CME dynamics. The backbone of unveiling the molecular control of CME is an imaging-based inventory of the full diversity of individual CCP behaviors, which requires detection and tracking of structural fiduciaries and regulatory proteins with an accuracy of >99.9%, despite very low signals. This level of confidence can only be achieved by combining appropriate imaging modalities with self-diagnostic computational algorithms for image analysis and data mining. PMID:25167858

  17. Endocytosis of simian virus 40 into the endoplasmic reticulum

    SciTech Connect

    Kartenbeck, J.; Stukenbrok, H.; Helenius, A. )

    1989-12-01

    The endocytosis of SV-40 into CV-1 cells we studied using biochemical and ultrastructural techniques. The half-time of binding of ({sup 35}S)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.

  18. Ikarugamycin: A Natural Product Inhibitor of Clathrin-Mediated Endocytosis

    PubMed Central

    Elkin, Sarah R.; Oswald, Nathaniel W.; Reed, Dana K.; Mettlen, Marcel; MacMillan, John B.; Schmid, Sandra L.

    2017-01-01

    Ikarugamycin (IKA) is a previously discovered antibiotic, which has been shown to inhibit the uptake of oxidized low-density lipoproteins in macrophages. Furthermore, several groups have previously used IKA to inhibit clathrin-mediated endocytosis (CME) in plant cell lines. However, detailed characterization of IKA has yet to be performed. Consequently, we performed biochemistry and microscopy experiments to further characterize the effects of IKA on CME. We show that IKA has an IC50 of 2.7 µm in H1299 cells and acutely inhibits CME, but not other endocytic pathways, in a panel of cell lines. Although long-term incubation with IKA has cytotoxic effects, the short-term inhibitory effects on CME are reversible. Thus, IKA can be a useful tool for probing routes of endocytic trafficking. PMID:27392092

  19. Endocytosis of simian virus 40 into the endoplasmic reticulum

    PubMed Central

    1989-01-01

    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. PMID:2556405

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

    SciTech Connect

    Tomoda, H.; Kishimoto, Y.; Lee, Y.C. )

    1989-09-15

    Endocytosis of 125I-mannose-bovine serum albumin (BSA) and exocytosis of {sup 125}I-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 {sup 125}I-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. {sup 125}I-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 {sup 125}I-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 {sup 125}I-labeled ligand is quite different from that of endocytosis, suggesting that the two processes are controlled by different mechanisms. Stimulation of secretion of preloaded {sup 125}I-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 {sup 125}I-mannose-N-acetyl-poly-D-lysine from lysosomes.

  1. Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1.

    PubMed

    Renigunta, Vijay; Fischer, Thomas; Zuzarte, Marylou; Kling, Stefan; Zou, Xinle; Siebert, Kai; Limberg, Maren M; Rinné, Susanne; Decher, Niels; Schlichthörl, Günter; Daut, Jürgen

    2014-06-15

    The endosomal SNARE protein syntaxin-8 interacts with the acid-sensitive potassium channel TASK-1. The functional relevance of this interaction was studied by heterologous expression of these proteins (and mutants thereof) in Xenopus oocytes and in mammalian cell lines. Coexpression of syntaxin-8 caused a fourfold reduction in TASK-1 current, a corresponding reduction in the expression of TASK-1 at the cell surface, and a marked increase in the rate of endocytosis of the channel. TASK-1 and syntaxin-8 colocalized in the early endosomal compartment, as indicated by the endosomal markers 2xFYVE and rab5. The stimulatory effect of the SNARE protein on the endocytosis of the channel was abolished when both an endocytosis signal in TASK-1 and an endocytosis signal in syntaxin-8 were mutated. A syntaxin-8 mutant that cannot assemble with other SNARE proteins had virtually the same effect as wild-type syntaxin-8. Total internal reflection fluorescence microscopy showed formation and endocytosis of vesicles containing fluorescence-tagged clathrin, TASK-1, and/or syntaxin-8. Our results suggest that the unassembled form of syntaxin-8 and the potassium channel TASK-1 are internalized via clathrin-mediated endocytosis in a cooperative manner. This implies that syntaxin-8 regulates the endocytosis of TASK-1. Our study supports the idea that endosomal SNARE proteins can have functions unrelated to membrane fusion.

  2. Endocytosis and membrane receptor internalization: implication of F-BAR protein Carom.

    PubMed

    Xu, Yanjie; Xia, Jixiang; Liu, Suxuan; Stein, Sam; Ramon, Cueto; Xi, Hang; Wang, Luqiao; Xiong, Xinyu; Zhang, Lixiao; He, Dingwen; Yang, William; Zhao, Xianxian; Cheng, Xiaoshu; Yang, Xiaofeng; Wang, Hong

    2017-03-01

    Endocytosis is a cellular process mostly responsible for membrane receptor internalization. Cell membrane receptors bind to their ligands and form a complex which can be internalized. We previously proposed that F-BAR protein initiates membrane curvature and mediates endocytosis via its binding partners. However, F-BAR protein partners involved in membrane receptor endocytosis and the regulatory mechanism remain unknown. In this study, we established database mining strategies to explore mechanisms underlying receptor-related endocytosis. We identified 34 endocytic membrane receptors and 10 regulating proteins in clathrin-dependent endocytosis (CDE), a major process of membrane receptor internalization. We found that F-BAR protein FCHSD2 (Carom) may facilitate endocytosis via 9 endocytic partners. Carom is highly expressed, along with highly expressed endocytic membrane receptors and partners, in endothelial cells and macrophages. We established 3 models of Carom-receptor complexes and their intracellular trafficking based on protein interaction and subcellular localization. We conclude that Carom may mediate receptor endocytosis and transport endocytic receptors to the cytoplasm for receptor signaling and lysosome/proteasome degradation, or to the nucleus for RNA processing, gene transcription and DNA repair.

  3. Isoform-specific monoubiquitination, endocytosis, and degradation of alternatively spliced ErbB4 isoforms.

    PubMed

    Sundvall, Maria; Korhonen, Anna; Paatero, Ilkka; Gaudio, Eugenio; Melino, Gerry; Croce, Carlo M; Aqeilan, Rami I; Elenius, Klaus

    2008-03-18

    Endocytosis and subsequent lysosomal degradation serve as a well characterized mechanism to fine-tune and down-regulate EGFR signaling. However, other members of the EGFR/ErbB receptor family have been reported to be endocytosis-impaired. Here we demonstrate that endocytosis of ErbB4 is regulated in an isoform-specific manner: CYT-1 isoforms were efficiently endocytosed whereas CYT-2 isoforms were endocytosis-impaired. CYT-1 isoforms in endocytic vesicles colocalized with Rab5 and Rab7 indicating trafficking via early endosomes to late endosomal/lysosomal structures. A PPXY motif within the CYT-1-specific sequence that lacks from CYT-2 was necessary both for ubiquitination and endocytosis of CYT-1 isoforms and provided a binding site for a WW domain-containing ubiquitin ligase Itch. Itch catalyzed ubiquitination of ErbB4 CYT-1, promoted its localization into intracellular vesicles, and stimulated degradation of ErbB4 CYT-1. Dominant negative Itch suppressed ErbB4 CYT-1 endocytosis and degradation. These data indicate that ErbB4 isoforms differ in endocytosis and degradation by a mechanism mediated by CYT-1-specific PPXY motif interacting with a WW domain-containing E3 ubiquitin ligase.

  4. Ezetimibe-sensitive cholesterol uptake by NPC1L1 protein does not require endocytosis

    PubMed Central

    Johnson, Tory A.; Pfeffer, Suzanne R.

    2016-01-01

    Human NPC1L1 protein mediates cholesterol absorption in the intestine and liver and is the target of the drug ezetimibe, which is used to treat hypercholesterolemia. Previous studies concluded that NPC1L1-GFP protein trafficking is regulated by cholesterol binding and that ezetimibe blocks NPC1L1-GFP function by inhibiting its endocytosis. We used cell surface biotinylation to monitor NPC1L1-GFP endocytosis and show that ezetimibe does not alter the rate of NPC1L1-GFP endocytosis in cultured rat hepatocytes grown under normal growth conditions. As expected, NPC1L1-GFP endocytosis depends in part on C-terminal, cytoplasmically oriented sequences, but endocytosis does not require cholesterol binding to NPC1L1’s N-terminal domain. In addition, two small- molecule inhibitors of general (and NPC1L1-GFP) endocytosis failed to inhibit the ezetimibe-sensitive uptake of [3H]cholesterol from taurocholate micelles. These experiments demonstrate that cholesterol uptake by NPC1L1 does not require endocytosis; moreover, ezetimibe interferes with NPC1L1’s cholesterol adsorption activity without blocking NPC1L1 internalization in RH7777 cells. PMID:27075173

  5. WAVE/SCAR promotes endocytosis and early endosome morphology in polarized C. elegans epithelia

    PubMed Central

    Patel, Falshruti B.; Soto, Martha C.

    2013-01-01

    Cells can use the force of actin polymerization to drive intracellular transport, but the role of actin in endocytosis is not clear. Studies in single-celled yeast demonstrate the essential role of the branched actin nucleator, Arp2/3, and its activating nucleation promoting factors (NPFs) in the process of invagination from the cell surface through endocytosis. However, some mammalian studies have disputed the need for F-actin and Arp2/3 in Clathrin-Mediated Endocytosis (CME) in multicellular organisms. We investigate the role of Arp2/3 during endocytosis in C. elegans, a multicellular organism with polarized epithelia. Arp2/3 and its NPF, WAVE/SCAR, are essential for C. elegans embryonic morphogenesis. We show that WAVE/SCAR and Arp2/3 regulate endocytosis and early endosome morphology in diverse tissues of C. elegans. Depletion of WAVE/SCAR or Arp2/3, but not of the NPF Wasp, severely disrupts the distribution of molecules proposed to be internalized via CME, and alters the subcellular enrichment of the early endosome regulator RAB-5. Loss of WAVE/SCAR or of the GEFs that regulate RAB-5 results in similar defects in endocytosis in the intestine and coelomocyte cells. This study in a multicellular organism supports an essential role for branched actin regulators in endocytosis, and identifies WAVE/SCAR as a key NPF that promotes Arp2/3 endocytic function in C. elegans. PMID:23510716

  6. Calcium influx selects the fast mode of endocytosis in the synaptic terminal of retinal bipolar cells

    PubMed Central

    Neves, Guilherme; Gomis, Ana; Lagnado, Leon

    2001-01-01

    To investigate the regulation of endocytosis by Ca2+, we have made capacitance measurements in the synaptic terminal of depolarizing bipolar cells from the retina of goldfish. After a brief depolarization, all of the excess membrane was retrieved rapidly (τ ≈1 s). But when the rise in free [Ca2+] was reduced by the introduction of Ca2+ buffers [1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate (BAPTA) or EGTA], a large fraction of the membrane was retrieved by a second, slower mechanism (τ ≥ 10 s). The block of fast endocytosis by EGTA could be overcome by increasing the amplitude of the Ca2+ current, demonstrating that Ca2+ influx was the trigger for fast endocytosis. These manipulations of the Ca2+ signal altered the relative proportions of fast and slow endocytosis but did not modulate the rate constants of these processes. A brief stimulus that triggered fast endocytosis did not generate a significant rise in the spatially averaged [Ca2+], indicating that Ca2+ regulated endocytosis through an action close to the active zone. The slow mode of retrieval occurred at the resting [Ca2+]. These results demonstrate that Ca2+ influx couples fast endocytosis and exocytosis at this synapse. PMID:11734626

  7. Endocytosis and membrane receptor internalization: implication of F-BAR protein Carom

    PubMed Central

    Xu, Yanjie; Liu, Suxuan; Xia, Jixiang; Stein, Sam; Ramon, Cueto; Xi, Hang; Wang, Luqiao; Xiong, Xinyu; Zhang, Lixiao; He, Dingwen; Yang, William; Zhao, Xianxian; Cheng, Xiaoshu; Yang, Xiaofeng; Wang, Hong

    2016-01-01

    Endocytosis is a cellular process mostly responsible for membrane receptor internalization. Cell membrane receptors bind to their ligands and form a complex which can be internalized. We previously proposed that F-BAR protein initiates membrane curvature and mediates endocytosis via their binding partners. However, F-BAR protein partners involved in membrane receptor endocytosis and the regulatory mechanism remain unknown. In this study, we established a group of database mining strategies to explore mechanisms underlying receptor-related endocytosis. We identified 34 endocytic membrane receptors and 10 regulating proteins for vesicle formation in clathrin-dependent endocytosis (CDE), a major process of membrane receptor internalization. We found that F-BAR protein FCHSD2 (Carom) may facilitate endocytosis via 9 endocytic partners. Carom is highly expressed, along with highly expressed endocytic membrane receptors and partners, in endothelial cells and macrophages. We established 3 models of Carom-receptor complex and their intracellular trafficking based on protein-protein interaction and subcellular localization. We conclude that Carom may mediate receptor endocytosis and transport endocytic receptors to the cytoplasm for receptor signaling and lysosome/proteasome degradation, or to the nucleus for RNA processing, gene transcription and DNA repair. PMID:28199211

  8. Endocytosis is required for exocytosis and priming of respiratory burst activity in human neutrophils.

    PubMed

    Creed, T Michael; Tandon, Shweta; Ward, Richard A; McLeish, Kenneth R

    2017-06-21

    Neutrophil generation of reactive oxygen species (ROS) is enhanced by exposure to pro-inflammatory agents in a process termed priming. Priming is depending on exocytosis of neutrophil granules and p47(phox) phosphorylation-dependent translocation of cytosolic NADPH oxidase components. Clathrin-mediated endocytosis was recently reported to be necessary for priming, but the mechanism linking endocytosis to priming was not identified. The present study examined the hypothesis that endocytosis regulates neutrophil priming by controlling granule exocytosis. Clathrin-mediated endocytosis by isolated human neutrophils was inhibited by chlorpromazine, monodansylcadaverine, and sucrose. Exocytosis of granule subsets was measured as release of granule components by ELISA or chemiluminescence. ROS generation was measured as extracellular release of superoxide as reduction of ferrocytochrome c. p38 MAPK activation and p47(phox) phosphorylation were measured by immunoblot analysis. Statistical analysis was performed using a one-way ANOVA with the Tukey-Kramer multiple-comparison test. Inhibition of endocytosis prevented priming of superoxide release by TNFα and inhibited TNFα stimulation and priming of exocytosis of all four granule subsets. Inhibition of endocytosis did not reduce TNFα-stimulated p38 MAPK activation or p47(phox) phosphorylation. Inhibition of NADPH oxidase activity blocked TNFα stimulation of secretory vesicle and gelatinase granule exocytosis. Endocytosis is linked to priming of respiratory burst activity through ROS-mediated control of granule exocytosis.

  9. Notch1 endocytosis is induced by ligand and is required for signal transduction.

    PubMed

    Chapman, G; Major, J A; Iyer, K; James, A C; Pursglove, S E; Moreau, J L M; Dunwoodie, S L

    2016-01-01

    The Notch signalling pathway is widely utilised during embryogenesis in situations where cell-cell interactions are important for cell fate specification and differentiation. DSL ligand endocytosis into the ligand-expressing cell is an important aspect of Notch signalling because it is thought to supply the force needed to separate the Notch heterodimer to initiate signal transduction. A functional role for receptor endocytosis during Notch signal transduction is more controversial. Here we have used live-cell imaging to examine trafficking of the Notch1 receptor in response to ligand binding. Contact with cells expressing ligands induced internalisation and intracellular trafficking of Notch1. Notch1 endocytosis was accompanied by transendocytosis of ligand into the Notch1-expressing signal-receiving cell. Ligand caused Notch1 endocytosis into SARA-positive endosomes in a manner dependent on clathrin and dynamin function. Moreover, inhibition of endocytosis in the receptor-expressing cell impaired ligand-induced Notch1 signalling. Our findings resolve conflicting observations from mammalian and Drosophila studies by demonstrating that ligand-dependent activation of Notch1 signalling requires receptor endocytosis. Endocytosis of Notch1 may provide a force on the ligand:receptor complex that is important for potent signal transduction. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1

    PubMed Central

    Renigunta, Vijay; Fischer, Thomas; Zuzarte, Marylou; Kling, Stefan; Zou, Xinle; Siebert, Kai; Limberg, Maren M.; Rinné, Susanne; Decher, Niels; Schlichthörl, Günter; Daut, Jürgen

    2014-01-01

    The endosomal SNARE protein syntaxin-8 interacts with the acid-sensitive potassium channel TASK-1. The functional relevance of this interaction was studied by heterologous expression of these proteins (and mutants thereof) in Xenopus oocytes and in mammalian cell lines. Coexpression of syntaxin-8 caused a fourfold reduction in TASK-1 current, a corresponding reduction in the expression of TASK-1 at the cell surface, and a marked increase in the rate of endocytosis of the channel. TASK-1 and syntaxin-8 colocalized in the early endosomal compartment, as indicated by the endosomal markers 2xFYVE and rab5. The stimulatory effect of the SNARE protein on the endocytosis of the channel was abolished when both an endocytosis signal in TASK-1 and an endocytosis signal in syntaxin-8 were mutated. A syntaxin-8 mutant that cannot assemble with other SNARE proteins had virtually the same effect as wild-type syntaxin-8. Total internal reflection fluorescence microscopy showed formation and endocytosis of vesicles containing fluorescence-tagged clathrin, TASK-1, and/or syntaxin-8. Our results suggest that the unassembled form of syntaxin-8 and the potassium channel TASK-1 are internalized via clathrin-mediated endocytosis in a cooperative manner. This implies that syntaxin-8 regulates the endocytosis of TASK-1. Our study supports the idea that endosomal SNARE proteins can have functions unrelated to membrane fusion. PMID:24743596

  11. Co-regulation of Caveolar and Cdc42-dependent Fluid Phase Endocytosis by Phosphocaveolin-1*

    PubMed Central

    Cheng, Zhi-Jie; Singh, Raman Deep; Holicky, Eileen L.; Wheatley, Christine L.; Marks, David L.; Pagano, Richard E.

    2010-01-01

    Several clathrin-independent endocytosis mechanisms have been identified that can be distinguished by specific requirements for certain proteins, such as caveolin-1 (Cav1) and the Rho GTPases, RhoA and Cdc42, as well as by specific cargo. Some endocytic pathways may be co-regulated such that disruption of one pathway leads to the up-regulation of another; however, the underlying mechanisms for this are unclear. Cav1 has been reported to function as a guanine nucleotide dissociation inhibitor (GDI), which inhibits Cdc42 activation. We tested the hypothesis that Cav1 can regulate Cdc42-dependent, fluid phase endocytosis. We demonstrate that Cav1 overexpression decreases fluid phase endocytosis, whereas silencing of Cav1 enhances this pathway. Enhancement of Cav1 phosphorylation using a phosphatase inhibitor reduces Cdc42-regulated pinocytosis while stimulating caveolar endocytosis. Fluid phase endocytosis was inhibited by expression of a putative phosphomimetic mutant, Cav1-Y14E, but not by the phospho-deficient mutant, Cav1-Y14F. Overexpression of Cav2, or a Cav1 mutant in which the GDI region was altered to the corresponding sequence in Cav2, did not suppress fluid phase endocytosis. These results suggest that the Cav1 expression level and phosphorylation state regulates fluid phase endocytosis via the interaction between the Cav1 GDI region and Cdc42. These data define a novel molecular mechanism for co-regulation of two distinct clathrin-independent endocytic pathways. PMID:20228056

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

    PubMed

    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.

  13. Akt recruits Dab2 to albumin endocytosis in the proximal tubule.

    PubMed

    Koral, Kelly; Li, Hui; Ganesh, Nandita; Birnbaum, Morris J; Hallows, Kenneth R; Erkan, Elif

    2014-12-15

    Proximal tubule epithelial cells have a highly sophisticated endocytic machinery to retrieve the albumin in the glomerular filtrate. The megalin-cubilin complex and the endocytic adaptor disabled-2 (Dab2) play a pivotal role in albumin endocytosis. We previously demonstrated that protein kinase B (Akt) regulates albumin endocytosis in the proximal tubule through an interaction with Dab2. Here, we examined the nature of Akt-Dab2 interaction. The pleckstrin homology (PH) and catalytic domains (CD) of Akt interacted with the proline-rich domain (PRD) of Dab2 based on yeast-two hybrid (Y2H) experiments. Pull-down experiments utilizing the truncated constructs of Dab2 demonstrated that the initial 11 amino acids of Dab2-PRD were sufficient to mediate the interaction between Akt and Dab2. Endocytosis experiments utilizing Akt1- and Akt2-silencing RNA revealed that both Akt1 and Akt2 mediate albumin endocytosis in proximal tubule epithelial cells; therefore, Akt1 and Akt2 may play a compensatory role in albumin endocytosis. Furthermore, both Akt isoforms phosphorylated Dab2 at Ser residues 448 and 449. Ser-to-Ala mutations of these Dab2 residues inhibited albumin endocytosis and resulted in a shift in location of Dab2 from the peripheral to the perinuclear area, suggesting the physiological relevance of these phosphorylation sites in albumin endocytosis. We conclude that both Akt1 and Akt2 are involved in albumin endocytosis, and phosphorylation of Dab2 by Akt induces albumin endocytosis in proximal tubule epithelial cells. Further delineation of how Akt affects expression/phosphorylation of endocytic adaptors and receptors will enhance our understanding of the molecular network triggered by albumin overload in the proximal tubule.

  14. Developmental changes in Ca2+ channel subtypes regulating endocytosis at the calyx of Held

    PubMed Central

    Midorikawa, Mitsuharu; Okamoto, Yuji; Sakaba, Takeshi

    2014-01-01

    At the mammalian central synapse, Ca2+ influx through Ca2+ channels triggers neurotransmitter release by exocytosis of synaptic vesicles, which fuse with the presynaptic membrane and are subsequently retrieved by endocytosis. At the calyx of Held terminal, P/Q-type Ca2+ channels mainly mediate exocytosis, while N- and R-type channels have a minor role in young terminals (postnatal days 8–11). The role of each Ca2+ channel subtype in endocytosis remains to be elucidated; therefore, we examined the role of each type of Ca2+ channel in endocytosis, by using whole-cell patch-clamp recordings in conjunction with capacitance measurement techniques. We found that at the young calyx terminal, when R-type Ca2+ channels were blocked, the slow mode of endocytosis was further slowed, while blocking of either P/Q- or N-type Ca2+ channels had no major effect. In more mature terminals (postnatal days 14–17), the slow mode of endocytosis was mainly triggered by P/Q-type Ca2+ channels, suggesting developmental changes in the regulation of the slow mode of endocytosis by different Ca2+ channel subtypes. In contrast, a fast mode of endocytosis was observed after strong stimulation in young terminals that was mediated mainly by P/Q-type, but not R- or N-type Ca2+ channels. These results suggest that different types of Ca2+ channels regulate the two different modes of endocytosis. The results may also suggest that exo- and endocytosis are regulated independently at different sites in young animals but are more tightly coupled in older animals, allowing more efficient synaptic vesicle cycling adapted for fast signalling. PMID:24907302

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

    PubMed Central

    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

  16. Developmental changes in Ca2+ channel subtypes regulating endocytosis at the calyx of Held.

    PubMed

    Midorikawa, Mitsuharu; Okamoto, Yuji; Sakaba, Takeshi

    2014-08-15

    At the mammalian central synapse, Ca(2+) influx through Ca(2+) channels triggers neurotransmitter release by exocytosis of synaptic vesicles, which fuse with the presynaptic membrane and are subsequently retrieved by endocytosis. At the calyx of Held terminal, P/Q-type Ca(2+) channels mainly mediate exocytosis, while N- and R-type channels have a minor role in young terminals (postnatal days 8-11). The role of each Ca(2+) channel subtype in endocytosis remains to be elucidated; therefore, we examined the role of each type of Ca(2+) channel in endocytosis, by using whole-cell patch-clamp recordings in conjunction with capacitance measurement techniques. We found that at the young calyx terminal, when R-type Ca(2+) channels were blocked, the slow mode of endocytosis was further slowed, while blocking of either P/Q- or N-type Ca(2+) channels had no major effect. In more mature terminals (postnatal days 14-17), the slow mode of endocytosis was mainly triggered by P/Q-type Ca(2+) channels, suggesting developmental changes in the regulation of the slow mode of endocytosis by different Ca(2+) channel subtypes. In contrast, a fast mode of endocytosis was observed after strong stimulation in young terminals that was mediated mainly by P/Q-type, but not R- or N-type Ca(2+) channels. These results suggest that different types of Ca(2+) channels regulate the two different modes of endocytosis. The results may also suggest that exo- and endocytosis are regulated independently at different sites in young animals but are more tightly coupled in older animals, allowing more efficient synaptic vesicle cycling adapted for fast signalling.

  17. Visualization of clathrin-mediated endocytosis in live Drosophila egg chambers

    PubMed Central

    Jha, Anupma; Traub, Linton M.

    2015-01-01

    Summary In oviparous animals, clathrin-dependent endocytosis is often critical to stockpile a necessary supply of yolk within the maturing oocyte, which enables subsequent embryonic development. In the physically linked chains of maturing egg chambers within the Drosophila melanogaster ovary, a distinct, morphologically discernable, subset undergoes a massive burst clathrin-mediated endocytosis to accumulate yolk in a process termed vitellogenesis. Here, we describe how to prepare isolated ovaries to follow endocytosis, and detail approaches to follow live uptake of soluble reporters into vitellogenic Drosophila egg chambers. PMID:24947394

  18. The Measles Virus Receptor SLAMF1 Can Mediate Particle Endocytosis.

    PubMed

    Gonçalves-Carneiro, Daniel; McKeating, Jane A; Bailey, Dalan

    2017-04-01

    The signaling lymphocyte activation molecule F1 (SLAMF1) is both a microbial sensor and entry receptor for measles virus (MeV). Herein, we describe a new role for SLAMF1 to mediate MeV endocytosis that is in contrast with the alternative, and generally accepted, model that MeV genome enters cells only after fusion at the cell surface. We demonstrated that MeV engagement of SLAMF1 induces dramatic but transient morphological changes, most prominently in the formation of membrane blebs, which were shown to colocalize with incoming viral particles, and rearrangement of the actin cytoskeleton in infected cells. MeV infection was dependent on these dynamic cytoskeletal changes as well as fluid uptake through a macropinocytosis-like pathway as chemical inhibition of these processes inhibited entry. Moreover, we identified a role for the RhoA-ROCK-myosin II signaling axis in this MeV internalization process, highlighting a novel role for this recently characterized pathway in virus entry. Our study shows that MeV can hijack a microbial sensor normally involved in bacterial phagocytosis to drive endocytosis using a complex pathway that shares features with canonical viral macropinocytosis, phagocytosis, and mechanotransduction. This uptake pathway is specific to SLAMF1-positive cells and occurs within 60 min of viral attachment. Measles virus remains a significant cause of mortality in human populations, and this research sheds new light on the very first steps of infection of this important pathogen.IMPORTANCE Measles is a significant disease in humans and is estimated to have killed over 200 million people since records began. According to current World Health Organization statistics, it still kills over 100,000 people a year, mostly children in the developing world. The causative agent, measles virus, is a small enveloped RNA virus that infects a broad range of cells during infection. In particular, immune cells are infected via interactions between glycoproteins found

  19. The Measles Virus Receptor SLAMF1 Can Mediate Particle Endocytosis

    PubMed Central

    Gonçalves-Carneiro, Daniel; McKeating, Jane A.

    2017-01-01

    ABSTRACT The signaling lymphocyte activation molecule F1 (SLAMF1) is both a microbial sensor and entry receptor for measles virus (MeV). Herein, we describe a new role for SLAMF1 to mediate MeV endocytosis that is in contrast with the alternative, and generally accepted, model that MeV genome enters cells only after fusion at the cell surface. We demonstrated that MeV engagement of SLAMF1 induces dramatic but transient morphological changes, most prominently in the formation of membrane blebs, which were shown to colocalize with incoming viral particles, and rearrangement of the actin cytoskeleton in infected cells. MeV infection was dependent on these dynamic cytoskeletal changes as well as fluid uptake through a macropinocytosis-like pathway as chemical inhibition of these processes inhibited entry. Moreover, we identified a role for the RhoA-ROCK-myosin II signaling axis in this MeV internalization process, highlighting a novel role for this recently characterized pathway in virus entry. Our study shows that MeV can hijack a microbial sensor normally involved in bacterial phagocytosis to drive endocytosis using a complex pathway that shares features with canonical viral macropinocytosis, phagocytosis, and mechanotransduction. This uptake pathway is specific to SLAMF1-positive cells and occurs within 60 min of viral attachment. Measles virus remains a significant cause of mortality in human populations, and this research sheds new light on the very first steps of infection of this important pathogen. IMPORTANCE Measles is a significant disease in humans and is estimated to have killed over 200 million people since records began. According to current World Health Organization statistics, it still kills over 100,000 people a year, mostly children in the developing world. The causative agent, measles virus, is a small enveloped RNA virus that infects a broad range of cells during infection. In particular, immune cells are infected via interactions between

  20. Actin-Based Feedback Circuits in Cell Migration and Endocytosis

    NASA Astrophysics Data System (ADS)

    Wang, Xinxin

    In this thesis, we study the switch and pulse functions of actin during two important cellular processes, cell migration and endocytosis. Actin is an abundant protein that can polymerize to form a dendritic network. The actin network can exert force to push or bend the cell membrane. During cell migration, the actin network behaves like a switch, assembling mostly at one end or at the other end. The end with the majority of the actin network is the leading edge, following which the cell can persistently move in the same direction. The other end, with the minority of the actin network, is the trailing edge, which is dragged by the cell as it moves forward. When subjected to large fluctuations or external stimuli, the leading edge and the trailing edge can interchange and change the direction of motion, like a motion switch. Our model of the actin network in a cell reveals that mechanical force is crucial for forming the motion switch. We find a transition from single state symmetric behavior to switch behavior, when tuning parameters such as the force. The model is studied by both stochastic simulations, and a set of rate equations that are consistent with the simulations. Endocytosis is a process by which cells engulf extracellular substances and recycle the cell membrane. In yeast cells, the actin network is transiently needed to overcome the pressure difference across the cell membrane caused by turgor pressure. The actin network behaves like a pulse, which assembles and then disassembles within about 30 seconds. Using a stochastic model, we reproduce the pulse behaviors of the actin network and one of its regulatory proteins, Las17. The model matches green fluorescence protein (GFP) experiments for wild-type cells. The model also predicts some phenotypes that modify or diminish the pulse behavior. The phenotypes are verified with both experiments performed at Washington University and with other groups' experiments. We find that several feedback mechanisms are

  1. Bradykinin Release Avoids High Molecular Weight Kininogen Endocytosis

    PubMed Central

    Nascimento, Fabio D.; Souza, Daianne S. P.; Araujo, Mariana S.; Souza, Sinval E. G.; Sampaio, Misako U.; Nader, Helena B.; Tersariol, Ivarne L. S.; Motta, Guacyara

    2015-01-01

    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 kininogenases. The present

  2. Moderate concentrations of ethanol inhibit endocytosis of the yeast maltose transporter.

    PubMed Central

    Lucero, P; Peñalver, E; Moreno, E; Lagunas, R

    1997-01-01

    The maltose transporter in Saccharomyces cerevisiae is degraded in the vacuole after internalization by endocytosis upon nitrogen starvation in the presence of a fermentable substrate. This degradation, known as catabolite inactivation, is inhibited by the presence of moderate concentrations (2 to 6%, vol/vol) of ethanol. We have investigated the mechanism of this inactivation and have found that it is due to the inhibition of the internalization of the transporter by endocytosis. The results also indicate that this inhibition is due to alterations produced by ethanol in the organization of the plasma membrane which also affects to endocytosis of other plasma membrane proteins. Apparently, endocytosis is particularly sensitive to these alterations compared with other processes occurring at the plasma membrane. PMID:9327546

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

    PubMed Central

    Qu, M.; Mehrmohammadi, M.; Emelianov, S.Y.

    2015-01-01

    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. PMID:26640773

  4. A cell-free biochemical complementation assay reveals complex and redundant cytosolic requirements for LRP endocytosis.

    PubMed

    Miwako, Ishido; Schmid, Sandra L

    2006-05-01

    The low density lipoprotein receptor-related protein (LRP) binds multiple, distinct ligands and participates in constitutive endocytosis and signal transduction. Using an in vitro reconstitution system and a new biochemical complementation assay, we have explored the limiting cytosolic requirements for endocytosis of LRP from isolated plasma membranes. We find that clathrin, AP2 and dynamin do not support efficient LRP uptake and that additional factors present in a 30% ammonium sulfate supernatant fraction of bovine brain cytosol (AS supt) are required. Fractionation of the AS supt revealed that multiple and redundant factors are required to support LRP endocytosis. Among these, we identified Hsc70, synaptojanin1 and CRMP-2 by mass spectrometry. Our data suggest that LRP, which bears several distinct endocytic motifs in its cytoplasmic domain, may use multiple pathways for endocytosis in vitro.

  5. Molecular Mechanisms for the Coupling of Endocytosis to Exocytosis in Neurons

    PubMed Central

    Xie, Zhenli; Long, Jiangang; Liu, Jiankang; Chai, Zuying; Kang, Xinjiang; Wang, Changhe

    2017-01-01

    Neuronal communication and brain function mainly depend on the fundamental biological events of neurotransmission, including the exocytosis of presynaptic vesicles (SVs) for neurotransmitter release and the subsequent endocytosis for SV retrieval. Neurotransmitters are released through the Ca2+- and SNARE-dependent fusion of SVs with the presynaptic plasma membrane. Following exocytosis, endocytosis occurs immediately to retrieve SV membrane and fusion machinery for local recycling and thus maintain the homeostasis of synaptic structure and sustained neurotransmission. Apart from the general endocytic machinery, recent studies have also revealed the involvement of SNARE proteins (synaptobrevin, SNAP25 and syntaxin), synaptophysin, Ca2+/calmodulin, and members of the synaptotagmin protein family (Syt1, Syt4, Syt7 and Syt11) in the balance and tight coupling of exo-endocytosis in neurons. Here, we provide an overview of recent progress in understanding how these neuron-specific adaptors coordinate to ensure precise and efficient endocytosis during neurotransmission. PMID:28348516

  6. ALG-2 interacting protein-X (Alix) is essential for clathrin-independent endocytosis and signaling.

    PubMed

    Mercier, Vincent; Laporte, Marine H; Destaing, Olivier; Blot, Béatrice; Blouin, Cédric M; Pernet-Gallay, Karin; Chatellard, Christine; Saoudi, Yasmina; Albiges-Rizo, Corinne; Lamaze, Christophe; Fraboulet, Sandrine; Petiot, Anne; Sadoul, Rémy

    2016-05-31

    The molecular mechanisms and the biological functions of clathrin independent endocytosis (CIE) remain largely elusive. Alix (ALG-2 interacting protein X), has been assigned roles in membrane deformation and fission both in endosomes and at the plasma membrane. Using Alix ko cells, we show for the first time that Alix regulates fluid phase endocytosis and internalization of cargoes entering cells via CIE, but has no apparent effect on clathrin mediated endocytosis or downstream endosomal trafficking. We show that Alix acts with endophilin-A to promote CIE of cholera toxin and to regulate cell migration. We also found that Alix is required for fast endocytosis and downstream signaling of the interleukin-2 receptor giving a first indication that CIE is necessary for activation of at least some surface receptors. In addition to characterizing a new function for Alix, our results highlight Alix ko cells as a unique tool to unravel the biological consequences of CIE.

  7. ALG-2 interacting protein-X (Alix) is essential for clathrin-independent endocytosis and signaling

    PubMed Central

    Mercier, Vincent; Laporte, Marine H.; Destaing, Olivier; Blot, Béatrice; Blouin, Cédric M.; Pernet-Gallay, Karin; Chatellard, Christine; Saoudi, Yasmina; Albiges-Rizo, Corinne; Lamaze, Christophe; Fraboulet, Sandrine; Petiot, Anne; Sadoul, Rémy

    2016-01-01

    The molecular mechanisms and the biological functions of clathrin independent endocytosis (CIE) remain largely elusive. Alix (ALG-2 interacting protein X), has been assigned roles in membrane deformation and fission both in endosomes and at the plasma membrane. Using Alix ko cells, we show for the first time that Alix regulates fluid phase endocytosis and internalization of cargoes entering cells via CIE, but has no apparent effect on clathrin mediated endocytosis or downstream endosomal trafficking. We show that Alix acts with endophilin-A to promote CIE of cholera toxin and to regulate cell migration. We also found that Alix is required for fast endocytosis and downstream signaling of the interleukin-2 receptor giving a first indication that CIE is necessary for activation of at least some surface receptors. In addition to characterizing a new function for Alix, our results highlight Alix ko cells as a unique tool to unravel the biological consequences of CIE. PMID:27244115

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

    PubMed

    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.

  9. Recording the dynamic endocytosis of single gold nanoparticles by AFM-based force tracing

    NASA Astrophysics Data System (ADS)

    Ding, Bohua; Tian, Yongmei; Pan, Yangang; Shan, Yuping; Cai, Mingjun; Xu, Haijiao; Sun, Yingchun; Wang, Hongda

    2015-04-01

    We utilized force tracing to directly record the endocytosis of single gold nanoparticles (Au NPs) with different sizes, revealing the size-dependent endocytosis dynamics and the crucial role of membrane cholesterol. The force, duration and velocity of Au NP invagination are accurately determined at the single-particle and microsecond level unprecedentedly.We utilized force tracing to directly record the endocytosis of single gold nanoparticles (Au NPs) with different sizes, revealing the size-dependent endocytosis dynamics and the crucial role of membrane cholesterol. The force, duration and velocity of Au NP invagination are accurately determined at the single-particle and microsecond level unprecedentedly. Electronic supplementary information (ESI) available: Details of the experimental procedures and the results of the control experiments. See DOI: 10.1039/c5nr01020a

  10. Different dynamin blockers interfere with distinct phases of synaptic endocytosis during stimulation in motoneurones

    PubMed Central

    Linares-Clemente, Pedro; Rozas, José L; Mircheski, Josif; García-Junco-Clemente, Pablo; Martínez-López, José A; Nieto-González, José L; Vázquez, M Eugenio; Pintado, C Oscar; Fernández-Chacón, Rafael

    2015-01-01

    Key points Neurotransmitter release requires a tight coupling between synaptic vesicle exocytosis and endocytosis with dynamin being a key protein in that process. We used imaging techniques to examine the time course of endocytosis at mouse motor nerve terminals expressing synaptopHluorin, a genetically encoded reporter of the synaptic vesicle cycle. We separated two sequential phases of endocytosis taking place during the stimulation train: early and late endocytosis. Freshly released synaptic vesicle proteins are preferentially retrieved during the early phase, which is very sensitive to dynasore, an inhibitor of dynamin GTPase activity. Synaptic vesicle proteins pre-existing at the plasma membrane before the stimulation are preferentially retrieved during the late phase, which is very sensitive to myristyl trimethyl ammonium bromide (MitMAB), an inhibitor of the dynamin–phospholipid interaction. Abstract Synaptic endocytosis is essential at nerve terminals to maintain neurotransmitter release by exocytosis. Here, at the neuromuscular junction of synaptopHluorin (spH) transgenic mice, we have used imaging to study exo- and endocytosis occurring simultaneously during nerve stimulation. We observed two endocytosis components, which occur sequentially during stimulation. The early component of endocytosis apparently internalizes spH molecules freshly exocytosed. This component was sensitive to dynasore, a blocker of dynamin 1 GTPase activity. In contrast, this early component was resistant to myristyl trimethyl ammonium bromide (MiTMAB), a competitive agent that blocks dynamin binding to phospholipid membranes. The late component of endocytosis is likely to internalize spH molecules that pre-exist at the plasma membrane before stimulation starts. This component was blocked by MiTMAB, perhaps by impairing the binding of dynamin or other key endocytic proteins to phospholipid membranes. Our study suggests the co-existence of two sequential synaptic endocytosis

  11. Seesaw in the Bulk

    NASA Astrophysics Data System (ADS)

    Watanabe, A.; Yoshioka, K.

    2011-01-01

    A five-dimensional seesaw framework is analyzed with the lepton-number-violating propagator of bulk right-handed neutrinos. That can bypass summing up the effects of heavy Majorana particles whose masses and wavefunctions are not exactly known. The propagator method makes it easier to evaluate the seesaw-induced neutrino mass for various boundary conditions of bulk neutrinos and in a general background geometry, including the warped extra dimension. It is also found that the higher-dimensional seesaw gives a natural framework for the inverse seesaw suppression of low-energy neutrino masses.

  12. Ebola Virus Uses Clathrin-Mediated Endocytosis as an Entry Pathway

    DTIC Science & Technology

    2010-01-01

    clathrin-mediated endocytosis is not restricted to small spherical coated pits. Listeria has been shown to utilize clathrin- mediated endocytosis to...enter cells (Veiga and Cossart, 2005). The large size of the Listeria , which extends to 2 μm (Giardini and Theriot, 2001), suggests that clathrin coated... Listeria monocytogenes. Biophys. J. 81 (6), 3193–3203. Hanover, J.A., Willingham, M.C., Pastan, I., 1984. Kinetics of transit of transferrin and epidermal

  13. Characterization of Parameters Influencing Receptor-Mediated Endocytosis in Cultured Soybean Cells 1

    PubMed Central

    Horn, Mark A.; Heinstein, Peter F.; Low, Philip S.

    1992-01-01

    In a recent publication, we were able to demonstrate that biotin enters plant cells by receptor-mediated endocytosis and that impermeable macromolecules can be cotransported into cells by the same pathway if they are first covalently linked to biotin. In the present study, we have exploited the biotin endocytosis pathway to evaluate the variables in the cell wall and surrounding growth medium that influence the efficiency of endocytosis in plants. Under normal growth conditions, the major constraint limiting macromolecule endocytosis was found to be the size of the internalized macromolecule. Thus, a log-linear relationship with a negative slope exists between the molecular weight of the biotin-conjugated macromolecule and its rate of internalization by cultured soybean cells. This relationship, which extends from insulin (Mr approximately 5700) to immunoglobulin G (Mr approximately 160,000), is characterized by a slope of −1.04 × 105 molecules/cell/min per log Mr unit and an x intercept (no endocytosis detectable) of approximately log 160,000 daltons. Unfortunately, mild digestion with cell wall-degrading enzymes is unable to increase significantly the upper size limit of molecules that can be internalized, but uptake of lower molecular weight proteins can be enhanced by mild cell wall digestion. The optimal extracellular pH for endocytosis was found to be 4.6, i.e. near the normal pH of the cell culture medium. Furthermore, the osmotic strength at which endocytosis occurs most rapidly was observed to be isotonic to slightly hypotonic, suggesting that turgor pressure within the plant cell must not be a major determinant of endocytosis rates by cultured soybean (Glycine max) cells. Finally, cell age was found to impact significantly on the rate of macromolecule internalization, with maximal uptake rates occurring during early exponential growth and decreasing by a factor of 2 when the cells reach stationary growth phase. PMID:16668694

  14. The ARH adaptor protein regulates endocytosis of the ROMK potassium secretory channel in mouse kidney

    PubMed Central

    Fang, Liang; Garuti, Rita; Kim, Bo-Young; Wade, James B.; Welling, Paul A.

    2009-01-01

    Renal outer medullary potassium (ROMK) channels are exquisitely regulated to adjust renal potassium excretion and maintain potassium balance. Clathrin-dependent endocytosis plays a critical role, limiting urinary potassium loss in potassium deficiency. In renal disease, aberrant ROMK endocytosis may contribute to potassium retention and hyperkalemia. Previous work has indicated that ROMK endocytosis is stimulated by with-no-lysine (WNK) kinases, but the endocytotic signal and the internalization machinery have not been defined. Here, we found that ROMK bound directly to the clathrin adaptor molecule autosomal recessive hypercholesterolemia (ARH), and this interaction was mediated by what we believe to be a novel variant of the canonical “NPXY” endocytotic signal, YxNPxFV. ARH recruits ROMK to clathrin-coated pits for constitutive and WNK1-stimuated endocytosis, and ARH knockdown decreased basal rates of ROMK endocytosis, in a heterologous expression system, COS-7 cells. We found that ARH was predominantly expressed in the distal nephron where it coimmunoprecipitated and colocalized with ROMK. In mice, the abundance of kidney ARH protein was modulated by dietary potassium and inversely correlated with changes in ROMK. Furthermore, ARH-knockout mice exhibited an altered ROMK response to potassium intake. These data suggest that ARH marks ROMK for clathrin-dependent endocytosis, in concert with the demands of potassium homeostasis. PMID:19841541

  15. Molecular mediators for raft-dependent endocytosis of syndecan-1, a highly conserved, multifunctional receptor.

    PubMed

    Chen, Keyang; Williams, Kevin Jon

    2013-05-17

    Endocytosis via rafts has attracted considerable recent interest, but the molecular mediators remain incompletely characterized. Here, we focused on the syndecan-1 heparan sulfate proteoglycan, a highly conserved, multifunctional receptor that we previously showed to undergo raft-dependent endocytosis upon clustering. Alanine scanning mutagenesis of three to five consecutive cytoplasmic residues at a time revealed that a conserved juxtamembrane motif, MKKK, was the only region required for efficient endocytosis after clustering. Endocytosis of clustered syndecan-1 occurs in two phases, each requiring a kinase and a corresponding cytoskeletal partner. In the initial phase, ligands trigger rapid MKKK-dependent activation of ERK and the localization of syndecan-1 into rafts. Activation of ERK drives the dissociation of syndecan-1 from α-tubulin, a molecule that may act as an anchor for syndecan-1 at the plasma membrane in the basal state. In the second phase, Src family kinases phosphorylate tyrosyl residues within the transmembrane and cytoplasmic regions of syndecan-1, a process that also requires MKKK. Tyrosine phosphorylation of syndecan-1 triggers the robust recruitment of cortactin, which we found to be an essential mediator of efficient actin-dependent endocytosis. These findings represent the first detailed characterization of the molecular events that drive endocytosis of a raft-dependent receptor and identify a novel endocytic motif, MKKK. Moreover, the results provide new tools to study syndecan function and regulation during uptake of its biologically and medically important ligands, such as HIV-1, atherogenic postprandial remnant lipoproteins, and molecules implicated in Alzheimer disease.

  16. Optical Tweezers Studies on Notch: Single-molecule Interaction Strength is Independent of Ligand Endocytosis

    PubMed Central

    Shergill, Bhupinder; Meloty-Kapella, Laurence; Musse, Abdiwahab A.; Weinmaster, Gerry; Botvinick, Elliot

    2012-01-01

    SUMMARY Notch signaling controls diverse cellular processes critical to development and disease. Cell surface ligands bind Notch on neighboring cells yet require endocytosis to activate signaling. The role ligand endocytosis plays in Notch activation has not been established. Here we integrate optical tweezers with cell biological and biochemical methods to test the prevailing model that ligand endocytosis facilitates recycling to enhance ligand interactions with Notch necessary to trigger signaling. Specifically, single-molecule measurements indicate that interference of ligand endocytosis and/or recycling does not alter the force required to rupture bonds formed between cells expressing the Notch ligand Delta-like1 (Dll1) and laser-trapped Notch1-beads. Together, our analyses eliminate roles for ligand endocytosis and recycling in Dll1-Notch1 interactions, and indicate that recycling indirectly affects signaling by regulating the accumulation of cell-surface ligand. Importantly, our study demonstrates the utility of optical tweezers to test a role for ligand endocytosis in generating cell-mediated mechanical force. PMID:22658935

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

    PubMed

    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.

  18. TRAIL-death receptor endocytosis and apoptosis are selectively regulated by dynamin-1 activation

    PubMed Central

    Reis, Carlos R.; Chen, Ping-Hung; Bendris, Nawal; Schmid, Sandra L.

    2017-01-01

    Clathrin-mediated endocytosis (CME) constitutes the major pathway for uptake of signaling receptors into eukaryotic cells. As such, CME regulates signaling from cell-surface receptors, but whether and how specific signaling receptors reciprocally regulate the CME machinery remains an open question. Although best studied for its role in membrane fission, the GTPase dynamin also regulates early stages of CME. We recently reported that dynamin-1 (Dyn1), previously assumed to be neuron-specific, can be selectively activated in cancer cells to alter endocytic trafficking. Here we report that dynamin isoforms differentially regulate the endocytosis and apoptotic signaling downstream of TNF-related apoptosis-inducing ligand–death receptor (TRAIL–DR) complexes in several cancer cells. Whereas the CME of constitutively internalized transferrin receptors is mainly dependent on the ubiquitously expressed Dyn2, TRAIL-induced DR endocytosis is selectively regulated by activation of Dyn1. We show that TRAIL stimulation activates ryanodine receptor-mediated calcium release from endoplasmic reticulum stores, leading to calcineurin-mediated dephosphorylation and activation of Dyn1, TRAIL–DR endocytosis, and increased resistance to TRAIL-induced apoptosis. TRAIL–DR-mediated ryanodine receptor activation and endocytosis is dependent on early caspase-8 activation. These findings delineate specific mechanisms for the reciprocal crosstalk between signaling and the regulation of CME, leading to autoregulation of endocytosis and signaling downstream of surface receptors. PMID:28049841

  19. Receptor mediated endocytosis of vicilin in Callosobruchus maculatus (Coleoptera: Chrysomelidae) larval midgut epithelial cells.

    PubMed

    Kunz, Daniele; Oliveira, Gabriel B; Uchôa, Adriana F; Samuels, Richard I; Macedo, Maria Lígia R; Silva, Carlos P

    2017-08-01

    The transport of proteins across the intestinal epithelium of insects is still not well understood. There is evidence that vicilin, a major storage protein of cowpea seeds (Vigna unguiculata), is internalized in larvae of the seed-beetle Callosobruchus maculatus. It has been reported that this vicilin interacts with proteins present in the microvillar membranes of columnar cells along the digestive tract of the larvae. In the present work, we studied the cellular pathway involved in endocytosis of vicilin in larval C. maculatus by employing ex vivo experiments. In the ex vivo approach, we incubated FITC-labelled vicilin with isolated midgut wholemounts in the absence or in the presence of endocytosis inhibitors. The fate of labelled or non-labelled globulins was monitored by confocal microscopy and fluorescence measurement. Our results suggest that the internalization of vicilins is due to receptor-mediated endocytosis. Here we report the identity of a microvillar vicilin-binding protein that was purified using affinity chromatography on a vicilin-sepharose column. The putative vicilin receptor showed high homology to proteins with the CRAL-TRIO domain, specifically the Sec14 superfamily member α-tocopherol transfer protein. The precise mechanism involved in vicilin internalization was defined through the use of specific inhibitors of the endocytosis pathway. The inhibitors filipin III and nystatin significantly inhibited the endocytosis of vicilin, while chlorpromazine and phenylarsine oxide had a much lower effect on endocytosis, suggesting that the endocytic pathway is predominantly mediated by caveolin. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Inhibitor of endocytosis impairs gene electrotransfer to mouse muscle in vivo.

    PubMed

    Markelc, Bostjan; Skvarca, Eva; Dolinsek, Tanja; Kloboves, Veronika Prevodnik; Coer, Andrej; Sersa, Gregor; Cemazar, Maja

    2015-06-01

    Application of electric pulses (electroporation/electropermeabilization) is an effective method for gene transfer (i.e. gene electrotransfer (GET)) in vitro and in vivo. Currently, the mechanisms by which the DNA enters the cell are not yet fully understood. Experimental evidence is building up that endocytosis is the main mechanism by which the DNA, which is later expressed, enters the cell. Therefore the aim of our study was to elucidate whether inhibitors of endocytosis, methyl-β-cyclodextrin (MβCD), Concanavalin A (ConA) and Dynasore, can impair the transfection efficacy of GET in vitro in B16F1 murine melanoma and in vivo in m. tibialis cranialis in mice. We show that MβCD--general inhibitor of endocytosis--can almost prevent GET of EGFP-N1 plasmid in vitro, that ConA--inhibitor of clathrin mediated endocytosis--also abrogates GET but to a lesser extent, and when using Dynasore--reversible inhibitor of dynamin--there is no effect on GET efficacy, if endocytosis is blocked for only 5 min after GET. Moreover, MβCD also reduced GET efficacy in vivo in m. tibialis cranialis and this effect was long lasting. The results of this study show that endocytosis is probably the main mechanism of entrance of DNA after GET in vitro and also in vivo.

  1. Myosin VI and its cargo adaptors - linking endocytosis and autophagy.

    PubMed

    Tumbarello, David A; Kendrick-Jones, John; Buss, Folma

    2013-06-15

    The coordinated trafficking and tethering of membrane cargo within cells relies on the function of distinct cytoskeletal motors that are targeted to specific subcellular compartments through interactions with protein adaptors and phospholipids. The unique actin motor myosin VI functions at distinct steps during clathrin-mediated endocytosis and the early endocytic pathway - both of which are involved in cargo trafficking and sorting - through interactions with Dab2, GIPC, Tom1 and LMTK2. This multifunctional ability of myosin VI can be attributed to its cargo-binding tail region that contains two protein-protein interaction interfaces, a ubiquitin-binding motif and a phospholipid binding domain. In addition, myosin VI has been shown to be a regulator of the autophagy pathway, because of its ability to link the endocytic and autophagic pathways through interactions with the ESCRT-0 protein Tom1 and the autophagy adaptor proteins T6BP, NDP52 and optineurin. This function has been attributed to facilitating autophagosome maturation and subsequent fusion with the lysosome. Therefore, in this Commentary, we discuss the relationship between myosin VI and the different myosin VI adaptor proteins, particularly with regards to the spatial and temporal regulation that is required for the sorting of cargo at the early endosome, and their impact on autophagy.

  2. A Yeast t-SNARE Involved in Endocytosis

    PubMed Central

    Séron, Karin; Tieaho, Ville; Prescianotto-Baschong, Cristina; Aust, Thomas; Blondel, Marie-Odile; Guillaud, Philippe; Devilliers, Ginette; Rossanese, Olivia W.; Glick, Benjamin S.; Riezman, Howard; Keränen, Sirkka; Haguenauer-Tsapis, Rosine

    1998-01-01

    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 tlg2Δ cells, internalization was normal for two endocytic markers, the pheromone α-factor and the plasma membrane uracil permease. In contrast, degradation of α-factor and uracil permease was delayed in tlg2Δ 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

  3. Tryptophan within basic peptide sequences triggers glycosaminoglycan-dependent endocytosis.

    PubMed

    Bechara, Chérine; Pallerla, Manjula; Zaltsman, Yefim; Burlina, Fabienne; Alves, Isabel D; Lequin, Olivier; Sagan, Sandrine

    2013-02-01

    Deciphering the structural requirements and mechanisms for internalization of cell-penetrating peptides (CPPs) is required to improve their delivery efficiency. Herein, a unique role of tryptophan (Trp) residues in the interaction and structuring of cationic CPP sequences with glycosaminoglycans (GAGs) has been characterized, in relation with cell internalization. Using isothermal titration calorimetry, circular dichroism, NMR, mass spectrometry, and phase-contrast microscopy, we compared the interaction of 7 basic CPPs with 5 classes of GAGs. We found that the affinity of CPPs for GAGs increases linearly with the number of Trp residues, from 30 nM for a penetratin analog with 1 Trp residue to 1.5 nM for a penetratin analog with 6 Trp residues for heparin (HI); peptides with Trp residues adopt a predominantly β-strand structure in complex with HI and form large, stable β-sheet aggregates with GAGs; and in the absence of any cytotoxicity effect, the quantity of peptide internalized into CHO cells increased 2 times with 1 Trp residue, 10 times with 2 Trp residues, and 20 times with 3 Trp residues, compared with +6 peptides with no Trp residues. Therefore, Trp residues represent molecular determinants in basic peptide sequences not only for direct membrane translocation but also for efficient endocytosis through GAGs.

  4. Actin dynamics counteract membrane tension during clathrin-mediated endocytosis

    PubMed Central

    Boulant, Steeve; Kural, Comert; Zeeh, Jean-Christophe; Ubelmann, Florent; Kirchhausen, Tom

    2011-01-01

    Clathrin-mediated endocytosis is independent of actin dynamics in many circumstances but requires actin polymerization in others. We show that membrane tension determines the actin dependence of clathrin-coat assembly. As found previously, clathrin assembly supports formation of mature coated pits in the absence of actin polymerization on both dorsal and ventral surfaces of non-polarized mammalian cells, and also on basolateral surfaces of polarized cells. Actin engagement is necessary, however, to complete membrane deformation into a coated pit on apical surfaces of polarized cells and, more generally, on the surface of any cell in which the plasma membrane is under tension from osmotic swelling or mechanical stretching. We use these observations to alter actin dependence experimentally and show that resistance of the membrane to propagation of the clathrin lattice determines the distinction between "actin-dependent" and "actin-independent". We also find that light-chain bound Hip1R mediates actin engagement. These data thus provide a unifying explanation for the role of actin dynamics in coated-pit budding. PMID:21841790

  5. Clathrin- and caveolin-1–independent endocytosis

    PubMed Central

    Damm, Eva-Maria; Pelkmans, Lucas; Kartenbeck, Jürgen; Mezzacasa, Anna; Kurzchalia, Teymuras; Helenius, Ari

    2005-01-01

    Simian Virus 40 (SV40) has been shown to enter host cells by caveolar endocytosis followed by transport via caveosomes to the endoplasmic reticulum (ER). Using a caveolin-1 (cav-1)–deficient cell line (human hepatoma 7) and embryonic fibroblasts from a cav-1 knockout mouse, we found that in the absence of caveolae, but also in wild-type embryonic fibroblasts, the virus exploits an alternative, cav-1–independent pathway. Internalization was rapid (t1/2 = 20 min) and cholesterol and tyrosine kinase dependent but independent of clathrin, dynamin II, and ARF6. The viruses were internalized in small, tight-fitting vesicles and transported to membrane-bounded, pH-neutral organelles similar to caveosomes but devoid of cav-1 and -2. The viruses were next transferred by microtubule-dependent vesicular transport to the ER, a step that was required for infectivity. Our results revealed the existence of a virus-activated endocytic pathway from the plasma membrane to the ER that involves neither clathrin nor caveolae and that can be activated also in the presence of cav-1. PMID:15668298

  6. Effects of cholesterol and lipoproteins on endocytosis by a monocyte-like cell line.

    PubMed

    Esfahani, M; Scerbo, L; Lund-Katz, S; DePace, D M; Maniglia, R; Alexander, J K; Phillips, M C

    1986-12-19

    The human monocyte/macrophage-like cell line U937 is a cholesterol auxotroph. Incubation of these cells in the growth medium in which delipidated fetal calf serum has been substituted for fetal calf serum depletes cellular cholesterol and inhibits growth. The cholesterol requirement of these cells for growth can be satisfied by human low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL), but not by high-density lipoprotein (HDL). U937 cells can bind and degrade LDL via a high-affinity site and this recognition is altered by acetylation of LDL. This indicates that these cells express relatively high LDL receptor activity and low levels of the acetyl-LDL receptor. The cells were used to study the role of cholesterol in lectin-mediated and fluid-phase endocytosis. Growth of the cells in the medium containing delipidated fetal calf serum results in impairment of both concanavalin A-mediated endocytosis of horseradish peroxidase and concanavalin A-independent endocytosis of Lucifer Yellow. Supplementation of the medium with cholesterol prevents cellular cholesterol depletion, supports growth and stimulates Lucifer Yellow endocytosis but fails to restore horseradish peroxidase endocytosis. However, if the cells are incubated in the presence of no less than 40 micrograms LDL protein/ml to maintain normal cell cholesterol levels, concanavalin A-mediated endocytosis of horseradish peroxidase is activated. The effect of LDL is specific since neither VLDL nor HDL3 at the same protein concentration activates horseradish peroxidase uptake by the cells. Furthermore, the activation of endocytosis by LDL is not inhibited by the inclusion of heparin or acetylation of the LDL indicating that binding of LDL to the LDL receptor is not required for these effects. The mediation of activation of horseradish peroxidase endocytosis by the lectin is presumed to involve binding of LDL to concanavalin A associated with the cell surface which in turn stimulates horseradish

  7. Phosphorylation of Synaptojanin Differentially Regulates Endocytosis of Functionally Distinct Synaptic Vesicle Pools

    PubMed Central

    Geng, Junhua; Wang, Liping; Lee, Joo Yeun; Chen, Chun-Kan

    2016-01-01

    The rapid replenishment of synaptic vesicles through endocytosis is crucial for sustaining synaptic transmission during intense neuronal activity. Synaptojanin (Synj), a phosphoinositide phosphatase, is known to play an important role in vesicle recycling by promoting the uncoating of clathrin following synaptic vesicle uptake. Synj has been shown to be a substrate of the minibrain (Mnb) kinase, a fly homolog of the dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A); however, the functional impacts of Synj phosphorylation by Mnb are not well understood. Here we identify that Mnb phosphorylates Synj at S1029 in Drosophila. We find that phosphorylation of Synj at S1029 enhances Synj phosphatase activity, alters interaction between Synj and endophilin, and promotes efficient endocytosis of the active cycling vesicle pool (also referred to as exo-endo cycling pool) at the expense of reserve pool vesicle endocytosis. Dephosphorylated Synj, on the other hand, is deficient in the endocytosis of the active recycling pool vesicles but maintains reserve pool vesicle endocytosis to restore total vesicle pool size and sustain synaptic transmission. Together, our findings reveal a novel role for Synj in modulating reserve pool vesicle endocytosis and further indicate that dynamic phosphorylation and dephosphorylation of Synj differentially maintain endocytosis of distinct functional synaptic vesicle pools. SIGNIFICANCE STATEMENT Synaptic vesicle endocytosis sustains communication between neurons during a wide range of neuronal activities by recycling used vesicle membrane and protein components. Here we identify that Synaptojanin, a protein with a known role in synaptic vesicle endocytosis, is phosphorylated at S1029 in vivo by the Minibrain kinase. We further demonstrate that the phosphorylation status of Synaptojanin at S1029 differentially regulates its participation in the recycling of distinct synaptic vesicle pools. Our results reveal a new role for

  8. Endocytosis and Physiology: Insights from Disabled-2 Deficient Mice

    PubMed Central

    Tao, Wensi; Moore, Robert; Smith, Elizabeth R.; Xu, Xiang-Xi

    2016-01-01

    Disabled-2 (Dab2) is a clathrin and cargo binding endocytic adaptor protein, and cell biology studies revealed that Dab2 plays a role in cellular trafficking of a number of transmembrane receptors and signaling proteins. A PTB/PID domain located in the N-terminus of Dab2 binds the NPXY motif(s) present at the cytoplasmic tails of certain transmembrane proteins/receptors. The membrane receptors reported to bind directly to Dab2 include LDL receptor and its family members LRP1 and LRP2 (megalin), growth factor receptors EGFR and FGFR, and the cell adhesion receptor beta1 integrin. Dab2 also serves as an adaptor in signaling pathways. Particularly, Dab2 facilitates the endocytosis of the Ras activating Grb2/Sos1 signaling complex, controls its disassembly, and thereby regulates the Ras/MAPK signaling pathway. Cellular analyses have suggested several diverse functions for the widely expressed proteins, and Dab2 is also considered a tumor suppressor, as loss or reduced expression is found in several cancer types. Dab2 null mutant mice were generated and investigated to determine if the findings from cellular studies might be important and relevant in intact animals. Dab2 conditional knockout mice mediated through a Sox2-Cre transgene have no obvious developmental defects and have a normal life span despite that the Dab2 protein is essentially absent in the mutant mice. The conditional knockout mice were grossly normal, though more recent investigation of the Dab2-deficient mice revealed several phenotypes, which can be accounted for by several previously suggested mechanisms. The studies of mutant mice established that Dab2 plays multiple physiological roles through its endocytic functions and modulation of signal pathways. PMID:27933291

  9. Design principles for robust vesiculation in clathrin-mediated endocytosis

    PubMed Central

    Hassinger, Julian E.; Oster, George; Drubin, David G.; Rangamani, Padmini

    2017-01-01

    A critical step in cellular-trafficking pathways is the budding of membranes by protein coats, which recent experiments have demonstrated can be inhibited by elevated membrane tension. The robustness of processes like clathrin-mediated endocytosis (CME) across a diverse range of organisms and mechanical environments suggests that the protein machinery in this process has evolved to take advantage of some set of physical design principles to ensure robust vesiculation against opposing forces like membrane tension. Using a theoretical model for membrane mechanics and membrane protein interaction, we have systematically investigated the influence of membrane rigidity, curvature induced by the protein coat, area covered by the protein coat, membrane tension, and force from actin polymerization on bud formation. Under low tension, the membrane smoothly evolves from a flat to budded morphology as the coat area or spontaneous curvature increases, whereas the membrane remains essentially flat at high tensions. At intermediate, physiologically relevant, tensions, the membrane undergoes a “snap-through instability” in which small changes in the coat area, spontaneous curvature or membrane tension cause the membrane to “snap” from an open, U-shape to a closed bud. This instability can be smoothed out by increasing the bending rigidity of the coat, allowing for successful budding at higher membrane tensions. Additionally, applied force from actin polymerization can bypass the instability by inducing a smooth transition from an open to a closed bud. Finally, a combination of increased coat rigidity and force from actin polymerization enables robust vesiculation even at high membrane tensions. PMID:28126722

  10. The effect of substrate elasticity and actomyosin contractility on different forms of endocytosis.

    PubMed

    Missirlis, Dimitris

    2014-01-01

    Substrate mechanical properties have emerged as potent determinants of cell functions and fate. We here tested the hypothesis that different forms of endocytosis are regulated by the elasticity of the synthetic hydrogels cells are cultured on. Towards this objective, we quantified cell-associated fluorescence of the established endocytosis markers transferrin (Tf) and cholera toxin subunit B (CTb) using a flow-cytometry based protocol, and imaged marker internalization using microscopy techniques. Our results demonstrated that clathrin-mediated endocytosis of Tf following a 10-minute incubation with a fibroblast cell line was lower on the softer substrates studied (5 kPa) compared to those with elasticities of 40 and 85 kPa. This effect was cancelled after 1-hour incubation revealing that intracellular accumulation of Tf at this time point did not depend on substrate elasticity. Lipid-raft mediated endocytosis of CTb, on the other hand, was not affected by substrate elasticity in the studied range of time and substrate elasticity. The use of pharmacologic contractility inhibitors revealed inhibition of endocytosis for both Tf and CTb after a 10-minute incubation and a dissimilar effect after 1 hour depending on the inhibitor type. Further, the internalization of fluorescent NPs, used as model drug delivery systems, showed a dependence on substrate elasticity, while transfection efficiency was unaffected by it. Finally, an independence on substrate elasticity of Tf and CTb association with HeLa cells indicated that there are cell-type differences in this respect. Overall, our results suggest that clathrin-mediated but not lipid-raft mediated endocytosis is potentially influenced by substrate mechanics at the cellular level, while intracellular trafficking and accumulation show a more complex dependence. Our findings are discussed in the context of previous work on how substrate mechanics affect the fundamental process of endocytosis and highlight important

  11. Recruitment of endocytosis in sonopermeabilization-mediated drug delivery: a real-time study

    NASA Astrophysics Data System (ADS)

    Derieppe, Marc; Rojek, Katarzyna; Escoffre, Jean-Michel; de Senneville, Baudouin Denis; Moonen, Chrit; Bos, Clemens

    2015-07-01

    Microbubbles (MBs) in combination with ultrasound (US) can enhance cell membrane permeability, and have the potential to facilitate the cellular uptake of hydrophilic molecules. However, the exact mechanism behind US- and MB-mediated intracellular delivery still remains to be fully understood. Among the proposed mechanisms are formation of transient pores and endocytosis stimulation. In our study, we investigated whether endocytosis is involved in US- and MB-mediated delivery of small molecules. Dynamic fluorescence microscopy was used to investigate the effects of endocytosis inhibitors on the pharmacokinetic parameters of US- and MB-mediated uptake of SYTOX Green, a 600 Da hydrophilic model drug. C6 rat glioma cells, together with SonoVue® MBs, were exposed to 1.4 MHz US waves at 0.2 MPa peak-negative pressure. Collection of the signal intensity in each individual nucleus was monitored during and after US exposure by a fibered confocal fluorescence microscope designed for real-time imaging. Exposed to US waves, C6 cells pretreated with chlorpromazine, an inhibitor of clathrin-mediated endocytosis, showed up to a 2.5-fold significant increase of the uptake time constant, and a 1.1-fold increase with genistein, an inhibitor of caveolae-mediated endocytosis. Both inhibitors slowed down the US-mediated uptake of SYTOX Green. With C6 cells and our experimental settings, these quantitative data indicate that endocytosis plays a role in sonopermeabilization-mediated delivery of small molecules with a more predominant contribution of clathrin-mediated endocytosis.

  12. The Effect of Substrate Elasticity and Actomyosin Contractility on Different Forms of Endocytosis

    PubMed Central

    Missirlis, Dimitris

    2014-01-01

    Substrate mechanical properties have emerged as potent determinants of cell functions and fate. We here tested the hypothesis that different forms of endocytosis are regulated by the elasticity of the synthetic hydrogels cells are cultured on. Towards this objective, we quantified cell-associated fluorescence of the established endocytosis markers transferrin (Tf) and cholera toxin subunit B (CTb) using a flow-cytometry based protocol, and imaged marker internalization using microscopy techniques. Our results demonstrated that clathrin-mediated endocytosis of Tf following a 10-minute incubation with a fibroblast cell line was lower on the softer substrates studied (5 kPa) compared to those with elasticities of 40 and 85 kPa. This effect was cancelled after 1-hour incubation revealing that intracellular accumulation of Tf at this time point did not depend on substrate elasticity. Lipid-raft mediated endocytosis of CTb, on the other hand, was not affected by substrate elasticity in the studied range of time and substrate elasticity. The use of pharmacologic contractility inhibitors revealed inhibition of endocytosis for both Tf and CTb after a 10-minute incubation and a dissimilar effect after 1 hour depending on the inhibitor type. Further, the internalization of fluorescent NPs, used as model drug delivery systems, showed a dependence on substrate elasticity, while transfection efficiency was unaffected by it. Finally, an independence on substrate elasticity of Tf and CTb association with HeLa cells indicated that there are cell-type differences in this respect. Overall, our results suggest that clathrin-mediated but not lipid-raft mediated endocytosis is potentially influenced by substrate mechanics at the cellular level, while intracellular trafficking and accumulation show a more complex dependence. Our findings are discussed in the context of previous work on how substrate mechanics affect the fundamental process of endocytosis and highlight important

  13. Particulate endocytosis mediates biological responses of human mesenchymal stem cells to titanium wear debris.

    PubMed

    Okafor, Chukwuka C; Haleem-Smith, Hana; Laqueriere, Patrice; Manner, Paul A; Tuan, Rocky S

    2006-03-01

    Continual loading and articulation cycles undergone by metallic (e.g., titanium) alloy arthroplasty prostheses lead to liberation of a large number of metallic debris particulates, which have long been implicated as a primary cause of periprosthetic osteolysis and postarthroplasty aseptic implant loosening. Long-term stability of total joint replacement prostheses relies on proper integration between implant biomaterial and osseous tissue, and factors that interfere with this integration are likely to cause osteolysis. Because multipotent mesenchymal stem cells (MSCs) located adjacent to the implant have an osteoprogenitor function and are critical contributors to osseous tissue integrity, when their functions or activities are compromised, osteolysis will most likely occur. To date, it is not certain or sufficiently confirmed whether MSCs endocytose titanium particles, and if so, whether particulate endocytosis has any effect on cellular responses to wear debris. This study seeks to clarify the phenomenon of titanium endocytosis by human MSCs (hMSCs), and investigates the influence of endocytosis on their activities. hMSCs incubated with commercially pure titanium particles exhibited internalized particles, as observed by scanning electron microscopy and confocal laser scanning microscopy, with time-dependent reduction in the number of extracellular particles. Particulate endocytosis was associated with reduced rates of cellular proliferation and cell-substrate adhesion, suppressed osteogenic differentiation, and increased rate of apoptosis. These cellular effects of exposure to titanium particles were reduced when endocytosis was inhibited by treatment with cytochalasin D, and no significant effect was seen when hMSCs were treated only with conditioned medium obtained from particulate-treated cells. These findings strongly suggest that the biological responses of hMSCs to wear debris are triggered primarily by the direct endocytosis of titanium particulates, and

  14. Steering neuronal growth cones by shifting the imbalance between exocytosis and endocytosis.

    PubMed

    Tojima, Takuro; Itofusa, Rurika; Kamiguchi, Hiroyuki

    2014-05-21

    Extracellular molecular cues guide migrating growth cones along specific routes during development of axon tracts. Such processes rely on asymmetric elevation of cytosolic Ca(2+) concentrations across the growth cone that mediates its attractive or repulsive turning toward or away from the side with Ca(2+) elevation, respectively. Downstream of these Ca(2+) signals, localized activation of membrane trafficking steers the growth cone bidirectionally, with endocytosis driving repulsion and exocytosis causing attraction. However, it remains unclear how Ca(2+) can differentially regulate these opposite membrane-trafficking events. Here, we show that growth cone turning depends on localized imbalance between exocytosis and endocytosis and identify Ca(2+)-dependent signaling pathways mediating such imbalance. In embryonic chicken dorsal root ganglion neurons, repulsive Ca(2+) signals promote clathrin-mediated endocytosis through a 90 kDa splice variant of phosphatidylinositol-4-phosphate 5-kinase type-1γ (PIPKIγ90). In contrast, attractive Ca(2+) signals facilitate exocytosis but suppress endocytosis via Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (Cdk5) that can inactivate PIPKIγ90. Blocking CaMKII or Cdk5 leads to balanced activation of both exocytosis and endocytosis that causes straight growth cone migration even in the presence of guidance signals, whereas experimentally perturbing the balance restores the growth cone's turning response. Remarkably, the direction of this resumed turning depends on relative activities of exocytosis and endocytosis, but not on the type of guidance signals. Our results suggest that navigating growth cones can be redirected by shifting the imbalance between exocytosis and endocytosis, highlighting the importance of membrane-trafficking imbalance for axon guidance and, possibly, for polarized cell migration in general.

  15. Recruitment of endocytosis in sonopermeabilization-mediated drug delivery: a real-time study.

    PubMed

    Derieppe, Marc; Rojek, Katarzyna; Escoffre, Jean-Michel; de Senneville, Baudouin Denis; Moonen, Chrit; Bos, Clemens

    2015-06-29

    Microbubbles (MBs) in combination with ultrasound (US) can enhance cell membrane permeability, and have the potential to facilitate the cellular uptake of hydrophilic molecules. However, the exact mechanism behind US- and MB-mediated intracellular delivery still remains to be fully understood. Among the proposed mechanisms are formation of transient pores and endocytosis stimulation. In our study, we investigated whether endocytosis is involved in US- and MB-mediated delivery of small molecules. Dynamic fluorescence microscopy was used to investigate the effects of endocytosis inhibitors on the pharmacokinetic parameters of US- and MB-mediated uptake of SYTOX Green, a 600 Da hydrophilic model drug. C6 rat glioma cells, together with SonoVue(®) MBs, were exposed to 1.4 MHz US waves at 0.2 MPa peak-negative pressure. Collection of the signal intensity in each individual nucleus was monitored during and after US exposure by a fibered confocal fluorescence microscope designed for real-time imaging. Exposed to US waves, C6 cells pretreated with chlorpromazine, an inhibitor of clathrin-mediated endocytosis, showed up to a 2.5-fold significant increase of the uptake time constant, and a 1.1-fold increase with genistein, an inhibitor of caveolae-mediated endocytosis. Both inhibitors slowed down the US-mediated uptake of SYTOX Green. With C6 cells and our experimental settings, these quantitative data indicate that endocytosis plays a role in sonopermeabilization-mediated delivery of small molecules with a more predominant contribution of clathrin-mediated endocytosis.

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

    PubMed

    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

  17. Novel activity-dependent approaches to therapeutic hypnosis and psychotherapy: the general waking trance.

    PubMed

    Rossi, Ernest; Erickson-Klein, Roxanna; Rossi, Kathryn

    2008-10-01

    This paper presents a highly edited version of a videotape made in 1980 by Marion Moore, M.D., showing Milton H. Erickson and Moore demonstrating novel, activity-dependent approaches to hand-levitation and therapeutic hypnosis on their subject, Ernest Rossi. Erickson's naturalistic and utilization approach is described in his very direct and surprising induction in a trance challenged patient. These novel, and surprising inductions are examples of how Erickson was prescient in developing activity-dependent approaches to therapeutic hypnosis and psychotherapy several generations before modern neuroscience documented the activity-dependent molecular-genomic mechanisms of memory, learning, and behavior change. Erickson describes a case where he utilized what he called, "The General Waking Trance" when he "dared" not use an obvious hypnotic induction. It is proposed that the states of intense mental absorption and response attentiveness that are facilitated by the general waking trance are functionally related to the three conditions neuroscientists have identified as novelty, enrichment, and exercise (both mental and physical), which can turn on activity-dependent gene expression and activity-dependent brain plasticity, that are the molecular-genomic and neural basis ofmemory, learning, consciousness, and behavior change. We recommend that the next step in investigating the efficacy of therapeutic hypnosis will be in partnering with neuroscientists to explore the possibilities and limitations of utilizing the activity-dependent approaches to hypnotic induction and the general waking trance in facilitating activity-dependent gene expression and brain plasticity.

  18. Transmembrane-domain shape is a novel endocytosis signal for single-spanning membrane proteins.

    PubMed

    Montoro, Ayelén González; Bigliani, Gonzalo; Taubas, Javier Valdez

    2017-10-02

    Endocytosis is crucial for all cells as it allows them to incorporate material from the extracellular space and control the availability of transmembrane proteins at the plasma membrane. In yeast, endocytosis followed by recycling to the plasma membrane results in a polarised distribution of membrane proteins by a kinetic mechanism. Here we report that increasing the volume of the residues that constitute the exoplasmic half of the transmembrane domain in the yeast SNARE Sso1, a type II membrane protein, results in its polarised distribution at the plasma membrane. Expression of this chimera in strains affected in either endocytosis or recycling revealed that this polarisation is achieved by endocytic cycling. A bioinformatics search of the Saccharomyces cerevisiae proteome identified several proteins with high-volume exoplasmic hemi-TMDs. Our experiments indicate that TMDs from these proteins can confer a polarised distribution to the Sso1 cytoplasmic domain, indicating that the shape of the TMD can act as a novel endocytosis and polarity signal in yeast. Additionally, a high-volume exoplasmic hemi-TMD can act as an endocytosis signal in a mammalian cell line. © 2017. Published by The Company of Biologists Ltd.

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

    PubMed

    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.

  20. A critical role for PSD-95/AKAP interactions in endocytosis of synaptic AMPA receptors.

    PubMed

    Bhattacharyya, Samarjit; Biou, Virginie; Xu, Weifeng; Schlüter, Oliver; Malenka, Robert C

    2009-02-01

    The endocytosis of AMPA receptors (AMPARs) underlies several forms of synaptic plasticity, including NMDA receptor (NMDAR)-dependent long-term depression (LTD), but the molecular mechanisms responsible for this trafficking remain unknown. We found that PSD-95, a major postsynaptic density protein, is important for NMDAR-triggered endocytosis of synaptic AMPARs in rat neuron cultures because of its binding to A kinase-anchoring protein 150 (AKAP150), a scaffold for specific protein kinases and phosphatases. Knockdown of PSD-95 with shRNA blocked NMDAR-triggered, but not constitutive or mGluR-triggered, endocytosis of AMPARs. Deletion of PSD-95's Src homology 3 and guanylate kinase-like domains, as well as a point mutation (L460P), both of which inhibit binding of PSD-95 to AKAP150, also blocked NMDAR-triggered AMPAR endocytosis. Furthermore, expression of a mutant AKAP150 that does not bind calcineurin inhibited this NMDAR-triggered trafficking event. Our results suggest that PSD-95's interaction with AKAP150 is critical for NMDAR-triggered AMPAR endocytosis and LTD, possibly because these scaffolds position calcineurin in the appropriate subsynaptic domain.

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

    NASA Astrophysics Data System (ADS)

    Youta Dombu, Christophe; Kroubi, Maya; Zibouche, Rima; Matran, Regis; Betbeder, Didier

    2010-09-01

    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 °C compared with 37 °C, or by NaN3 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.

  2. Endophilin, Lamellipodin, and Mena cooperate to regulate F-actin-dependent EGF-receptor endocytosis.

    PubMed

    Vehlow, Anne; Soong, Daniel; Vizcay-Barrena, Gema; Bodo, Cristian; Law, Ah-Lai; Perera, Upamali; Krause, Matthias

    2013-10-16

    The epidermal growth factor receptor (EGFR) plays an essential role during development and diseases including cancer. Lamellipodin (Lpd) is known to control lamellipodia protrusion by regulating actin filament elongation via Ena/VASP proteins. However, it is unknown whether this mechanism supports endocytosis of the EGFR. Here, we have identified a novel role for Lpd and Mena in clathrin-mediated endocytosis (CME) of the EGFR. We have discovered that endogenous Lpd is in a complex with the EGFR and Lpd and Mena knockdown impairs EGFR endocytosis. Conversely, overexpressing Lpd substantially increases the EGFR uptake in an F-actin-dependent manner, suggesting that F-actin polymerization is limiting for EGFR uptake. Furthermore, we found that Lpd directly interacts with endophilin, a BAR domain containing protein implicated in vesicle fission. We identified a role for endophilin in EGFR endocytosis, which is mediated by Lpd. Consistently, Lpd localizes to clathrin-coated pits (CCPs) just before vesicle scission and regulates vesicle scission. Our findings suggest a novel mechanism in which Lpd mediates EGFR endocytosis via Mena downstream of endophilin.

  3. Endocytosis of Multiwalled Carbon Nanotubes in Bronchial Epithelial and Mesothelial Cells

    PubMed Central

    Maruyama, Kayo; Matsuda, Yoshikazu; Kobayashi, Shinsuke; Tanaka, Manabu; Aoki, Kaoru; Takanashi, Seiji; Okamoto, Masanori; Kato, Hiroyuki

    2015-01-01

    Bronchial epithelial cells and mesothelial cells are crucial targets for the safety assessment of inhalation of carbon nanotubes (CNTs), which resemble asbestos particles in shape. Intrinsic properties of multiwalled CNTs (MWCNTs) are known to cause potentially hazardous effects on intracellular and extracellular pathways. These interactions alter cellular signaling and affect major cell functions, resulting in cell death, lysosome injury, reactive oxygen species production, apoptosis, and cytokine release. Furthermore, CNTs are emerging as a novel class of autophagy inducers. Thus, in this study, we focused on the mechanisms of MWCNT uptake into the human bronchial epithelial cells (HBECs) and human mesothelial cells (HMCs). We verified that MWCNTs are actively internalized into HBECs and HMCs and were accumulated in the lysosomes of the cells after 24-hour treatment. Next, we determined which endocytosis pathways (clathrin-mediated, caveolae-mediated, and macropinocytosis) were associated with MWCNT internalization by using corresponding endocytosis inhibitors, in two nonphagocytic cell lines derived from bronchial epithelial cells and mesothelioma cells. Clathrin-mediated endocytosis inhibitors significantly suppressed MWCNT uptake, whereas caveolae-mediated endocytosis and macropinocytosis were also found to be involved in MWCNT uptake. Thus, MWCNTs were positively taken up by nonphagocytic cells, and their cytotoxicity was closely related to these three endocytosis pathways. PMID:26090445

  4. Counterintuitive cooperative endocytosis of like-charged nanoparticles in cellular internalization: computer simulation and experiment.

    PubMed

    Li, Ye; Yuan, Bing; Yang, Kai; Zhang, Xianren; Yan, Bing; Cao, Dapeng

    2017-02-24

    The nanoparticles (NPs) functionalized with charged ligands are of particular significance due to their potential drug/gene delivery and biomedical applications. However, the molecular mechanism of endocytosis of the charged NPs by cells, especially the effect of the NP-NP and NP-biomembrane interactions on the internalization pathways is still poorly understood. In this work, we systematically investigate the internalization behaviors of the positively charged NPs by combining experiment technology and dissipative particle dynamics (DPD) simulation. We experimentally find an interesting but highly counterintuitive phenomenon, i.e. the multiple positively charged NPs prefer to enter cells cooperatively although the like-charged NPs have obvious electrostatic repulsion. Furthermore, we adopt the DPD simulation to confirm the experimental findings, and reveal that the mechanism of the cooperative endocytosis between like-charged NPs is definitely caused by the interplay of particle size, the charged ligand density on particle surface and local concentration of NPs. Importantly, we not only observe the normal cooperative endocytosis of like-charged NPs in cell biomembrane like neutral NP case, but also predict the 'bud' cooperative endocytosis of like-charged NPs which is absence in the neutral NP case. The results indicate that electrostatic repulsion between the positively charged nanoparticles plays an important role in the 'bud' cooperative endocytosis of like-charged NPs.

  5. Initiation of clathrin-mediated endocytosis: all you need is two?

    PubMed

    Swan, Laura E

    2013-05-01

    Clathrin-mediated endocytosis is a major route for the retrieval of plasma-membrane cargoes, and defects of this process can cause catastrophic human dysfunctions. However, the processes governing how a clathrin-coated profile (ccp) is initiated are still murky. Despite an ever-growing cast of molecules proposed as triggers of ccp nucleation and increasingly sophisticated bioimaging techniques examining clathrin-mediated endocytosis, it is yet unknown if ccp formation is governed by a universal mechanism. A recent paper by Cocucci et al. has tracked single-molecule events to identify that stable accumulation of ccps requires the near-simultaneous arrival of two AP2 adaptors bridged by one clathrin triskelion. This commentary examines the role of AP2 in cargo-mediated endocytosis in the light of recent advances in biophotonics, chemical inhibitors and genetics, examines the claims of other molecules to be the initiators of ccp formation and proposes future directions in research into this topic. Editor's suggested further reading in BioEssays: The evolution of dynamin to regulate clathrin-mediated endocytosis Abstract Clathrin-mediated endocytosis: What works for small, also works for big Abstract.

  6. Mutation of the YXXL Endocytosis Motif in the Cytoplasmic Tail of Pseudorabies Virus gE

    PubMed Central

    Tirabassi, R. S.; Enquist, L. W.

    1999-01-01

    The role of alphaherpesvirus membrane protein internalization during the course of viral infection remains a matter of speculation. To determine the role of internalization of the pseudorabies virus (PRV) gE and gI proteins, we constructed viral mutants encoding specific mutations in the cytoplasmic tail of the gE gene that inhibited internalization of the gE-gI complex. We used these mutants to assess the role of gE-gI endocytosis in incorporation of the proteins into the viral envelope and in gE-mediated spread or gE-promoted virulence. In addition, we report that another viral mutant, PRV 25, which encodes a gE protein defective in endocytosis, contains an additional, previously uncharacterized mutation in the gE gene. We compared PRV 25 to another viral mutant, PRV 107, that does not express the cytoplasmic tail of the gE protein. The gE protein encoded by PRV 107 is also defective in endocytosis. We conclude that efficient endocytosis of gE is not required for gE incorporation into virions, gE-mediated virulence, or spread of virus in the rat central nervous system. However, we do correlate the defect in endocytosis to a small-plaque phenotype in cultured cells. PMID:10074118

  7. Counterintuitive cooperative endocytosis of like-charged nanoparticles in cellular internalization: computer simulation and experiment

    NASA Astrophysics Data System (ADS)

    Li, Ye; Yuan, Bing; Yang, Kai; Zhang, Xianren; Yan, Bing; Cao, Dapeng

    2017-02-01

    The nanoparticles (NPs) functionalized with charged ligands are of particular significance due to their potential drug/gene delivery and biomedical applications. However, the molecular mechanism of endocytosis of the charged NPs by cells, especially the effect of the NP-NP and NP-biomembrane interactions on the internalization pathways is still poorly understood. In this work, we systematically investigate the internalization behaviors of the positively charged NPs by combining experiment technology and dissipative particle dynamics (DPD) simulation. We experimentally find an interesting but highly counterintuitive phenomenon, i.e. the multiple positively charged NPs prefer to enter cells cooperatively although the like-charged NPs have obvious electrostatic repulsion. Furthermore, we adopt the DPD simulation to confirm the experimental findings, and reveal that the mechanism of the cooperative endocytosis between like-charged NPs is definitely caused by the interplay of particle size, the charged ligand density on particle surface and local concentration of NPs. Importantly, we not only observe the normal cooperative endocytosis of like-charged NPs in cell biomembrane like neutral NP case, but also predict the ‘bud’ cooperative endocytosis of like-charged NPs which is absence in the neutral NP case. The results indicate that electrostatic repulsion between the positively charged nanoparticles plays an important role in the ‘bud’ cooperative endocytosis of like-charged NPs.

  8. EphA2 signalling following endocytosis: role of Tiam1

    PubMed Central

    Boissier, Pomme; Chen, Jin; Huynh-Do, Uyen

    2013-01-01

    Eph receptors and their membrane-bound ligands, the ephrins, represent a complex subfamily of receptor tyrosine kinases (RTKs). Eph/ephrin binding can lead to various and opposite cellular behaviours such as adhesion versus repulsion, or cell migration versus cell adhesion. Recently, Eph endocytosis has been identified as one of the critical steps responsible for such diversity. Eph receptors, as many RTKs, are rapidly endocytosed following ligand-mediated activation and traffic through endocytic compartments prior to degradation. However, it is becoming obvious that endocytosis controls signalling in many different manners. Here we showed that activated EphA2 are degraded in the lysosomes and that about 35% of internalized receptors are recycled back to the plasma membrane. Our study is also the first to demonstrate that EphA2 retains the capacity to signal in endosomes. In particular, activated EphA2 interacted with the Rho family GEF Tiam1 in endosomes. This association led to Tiam1 activation, which in turn increased Rac1 activity and facilitated Eph/ephrin endocytosis. Disrupting Tiam1 function with RNA interference impaired both ephrinA1-dependent Rac1 activation and ephrinA1-induced EphA2 endocytosis. In summary, our findings shed new light on the regulation of EphA2 endocytosis, intracellular trafficking and signal termination and establish Tiam1 as an important modulator of EphA2 signalling. PMID:24112471

  9. ER network homeostasis is critical for plant endosome streaming and endocytosis

    PubMed Central

    Stefano, Giovanni; Renna, Luciana; Lai, YaShiuan; Slabaugh, Erin; Mannino, Nicole; Buono, Rafael A; Otegui, Marisa S; Brandizzi, Federica

    2015-01-01

    Eukaryotic cells internalize cargo at the plasma membrane via endocytosis, a vital process that is accomplished through a complex network of endosomal organelles. In mammalian cells, the ER is in close association with endosomes and regulates their fission. Nonetheless, the physiological role of such interaction on endocytosis is yet unexplored. Here, we probed the existence of ER–endosome association in plant cells and assayed its physiological role in endocytosis. Through live-cell imaging and electron microscopy studies, we established that endosomes are extensively associated with the plant ER, supporting conservation of interaction between heterotypic organelles in evolutionarily distant kingdoms. Furthermore, by analyzing ER–endosome dynamics in genetic backgrounds with defects in ER structure and movement, we also established that the ER network integrity is necessary for homeostasis of the distribution and streaming of various endosome populations as well as for efficient endocytosis. These results support a novel model that endocytosis homeostasis depends on a spatiotemporal control of the endosome dynamics dictated by the ER membrane network. PMID:27462431

  10. ER network homeostasis is critical for plant endosome streaming and endocytosis.

    PubMed

    Stefano, Giovanni; Renna, Luciana; Lai, YaShiuan; Slabaugh, Erin; Mannino, Nicole; Buono, Rafael A; Otegui, Marisa S; Brandizzi, Federica

    2015-01-01

    Eukaryotic cells internalize cargo at the plasma membrane via endocytosis, a vital process that is accomplished through a complex network of endosomal organelles. In mammalian cells, the ER is in close association with endosomes and regulates their fission. Nonetheless, the physiological role of such interaction on endocytosis is yet unexplored. Here, we probed the existence of ER-endosome association in plant cells and assayed its physiological role in endocytosis. Through live-cell imaging and electron microscopy studies, we established that endosomes are extensively associated with the plant ER, supporting conservation of interaction between heterotypic organelles in evolutionarily distant kingdoms. Furthermore, by analyzing ER-endosome dynamics in genetic backgrounds with defects in ER structure and movement, we also established that the ER network integrity is necessary for homeostasis of the distribution and streaming of various endosome populations as well as for efficient endocytosis. These results support a novel model that endocytosis homeostasis depends on a spatiotemporal control of the endosome dynamics dictated by the ER membrane network.

  11. Endocytosis of the anthrax toxin is mediated by clathrin, actin and unconventional adaptors.

    PubMed

    Abrami, Laurence; Bischofberger, Mirko; Kunz, Béatrice; Groux, Romain; van der Goot, F Gisou

    2010-03-05

    The anthrax toxin is a tripartite toxin, where the two enzymatic subunits require the third subunit, the protective antigen (PA), to interact with cells and be escorted to their cytoplasmic targets. PA binds to cells via one of two receptors, TEM8 and CMG2. Interestingly, the toxin times and triggers its own endocytosis, in particular through the heptamerization of PA. Here we show that PA triggers the ubiquitination of its receptors in a beta-arrestin-dependent manner and that this step is required for clathrin-mediated endocytosis. In addition, we find that endocytosis is dependent on the heterotetrameric adaptor AP-1 but not the more conventional AP-2. Finally, we show that endocytosis of PA is strongly dependent on actin. Unexpectedly, actin was also found to be essential for efficient heptamerization of PA, but only when bound to one of its 2 receptors, TEM8, due to the active organization of TEM8 into actin-dependent domains. Endocytic pathways are highly modular systems. Here we identify some of the key players that allow efficient heptamerization of PA and subsequent ubiquitin-dependent, clathrin-mediated endocytosis of the anthrax toxin.

  12. Meeting after meeting: 20 years of discoveries by the members of the Exocytosis-Endocytosis Club.

    PubMed

    Niedergang, Florence; Gasman, Stéphane; Vitale, Nicolas; Desnos, Claire; Lamaze, Christophe

    2017-09-01

    Twenty years ago, a group of French cell biologists merged two scientific clubs with the aim of bringing together researchers in the fields of Endocytosis and Exocytosis. Founded in 1997, the first annual meeting of the Exocytosis Club was held in 1998. The Endocytosis Club held quarterly meetings from its founding in 1999. The first joint annual meeting of the Exocytosis-Endocytosis Club took place in Paris in April, 2001. What started as a modest gathering of enthusiastic scientists working in the field of cell trafficking has gone from strength to strength, rapidly becoming an unmissable yearly meeting, vividly demonstrating the high quality of science performed in our community and beyond. On the occasion of the 20th meeting of our club, we want to provide historic insight into the fields of exocytosis and endocytosis, and by extension, to subcellular trafficking, highlighting how French scientists have contributed to major advances in these fields. Today, the Exocytosis-Endocytosis Club represents a vibrant and friendly community that will hold its 20th meeting at the Presqu'Ile de Giens, near Toulon in the South of France, on May 11-13, 2017. © 2017 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

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

    PubMed

    Campa, Víctor M; Capilla, Almudena; Varela, María J; de la Rocha, Arlet M Acanda; Fernandez-Troyano, Juan C; Barreiro, R Belén; Lopez-Gimenez, Juan F

    2015-01-01

    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.

  14. EphA2 signaling following endocytosis: role of Tiam1.

    PubMed

    Boissier, Pomme; Chen, Jin; Huynh-Do, Uyen

    2013-12-01

    Eph receptors and their membrane-bound ligands, the ephrins, represent a complex subfamily of receptor tyrosine kinases (RTKs). Eph/ephrin binding can lead to various and opposite cellular behaviors such as adhesion versus repulsion, or cell migration versus cell-adhesion. Recently, Eph endocytosis has been identified as one of the critical steps responsible for such diversity. Eph receptors, as many RTKs, are rapidly endocytosed following ligand-mediated activation and traffic through endocytic compartments prior to degradation. However, it is becoming obvious that endocytosis controls signaling in many different manners. Here we showed that activated EphA2 are degraded in the lysosomes and that about 35% of internalized receptors are recycled back to the plasma membrane. Our study is also the first to demonstrate that EphA2 retains the capacity to signal in endosomes. In particular, activated EphA2 interacted with the Rho family GEF Tiam1 in endosomes. This association led to Tiam1 activation, which in turn increased Rac1 activity and facilitated Eph/ephrin endocytosis. Disrupting Tiam1 function with RNA interference impaired both ephrinA1-dependent Rac1 activation and ephrinA1-induced EphA2 endocytosis. In summary, our findings shed new light on the regulation of EphA2 endocytosis, intracellular trafficking and signal termination and establish Tiam1 as an important modulator of EphA2 signaling.

  15. Clathrin to Lipid Raft-Endocytosis via Controlled Surface Chemistry and Efficient Perinuclear Targeting of Nanoparticle.

    PubMed

    Chakraborty, Atanu; Jana, Nikhil R

    2015-09-17

    Nanoparticle interacts with live cells depending on their surface chemistry, enters into cell via endocytosis, and is commonly trafficked to an endosome/lysozome that restricts subcellular targeting options. Here we show that nanoparticle surface chemistry can be tuned to alter their cell uptake mechanism and subcellular trafficking. Quantum dot based nanoprobes of 20-30 nm hydrodynamic diameters have been synthesized with tunable surface charge (between +15 mV to -25 mV) and lipophilicity to influence their cellular uptake processes and subcellular trafficking. It is observed that cationic nanoprobe electrostatically interacts with cell membrane and enters into cell via clathrin-mediated endocytosis. At lower surface charge (between +10 mV to -10 mV), the electrostatic interaction with cell membrane becomes weaker, and additional lipid raft endocytosis is initiated. If a lipophilic functional group is introduced on a weakly anionic nanoparticle surface, the uptake mechanism shifts to predominant lipid raft-mediated endocytosis. In particular, the zwitterionic-lipophilic nanoprobe has the unique advantage as it weakly interacts with anionic cell membrane, migrates toward lipid rafts for interaction through lipophilic functional group, and induces lipid raft-mediated endocytosis. While predominate or partial clathrin-mediated entry traffics most of the nanoprobes to lysozome, predominate lipid raft-mediated entry traffics them to perinuclear region, particularly to the Golgi apparatus. This finding would guide in designing appropriate nanoprobe for subcellular targeting and delivery.

  16. A Wiskott-Aldrich syndrome protein is involved in endocytosis in Aspergillus nidulans.

    PubMed

    Hoshi, Hiro-Omi; Zheng, Lu; Ohta, Akinori; Horiuchi, Hiroyuki

    2016-09-01

    Endocytosis is vital for hyphal tip growth in filamentous fungi and is involved in the tip localization of various membrane proteins. To investigate the function of a Wiskott-Aldrich syndrome protein (WASP) in endocytosis of filamentous fungi, we identified a WASP ortholog-encoding gene, wspA, in Aspergillus nidulans and characterized it. The wspA product, WspA, localized to the tips of germ tubes during germination and actin rings in the subapical regions of mature hyphae. wspA is essential for the growth and functioned in the polarity establishment and maintenance during germination of conidia. We also investigated its function in endocytosis and revealed that endocytosis of SynA, a synaptobrevin ortholog that is known to be endocytosed at the subapical regions of hyphal tips in A. nidulans, did not occur when wspA expression was repressed. These results suggest that WspA plays roles in endocytosis at hyphal tips and polarity establishment during germination.

  17. Pan1 regulates transitions between stages of clathrin-mediated endocytosis.

    PubMed

    Bradford, Mary Katherine; Whitworth, Karen; Wendland, Beverly

    2015-04-01

    Endocytosis is a well-conserved process by which cells invaginate small portions of the plasma membrane to create vesicles containing extracellular and transmembrane cargo proteins. Dozens of proteins and hundreds of specific binding interactions are needed to coordinate and regulate these events. Saccharomyces cerevisiae is a powerful model system with which to study clathrin-mediated endocytosis (CME). Pan1 is believed to be a scaffolding protein due to its interactions with numerous proteins that act throughout the endocytic process. Previous research characterized many Pan1 binding interactions, but due to Pan1's essential nature, the exact mechanisms of Pan1's function in endocytosis have been difficult to define. We created a novel Pan1-degron allele, Pan1-AID, in which Pan1 can be specifically and efficiently degraded in <1 h upon addition of the plant hormone auxin. The loss of Pan1 caused a delay in endocytic progression and weakened connections between the coat/actin machinery and the membrane, leading to arrest in CME. In addition, we determined a critical role for the central region of Pan1 in endocytosis and viability. The regions important for endocytosis and viability can be separated, suggesting that Pan1 may have a distinct role in the cell that is essential for viability.

  18. The miR-199-dynamin regulatory axis controls receptor-mediated endocytosis.

    PubMed

    Aranda, Juan F; Canfrán-Duque, Alberto; Goedeke, Leigh; Suárez, Yajaira; Fernández-Hernando, Carlos

    2015-09-01

    Small non-coding RNAs (microRNAs) are important regulators of gene expression that modulate many physiological processes; however, their role in regulating intracellular transport remains largely unknown. Intriguingly, we found that the dynamin (DNM) genes, a GTPase family of proteins responsible for endocytosis in eukaryotic cells, encode the conserved miR-199a and miR-199b family of miRNAs within their intronic sequences. Here, we demonstrate that miR-199a and miR-199b regulate endocytic transport by controlling the expression of important mediators of endocytosis such as clathrin heavy chain (CLTC), Rab5A, low-density lipoprotein receptor (LDLR) and caveolin-1 (Cav-1). Importantly, miR-199a-5p and miR-199b-5p overexpression markedly inhibits CLTC, Rab5A, LDLR and Cav-1 expression, thus preventing receptor-mediated endocytosis in human cell lines (Huh7 and HeLa). Of note, miR-199a-5p inhibition increases target gene expression and receptor-mediated endocytosis. Taken together, our work identifies a new mechanism by which microRNAs regulate intracellular trafficking. In particular, we demonstrate that the DNM, miR-199a-5p and miR-199b-5p genes act as a bifunctional locus that regulates endocytosis, thus adding an unexpected layer of complexity in the regulation of intracellular trafficking.

  19. An organized co-assembly of clathrin adaptors is essential for endocytosis.

    PubMed

    Skruzny, Michal; Desfosses, Ambroise; Prinz, Simone; Dodonova, Svetlana O; Gieras, Anna; Uetrecht, Charlotte; Jakobi, Arjen J; Abella, Marc; Hagen, Wim J H; Schulz, Joachim; Meijers, Rob; Rybin, Vladimir; Briggs, John A G; Sachse, Carsten; Kaksonen, Marko

    2015-04-20

    Clathrin-mediated endocytosis, the main trafficking route from the plasma membrane to the cytoplasm, is critical to many fundamental cellular processes. Clathrin, coupled to the membrane by adaptor proteins, is thought to play a major structural role in endocytosis by self-assembling into a cage-like lattice around the forming vesicle. Although clathrin adaptors are essential for endocytosis, little is known about their structural role in this process. Here we show that the membrane-binding domains of two conserved clathrin adaptors, Sla2 and Ent1, co-assemble in a PI(4,5)P2-dependent manner to form organized lattices on membranes. We determined the structure of the co-assembled lattice by electron cryo-microscopy and designed mutations that specifically impair the lattice formation in vitro. We show that these mutations block endocytosis in vivo. We suggest that clathrin adaptors not only link the polymerized clathrin to the membrane but also form an oligomeric structure, which is essential for membrane remodeling during endocytosis.

  20. Active dependency.

    PubMed

    Bornstein, R F

    1995-02-01

    Although dependency has long been associated with passivity, weakness, and submissiveness, a review of the empirical literature reveals that, in certain situations and settings, dependent persons actually exhibit a variety of active, assertive behaviors. In this article, I: a) trace the historical roots of the dependency-passivity link; b) review empirical studies from developmental, social, and clinical psychology which indicate that, in certain circumstances, dependency is associated with active, assertive behavior on the part of the dependent person; c) offer an alternative conceptual model of dependency that accounts for the entire range of behaviors-both passive and active-that are exhibited by the dependent person; and d) discuss the diagnostic and therapeutic implications of this alternative conceptual model of dependency.

  1. Specific analogues uncouple transport, signalling, oligo-ubiquitination and endocytosis in the yeast Gap1 amino acid transceptor

    PubMed Central

    Van Zeebroeck, Griet; Rubio-Texeira, Marta; Schothorst, Joep; Thevelein, Johan M

    2014-01-01

    The Saccharomyces cerevisiae amino acid transceptor Gap1 functions as receptor for signalling to the PKA pathway and concomitantly undergoes substrate-induced oligo-ubiquitination and endocytosis. We have identified specific amino acids and analogues that uncouple to certain extent signalling, transport, oligo-ubiquitination and endocytosis. l-lysine, l-histidine and l-tryptophan are transported by Gap1 but do not trigger signalling. Unlike l-histidine, l-lysine triggers Gap1 oligo-ubiquitination without substantial induction of endocytosis. Two transported, non-metabolizable signalling agonists, β-alanine and d-histidine, are strong and weak inducers of Gap1 endocytosis, respectively, but both causing Gap1 oligo-ubiquitination. The non-signalling agonist, non-transported competitive inhibitor of Gap1 transport, l-Asp-γ-l-Phe, induces oligo-ubiquitination but no discernible endocytosis. The Km of l-citrulline transport is much lower than the threshold concentration for signalling and endocytosis. These results show that molecules can be transported without triggering signalling or substantial endocytosis, and that oligo-ubiquitination and endocytosis do not require signalling nor metabolism. Oligo-ubiquitination is required, but apparently not sufficient to trigger endocytosis. In addition, we demonstrate intracellular cross-induction of endocytosis of transport-defective Gap1Y395C by ubiquitination- and endocytosis-deficient Gap1K9R,K16R. Our results support the concept that different substrates bind to partially overlapping binding sites in the same general substrate-binding pocket of Gap1, triggering divergent conformations, resulting in different conformation-induced downstream processes. PMID:24852066

  2. Specific analogues uncouple transport, signalling, oligo-ubiquitination and endocytosis in the yeast Gap1 amino acid transceptor.

    PubMed

    Van Zeebroeck, Griet; Rubio-Texeira, Marta; Schothorst, Joep; Thevelein, Johan M

    2014-07-01

    The Saccharomyces cerevisiae amino acid transceptor Gap1 functions as receptor for signalling to the PKA pathway and concomitantly undergoes substrate-induced oligo-ubiquitination and endocytosis. We have identified specific amino acids and analogues that uncouple to certain extent signalling, transport, oligo-ubiquitination and endocytosis. L-lysine, L-histidine and L-tryptophan are transported by Gap1 but do not trigger signalling. Unlike L-histidine, L-lysine triggers Gap1 oligo-ubiquitination without substantial induction of endocytosis. Two transported, non-metabolizable signalling agonists, β-alanine and D-histidine, are strong and weak inducers of Gap1 endocytosis, respectively, but both causing Gap1 oligo-ubiquitination. The non-signalling agonist, non-transported competitive inhibitor of Gap1 transport, L-Asp-γ-L-Phe, induces oligo-ubiquitination but no discernible endocytosis. The Km of L-citrulline transport is much lower than the threshold concentration for signalling and endocytosis. These results show that molecules can be transported without triggering signalling or substantial endocytosis, and that oligo-ubiquitination and endocytosis do not require signalling nor metabolism. Oligo-ubiquitination is required, but apparently not sufficient to trigger endocytosis. In addition, we demonstrate intracellular cross-induction of endocytosis of transport-defective Gap1(Y395C) by ubiquitination- and endocytosis-deficient Gap1(K9R,K16R). Our results support the concept that different substrates bind to partially overlapping binding sites in the same general substrate-binding pocket of Gap1, triggering divergent conformations, resulting in different conformation-induced downstream processes.

  3. Imaging galectin-3 dependent endocytosis with lattice light-sheet microscopy

    NASA Astrophysics Data System (ADS)

    Baek, Jongho; Lou, Jieqiong; Coelho, Simao; Lim, Yean Jin; Seidlitz, Silvia; Nicovich, Philip R.; Wunder, Christian; Johannes, Ludger; Gaus, Katharina

    2017-04-01

    Lattice light-sheet (LLS) microscopy provides ultrathin light sheets of a two-dimensional optical lattice that allows us imaging three-dimensional (3D) objects for hundreds of time points at sub-second intervals and at or below the diffraction limit. Galectin-3 (Gal3), a carbohydrate-binding protein, triggers glycosphingolipid (GSL)-dependent biogenesis of morphologically distinct endocytic vesicles that are cargo specific and clathrin independent. In this study, we apply LLS microscopy to study the dynamics of Gal3 dependent endocytosis in live T cells. This will allow us to observe Gal3-mediated endocytosis at high temporal and excellent 3D spatial resolution, which may shed light on our understanding of the mechanism and physiological function of Gal3-induced endocytosis.

  4. Clathrin and synaptic vesicle endocytosis: studies at the squid giant synapse

    PubMed Central

    Augustine, G.J.; Morgan, J.R.; Villalba-Galea, C.A.; Jin, S.; Prasad, K.; Lafer, E.M.

    2015-01-01

    The role of clathrin-mediated endocytosis in SV (synaptic vesicle) recycling has been studied by combining molecular biology, physiology and electron microscopy at the squid giant synapse. Procedures that prevent clathrin from assembling into membrane coats, such as impairment of binding of the AP180 and AP-2 adaptor proteins, completely prevent membrane budding during endocytosis. These procedures also reduce exocytosis, presumably an indirect effect of a reduction in the number of SVs following block of endocytosis. Disrupting the binding of auxilin to Hsc70 (heat-shock cognate 70) prevents clathrin-coated vesicles from uncoating and also disrupts SV recycling. Taken together, these results indicate that a clathrin-dependent pathway is the primary means of SV recycling at this synapse under physiological conditions. PMID:16417485

  5. Clathrin and synaptic vesicle endocytosis: studies at the squid giant synapse.

    PubMed

    Augustine, G J; Morgan, J R; Villalba-Galea, C A; Jin, S; Prasad, K; Lafer, E M

    2006-02-01

    The role of clathrin-mediated endocytosis in SV (synaptic vesicle) recycling has been studied by combining molecular biology, physiology and electron microscopy at the squid giant synapse. Procedures that prevent clathrin from assembling into membrane coats, such as impairment of binding of the AP180 and AP-2 adaptor proteins, completely prevent membrane budding during endocytosis. These procedures also reduce exocytosis, presumably an indirect effect of a reduction in the number of SVs following block of endocytosis. Disrupting the binding of auxilin to Hsc70 (heat-shock cognate 70) prevents clathrin-coated vesicles from uncoating and also disrupts SV recycling. Taken together, these results indicate that a clathrin-dependent pathway is the primary means of SV recycling at this synapse under physiological conditions.

  6. Asynchronous transmitter release: control of exocytosis and endocytosis at the salamander rod synapse.

    PubMed Central

    Rieke, F; Schwartz, E A

    1996-01-01

    1. We have studied exocytosis and endocytosis in the synaptic terminal of salamander rods using a combination of Ca2+ imaging, capacitance measurement and the photolysis of Ca2+ buffers. 2. The average cytoplasmic Ca2+ concentration at the dark resting potential was 2-4 microM. 3. An average cytoplasmic Ca2+ concentration of 2-4 microM maintained a high rate of continuous exocytosis and endocytosis. 4. Changes in the rate of exocytosis were followed in less than 0.7 s by compensatory changes in the rate of endocytosis. 5. Vesicle cycling in the rod synapse is specialized for graded transmission and differs from that previously described for synapses that release synchronized bursts of transmitter. Images Figure 3 PMID:8735690

  7. Asynchronous transmitter release: control of exocytosis and endocytosis at the salamander rod synapse.

    PubMed

    Rieke, F; Schwartz, E A

    1996-05-15

    1. We have studied exocytosis and endocytosis in the synaptic terminal of salamander rods using a combination of Ca2+ imaging, capacitance measurement and the photolysis of Ca2+ buffers. 2. The average cytoplasmic Ca2+ concentration at the dark resting potential was 2-4 microM. 3. An average cytoplasmic Ca2+ concentration of 2-4 microM maintained a high rate of continuous exocytosis and endocytosis. 4. Changes in the rate of exocytosis were followed in less than 0.7 s by compensatory changes in the rate of endocytosis. 5. Vesicle cycling in the rod synapse is specialized for graded transmission and differs from that previously described for synapses that release synchronized bursts of transmitter.

  8. Imaging Synaptic Vesicle Exocytosis-Endocytosis with pH-Sensitive Fluorescent Proteins.

    PubMed

    Afuwape, Olusoji A T; Kavalali, Ege T

    2016-01-01

    The introduction of pHluorin, a pH-sensitive GFP, by Miesenbock and colleagues provided a versatile tool to studies of vesicle trafficking, in particular synaptic vesicle exocytosis and endocytosis. By tagging pHluorin to the luminal region of the synaptic vesicular protein synaptobrevin (also called VAMP, vesicle-associated membrane protein) or other synaptic vesicle-specific proteins such as the vesicular glutamate transporter-1, we are able to directly track synaptic vesicle endocytosis in response to stimuli in a molecularly specific manner. Here, we describe the process of imaging synaptic vesicle endocytosis in response to extracellular stimulation in dissociated neuronal cultures of hippocampal neurons obtained from rats-also applicable to mice-using pHluorin-tagged vesicular glutamate transporter-1 as a reporter.

  9. Capacitance-based assay for real-time monitoring of endocytosis and cell viability.

    PubMed

    Lee, Rimi; Kim, Jihun; Kim, Sook Young; Jang, Seon Mi; Lee, Sun-Mi; Choi, In-Hong; Park, Seung Woo; Shin, Jeon-Soo; Yoo, Kyung-Hwa

    2012-07-07

    Label-free cell-based assays have emerged as a promising means for high-throughput screening. Most label-free sensors are based on impedance measurements that reflect the passive electrical properties of cells. Here we introduce a capacitance-based assay that measures the dielectric constant (capacitance) of biological cells, and demonstrate the feasibility of analyzing endocytosis and screening chemotherapeutic agents with this assay. Endocytosis induces a change in the zeta potential, leading to a change in the dielectric constant which enables real-time endocytosis monitoring using the capacitance sensor. Additionally, since the dielectric constant is proportional to cell radius and cell volume, cell viability can be estimated from the change in capacitance. Therefore, the capacitance sensor array can also be used for cytotoxicity testing for large-scale chemotherapeutic screening.

  10. Loss of gamma-secretase function impairs endocytosis of lipoprotein particles and membrane cholesterol homeostasis.

    PubMed

    Tamboli, Irfan Y; Prager, Kai; Thal, Dietmar R; Thelen, Karin M; Dewachter, Ilse; Pietrzik, Claus U; St George-Hyslop, Peter; Sisodia, Sangram S; De Strooper, Bart; Heneka, Michael T; Filippov, Mikhail A; Müller, Ulrike; van Leuven, Fred; Lütjohann, Dieter; Walter, Jochen

    2008-11-12

    Presenilins (PSs) are components of the gamma-secretase complex that mediates intramembranous cleavage of type I membrane proteins. We show that gamma-secretase is involved in the regulation of cellular lipoprotein uptake. Loss of gamma-secretase function decreased endocytosis of low-density lipoprotein (LDL) receptor. The decreased uptake of lipoproteins led to upregulation of cellular cholesterol biosynthesis by increased expression of CYP51 and enhanced metabolism of lanosterol. Genetic deletion of PS1 or transgenic expression of PS1 mutants that cause early-onset Alzheimer's disease led to accumulation of gamma-secretase substrates and mistargeting of adaptor proteins that regulate endocytosis of the LDL receptor. Consistent with decreased endocytosis of these receptors, PS1 mutant mice have elevated levels of apolipoprotein E in the brain. Thus, these data demonstrate a functional link between two major genetic factors that cause early-onset and late-onset Alzheimer's disease.

  11. The Wurst protein: a novel endocytosis regulator involved in airway clearance and respiratory tube size control.

    PubMed

    Wingen, Christian; Aschenbrenner, Anna C; Stümpges, Birgit; Hoch, Michael; Behr, Matthias

    2009-01-01

    The mammalian lung and the Drosophila airways are composed of an intricate network of epithelial tubes that transports fluids or gases and converts during late embryogenesis from liquid- to air-filling. Conserved growth factor pathways have been characterized in model organisms such as Drosophila or the mouse that control patterning and branching of tubular networks. In contrast, knowledge of the coordination of respiratory tube size and physiology is still limited. Latest studies have shown that endocytosis plays a major role in size determination and liquid clearance of the respiratory tubes and a new key regulator of these processes was identified, the Drosophila Wurst protein. wurst encodes a J-domain transmembrane protein which is essential for Clathrin-mediated endocytosis. It is evolutionary conserved and single Wurst orthologs are found in mammals (termed DNAJC22). In this commentary, we discuss the role of Wurst/DNAJC22 and address whether these proteins may be general regulators of Clathrin-mediated endocytosis.

  12. Flow cytometric determination of endocytosis of viable labelled Legionella pneumophila by Acanthamoeba palestinensis.

    PubMed

    Harf, C; Goffinet, S; Meunier, O; Monteil, H; Colin, D A

    1997-03-01

    Endocytosis of fluorescently labelled cells of Legionella pneumophila (L. pneumophila) by free-living Acanthamoeba palestinensis (A. palestinensis) has been studied using flow cytometry. L. pneumophila cells were labelled with CM-DiI, a lipophilic fluorescent probe under conditions that did not modify viability. Coculturing the bacteria with amoebae was accompanied by rapid endocytosis; after 5 min, 90% of the amoebae had internalized bacteria. This percentage remained unchanged during further coculture, but the number of bacteria ingested per amoeba increased. Moreover, the number of ingested bacteria was found to be dependent on the size of the amoeba. The validity of the internalization analyzed by flow cytometry was confirmed by observation using epifluorescence and phase contrast microscopy. CM-DiI labelling associated with flow cytometry provides a very valuable technique for the determination of bacteria endocytosis by free-living amoeba.

  13. Clathrin in Chara australis: Molecular Analysis and Involvement in Charasome Degradation and Constitutive Endocytosis

    PubMed Central

    Hoepflinger, Marion C.; Hoeftberger, Margit; Sommer, Aniela; Hametner, Christina; Foissner, Ilse

    2017-01-01

    Charasomes are convoluted plasma membrane domains in characean green algae. They are known to form in response to light via secretion of trans-Golgi network (TGN) vesicles and local inhibition of endocytosis. Charasomes are involved in the acidification of their aqueous environment, thereby facilitating photosynthesis-dependent carbon uptake. Charasome formation is reversible to allow cells to adapt to different light conditions. Here, we show that darkness-induced degradation of charasomes involves the formation of coated pits and coated vesicles. The darkness-induced degradation of charasomes can be inhibited by 1–2 μM ikarugamycin (IKA), which is considered to be a specific inhibitor of clathrin-dependent endocytosis. At a much higher concentration (100 μM), IKA also significantly reduces the internalization of styryl dyes, indicating uptake via clathrin-coated vesicles (CV). We are the first to present evidence, based on fine structure investigation, that IKA does not interfere with the formation of clathrin coat, but inhibits the detachment and/or further processing of coated vesicles. Both charasome degradation and constitutive endocytosis are also significantly inhibited by sterol complexing agents (methyl-ß-cyclodextrin and filipin). The absence of an additive effect, when applied together with IKA, suggests that charasome degradation and constitutive endocytosis (measured via styryl dye uptake) is not inhibited due to membrane retrieval via lipid rafts, but due to clathrin coat formation requirement of a specific set of sterols. Analysis of Chara australis clathrin proteins revealed two heavy chains and several light chains with sequence peculiarities, suggesting functional and/or species specific differences. The data obtained indicate that clathrin plays a central role not only in constitutive endocytosis but also in the degradation of charasomes, thereby representing a valuable system for studying targeted exo- and endocytosis. PMID:28184226

  14. Endocytosis Plays a Critical Role in Proteolytic Processing of the Hendra Virus Fusion Protein

    PubMed Central

    Meulendyke, Kelly Ann; Wurth, Mark Allen; McCann, Richard O.; Dutch, Rebecca Ellis

    2005-01-01

    The Hendra virus fusion (F) protein is synthesized as a precursor protein, F0, which is proteolytically processed to the mature form, F1+F2. Unlike the case for the majority of paramyxovirus F proteins, the processing event is furin independent, does not require the addition of exogenous proteases, is not affected by reductions in intracellular Ca2+, and is strongly affected by conditions that raise the intracellular pH (C. T. Pager, M. A. Wurth, and R. E. Dutch, J. Virol. 78:9154-9163, 2004). The Hendra virus F protein cytoplasmic tail contains a consensus motif for endocytosis, YXXΦ. To analyze the potential role of endocytosis in the processing and membrane fusion promotion of the Hendra virus F protein, mutation of tyrosine 525 to alanine (Hendra virus F Y525A) or phenylalanine (Hendra virus F Y525F) was performed. The rate of endocytosis of Hendra virus F Y525A was significantly reduced compared to that of the wild-type (wt) F protein, confirming the functional importance of the endocytosis motif. An intermediate level of endocytosis was observed for Hendra virus F Y525F. Surprisingly, dramatic reductions in the rate of proteolytic processing were observed for Hendra virus F Y525A, although initial transport to the cell surface was not affected. The levels of surface expression for both Hendra virus F Y525A and Hendra virus F Y525F were higher than that of the wt protein, and these mutants displayed enhanced syncytium formation. These results suggest that endocytosis is critically important for Hendra virus F protein cleavage, representing a new paradigm for proteolytic processing of paramyxovirus F proteins. PMID:16188966

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

    PubMed Central

    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 and endocytosis. We first identified the region of the FcRII-B2 cytoplasmic domain that is required for rapid internalization. Using a biochemical assay for endocytosis and an immuno-EM assay to determine coated pit localization directly, we found that the distal half of the cytoplasmic domain, particularly a region including residues 18-31, as needed for coated pit-mediated endocytosis. Elimination of the tyrosine residues at position 26 and 43, separately or together, had little effect on coated pit localization and a partial effect on endocytosis of ligand. Since the FcRII-B1 insertion occurs in the membrane-proximal region of the cytoplasmic domain (residue 6) not required for internalization, it is unlikely to act by physically disrupting the coated pit localization determinant. In fact, the insertion was found to prevent endocytosis irrespective of its position in the cytoplasmic tail and appeared to selectively exclude the receptor from coated regions. Moreover, receptors bearing the insertion exhibited a temperature- and ligand-dependent association with a detergent-insoluble fraction and with actin filaments, perhaps in part explaining the inability of FcRII-B1 to enter coated pits. PMID:1734021

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

    SciTech Connect

    Singh, Raman Deep Schroeder, Andreas S.; Scheffer, Luana; Holicky, Eileen L.; Wheatley, Christine L.; Marks, David L. 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

  17. Functional equivalence of the clathrin heavy chains CHC17 and CHC22 in endocytosis and mitosis.

    PubMed

    Hood, Fiona E; Royle, Stephen J

    2009-07-01

    Clathrin is crucial for endocytosis and plays a recently described role in mitosis. Two clathrin heavy chains (CHCs) are found in humans: the ubiquitous CHC17, and CHC22, a CHC that is enriched in skeletal muscle. Functional differences have been proposed for these clathrins despite high sequence similarity. Here, we compared each paralogue in functional assays of endocytosis and mitosis. We find that CHC17 and CHC22 are functionally equivalent. We also describe how previous work on CHC22 has involved a splice variant that is not usually expressed in cells.

  18. Water Soluble Single-Walled Carbon Nanotubes Inhibit Stimulated Endocytosis in Neurons

    PubMed Central

    Malarkey, Erik B.; Reyes, Reno C.; Zhao, Bin; Haddon, Robert C.; Parpura, Vladimir

    2009-01-01

    We report the use of chemically-functionalized water soluble single-walled carbon nanotube (SWNT) graft copolymers to inhibit endocytosis. The graft copolymers were prepared by the functionalization of SWNTs with poly-ethylene glycol. When added to the culturing medium, these functionalized water soluble SWNTs were able to increase the length of various neuronal processes, neurites, as previously reported. Here we have determined that SWNTs are able to block stimulated membrane endocytosis in neurons, which could then explain the previously noted extended neurite length. PMID:18759491

  19. Receptor-mediated endocytosis for drug delivery in African trypanosomes: fulfilling Paul Ehrlich's vision of chemotherapy.

    PubMed

    Alsford, Sam; Field, Mark C; Horn, David

    2013-05-01

    Bloodstream-form cells of Trypanosoma brucei exhibit massively increased endocytic activity relative to the insect midgut stage, enabling rapid recycling of variant surface glycoprotein and antibody clearance from the surface. In addition, recent advances have identified a role for receptor-mediated endocytosis in the uptake of the antitrypanosomal drug, suramin, via invariant surface glycoprotein 75, and in the uptake of trypanosome lytic factor 1 via haptoglobin-haemoglobin receptor. Here, we argue that receptor-mediated endocytosis represents both a validated drug target and a promising route for the delivery of novel therapeutics into trypanosomes.

  20. Rab5 Isoforms Specifically Regulate Different Modes of Endocytosis in Leishmania.

    PubMed

    Rastogi, Ruchir; Verma, Jitender Kumar; Kapoor, Anjali; Langsley, Gordon; Mukhopadhyay, Amitabha

    2016-07-08

    Differential functions of Rab5 isoforms in endocytosis are not well characterized. Here, we cloned, expressed, and characterized Rab5a and Rab5b from Leishmania and found that both of them are localized in the early endosome. To understand the role of LdRab5 isoforms in different modes of endocytosis in Leishmania, we generated transgenic parasites overexpressing LdRab5a, LdRab5b, or their dominant-positive (LdRab5a:Q93L and LdRab5b:Q80L) or dominant-negative mutants (LdRab5a:N146I and LdRab5b:N133I). Using LdRab5a or its mutants overexpressing parasites, we found that LdRab5a specifically regulates the fluid-phase endocytosis of horseradish peroxidase and also specifically induced the transport of dextran-Texas Red to the lysosomes. In contrast, cells overexpressing LdRab5b or its mutants showed that LdRab5b explicitly controls receptor-mediated endocytosis of hemoglobin, and overexpression of LdRab5b:WT enhanced the transport of internalized Hb to the lysosomes in comparison with control cells. To unequivocally demonstrate the role of Rab5 isoforms in endocytosis in Leishmania, we tried to generate null-mutants of LdRab5a and LdRab5b parasites, but both were lethal indicating their essential functions in parasites. Therefore, we used heterozygous LdRab5a(+/-) and LdRab5b(+/-) cells. LdRab5a(+/-) Leishmania showed 50% inhibition of HRP uptake, but hemoglobin endocytosis was uninterrupted. In contrast, about 50% inhibition of Hb endocytosis was observed in LdRab5b(+/-) cells without any significant effect on HRP uptake. Finally, we tried to identify putative LdRab5a and LdRab5b effectors. We found that LdRab5b interacts with clathrin heavy chain and hemoglobin receptor. However, LdRab5a failed to interact with the clathrin heavy chain, and interaction with hemoglobin receptor was significantly less. Thus, our results showed that LdRab5a and LdRab5b differentially regulate fluid phase and receptor-mediated endocytosis in Leishmania.

  1. Different contributions of clathrin- and caveolae-mediated endocytosis of vascular endothelial cadherin to lipopolysaccharide-induced vascular hyperpermeability.

    PubMed

    Zhang, Ye; Zhang, Lianyang; Li, Yang; Sun, Shijin; Tan, Hao

    2014-01-01

    Vascular hyperpermeability induced by lipopolysaccharide (LPS) is a common pathogenic process in cases of severe trauma and sepsis. Vascular endothelial cadherin (VE-cad) is a key regulatory molecule involved in this process, although the detailed mechanism through which this molecule acts remains unclear. We assessed the role of clathrin-mediated and caveolae-mediated endocytosis of VE-cad in LPS-induced vascular hyperpermeability in the human vascular endothelial cell line CRL-2922 and determined that vascular permeability and VE-cad localization at the plasma membrane were negatively correlated after LPS treatment. Additionally, the loss of VE-cad at the plasma membrane was caused by both clathrin-mediated and caveolae-mediated endocytosis. Clathrin-mediated endocytosis was dominant early after LPS treatment, and caveolae-mediated endocytosis was dominant hours after LPS treatment. The caveolae-mediated endocytosis of VE-cad was activated through the LPS-Toll-like receptor 4 (TLR4)-Src signaling pathway. Structural changes in the actin cytoskeleton, specifically from polymerization to depolymerization, were important reasons for the switching of the VE-cad endocytosis pathway from clathrin-mediated to caveolae-mediated. Our findings suggest that clathrin-mediated and caveolae-mediated endocytosis of VE-cad contribute to LPS-induced vascular hyperpermeability, although they contribute via different mechanism. The predominant means of endocytosis depends on the time since LPS treatment.

  2. PI3K regulates endocytosis after insulin secretion by mediating signaling crosstalk between Arf6 and Rab27a.

    PubMed

    Yamaoka, Mami; Ando, Tomomi; Terabayashi, Takeshi; Okamoto, Mitsuhiro; Takei, Masahiro; Nishioka, Tomoki; Kaibuchi, Kozo; Matsunaga, Kohichi; Ishizaki, Ray; Izumi, Tetsuro; Niki, Ichiro; Ishizaki, Toshimasa; Kimura, Toshihide

    2016-02-01

    In secretory cells, endocytosis is coupled to exocytosis to enable proper secretion. Although endocytosis is crucial to maintain cellular homeostasis before and after secretion, knowledge about secretagogue-induced endocytosis in secretory cells is still limited. Here, we searched for proteins that interacted with the Rab27a GTPase-activating protein (GAP) EPI64 (also known as TBC1D10A) and identified the Arf6 guanine-nucleotide-exchange factor (GEF) ARNO (also known as CYTH2) in pancreatic β-cells. We found that the insulin secretagogue glucose promotes phosphatidylinositol (3,4,5)-trisphosphate (PIP3) generation through phosphoinositide 3-kinase (PI3K), thereby recruiting ARNO to the intracellular side of the plasma membrane. Peripheral ARNO promotes clathrin assembly through its GEF activity for Arf6 and regulates the early stage of endocytosis. We also found that peripheral ARNO recruits EPI64 to the same area and that the interaction requires glucose-induced endocytosis in pancreatic β-cells. Given that GTP- and GDP-bound Rab27a regulate exocytosis and the late stage of endocytosis, our results indicate that the glucose-induced activation of PI3K plays a pivotal role in exocytosis-endocytosis coupling, and that ARNO and EPI64 regulate endocytosis at distinct stages.

  3. Explosive bulk charge

    DOEpatents

    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.

  4. Bulk amorphous materials

    SciTech Connect

    Schwarz, R.B.; Archuleta, J.I.; Sickafus, K.E.

    1998-12-01

    This is the final report for a three-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The objective of this work was to develop the competency for the synthesis of novel bulk amorphous alloys. The authors researched their synthesis methods and alloy properties, including thermal stability, mechanical, and transport properties. The project also addressed the development of vanadium-spinel alloys for structural applications in hostile environments, the measurement of elastic constants and thermal expansion in single-crystal TiAl from 300 to 750 K, the measurement of elastic constants in gallium nitride, and a study of the shock-induced martensitic transformations in NiTi alloys.

  5. Bulk material handling system

    DOEpatents

    Kleysteuber, William K.; Mayercheck, William D.

    1979-01-01

    This disclosure relates to a bulk material handling system particularly adapted for underground mining and includes a monorail supported overhead and carrying a plurality of conveyors each having input and output end portions with the output end portion of a first of the conveyors positioned above an input end portion of a second of the conveyors, a device for imparting motion to the conveyors to move the material from the input end portions toward the output end portions thereof, a device for supporting at least one of the input and output end portions of the first and second conveyors from the monorail, and the supporting device including a plurality of trolleys rollingly supported by the monorail whereby the conveyors can be readily moved therealong.

  6. Bulk muscles, loose cables

    PubMed Central

    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

  7. Bulk Site Reference Materials

    SciTech Connect

    Barich, J.J. III; Jones, R.R. Sr.

    1996-12-31

    The selection, manufacture and use of Bulk Site Reference Materials (BSRMs) at hazardous waste sites is discussed. BSRMs are useful in preparing stabilization/solidification (S/S) formulations for soils, ranking competing S/S processes, comparing S/S alternatives to other technologies, and in interpreting data from different test types. BSRMs are large volume samples that are representative of the physical and chemical characteristics of a site soil, and that contain contaminants at reasonably high levels. A successful BSRM is extremely homogeneous and well-characterized. While not representative of any point on the site, they contain the contaminants of the site in the matrices of the site. Design objectives for a BSRM are to produce a material that (1) maintains good fidelity to site matrices and contaminants, and (2) exhibits the lowest possible relative standard deviation.

  8. Creating bulk nanocrystalline metal.

    SciTech Connect

    Fredenburg, D. Anthony; Saldana, Christopher J.; Gill, David D.; Hall, Aaron Christopher; Roemer, Timothy John; 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. Specific Endocytosis Blockade of Trypanosoma cruzi Exposed to a Poly-LAcNAc Binding Lectin Suggests that Lectin-Sugar Interactions Participate to Receptor-Mediated Endocytosis

    PubMed Central

    Brosson, Sébastien; Fontaine, Frédéric; Vermeersch, Marjorie; Perez-Morga, David; Pays, Etienne; Bousbata, Sabrina; Salmon, Didier

    2016-01-01

    Trypanosoma cruzi is a protozoan parasite transmitted by a triatomine insect, and causing human Chagas disease in South America. This parasite undergoes a complex life cycle alternating between non-proliferative and dividing forms. Owing to their high energy requirement, replicative epimastigotes of the insect midgut display high endocytic activity. This activity is mainly restricted to the cytostome, by which the cargo is taken up and sorted through the endosomal vesicular network to be delivered to reservosomes, the final lysosomal-like compartments. In African trypanosomes tomato lectin (TL) and ricin, respectively specific to poly-N-acetyllactosamine (poly-LacNAc) and β-D-galactose, allowed the identification of giant chains of poly-LacNAc in N-glycoproteins of the endocytic pathway. We show that in T. cruzi epimastigote forms also, glycoproteins of the endocytic pathway are characterized by the presence of N-linked glycans binding to both ricin and TL. Affinity chromatography using both TL and Griffonia simplicifolia lectin II (GSLII), specific to non-reducing terminal residue of N-acetylglucosamine (GlcNAc), led to an enrichment of glycoproteins of the trypanosomal endocytic pathway. Incubation of live parasites with TL, which selectively bound to the cytostome/cytopharynx, specifically inhibited endocytosis of transferrin (Tf) but not dextran, a marker of fluid endocytosis. Taken together, our data suggest that N-glycan modification of endocytic components plays a crucial role in receptor-mediated endocytosis of T. cruzi. PMID:27685262

  10. The kinetics of exocytosis and endocytosis in the synaptic terminal of goldfish retinal bipolar cells

    PubMed Central

    Neves, Guilherme; Lagnado, Leon

    1999-01-01

    The kinetics of exocytosis and endocytosis were studied in the giant synaptic terminal of depolarizing bipolar cells from the goldfish retina. Two techniques were applied: capacitance measurements of changes in membrane surface area, and fluorescence measurements of exocytosis using the membrane dye FM1-43. Three phases of exocytosis occurred during maintained depolarization to 0 mV. The first component was complete within about 10 ms and involved a pool of 1200–1800 vesicles (with a total membrane area equivalent to about 1.6% of the surface of the terminal). The second component of exocytosis involved the release of about 4400 vesicles over 1 s. The third component of exocytosis was stimulated continuously at a rate of about 1000 vesicles s−1. After short depolarizations (< 200 ms), neither the FM1-43 signal nor the capacitance signal continued to rise, indicating that exocytosis stopped rapidly after closure of Ca2+ channels. The fall in capacitance could therefore be used to monitor endocytosis independently of exocytosis. The capacitance measured after brief stimuli began to fall immediately, recovering to the pre-stimulus baseline with a rate constant of 0.8 s−1. The amount of exocytosis measured using the capacitance and FM1-43 techniques was similar during the first 200 ms of depolarization, suggesting that the most rapidly released vesicles could be detected by either method. After a few seconds of continuous stimulation, the net increase in membrane surface area reached a plateau at about 5%, even though continuous exocytosis occurred at a rate of 0.9% s−1. Under these conditions of balanced exocytosis and endocytosis, the rate constant of endocytosis was about 0.2 s−1. The average rate of endocytosis during maintained depolarization was therefore considerably slower than the rate observed after a brief stimulus. After longer depolarizations (> 500 ms), both the capacitance and FM1-43 signals continued to rise for periods of seconds after

  11. Molecular components required for resting and stimulated endocytosis of botulinum neurotoxins by glutamatergic and peptidergic neurons.

    PubMed

    Meng, Jianghui; Wang, Jiafu; Lawrence, Gary W; Dolly, J Oliver

    2013-08-01

    Proteins responsible for basal and stimulated endocytosis in nerves containing small clear synaptic vesicles (SCSVs) or large dense-core vesicles (LDCVs) are revealed herein, using probes that exploit surface-exposed vesicle proteins as acceptors for internalization. Basal uptake of botulinum neurotoxins (BoNTs) by both SCSV-releasing cerebellar granule neurons (CGNs) and LDCV-enriched trigeminal ganglionic neurons (TGNs) was found to require protein acceptors and acidic compartments. In addition, dynamin, clathrin, adaptor protein complex-2 (AP2), and amphiphysin contribute to the depolarization-evoked entry. For fast recycling of SCSVs, knockdown and knockout strategies demonstrated that CGNs use predominantly dynamin 1, whereas isoform 2 and, to a smaller extent, isoform 3 support a less rapid mode of stimulated endocytosis. Accordingly, proximity ligation assay confirmed that dynamin 1 and 2 colocalize with amphiphysin 1 in CGNs, and the latter copurified with both dynamins from cell extracts. In contrast, LDCV-releasing TGNs preferentially employ dynamins 2 and 3 and amphiphysin 1 for evoked endocytosis and lack the fast phase. Hence, stimulation recruits dynamin, clathrin, AP2, and amphiphysin to augment BoNT internalization, and neurons match endocytosis mediators to the different demands for locally recycling SCSVs or replenishing distally synthesized LDCVs.

  12. Polarized Exocytosis Induces Compensatory Endocytosis by Sec4p-Regulated Cortical Actin Polymerization

    PubMed Central

    Johansen, Jesper; Alfaro, Gabriel; Beh, Christopher T.

    2016-01-01

    Polarized growth is maintained by both polarized exocytosis, which transports membrane components to specific locations on the cell cortex, and endocytosis, which retrieves these components before they can diffuse away. Despite functional links between these two transport pathways, they are generally considered to be separate events. Using live cell imaging, in vivo and in vitro protein binding assays, and in vitro pyrene-actin polymerization assays, we show that the yeast Rab GTPase Sec4p couples polarized exocytosis with cortical actin polymerization, which induces endocytosis. After polarized exocytosis to the plasma membrane, Sec4p binds Las17/Bee1p (yeast Wiskott—Aldrich Syndrome protein [WASp]) in a complex with Sla1p and Sla2p during actin patch assembly. Mutations that inactivate Sec4p, or its guanine nucleotide exchange factor (GEF) Sec2p, inhibit actin patch formation, whereas the activating sec4-Q79L mutation accelerates patch assembly. In vitro assays of Arp2/3-dependent actin polymerization established that GTPγS-Sec4p overrides Sla1p inhibition of Las17p-dependent actin nucleation. These results support a model in which Sec4p relocates along the plasma membrane from polarized sites of exocytic vesicle fusion to nascent sites of endocytosis. Activated Sec4p then promotes actin polymerization and triggers compensatory endocytosis, which controls surface expansion and kinetically refines cell polarization. PMID:27526190

  13. Internal Trehalose Protects Endocytosis from Inhibition by Ethanol in Saccharomyces cerevisiae

    PubMed Central

    Lucero, P.; Peñalver, E.; Moreno, E.; Lagunas, R.

    2000-01-01

    Endocytosis in Saccharomyces cerevisiae is inhibited by concentrations of ethanol of 2 to 6% (vol/vol), which are lower than concentrations commonly present in its natural habitats. In spite of this inhibition, endocytosis takes place under enological conditions when high concentrations of ethanol are present. Therefore, it seems that yeast has developed some means to circumvent the inhibition. In this work we have investigated this possibility. We identified two stress conditions under which endocytosis was resistant to inhibition by ethanol: fermentation during nitrogen starvation and growth on nonfermentable substrates. Under these conditions, yeast accumulates stress protectors, primarily trehalose and Hsp104, a protein required for yeast to survive ethanol stress. We found the following. (i) The appearance of ethanol resistance was accompanied by trehalose accumulation. (ii) Mutant cells unable to synthesize trehalose also were unable to develop resistance. (iii) Mutant cells that accumulated trehalose during growth on sugars were resistant to ethanol even under this nonstressing condition. (iv) Mutant cells unable to synthesize Hsp104 were able to develop resistance. We conclude that trehalose is the major factor in the protection of endocytosis from ethanol. Our results suggest another important physiological role for trehalose in yeast. PMID:11010898

  14. Endocytosis of Ubiquitylation-Deficient EGFR Mutants via Clathrin-Coated Pits is Mediated by Ubiquitylation.

    PubMed

    Fortian, Arola; Dionne, Lai K; Hong, Sun H; Kim, Woong; Gygi, Steven P; Watkins, Simon C; Sorkin, Alexander

    2015-11-01

    Signaling by epidermal growth factor receptor (EGFR) is controlled by endocytosis. However, mechanisms of EGFR endocytosis remain poorly understood. Here, we found that the EGFR mutant lacking known ubiquitylation, acetylation and clathrin adaptor AP-2-binding sites (21KRΔAP2) was internalized at relatively high rates via the clathrin-dependent pathway in human duodenal adenocarcinoma HuTu-80 cells. RNA interference analysis revealed that this residual internalization is strongly inhibited by depletion of Grb2 and the E2 ubiquitin-conjugating enzyme UbcH5b/c, and partially affected by depletion of the E3 ubiquitin ligase Cbl and ubiquitin-binding adaptors, indicating that an ubiquitylation process is involved. Several new ubiquitin conjugation sites were identified by mass spectrometry in the 21KRΔAP2 mutant, suggesting that cryptic ubiquitylation may mediate endocytosis of this mutant. Total internal reflection fluorescence microscopy imaging of HuTu-80 cells transfected with labeled ubiquitin adaptor epsin1 demonstrated that the ubiquitylation-deficient EGFR mutant was endocytosed through a limited population of epsin-enriched clathrin-coated pits (CCPs), although with a prolonged CCP lifetime. Native EGFR was recruited with the same efficiency into CCPs containing either AP-2 or epsin1 that were tagged with fluorescent proteins by genome editing of MDA-MD-231 cells. We propose that two redundant mechanisms, ubiquitylation and interaction with AP-2, contribute to EGFR endocytosis via CCPs in a stochastic fashion.

  15. Endocytosis and serpentine filopodia drive blebbishield-mediated resurrection of apoptotic cancer stem cells

    PubMed Central

    Jinesh, G G; Kamat, A M

    2016-01-01

    The blebbishield emergency program helps to resurrect apoptotic cancer stem cells (CSCs) themselves. Understanding the mechanisms behind this program is essential to block resurrection of CSCs during cancer therapy. Here we demonstrate that endocytosis drives serpentine filopodia to construct blebbishields from apoptotic bodies and that a VEGF-VEGFR2-endocytosis-p70S6K axis governs subsequent transformation. Disengagement of RalGDS from E-cadherin initiates endocytosis of RalGDS and its novel interaction partners cdc42, VEGFR2, cleaved β-catenin, and PKC-ζ as well as its known interaction partner K-Ras. We also report novel interactions of p45S6K (cleaved p70S6K) and PKM-ζ with PAK-1 filopodia-forming machinery specifically in blebbishields. Thus, a RalGDS-endocytosis-filopodia-VEGFR2-K-Ras-p70S6K axis drives the blebbishield emergency program, and therapeutic targeting of this axis might prevent resurrection of CSCs during cancer therapy. PMID:27226900

  16. Megalin acts in concert with cubilin to mediate endocytosis of high density lipoproteins.

    PubMed

    Hammad, S M; Barth, J L; Knaak, C; Argraves, W S

    2000-04-21

    Cubilin has recently been shown to function as an endocytic receptor for high density lipoproteins (HDL). The lack of apparent transmembrane and cytoplasmic domains in cubilin raises questions as to the means by which it can mediate endocytosis. Since cubilin has been reported to bind the endocytic receptor megalin, we explored the possibility that megalin acts in conjunction with cubilin to mediate HDL endocytosis. While megalin did not bind to HDL, delipidated HDL, or apoA-I, it was found to copurify with cubilin isolated by HDL-Sepharose affinity chromatography. Cubilin and megalin exhibited coincident patterns of mRNA expression in mouse tissues including the kidney, ileum, thymus, placenta, and yolk sac endoderm. The expression of both receptors in yolk sac endoderm-like cells was inducible by retinoic acid treatment but not by conditions of sterol depletion. Suppression of megalin activity or expression by treatment with either megalin antibodies or megalin antisense oligodeoxynucleotides resulted in inhibition of cubilin-mediated endocytosis of HDL. Furthermore, megalin antisense oligodeoxynucleotide treatment resulted in reduced cell surface expression of cubilin. These data demonstrate that megalin acts together with cubilin to mediate HDL endocytosis and further suggest that megalin may play a role in the intracellular trafficking of cubilin.

  17. Angiotensin II increases glomerular permeability by β-arrestin mediated nephrin endocytosis

    PubMed Central

    Königshausen, Eva; Zierhut, Ulf M.; Ruetze, Martin; Potthoff, Sebastian A.; Stegbauer, Johannes; Woznowski, Magdalena; Quack, Ivo; Rump, Lars C.; Sellin, Lorenz

    2016-01-01

    Glomerular permeability and subsequent albuminuria are early clinical markers for glomerular injury in hypertensive nephropathy. Albuminuria predicts mortality and cardiovascular morbidity. AT1 receptor blockers protect from albuminuria, cardiovascular morbidity and mortality. A blood pressure independent, molecular mechanism for angiotensin II (Ang II) dependent albuminuria has long been postulated. Albuminuria results from a defective glomerular filter. Nephrin is a major structural component of the glomerular slit diaphragm and its endocytosis is mediated by β-arrestin2. Ang II stimulation increases nephrin-β-arrestin2 binding, nephrin endocytosis and glomerular permeability in mice. This Ang II effect is mediated by AT1-receptors. AT1-receptor mutants identified G-protein signaling to be essential for this Ang II effect. Gαq knockdown and phospholipase C inhibition block Ang II mediated enhanced nephrin endocytosis. Nephrin Y1217 is the critical residue controlling nephrin binding to β-arrestin under Ang II stimulation. Nephrin Y1217 also mediates cytoskeletal anchoring to actin via nck2. Ang II stimulation decreases nephrin nck2 binding. We conclude that Ang II weakens the structural integrity of the slit diaphragm by increased nephrin endocytosis and decreased nephrin binding to nck2, which leads to increased glomerular permeability. This novel molecular mechanism of Ang II supports the use of AT1-receptor blockers to prevent albuminuria even in normotensives. PMID:28004760

  18. Defective calmodulin-dependent rapid apical endocytosis in zebrafish sensory hair cell mutants.

    PubMed

    Seiler, C; Nicolson, T

    1999-11-15

    Vertebrate mechanosensory hair cells contain a narrow "pericuticular" zone which is densely populated with small vesicles between the cuticular plate and cellular junctions near the apical surface. The presence of many cytoplasmic vesicles suggests that the apical surface of hair cells has a high turnover rate. The significance of intense membrane trafficking at the apical surface is not known. Using a marker of endocytosis, the styryl dye FM1-43, this report shows that rapid apical endocytosis in zebrafish lateral line sensory hair cells is calcium and calmodulin dependent and is partially blocked by the presence of amiloride and dihydrostreptomycin, known inhibitors of mechanotransduction channels. As seen in lateral line hair cells, sensory hair cells within the larval otic capsule also exhibit rapid apical endocytosis. Defects in internalization of the dye in both lateral line and inner ear hair cells were found in five zebrafish auditory/vestibular mutants: sputnik, mariner, orbiter, mercury, and skylab. In addition, lateral line hair cells in these mutants were not sensitive to prolonged exposure to streptomycin, which is toxic to hair cells. The presence of endocytic defects in the majority of zebrafish mechanosensory mutants points to a important role of apical endocytosis in hair cell function. Copyright 1999 John Wiley & Sons, Inc.

  19. Kainate receptor activation induces glycine receptor endocytosis through PKC deSUMOylation

    PubMed Central

    Sun, Hao; Lu, Li; Zuo, Yong; Wang, Yan; Jiao, Yingfu; Zeng, Wei-Zheng; Huang, Chao; Zhu, Michael X.; Zamponi, Gerald W.; Zhou, Tong; Xu, Tian-Le; Cheng, Jinke; Li, Yong

    2014-01-01

    Surface expression and regulated endocytosis of glycine receptors (GlyRs) play a critical function in balancing neuronal excitability. SUMOylation (SUMO modification) is of critical importance for maintaining neuronal function in the central nervous system. Here we show that activation of kainate receptors (KARs) causes GlyR endocytosis in a calcium- and protein kinase C (PKC)-dependent manner, leading to reduced GlyR-mediated synaptic activity in cultured spinal cord neurons and the superficial dorsal horn of rat spinal cord slices. This effect requires SUMO1/sentrin-specific peptidase 1 (SENP1)-mediated deSUMOylation of PKC, indicating that the crosstalk between KARs and GlyRs relies on the SUMOylation status of PKC. SENP1-mediated deSUMOylation of PKC is involved in the kainate-induced GlyR endocytosis and thus plays an important role in the anti-homeostatic regulation between excitatory and inhibitory ligand-gated ion channels. Altogether, we have identified a SUMOylation-dependent regulatory pathway for GlyR endocytosis, which may have important physiological implications for proper neuronal excitability. PMID:25236484

  20. Receptor-mediated endocytosis of carcinoembryonic antigen by rat liver Kupffer cells.

    PubMed

    Toth, C A; Thomas, P; Broitman, S A; Zamcheck, N

    1985-01-01

    In vivo, carcinoembryonic antigen (CEA) is removed from the circulation by the liver Kupffer cells. Immunologically identifiable CEA is transferred from these macrophages to the hepatocytes, where degradation is completed. Circulatory clearance of CEA is specific, rapid [t1/2 = 3.7 +/- 0.9 (S.D.) min], and saturable. In vitro, Kupffer cells take up CEA by a saturable process which is time/temperature dependent and colchicine sensitive. Isolated Kupffer cells endocytose CEA with an apparent Km of 6 X 10(-8) M. There are approximately 16,000 CEA binding sites per cell. Nonspecific cross-reacting antigen (NCA), a glycoprotein structurally similar to CEA, is recognized with lower affinity by the same receptor. Endocytosis is independent of the nonreducing terminal sugars on the molecule: CEA modified by Smith degradation inhibits Kupffer cell recognition of native CEA. Since performic acid oxidized CEA also inhibits endocytosis, receptor binding is similarly independent of intact protein conformation. Isolated Kupffer cells have mannose and/or N-acetyl glucosamine receptor activity but do not internalize CEA by that mechanism. Galactose-terminated glycoproteins impede CEA and NCA clearance in vivo but not Kupffer cell endocytosis in vitro. Radiolabeled CEA released from isolated Kupffer cells following endocytosis shows no apparent molecular weight change. However, the released CEA contains species with higher isoelectric points, suggesting that perhaps the removal of sialic acid and the resulting exposure of galactose residues mediate the subsequent transfer to the hepatocyte.

  1. The Sla2p talin domain plays a role in endocytosis in Saccharomyces cerevisiae.

    PubMed Central

    Baggett, Jennifer J; D'Aquino, Katharine E; Wendland, Beverly

    2003-01-01

    Clathrin-binding adaptors play critical roles for endocytosis in multicellular organisms, but their roles in budding yeast have remained unclear. To address this question, we created a quadruple mutant yeast strain lacking the genes encoding the candidate clathrin adaptors Yap1801p, Yap1802p, and Ent2p and containing a truncated version of Ent1p, Ent1DeltaCBMp, missing its clathrin-binding motif. This strain was viable and competent for endocytosis, suggesting the existence of other redundant adaptor-like factors. To identify these factors, we mutagenized the quadruple clathrin adaptor mutant strain and selected cells that were viable in the presence of full-length Ent1p, but inviable with only Ent1DeltaCBMp; these strains were named Rcb (requires clathrin binding). One mutant strain, rcb432, contained a mutation in SLA2 that resulted in lower levels of a truncated protein lacking the F-actin binding talin homology domain. Analyses of this sla2 mutant showed that the talin homology domain is required for endocytosis at elevated temperature, that SLA2 exhibits genetic interactions with both ENT1 and ENT2, and that the clathrin adaptors and Sla2p together regulate the actin cytoskeleton and revealed conditions under which Yap1801p and Yap1802p contribute to viability. Together, our data support the view that Sla2p is an adaptor that links actin to clathrin and endocytosis. PMID:14704157

  2. Endocytosis and serpentine filopodia drive blebbishield-mediated resurrection of apoptotic cancer stem cells.

    PubMed

    Jinesh, Goodwin G; Kamat, Ashish M

    The blebbishield emergency program helps to resurrect apoptotic cancer stem cells (CSCs) themselves. Understanding the mechanisms behind this program is essential to block resurrection of CSCs during cancer therapy. Here we demonstrate that endocytosis drives serpentine filopodia to construct blebbishields from apoptotic bodies and that a VEGF-VEGFR2-endocytosis-p70S6K axis governs subsequent transformation. Disengagement of RalGDS from E-cadherin initiates endocytosis of RalGDS and its novel interaction partners cdc42, VEGFR2, cleaved β-catenin, and PKC-ζ as well as its known interaction partner K-Ras. We also report novel interactions of p45S6K (cleaved p70S6K) and PKM-ζ with PAK-1 filopodia-forming machinery specifically in blebbishields. Thus, a RalGDS-endocytosis-filopodia-VEGFR2-K-Ras-p70S6K axis drives the blebbishield emergency program, and therapeutic targeting of this axis might prevent resurrection of CSCs during cancer therapy.

  3. Long-term potentiation decay and memory loss are mediated by AMPAR endocytosis.

    PubMed

    Dong, Zhifang; Han, Huili; Li, Hongjie; Bai, Yanrui; Wang, Wei; Tu, Man; Peng, Yan; Zhou, Limin; He, Wenting; Wu, Xiaobin; Tan, Tao; Liu, Mingjing; Wu, Xiaoyan; Zhou, Weihui; Jin, Wuyang; Zhang, Shu; Sacktor, Todd Charlton; Li, Tingyu; Song, Weihong; Wang, Yu Tian

    2015-01-01

    Long-term potentiation (LTP) of synaptic strength between hippocampal neurons is associated with learning and memory, and LTP dysfunction is thought to underlie memory loss. LTP can be temporally and mechanistically classified into decaying (early-phase) LTP and nondecaying (late-phase) LTP. While the nondecaying nature of LTP is thought to depend on protein synthesis and contribute to memory maintenance, little is known about the mechanisms and roles of decaying LTP. Here, we demonstrated that inhibiting endocytosis of postsynaptic α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptors (AMPARs) prevents LTP decay, thereby converting it into nondecaying LTP. Conversely, restoration of AMPAR endocytosis by inhibiting protein kinase Mζ (PKMζ) converted nondecaying LTP into decaying LTP. Similarly, inhibition of AMPAR endocytosis prolonged memory retention in normal animals and reduced memory loss in a murine model of Alzheimer's disease. These results strongly suggest that an active process that involves AMPAR endocytosis mediates the decay of LTP and that inhibition of this process can prolong the longevity of LTP as well as memory under both physiological and pathological conditions.

  4. Buckwheat trypsin inhibitor enters Hep G2 cells by clathrin-dependent endocytosis.

    PubMed

    Cui, Xiaodong; Wang, Zhuanhua; Li, Yuying; Li, Chen

    2013-12-01

    Recombinant buckwheat trypsin inhibitor (rBTI) was studied to evaluate if it could enter cancer cells and to determine the mechanism. Fluorescein isothiocyanate-labelled buckwheat trypsin inhibitor (FITC-BTI) entered Hep G2 cells in a concentration-dependent manner. FITC-BTI colocalised with labelled transferrin (Tf) in the punctate structure, implying that rBTI enters Hep G2 cells by clathrin-dependent endocytosis. Incubation of Hep G2 cells with different chemical inhibitors abolished diffuse, but not punctate fluorescence, thus indicating that membrane potential plays a critical role in this process. Impairment of clathrin-mediated endocytosis by RNAi with clathrin heavy chain greatly reduced or completely abolished both diffuse and punctate fluorescence, further supporting a theory of a single route of endocytosis. Consistent with our working hypothesis, Hep G2 cells which were arrested in the M phase did not show any vesicular or diffuse FITC-BTI. We conclude from these results that both endocytosis and membrane potential are required for rBTI entry into Hep G2 cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. AMPA Receptor Endocytosis in Rat Perirhinal Cortex Underlies Retrieval of Object Memory

    ERIC Educational Resources Information Center

    Cazakoff, Brittany N.; Howland, John G.

    2011-01-01

    Mechanisms consistent with long-term depression in the perirhinal cortex (PRh) play a fundamental role in object recognition memory; however, whether AMPA receptor endocytosis is involved in distinct phases of recognition memory is not known. To address this question, we used local PRh infusions of the cell membrane-permeable Tat-GluA2[subscript…

  6. Dynasore impairs VEGFR2 signalling in an endocytosis-independent manner

    PubMed Central

    Basagiannis, Dimitris; Zografou, Sofia; Galanopoulou, Katerina; Christoforidis, Savvas

    2017-01-01

    VEGFR2 is a critical angiogenic receptor playing a key role in vascular homeostasis. Upon activation by VEGF, VEGFR2 becomes endocytosed. Internalisation of VEGFR2 is facilitated, in part, through clathrin mediated endocytosis (CME), the role of which in VEGFR2 function is debated. Here, we confirm the contribution of CME in VEGFR2 uptake. However, curiously, we find that different approaches of inhibition of CME exert contradictory effects on VEGF signalling; knockdown of clathrin, or of dynamin, or overexpression of dynamin K44A, do not affect VEGF-induced phosphorylation of ERK1/2, while dynasore causes strong inhibition. We resolve this discrepancy by showing that although dynasore inhibits CME of VEGFR2, its inhibitory action in ERK1/2 phosphorylation is not related to attenuation of VEGFR2 endocytosis; it is rather due to an off-target effect of the drug. Dynasore inhibits VEGF-induced calcium release, a signalling event that lies upstream of ERK1/2, which implies that this effect could be responsible, at least in part, for the inhibitory action of the drug on VEGF-to-ERK1/2 signalling. These results raise caution that although dynasore is specific in inhibiting clathrin- and dynamin-mediated endocytosis, it may also exert off-target effects on signalling molecules, hence influencing the interpretation of the role of endocytosis in signalling. PMID:28327657

  7. Polarized E-cadherin endocytosis directs actomyosin remodeling during embryonic wound repair.

    PubMed

    Hunter, Miranda V; Lee, Donghoon M; Harris, Tony J C; Fernandez-Gonzalez, Rodrigo

    2015-08-31

    Embryonic epithelia have a remarkable ability to rapidly repair wounds. A supracellular actomyosin cable around the wound coordinates cellular movements and promotes wound closure. Actomyosin cable formation is accompanied by junctional rearrangements at the wound margin. We used in vivo time-lapse quantitative microscopy to show that clathrin, dynamin, and the ADP-ribosylation factor 6, three components of the endocytic machinery, accumulate around wounds in Drosophila melanogaster embryos in a process that requires calcium signaling and actomyosin contractility. Blocking endocytosis with pharmacological or genetic approaches disrupted wound repair. The defect in wound closure was accompanied by impaired removal of E-cadherin from the wound edge and defective actomyosin cable assembly. E-cadherin overexpression also resulted in reduced actin accumulation around wounds and slower wound closure. Reducing E-cadherin levels in embryos in which endocytosis was blocked rescued actin localization to the wound margin. Our results demonstrate a central role for endocytosis in wound healing and indicate that polarized E-cadherin endocytosis is necessary for actomyosin remodeling during embryonic wound repair.

  8. Polarized E-cadherin endocytosis directs actomyosin remodeling during embryonic wound repair

    PubMed Central

    Hunter, Miranda V.; Lee, Donghoon M.; Harris, Tony J.C.

    2015-01-01

    Embryonic epithelia have a remarkable ability to rapidly repair wounds. A supracellular actomyosin cable around the wound coordinates cellular movements and promotes wound closure. Actomyosin cable formation is accompanied by junctional rearrangements at the wound margin. We used in vivo time-lapse quantitative microscopy to show that clathrin, dynamin, and the ADP-ribosylation factor 6, three components of the endocytic machinery, accumulate around wounds in Drosophila melanogaster embryos in a process that requires calcium signaling and actomyosin contractility. Blocking endocytosis with pharmacological or genetic approaches disrupted wound repair. The defect in wound closure was accompanied by impaired removal of E-cadherin from the wound edge and defective actomyosin cable assembly. E-cadherin overexpression also resulted in reduced actin accumulation around wounds and slower wound closure. Reducing E-cadherin levels in embryos in which endocytosis was blocked rescued actin localization to the wound margin. Our results demonstrate a central role for endocytosis in wound healing and indicate that polarized E-cadherin endocytosis is necessary for actomyosin remodeling during embryonic wound repair. PMID:26304727

  9. Inter-Cellular Exchange of Cellular Components via VE-Cadherin-Dependent Trans-Endocytosis

    PubMed Central

    Sakurai, Takashi; Woolls, Melissa J.; Jin, Suk-Won

    2014-01-01

    Cell-cell communications typically involve receptor-mediated signaling initiated by soluble or cell-bound ligands. Here, we report a unique mode of endocytosis: proteins originating from cell-cell junctions and cytosolic cellular components from the neighboring cell are internalized, leading to direct exchange of cellular components between two adjacent endothelial cells. VE-cadherins form transcellular bridges between two endothelial cells that are the basis of adherence junctions. At such adherens junction sites, we observed the movement of the entire VE-cadherin molecule from one endothelial cell into the other with junctional and cytoplasmic components. This phenomenon, here termed trans-endocytosis, requires the establishment of a VE-cadherin homodimer in trans with internalization proceeding in a Rac1-, and actomyosin-dependent manner. Importantly, the trans-endocytosis is not dependent on any known endocytic pathway including clathrin-dependent endocytosis, macropinocytosis or phagocytosis. This novel form of cell-cell communications, leading to a direct exchange of cellular components, was observed in 2D and 3D-cultured endothelial cells as well as in the developing zebrafish vasculature. PMID:24603875

  10. Synergistic effect of electrical and chemical factors on endocytosis in micro-discharge plasma gene transfection

    NASA Astrophysics Data System (ADS)

    Jinno, M.; Ikeda, Y.; Motomura, H.; Isozaki, Y.; Kido, Y.; Satoh, S.

    2017-06-01

    We have developed a new micro-discharge plasma (MDP)-based gene transfection method, which transfers genes into cells with high efficiency and low cytotoxicity; however, the mechanism underlying the method is still unknown. Studies revealed that the N-acetylcysteine-mediated inhibition of reactive oxygen species (ROS) activity completely abolished gene transfer. In this study, we used laser-produced plasma to demonstrate that gene transfer does not occur in the absence of electrical factors. Our results show that both electrical and chemical factors are necessary for gene transfer inside cells by microplasma irradiation. This indicates that plasma-mediated gene transfection utilizes the synergy between electrical and chemical factors. The electric field threshold required for transfection was approximately 1 kV m-1 in our MDP system. This indicates that MDP irradiation supplies sufficient concentrations of ROS, and the stimulation intensity of the electric field determines the transfection efficiency in our system. Gene transfer by plasma irradiation depends mainly on endocytosis, which accounts for at least 80% of the transfer, and clathrin-mediated endocytosis is a dominant endocytosis. In plasma-mediated gene transfection, alterations in electrical and chemical factors can independently regulate plasmid DNA adhesion and triggering of endocytosis, respectively. This implies that plasma characteristics can be adjusted according to target cell requirements, and the transfection process can be optimized with minimum damage to cells and maximum efficiency. This may explain how MDP simultaneously achieves high transfection efficiency with minimal cell damage.

  11. Exocytosis of acid sphingomyelinase by wounded cells promotes endocytosis and plasma membrane repair

    PubMed Central

    Tam, Christina; Idone, Vincent; Devlin, Cecilia; Fernandes, Maria Cecilia; Flannery, Andrew; He, Xingxuan; Schuchman, Edward; Tabas, Ira

    2010-01-01

    Rapid plasma membrane resealing is essential for cellular survival. Earlier studies showed that plasma membrane repair requires Ca2+-dependent exocytosis of lysosomes and a rapid form of endocytosis that removes membrane lesions. However, the functional relationship between lysosomal exocytosis and the rapid endocytosis that follows membrane injury is unknown. In this study, we show that the lysosomal enzyme acid sphingomyelinase (ASM) is released extracellularly when cells are wounded in the presence of Ca2+. ASM-deficient cells, including human cells from Niemann-Pick type A (NPA) patients, undergo lysosomal exocytosis after wounding but are defective in injury-dependent endocytosis and plasma membrane repair. Exogenously added recombinant human ASM restores endocytosis and resealing in ASM-depleted cells, suggesting that conversion of plasma membrane sphingomyelin to ceramide by this lysosomal enzyme promotes lesion internalization. These findings reveal a molecular mechanism for restoration of plasma membrane integrity through exocytosis of lysosomes and identify defective plasma membrane repair as a possible component of the severe pathology observed in NPA patients. PMID:20530211

  12. Endocytosis restricts Arabidopsis KNOLLE syntaxin to the cell division plane during late cytokinesis.

    PubMed

    Boutté, Yohann; Frescatada-Rosa, Márcia; Men, Shuzhen; Chow, Cheung-Ming; Ebine, Kazuo; Gustavsson, Anna; Johansson, Lenore; Ueda, Takashi; Moore, Ian; Jürgens, Gerd; Grebe, Markus

    2010-02-03

    Cytokinesis represents the final stage of eukaryotic cell division during which the cytoplasm becomes partitioned between daughter cells. The process differs to some extent between animal and plant cells, but proteins of the syntaxin family mediate membrane fusion in the plane of cell division in diverse organisms. How syntaxin localization is kept in check remains elusive. Here, we report that localization of the Arabidopsis KNOLLE syntaxin in the plane of cell division is maintained by sterol-dependent endocytosis involving a clathrin- and DYNAMIN-RELATED PROTEIN1A-dependent mechanism. On genetic or pharmacological interference with endocytosis, KNOLLE mis-localizes to lateral plasma membranes after cell-plate fusion. Fluorescence-loss-in-photo-bleaching and fluorescence-recovery-after-photo-bleaching experiments reveal lateral diffusion of GFP-KNOLLE from the plane of division to lateral membranes. In an endocytosis-defective sterol biosynthesis mutant displaying lateral KNOLLE diffusion, KNOLLE secretory trafficking remains unaffected. Thus, restriction of lateral diffusion by endocytosis may serve to maintain specificity of syntaxin localization during late cytokinesis.

  13. Otoferlin couples to clathrin-mediated endocytosis in mature cochlear inner hair cells

    PubMed Central

    Duncker, Susanne V.; Franz, Christoph; Kuhn, Stephanie; Schulte, Uwe; Campanelli, Dario; Brandt, Niels; Hirt, Bernhard; Fakler, Bernd; Blin, Nikolaus; Ruth, Peter; Engel, Jutta; Marcotti, Walter; Zimmermann, Ulrike; Knipper, Marlies

    2013-01-01

    The encoding of auditory information with indefatigable precision requires efficient re-supply of vesicles at inner hair cell (IHC) ribbon synapses. Otoferlin, a transmembrane protein responsible for deafness in DFNB9 families, has been postulated to act as a calcium sensor for exocytosis as well as to be involved in rapid vesicle replenishment of IHCs. However, the molecular basis of vesicle recycling in IHCs is largely unknown. In the present study, we used high-resolution liquid chromatography coupled with mass spectrometry to co-purify otoferlin interaction partners in the mammalian cochlea. We identified multiple subunits of the adaptor protein complex AP-2 (CLAP), an essential component of clathrin-mediated endocytosis, as binding partners of otoferlin in rats and mice. The interaction between otoferlin and AP-2 was confirmed by co-immunoprecipitation. We also found that AP-2 interacts with myosin VI, another otoferlin binding partner important for clathrin-mediated endocytosis. The expression of AP-2 in IHCs was verified by RT-PCR. Confocal microscopy experiments revealed that the expression of AP-2 and its co-localization with otoferlin is confined to mature IHCs. When clathrin-mediated endocytosis was inhibited by blocking dynamin action, real-time changes in membrane capacitance showed impaired synaptic vesicle replenishment in mature but not immature IHCs. We suggest that an otoferlin–AP-2 interaction drives Ca2+- and stimulus-dependent compensating clathrin-mediated endocytosis in mature IHCs. PMID:23719817

  14. A morphological and functional study on antigen binding and endocytosis by immunocytes.

    PubMed Central

    Goud, B; Antoine, J C; Gonatas, N K; Stieber, A; Avrameas, S

    1980-01-01

    Immunoenzymatic techniques were used to study antigen binding and endocytosis by lymph node cells of rats immunized against horseradish peroxidase, hen ovalbumin and rabbit IgG. The number of antigen-binding cells varied and depended on the type of antigen used, the time after immunization, and was higher after a booster injection. In secondary responses (4 days after booster), about 80% of antigen-binding cells were proplasmocytes and plasmocytes; by a double staining procedure it was found that 82% of these cells bore in addition to surface antigen, specific intracytoplasmic antibody as well. About 20% of antigen-binding cells were small and medium lymphocytes which did not contain detectable intracytoplasmic antibody. For ultrastructural studies of the endocytosis, peroxidase was used as the antigen. This antigen was found in cytoplasmic compartments which consisted of vesicles, cisternae and large round bodies (lysosomes?) often located near the Golgi apparatus. However, the cisternae of the Golgi apparatus, involved in the synthesis of specific antibody were not sites of retrieval of endocytosed antigen. The effect of endocytosis of antigen on the secretion and synthesis of antibody was studied by the local haemolysis plaque assay and biosynthetic labelling. No change was detected in antibody secretion and synthesis as a result of antigen endocytosis. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7007216

  15. Timely Endocytosis of Cytokinetic Enzymes Prevents Premature Spindle Breakage during Mitotic Exit

    PubMed Central

    Onishi, Masayuki; Yeong, Foong May

    2016-01-01

    Cytokinesis requires the spatio-temporal coordination of membrane deposition and primary septum (PS) formation at the division site to drive acto-myosin ring (AMR) constriction. It has been demonstrated that AMR constriction invariably occurs only after the mitotic spindle disassembly. It has also been established that Chitin Synthase II (Chs2p) neck localization precedes mitotic spindle disassembly during mitotic exit. As AMR constriction depends upon PS formation, the question arises as to how chitin deposition is regulated so as to prevent premature AMR constriction and mitotic spindle breakage. In this study, we propose that cells regulate the coordination between spindle disassembly and AMR constriction via timely endocytosis of cytokinetic enzymes, Chs2p, Chs3p, and Fks1p. Inhibition of endocytosis leads to over accumulation of cytokinetic enzymes during mitotic exit, which accelerates the constriction of the AMR, and causes spindle breakage that eventually could contribute to monopolar spindle formation in the subsequent round of cell division. Intriguingly, the mitotic spindle breakage observed in endocytosis mutants can be rescued either by deleting or inhibiting the activities of, CHS2, CHS3 and FKS1, which are involved in septum formation. The findings from our study highlight the importance of timely endocytosis of cytokinetic enzymes at the division site in safeguarding mitotic spindle integrity during mitotic exit. PMID:27447488

  16. Endocytosis of PEGylated nanoparticles accompanied by structural and free energy changes of the grafted polyethylene glycol.

    PubMed

    Li, Ying; Kröger, Martin; Liu, Wing Kam

    2014-10-01

    Nanoparticles (NPs) are in use to efficiently deliver drug molecules into diseased cells. The surfaces of NPs are usually grafted with polyethylene glycol (PEG) polymers, during so-called PEGylation, to improve water solubility, avoid aggregation, and prevent opsonization during blood circulation. The interplay between grafting density σp and grafted PEG polymerization degree N makes cellular uptake of PEGylated NPs distinct from that of bare NPs. To understand the role played by grafted PEG polymers, we study the endocytosis of 8 nm sized PEGylated NPs with different σp and N through large scale dissipative particle dynamics (DPD) simulations. The free energy change Fpolymer of grafted PEG polymers, before and after endocytosis, is identified to have an effect which is comparable to, or even larger than, the bending energy of the membrane during endocytosis. Based on self-consistent field theory Fpolymer is found to be dependent on both σp and N. By incorporating Fpolymer, the critical ligand-receptor binding strength for PEGylated NPs to be internalized can be correctly predicted by a simple analytical equation. Without considering Fpolymer, it turns out impossible to predict whether the PEGylated NPs will be delivered into the diseased cells. These simulation results and theoretical analysis not only provide new insights into the endocytosis process of PEGylated NPs, but also shed light on the underlying physical mechanisms, which can be utilized for designing efficient PEGylated NP-based therapeutic carriers with improved cellular targeting and uptake.

  17. Suppression of dynamin GTPase activity by sertraline leads to inhibition of dynamin-dependent endocytosis.

    PubMed

    Takahashi, Kiyofumi; Miyoshi, Hiroshi; Otomo, Masahiro; Osada, Kenichi; Yamaguchi, Noboru; Nakashima, Hideki

    2010-01-01

    Dynamin (Dyn) 1 plays a role in recycling of synaptic vesicles, and thus in nervous system function. We previously showed that sertraline, a selective serotonin reuptake inhibitor (SSRI), is a mixed-type inhibitor of Dyn 1 with respect to both GTP and L-alpha-phosphatidyl-L-serine (PS) in vitro, and we suggested that it may regulate the neurotransmitter transport by modulating synaptic vesicle endocytosis via inhibition of Dyn 1 GTPase. Here, we investigated the effect of sertraline on endocytosis of marker proteins in human neuroblastoma SH-Sy5Y cells and HeLa cells. Sertraline inhibited endocytosis in both cell lines. Western blotting showed that SH-Sy5Y expresses Dyn 1 and Dyn 2, while HeLa expresses only Dyn 2. GTPase assay showed that sertraline inhibited Dyn 2 as well as Dyn 1. Therefore, the effect of sertraline on endocytosis was mediated by Dyn 2, at least in HeLa cells, as well as by Dyn 1 in cell lines that express it. Moreover, the inhibition mechanism of transferrin (Tf) uptake by sertraline differed from that in cells expressing Dyn 1 K44A, a GTP binding-defective variant, and sertraline did not interfere with the interaction between Dyn 1 and PS-liposomes.

  18. Tumor-Derived Microvesicles Induce Proangiogenic Phenotype in Endothelial Cells via Endocytosis

    PubMed Central

    Kawamoto, Taisuke; Ohga, Noritaka; Akiyama, Kosuke; Hirata, Naoya; Kitahara, Shuji; Maishi, Nako; Osawa, Takahiro; Yamamoto, Kazuyuki; Kondoh, Miyako; Shindoh, Masanobu; Hida, Yasuhiro; Hida, Kyoko

    2012-01-01

    Background Increasing evidence indicates that tumor endothelial cells (TEC) differ from normal endothelial cells (NEC). Our previous reports also showed that TEC were different from NEC. For example, TEC have chromosomal abnormality and proangiogenic properties such as high motility and proliferative activity. However, the mechanism by which TEC acquire a specific character remains unclear. To investigate this mechanism, we focused on tumor-derived microvesicles (TMV). Recent studies have shown that TMV contain numerous types of bioactive molecules and affect normal stromal cells in the tumor microenvironment. However, most of the functional mechanisms of TMV remain unclear. Methodology/Principal Findings Here we showed that TMV isolated from tumor cells were taken up by NEC through endocytosis. In addition, we found that TMV promoted random motility and tube formation through the activation of the phosphoinositide 3-kinase/Akt pathway in NEC. Moreover, the effects induced by TMV were inhibited by the endocytosis inhibitor dynasore. Our results indicate that TMV could confer proangiogenic properties to NEC partly via endocytosis. Conclusion We for the first time showed that endocytosis of TMV contributes to tumor angiogenesis. These findings offer new insights into cancer therapies and the crosstalk between tumor and endothelial cells mediated by TMV in the tumor microenvironment. PMID:22479517

  19. Receptor-mediated endocytosis of macromolecules and strategy to enhance their transport in alveolar epithelial cells.

    PubMed

    Takano, Mikihisa; Kawami, Masashi; Aoki, Ayako; Yumoto, Ryoko

    2015-05-01

    Pulmonary delivery is an attractive administration route for therapeutic proteins and peptides. In this context, endocytosis/transcytosis at the distal lung epithelial barrier is an important process in the pulmonary absorption of therapeutic macromolecules. The alveolar epithelium is comprised of type I and type II cells. Understanding the transport mechanisms in these cells is essential for the development of efficient pulmonary delivery systems of therapeutic macromolecules. Endocytic pathways for albumin and insulin in alveolar epithelial cells and possible receptors for the endocytosis are discussed. Strategies to enhance the endocytosis and pulmonary absorption of macromolecules are also discussed, by focusing on the effects of cationic poly(amino acid)s. Although the surface area occupied by type II cells in alveoli is much smaller than that covered by type I cells, type II cells may significantly contribute to the endocytosis/transcytosis of macromolecules such as albumin. Identification of the receptors involved in the cellular uptake of each macromolecule is prerequisite for the understanding and regulation of its transport into and across alveolar epithelial cells. Establishment of novel in-vitro culture cell models of type I and type II cells would be a great help for the future advance of this research field.

  20. Membrane protrusion powers clathrin-independent endocytosis of interleukin-2 receptor

    PubMed Central

    Basquin, Cyril; Trichet, Michaël; Vihinen, Helena; Malardé, Valérie; Lagache, Thibault; Ripoll, Léa; Jokitalo, Eija; Olivo-Marin, Jean-Christophe; Gautreau, Alexis; Sauvonnet, Nathalie

    2015-01-01

    Endocytosis controls many functions including nutrient uptake, cell division, migration and signal transduction. A clathrin- and caveolin-independent endocytosis pathway is used by important physiological cargos, including interleukin-2 receptors (IL-2R). However, this process lacks morphological and dynamic data. Our electron microscopy (EM) and tomography studies reveal that IL-2R-pits and vesicles are initiated at the base of protrusions. We identify the WAVE complex as a specific endocytic actor. The WAVE complex interacts with IL-2R, via a WAVE-interacting receptor sequence (WIRS) present in the receptor polypeptide, and allows for receptor clustering close to membrane protrusions. In addition, using total internal reflection fluorescent microscopy (TIRF) and automated analysis we demonstrate that two timely distinct bursts of actin polymerization are required during IL-2R uptake, promoted first by the WAVE complex and then by N-WASP. Finally, our data reveal that dynamin acts as a transition controller for the recruitment of Arp2/3 activators required for IL-2R endocytosis. Altogether, our work identifies the spatio-temporal specific role of factors initiating clathrin-independent endocytosis by a unique mechanism that does not depend on the deformation of a flat membrane, but rather on that of membrane protrusions. PMID:26124312

  1. AMPA Receptor Endocytosis in Rat Perirhinal Cortex Underlies Retrieval of Object Memory

    ERIC Educational Resources Information Center

    Cazakoff, Brittany N.; Howland, John G.

    2011-01-01

    Mechanisms consistent with long-term depression in the perirhinal cortex (PRh) play a fundamental role in object recognition memory; however, whether AMPA receptor endocytosis is involved in distinct phases of recognition memory is not known. To address this question, we used local PRh infusions of the cell membrane-permeable Tat-GluA2[subscript…

  2. Scavenger receptor-mediated endocytosis by sinusoidal cells in rat bone marrow

    SciTech Connect

    Geoffroy, J.S.

    1987-01-01

    Endocytosis of serum albumin by sinusoidal endothelial cells in rat bone marrow was investigated initially at the ultrastructural level with subsequent biochemical investigation of the specificity mediating this event. Bovine serum albumin adsorbed to 20nm colloidal gold particles (AuBSA) was chosen as the electron microscopic probe. Morphological data strongly suggested that a receptor was involved in uptake of AuBSA. Confirmation of receptor involvement in the uptake of AuBSA by marrow sinusoidal endothelial cells was achieved utilizing an in situ isolated hind limb perfusion protocol in conjunction with unlabeled, radiolabeled, and radio-/colloidal gold labeled probes. The major findings of competition and saturation experiments were: (1) endocytosis of AuBSA was mediated by a receptor for modified/treated serum albumin; (2) endocytosis of formaldehyde-treated serum albumin was mediated by a binding site which may be the same or closely related to the site responsible for the uptake of AuBSA; and (3) endocytosis of native untreated albumin was not mediated by receptor and probably represents fluid-phase pinocitosis.

  3. Osmotic Stress Modulates the Balance between Exocytosis and Clathrin-Mediated Endocytosis in Arabidopsis thaliana.

    PubMed

    Zwiewka, Marta; Nodzyński, Tomasz; Robert, Stéphanie; Vanneste, Steffen; Friml, Jiří

    2015-08-01

    The sessile life style of plants creates the need to deal with an often adverse environment, in which water availability can change on a daily basis, challenging the cellular physiology and integrity. Changes in osmotic conditions disrupt the equilibrium of the plasma membrane: hypoosmotic conditions increase and hyperosmotic environment decrease the cell volume. Here, we show that short-term extracellular osmotic treatments are closely followed by a shift in the balance between endocytosis and exocytosis in root meristem cells. Acute hyperosmotic treatments (ionic and nonionic) enhance clathrin-mediated endocytosis simultaneously attenuating exocytosis, whereas hypoosmotic treatments have the opposite effects. In addition to clathrin recruitment to the plasma membrane, components of early endocytic trafficking are essential during hyperosmotic stress responses. Consequently, growth of seedlings defective in elements of clathrin or early endocytic machinery is more sensitive to hyperosmotic treatments. We also found that the endocytotic response to a change of osmotic status in the environment is dominant over the presumably evolutionary more recent regulatory effect of plant hormones, such as auxin. These results imply that osmotic perturbation influences the balance between endocytosis and exocytosis acting through clathrin-mediated endocytosis. We propose that tension on the plasma membrane determines the addition or removal of membranes at the cell surface, thus preserving cell integrity.

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

    SciTech Connect

    Xiong Xiong; Zhou Keming; Wu Zhengxing . E-mail: xutao@ibp.ac.cn; Xu Tao . E-mail: ibbwuzx@mail.hust.edu.cn

    2006-08-18

    Synaptotagmin I (Syt I) is a Ca{sup 2+} 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 [Ca{sup 2+}] {sub 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 Ca{sup 2+}-sensing of LDCV fusion with plasmalemma, but also plays a crucial role in fast endocytosis in INS-1 cells.

  5. Live Cell Imaging of the Endocytosis and the Intracellular Trafficking of Multifunctional Lipid Nanoparticles

    SciTech Connect

    Zhang, Tieqiao; Danthi, S. N.; Xie, Jianwu; Hu, Dehong; Lu, H. Peter; Li, King H.

    2006-12-01

    Artificial lipid nanoparticles have drawn great attention due to their potential in medicine. Linked with targeting ligands, they can be used as probes and/or gene delivery vectors for specific types of target cells. Therefore, they are very promising agents in early detection, diagnosis and treatment of cancers and other genetic diseases. However, there are several barriers blocking the applications. Controlling the cellular uptake of the lipid nanoparticles is an important technical challenge to overcome. Understanding the mechanism of the endocytosis and the following intracellular trafficking is very important for improving the design and therefore the efficiency as a drug delivery system. By using fluorescence microscopy methods, we studied the endocytosis of lipid nanoparticles by live M21 cells. The movements of the nanoparticles inside the cell were quantitatively characterized and classified based on the diffusion behavior. The trajectories of nanoparticles movement over the cell membrane revealed hop-diffusion behavior prior to the endocytosis. Fast movement in large steps is observed in intracellular trafficking and is attributed to active movement along microtubule. These observations help to understand the mechanism of the endocytosis and the pathway of the particles in cells.

  6. Loss of PiT-1 results in abnormal endocytosis in the yolk sac visceral endoderm.

    PubMed

    Wallingford, Mary C; Giachelli, Cecilia M

    2014-08-01

    PiT-1 protein is a transmembrane sodium-dependent phosphate (Pi) transporter. PiT-1 knock out (KO) embryos die from largely unknown causes by embryonic day (E) 12.5. We tested the hypothesis that PiT-1 is required for endocytosis in the embryonic yolk sac (YS) visceral endoderm (VE). Here we present data supporting that PiT-1 KO results in a YS remodeling defect and decreased endocytosis in the YS VE. The remodeling defect is not due to an upstream cardiomyocyte requirement for PiT-1, as SM22αCre-specific KO of PiT-1 in the developing heart and the YS mesodermal layer (ME) does not recapitulate the PiT-1 global KO phenotype. Furthermore, we find that high levels of PiT-1 protein localize to the YS VE apical membrane. Together these data support that PiT-1 is likely required in YS VE. During normal development maternal immunoglobulin (IgG) is endocytosed into YS VE and accumulates in the apical side of the VE in a specialized lysosome termed the apical vacuole (AV). We have identified a reduction in PiT-1 KO VE cell height and a striking loss of IgG accumulation in the PiT-1 KO VE. The endocytosis genes Tfeb, Lamtor2 and Snx2 are increased at the RNA level. Lysotracker Red staining reveals a loss of distinct AVs, and yolk sacs incubated ex vivo with phRODO Green Dextran for Endocytosis demonstrate a functional loss of endocytosis. As yolk sac endocytosis is controlled in part by microautophagy, but expression of LC3 had not been examined, we investigated LC3 expression during yolk sac development and found stage-specific LC3 RNA expression that is predominantly from the YS VE layer at E9.5. Normalized LC3-II protein levels are decreased in the PiT-1 KO YS, supporting a requirement for PiT-1 in autophagy in the YS. Therefore, we propose the novel idea that PiT-1 is central to the regulation of endocytosis and autophagy in the YS VE.

  7. Dual single-scission event analysis of constitutive transferrin receptor (TfR) endocytosis and ligand-triggered β2-adrenergic receptor (β2AR) or Mu-opioid receptor (MOR) endocytosis

    PubMed Central

    Lampe, Marko; Pierre, Fabienne; Al-Sabah, Suleiman; Krasel, Cornelius; Merrifield, Christien J.

    2014-01-01

    The dynamic relationship between constitutive and ligand-triggered clathrin-mediated endocytosis is only poorly characterized, and it remains controversial whether clathrin-coated pits specialize to internalize particular receptor cargo. Here we analyzed the ligand-triggered endocytosis of the model G-protein–coupled receptors (GPCRs) β2-adrenergic receptor (β2AR) and Mu-opioid receptor (MOR) at the level of individual endocytic events using a total internal reflection fluorescence microscopy (TIRFM)–based assay. Similar to the constitutive endocytosis of transferrin receptor (TfR), ligand- triggered endocytosis of β2AR occurs via quantized scission events hosted by clathrin spots and plaques of variable size and persistence. To address whether clathrin-coated structures (CCSs) specialize to internalize particular GPCRs, we adapted the TIRFM imaging assay to simultaneously quantify the internalization of TfR and the ligand- triggered endocytosis of the β2AR or MOR. Agonist-triggered β2AR or MOR endocytosis extended the maturation time of CCSs, as shown previously, but did not affect the rate of constitutive TfR endocytosis or loading of TfR into individual endocytic vesicles. Both the β2AR and the MOR receptors entered cells in the same vesicles as TfR, and the overall evidence for CCS specialization was weak. These data support a simple model in which different cargoes internalize through common CCSs. PMID:25079691

  8. Dual single-scission event analysis of constitutive transferrin receptor (TfR) endocytosis and ligand-triggered β2-adrenergic receptor (β2AR) or Mu-opioid receptor (MOR) endocytosis.

    PubMed

    Lampe, Marko; Pierre, Fabienne; Al-Sabah, Suleiman; Krasel, Cornelius; Merrifield, Christien J

    2014-10-01

    The dynamic relationship between constitutive and ligand-triggered clathrin-mediated endocytosis is only poorly characterized, and it remains controversial whether clathrin-coated pits specialize to internalize particular receptor cargo. Here we analyzed the ligand-triggered endocytosis of the model G-protein-coupled receptors (GPCRs) β2-adrenergic receptor (β2AR) and Mu-opioid receptor (MOR) at the level of individual endocytic events using a total internal reflection fluorescence microscopy (TIRFM)-based assay. Similar to the constitutive endocytosis of transferrin receptor (TfR), ligand- triggered endocytosis of β2AR occurs via quantized scission events hosted by clathrin spots and plaques of variable size and persistence. To address whether clathrin-coated structures (CCSs) specialize to internalize particular GPCRs, we adapted the TIRFM imaging assay to simultaneously quantify the internalization of TfR and the ligand- triggered endocytosis of the β2AR or MOR. Agonist-triggered β2AR or MOR endocytosis extended the maturation time of CCSs, as shown previously, but did not affect the rate of constitutive TfR endocytosis or loading of TfR into individual endocytic vesicles. Both the β2AR and the MOR receptors entered cells in the same vesicles as TfR, and the overall evidence for CCS specialization was weak. These data support a simple model in which different cargoes internalize through common CCSs.

  9. Developing bulk exchange spring magnets

    DOEpatents

    Mccall, Scott K.; Kuntz, Joshua D.

    2017-06-27

    A method of making a bulk exchange spring magnet by providing a magnetically soft material, providing a hard magnetic material, and producing a composite of said magnetically soft material and said hard magnetic material to make the bulk exchange spring magnet. The step of producing a composite of magnetically soft material and hard magnetic material is accomplished by electrophoretic deposition of the magnetically soft material and the hard magnetic material to make the bulk exchange spring magnet.

  10. Phosphorylation of Complexin by PKA Regulates Activity-dependent Spontaneous Neurotransmitter Release and Structural Synaptic Plasticity

    PubMed Central

    Cho, Richard W.; Buhl, Lauren K.; Volfson, Dina; Tran, Adrienne; Li, Feng; Akbergenova, Yulia; Littleton, J. Troy

    2016-01-01

    Summary Synaptic plasticity is a fundamental feature of the nervous system that allows adaptation to changing behavioral environments. Most studies of synaptic plasticity have examined the regulated trafficking of postsynaptic glutamate receptors that generates alterations in synaptic transmission. Whether and how changes in the presynaptic release machinery contribute to neuronal plasticity is less clear. The SNARE complex mediates neurotransmitter release in response to presynaptic Ca++ entry. Here we show that the SNARE fusion clamp Complexin undergoes activity-dependent phosphorylation that alters the basic properties of neurotransmission in Drosophila. Retrograde signaling following stimulation activates PKA-dependent phosphorylation of the Complexin C-terminus that selectively and transiently enhances spontaneous release. Enhanced spontaneous release is required for activity-dependent synaptic growth. These data indicate that SNARE-dependent fusion mechanisms can be regulated in an activity-dependent manner and highlight the key role of spontaneous neurotransmitter release as a mediator of functional and structural plasticity. PMID:26590346

  11. Rift Valley fever virus strain MP-12 enters mammalian host cells via caveola-mediated endocytosis.

    PubMed

    Harmon, Brooke; Schudel, Benjamin R; Maar, Dianna; Kozina, Carol; Ikegami, Tetsuro; Tseng, Chien-Te Kent; Negrete, Oscar A

    2012-12-01

    Rift Valley fever virus (RVFV) is a zoonotic pathogen capable of causing serious morbidity and mortality in both humans and livestock. The lack of efficient countermeasure strategies, the potential for dispersion into new regions, and the pathogenesis in humans and livestock make RVFV a serious public health concern. The receptors, cellular factors, and entry pathways used by RVFV and other members of the family Bunyaviridae remain largely uncharacterized. Here we provide evidence that RVFV strain MP-12 uses dynamin-dependent caveola-mediated endocytosis for cell entry. Caveolae are lipid raft domains composed of caveolin (the main structural component), cholesterol, and sphingolipids. Caveola-mediated endocytosis is responsible for the uptake of a wide variety of host ligands, as well as bacteria, bacterial toxins, and a number of viruses. To determine the cellular entry mechanism of RVFV, we used small-molecule inhibitors, RNA interference (RNAi), and dominant negative (DN) protein expression to inhibit the major mammalian cell endocytic pathways. Inhibitors and RNAi specific for macropinocytosis and clathrin-mediated endocytosis had no effect on RVFV infection. In contrast, inhibitors of caveola-mediated endocytosis, and RNAi targeted to caveolin-1 and dynamin, drastically reduced RVFV infection in multiple cell lines. Expression of DN caveolin-1 also reduced RVFV infection significantly, while expression of DN EPS15, a protein required for the assembly of clathrin-coated pits, and DN PAK-1, an obligate mediator of macropinocytosis, had no significant impact on RVFV infection. These results together suggest that the primary mechanism of RVFV MP-12 uptake is dynamin-dependent, caveolin-1-mediated endocytosis.

  12. IGF1R Signaling in Ewing Sarcoma Is Shaped by Clathrin-/Caveolin-Dependent Endocytosis

    PubMed Central

    Martins, Ana Sofia; Ordóñez, José Luis; Amaral, Ana Teresa; Prins, Frans; Floris, Giuseppe; Debiec-Rychter, Maria; Hogendoorn, Pancras C. W.; de Alava, Enrique

    2011-01-01

    Receptor endocytosis is critical for cell signaling. IGF1R mediates an autocrine loop that is de-regulated in Ewing Sarcoma (ES) cells. Here we study the impact of IGF1R internalization, mediated by clathrin and caveolin-1 (CAV1), in ES signaling. We used clathrin and CAV1-siRNA to interfere in clathrin- and caveolin-dependent endocytosis. Chlorpromazine (CPMZ) and methyl-beta-cyclo-dextrin (MCD) were also used in order to inhibit clathrin- and caveolin-dependent endocytosis, respectively. We analyzed IGF1R internalization and co-localization with clathrin and CAV1 upon ligand binding, as well as the status of the IGF1R pathway, cellular proliferation, and the apoptosis of interfered and inhibited ES cells. We performed a high-throughput tyrosine kinase phosphorylation assay to analyze the effects of combining the IGF1R tyrosine kinase inhibitor AEW541 (AEW) with CPMZ or MCD on the intracellular phospho-proteome. We observed that IGF1R is internalized upon ligand binding in ES cells and that this process is dependent on clathrin or CAV1. The blockage of receptor internalization inhibited AKT and MAPK phosphorylation, reducing the proliferative rate of ES cells and increasing the levels of apoptosis. Combination of AEW with CPMZ or MCD largely enhanced these effects. CAV1 and clathrin endocytosis controls IGF1R internalization and signaling and has a profound impact on ES IGF1R-promoted survival signaling. We propose the combination of tyrosine-kinase inhibitors with endocytosis inhibitors as a new therapeutic approach to achieve a stronger degree of receptor inhibition in this, or other neoplasms dependent on IGF1R signaling. PMID:21611203

  13. Phosphorylation Decreases Ubiquitylation of the Thiazide-sensitive Cotransporter NCC and Subsequent Clathrin-mediated Endocytosis*

    PubMed Central

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

    2014-01-01

    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

  14. Two threshold values of low pH block endocytosis at different stages.

    PubMed Central

    Davoust, J; Gruenberg, J; Howell, K E

    1987-01-01

    The influence of low extracellular pH on endocytosis was studied in baby hamster kidney cells. When the extracellular medium was adjusted to pH 5.7, the intracellular pH decreased within 2 min to pH 6.2 and the endocytosis of horseradish peroxidase (HRP) in the fluid phase dropped to an undetectable level. With an external pH of 6.3, the internal pH dropped to pH 6.8 and HRP was internalized at a normal rate for 5 min but accumulation during longer incubation times did not occur. Morphologically, HRP was visualized in the lumen of a subpopulation of tubular and vesicular endosomes. These observations were confirmed by subcellular fractionation studies using free flow electrophoresis. Low extracellular pH also had an effect on the endocytosis of the membrane-spanning glycoprotein G of vesicular stomatitis virus which was implanted into the plasma membrane. The internalization of G-protein was quantitated by a surface fluoroimmunoassay. The endocytosis of G-protein was not affected when the external pH was dropped to 6.3, but was reduced at an external pH of 5.7. The intracellular ATP was not depleted and the reduction of endocytosis was reversible upon return to physiological pH. Clathrin coated pits were detected by electron microscopy at the plasma membrane of the low-pH-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS) Images Fig. 3. Fig. 4. Fig. 8. PMID:3428267

  15. α-Synuclein Mutation Inhibits Endocytosis at Mammalian Central Nerve Terminals

    PubMed Central

    Wu, Xin-Sheng; Sheng, Jiansong; Zhang, Zhen; Yue, Hai-Yuan; Sun, Lixin; Sgobio, Carmelo; Lin, Xian; Peng, Shiyong; Jin, Yinghui; Gan, Lin; Wu, Ling-Gang

    2016-01-01

    α-Synuclein (α-syn) missense and multiplication mutations have been suggested to cause neurodegenerative diseases, including Parkinson's disease (PD) and dementia with Lewy bodies. Before causing the progressive neuronal loss, α-syn mutations impair exocytosis, which may contribute to eventual neurodegeneration. To understand how α-syn mutations impair exocytosis, we developed a mouse model that selectively expressed PD-related human α-syn A53T (h-α-synA53T) mutation at the calyx of Held terminals, where release mechanisms can be dissected with a patch-clamping technique. With capacitance measurement of endocytosis, we reported that h-α-synA53T, either expressed transgenically or dialyzed in the short term in calyces, inhibited two of the most common forms of endocytosis, the slow and rapid vesicle endocytosis at mammalian central synapses. The expression of h-α-synA53T in calyces also inhibited vesicle replenishment to the readily releasable pool. These findings may help to understand how α-syn mutations impair neurotransmission before neurodegeneration. SIGNIFICANCE STATEMENT α-Synuclein (α-syn) missense or multiplication mutations may cause neurodegenerative diseases, such as Parkinson's disease and dementia with Lewy bodies. The initial impact of α-syn mutations before neuronal loss is impairment of exocytosis, which may contribute to eventual neurodegeneration. The mechanism underlying impairment of exocytosis is poorly understood. Here we report that an α-syn mutant, the human α-syn A53T, inhibited two of the most commonly observed forms of endocytosis, slow and rapid endocytosis, at a mammalian central synapse. We also found that α-syn A53T inhibited vesicle replenishment to the readily releasable pool. These results may contribute to accounting for the widely observed early synaptic impairment caused by α-syn mutations in the progression toward neurodegeneration. PMID:27098685

  16. Mouse early extra-embryonic lineages activate compensatory endocytosis in response to poor maternal nutrition.

    PubMed

    Sun, Congshan; Velazquez, Miguel A; Marfy-Smith, Stephanie; Sheth, Bhavwanti; Cox, Andy; Johnston, David A; Smyth, Neil; Fleming, Tom P

    2014-03-01

    Mammalian extra-embryonic lineages perform the crucial role of nutrient provision during gestation to support embryonic and fetal growth. These lineages derive from outer trophectoderm (TE) and internal primitive endoderm (PE) in the blastocyst and subsequently give rise to chorio-allantoic and visceral yolk sac placentae, respectively. We have shown maternal low protein diet exclusively during mouse preimplantation development (Emb-LPD) is sufficient to cause a compensatory increase in fetal and perinatal growth that correlates positively with increased adult-onset cardiovascular, metabolic and behavioural disease. Here, to investigate early mechanisms of compensatory nutrient provision, we assessed the influence of maternal Emb-LPD on endocytosis within extra-embryonic lineages using quantitative imaging and expression of markers and proteins involved. Blastocysts collected from Emb-LPD mothers within standard culture medium displayed enhanced TE endocytosis compared with embryos from control mothers with respect to the number and collective volume per cell of vesicles with endocytosed ligand and fluid and lysosomes, plus protein expression of megalin (Lrp2) LDL-family receptor. Endocytosis was also stimulated using similar criteria in the outer PE-like lineage of embryoid bodies formed from embryonic stem cell lines generated from Emb-LPD blastocysts. Using an in vitro model replicating the depleted amino acid (AA) composition found within the Emb-LPD uterine luminal fluid, we show TE endocytosis response is activated through reduced branched-chain AAs (leucine, isoleucine, valine). Moreover, activation appears mediated through RhoA GTPase signalling. Our data indicate early embryos regulate and stabilise endocytosis as a mechanism to compensate for poor maternal nutrient provision.

  17. Real-time monitoring of NKCC2 endocytosis by total internal reflection fluorescence (TIRF) microscopy.

    PubMed

    Jaykumar, Ankita Bachhawat; Caceres, Paulo S; Sablaban, Ibrahim; Tannous, Bakhos A; Ortiz, Pablo A

    2016-01-15

    The apical Na-K-2Cl cotransporter (NKCC2) mediates NaCl reabsorption by the thick ascending limb (TAL). The amount of NKCC2 at the apical membrane of TAL cells is determined by exocytic delivery, recycling, and endocytosis. Surface biotinylation allows measurement of NKCC2 endocytosis, but it has low time resolution and does not allow imaging of the dynamic process of endocytosis. We hypothesized that total internal reflection fluorescence (TIRF) microscopy imaging of labeled NKCC2 would allow monitoring of NKCC2 endocytosis in polarized Madin-Darby canine kidney (MDCK) and TAL cells. Thus we generated a NKCC2 construct containing a biotin acceptor domain (BAD) sequence between the transmembrane domains 5 and 6. Once expressed in polarized MDCK or TAL cells, surface NKCC2 was specifically biotinylated by exogenous biotin ligase (BirA). We also demonstrate that expression of a secretory form of BirA in TAL cells induces metabolic biotinylation of NKCC2. Labeling biotinylated surface NKCC2 with fluorescent streptavidin showed that most apical NKCC2 was located within small discrete domains or clusters referred to as "puncta" on the TIRF field. NKCC2 puncta were observed to disappear from the TIRF field, indicating an endocytic event which led to a decrease in the number of surface puncta at a rate of 1.18 ± 0.16%/min in MDCK cells, and a rate 1.09 ± 0.08%/min in TAL cells (n = 5). Treating cells with a cholesterol-chelating agent (methyl-β-cyclodextrin) completely blocked NKCC2 endocytosis. We conclude that TIRF microscopy of labeled NKCC2 allows the dynamic imaging of individual endocytic events at the apical membrane of TAL cells.

  18. Rab5-regulated endocytosis plays a crucial role in apical extrusion of transformed cells.

    PubMed

    Saitoh, Sayaka; Maruyama, Takeshi; Yako, Yuta; Kajita, Mihoko; Fujioka, Yoichiro; Ohba, Yusuke; Kasai, Nobuhiro; Sugama, Natsu; Kon, Shunsuke; Ishikawa, Susumu; Hayashi, Takashi; Yamazaki, Tomohiro; Tada, Masazumi; Fujita, Yasuyuki

    2017-03-21

    Newly emerging transformed cells are often eliminated from epithelial tissues. Recent studies have revealed that this cancer-preventive process involves the interaction with the surrounding normal epithelial cells; however, the molecular mechanisms underlying this phenomenon remain largely unknown. In this study, using mammalian cell culture and zebrafish embryo systems, we have elucidated the functional involvement of endocytosis in the elimination of RasV12-transformed cells. First, we show that Rab5, a crucial regulator of endocytosis, is accumulated in RasV12-transformed cells that are surrounded by normal epithelial cells, which is accompanied by up-regulation of clathrin-dependent endocytosis. Addition of chlorpromazine or coexpression of a dominant-negative mutant of Rab5 suppresses apical extrusion of RasV12 cells from the epithelium. We also show in zebrafish embryos that Rab5 plays an important role in the elimination of transformed cells from the enveloping layer epithelium. In addition, Rab5-mediated endocytosis of E-cadherin is enhanced at the boundary between normal and RasV12 cells. Rab5 functions upstream of epithelial protein lost in neoplasm (EPLIN), which plays a positive role in apical extrusion of RasV12 cells by regulating protein kinase A. Furthermore, we have revealed that epithelial defense against cancer (EDAC) from normal epithelial cells substantially impacts on Rab5 accumulation in the neighboring transformed cells. This report demonstrates that Rab5-mediated endocytosis is a crucial regulator for the competitive interaction between normal and transformed epithelial cells in mammals.

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

    SciTech Connect

    Skornik, W.A.; Brain, J.D. )

    1990-09-01

    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.

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

    PubMed

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

    2014-05-09

    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.

  1. End4p/Sla2p interacts with actin-associated proteins for endocytosis in Saccharomyces cerevisiae.

    PubMed

    Wesp, A; Hicke, L; Palecek, J; Lombardi, R; Aust, T; Munn, A L; Riezman, H

    1997-11-01

    end4-1 was isolated as a temperature-sensitive endocytosis mutant. We cloned and sequenced END4 and found that it is identical to SLA2/MOP2. This gene is required for growth at high temperature, viability in the absence of Abp1p, polarization of the cortical actin cytoskeleton, and endocytosis. We used a mutational analysis of END4 to correlate in vivo functions with regions of End4p and we found that two regions of End4p participate in endocytosis but that the talin-like domain of End4p is dispensable. The N-terminal domain of End4p is required for growth at high temperature, endocytosis, and actin organization. A central coiled-coil domain of End4p is necessary for formation of a soluble sedimentable complex. Furthermore, this domain has an endocytic function that is redundant with the function(s) of ABP1 and SRV2. The endocytic function of Abp1p depends on its SH3 domain. In addition we have isolated a recessive negative allele of SRV2 that is defective for endocytosis. Combined biochemical, functional, and genetic analysis lead us to propose that End4p may mediate endocytosis through interaction with other actin-associated proteins, perhaps Rvs167p, a protein essential for endocytosis.

  2. End4p/Sla2p Interacts with Actin-associated Proteins for Endocytosis in Saccharomyces cerevisiae

    PubMed Central

    Wesp, A.; Hicke, L.; Palecek, J.; Lombardi, R.; Aust, T.; Munn, A.L.; Riezman, H.

    1997-01-01

    end4–1 was isolated as a temperature-sensitive endocytosis mutant. We cloned and sequenced END4 and found that it is identical to SLA2/MOP2. This gene is required for growth at high temperature, viability in the absence of Abp1p, polarization of the cortical actin cytoskeleton, and endocytosis. We used a mutational analysis of END4 to correlate in vivo functions with regions of End4p and we found that two regions of End4p participate in endocytosis but that the talin-like domain of End4p is dispensable. The N-terminal domain of End4p is required for growth at high temperature, endocytosis, and actin organization. A central coiled-coil domain of End4p is necessary for formation of a soluble sedimentable complex. Furthermore, this domain has an endocytic function that is redundant with the function(s) of ABP1 and SRV2. The endocytic function of Abp1p depends on its SH3 domain. In addition we have isolated a recessive negative allele of SRV2 that is defective for endocytosis. Combined biochemical, functional, and genetic analysis lead us to propose that End4p may mediate endocytosis through interaction with other actin-associated proteins, perhaps Rvs167p, a protein essential for endocytosis. PMID:9362070

  3. Phobos: Observed bulk properties

    NASA Astrophysics Data System (ADS)

    Pätzold, Martin; Andert, Tom; Jacobson, Robert; Rosenblatt, Pascal; Dehant, Véronique

    2014-11-01

    This work is a review of the mass determinations of the Mars moon Phobos by spacecraft close flybys, by solving for the Martian gravity field and by the analysis of secular orbit perturbations. The absolute value and accuracy is sensitive on the knowledge and accuracy of the Phobos ephemeris, of the spacecraft orbit, other perturbing forces acting on the spacecraft and the resolution of the Martian gravity field besides the measurement accuracy of the radio tracking data. The mass value and its error improved from spacecraft mission to mission or from the modern analysis of “old” tracking data but these solutions depend on the accuracy of the ephemeris at the time of observation. The mass value seems to settle within the range of GMPh=(7.11±0.09)×10-4 km3 s-2 which covers almost all mass values from close flybys and “distant” encounters within its 3-σ error (1.5%). Using the volume value determined from MEX HRSC imaging, the bulk density is (1873±31) kg m-3 (3-σ error or 1.7%), a low value which suggests that Phobos is either highly porous, is composed partially of light material or both. The determination of the gravity coefficients C20 and C22 from the Mars Express 2010 close flyby does not allow to draw conclusion on the internal structure. The large errors do not distinguish whether Phobos is homogeneous or not. In view of theories of the Phobos' origin, one possibility is that Phobos is not a captured asteroid but accreted from a debris disk in Mars orbit as a second generation solar system object.

  4. The ankyrin repeat-containing protein Akr1p is required for the endocytosis of yeast pheromone receptors.

    PubMed Central

    Givan, S A; Sprague, G F

    1997-01-01

    The Saccharomyces cerevisiae a-factor receptor (Ste3p) requires its C-terminal cytoplasmic tail for endocytosis. Wild-type receptor is delivered to the cell surface via the secretory pathway but remains there only briefly before being internalized and delivered to the vacuole for degradation. Receptors lacking all or part of the cytoplasmic tail are not subject to this constitutive endocytosis. We used the cytoplasmic tail of Ste3p as bait in the two-hybrid system in an effort to identify other proteins involved in endocytosis. One protein identified was Akr1p, an ankyrin repeat-containing protein. We applied three criteria to demonstrate that Akr1p is involved in the constitutive endocytosis of Ste3p. First, when receptor synthesis is shut off, akr1 delta cells retain the ability to mate longer than do AKR1 cells. Second, Ste3p half-life is increased by greater than 5-fold in akr1 delta cells compared with AKR1 cells. Third, after a pulse of synthesis, newly synthesized receptor remains at the cell surface in akr1 delta mutants, whereas it is rapidly internalized in AKR1 cells. Specifically, in akr1 delta mutants, newly synthesized receptor is accessible to exogenous protease, and by indirect immunofluorescence, the receptor is located at the cell surface. akr1 delta cells are also defective for endocytosis of the alpha-factor receptor (Ste2p). Despite the block to constitutive endocytosis exhibited by akr1 delta cells, they are competent to carry out ligand-mediated endocytosis of Ste3p. In contrast, akr1 delta cells cannot carry out ligand-mediated endocytosis of Ste2p. We discuss the implications for Akr1p function in endocytosis and suggest a link to the regulation of ADP-ribosylation proteins (Arf proteins). Images PMID:9243510

  5. Constitutive expression of clathrin hub hinders elicitor-induced clathrin-mediated endocytosis and defense gene expression in plant cells.

    PubMed

    Adam, T; Bouhidel, K; Der, C; Robert, F; Najid, A; Simon-Plas, F; Leborgne-Castel, N

    2012-09-21

    Endocytosis has been recently implicated in the signaling network associated with the recognition of microbes by plants. In a previous study, we showed that the elicitor cryptogein was able to induce clathrin-mediated endocytosis (CME) in tobacco suspension cells. Herein, we investigate further the induced CME by means of a GFP-tagged clathrin light chain and a CME inhibitor, the hub domain of clathrin heavy chain. Hub constitutive expression does affect neither cell growth nor constitutive endocytosis but abolishes cryptogein-induced CME. Such an inhibition has no impact on early events in the cryptogein signaling pathway but reduces the expression of defense-associated genes.

  6. Activity-dependent fluorescent labeling of bacterial cells expressing the TOL pathway

    SciTech Connect

    William K. Keener; Mary E. Watwood

    2005-01-01

    3-Ethynylbenzoate functions as an activity-dependent, fluorogenic and chromogenic probe for Pseudomonas putida mt-2, which is known to degrade toluene via conversion to benzoate, followed by meta ring fission of the intermediate, catechol. This direct physiological analysis allows the fluorescent labeling of cells whose toluene-degrading enzymes have been induced by an aromatic substrate.

  7. Activity-dependent Protein Dynamics Define Interconnected Cores of Co-regulated Postsynaptic Proteins*

    PubMed Central

    Trinidad, Jonathan C.; Thalhammer, Agnes; Burlingame, Alma L.; Schoepfer, Ralf

    2013-01-01

    Synapses are highly dynamic structures that mediate cell–cell communication in the central nervous system. Their molecular composition is altered in an activity-dependent fashion, which modulates the efficacy of subsequent synaptic transmission events. Whereas activity-dependent trafficking of individual key synaptic proteins into and out of the synapse has been characterized previously, global activity-dependent changes in the synaptic proteome have not been studied. To test the feasibility of carrying out an unbiased large-scale approach, we investigated alterations in the molecular composition of synaptic spines following mass stimulation of the central nervous system induced by pilocarpine. We observed widespread changes in relative synaptic abundances encompassing essentially all proteins, supporting the view that the molecular composition of the postsynaptic density is tightly regulated. In most cases, we observed that members of gene families displayed coordinate regulation even when they were not known to physically interact. Analysis of correlated synaptic localization revealed a tightly co-regulated cluster of proteins, consisting of mainly glutamate receptors and their adaptors. This cluster constitutes a functional core of the postsynaptic machinery, and changes in its size affect synaptic strength and synaptic size. Our data show that the unbiased investigation of activity-dependent signaling of the postsynaptic density proteome can offer valuable new information on synaptic plasticity. PMID:23035237

  8. DREAM Controls the On/Off Switch of Specific Activity-Dependent Transcription Pathways

    PubMed Central

    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

    2014-01-01

    Changes in nuclear Ca2+ 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 Ca2+-binding protein that binds DNA and represses transcription in a Ca2+-dependent manner. To study the function of DREAM in the brain, we used transgenic mice expressing a Ca2+-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

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

    PubMed

    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.

  10. Activity-dependent facilitation of Synaptojanin and synaptic vesicle recycling by the Minibrain kinase

    PubMed Central

    Chen, Chun-Kan; Bregere, Catherine; Paluch, Jeremy; Lu, Jason; Dickman, Dion K.; Chang, Karen T.

    2014-01-01

    Phosphorylation has emerged as a crucial regulatory mechanism in the nervous system to integrate the dynamic signaling required for proper synaptic development, function, and plasticity, particularly during changes in neuronal activity. Here we present evidence that Minibrain (Mnb; also known as Dyrk1A), a serine/threonine kinase implicated in autism spectrum disorder and Down syndrome, is required presynaptically for normal synaptic growth and rapid synaptic vesicle endocytosis at the Drosophila neuromuscular junction (NMJ). We find that Mnb-dependent phosphorylation of synaptojanin (Synj) is required, in vivo, for complex endocytic protein interactions and to enhance Synj activity. Neuronal stimulation drives Mnb mobilization to endocytic zones and triggers Mnb-dependent phosphorylation of Synj. Our data identify Mnb as a synaptic kinase that promotes efficient synaptic vesicle recycling by dynamically calibrating Synj function at the Drosophila NMJ, and in turn endocytic capacity, to adapt to conditions of high synaptic activity. PMID:24977345

  11. Activity-dependent facilitation of Synaptojanin and synaptic vesicle recycling by the Minibrain kinase.

    PubMed

    Chen, Chun-Kan; Bregere, Catherine; Paluch, Jeremy; Lu, Jason F; Dickman, Dion K; Chang, Karen T

    2014-06-30

    Phosphorylation has emerged as a crucial regulatory mechanism in the nervous system to integrate the dynamic signalling required for proper synaptic development, function and plasticity, particularly during changes in neuronal activity. Here we present evidence that Minibrain (Mnb; also known as Dyrk1A), a serine/threonine kinase implicated in autism spectrum disorder and Down syndrome, is required presynaptically for normal synaptic growth and rapid synaptic vesicle endocytosis at the Drosophila neuromuscular junction (NMJ). We find that Mnb-dependent phosphorylation of Synaptojanin (Synj) is required, in vivo, for complex endocytic protein interactions and to enhance Synj activity. Neuronal stimulation drives Mnb mobilization to endocytic zones and triggers Mnb-dependent phosphorylation of Synj. Our data identify Mnb as a synaptic kinase that promotes efficient synaptic vesicle recycling by dynamically calibrating Synj function at the Drosophila NMJ, and in turn endocytic capacity, to adapt to conditions of high synaptic activity.

  12. Mobility of tethering factor EEA1 on endosomes is decreased upon stimulation of EGF receptor endocytosis in HeLa cells

    SciTech Connect

    Kosheverova, Vera V.; Kamentseva, Rimma S.; Gonchar, Ilya V.; Kharchenko, Marianna V.; Kornilova, Elena S.

    2016-04-22

    Tethering factor EEA1, mediating homotypic fusion of early endosomes, was shown to be localized in membrane-bound state both in serum-deprived and stimulated for EGF receptor endocytosis cells. However, it is not known whether dynamics behavior of EEA1 is affected by EGF stimulation. We investigated EEA1 cytosol-to-membrane exchange rate in interphase HeLa cells by FRAP analysis. The data obtained fitted two-states binding model, with the bulk of membrane-associated EEA1 protein represented by the mobile fraction both in serum-starved and EGF-stimulated cells. Fast recovery state had similar half-times in the two cases: about 1.6 s and 2.8 s, respectively. However, the recovery half-time of slowly cycled EEA1 fraction significantly increased in EGF-stimulated comparing to serum-starved cells (from 21 to 99 s). We suppose that the retardation of EEA1 fluorescence recovery upon EGF-stimulation may be due to the increase of activated Rab5 on endosomal membranes, the growth of the number of tethering events between EEA1-positive vesicles and their clustering. - Highlights: • EEA1 mobility was compared in serum-starved and EGF-stimulated interphase HeLa cells. • FRAP analysis revealed fast and slow components of EEA1 recovery in both cases. • Stimulation of EGFR endocytosis did not affect fast EEA1 turnover. • EGF stimulation significantly increased half-time of slowly exchanged EEA1 fraction.

  13. Endocytosis as a mechanism for tyrosine kinase-dependent suppression of a voltage-gated potassium channel.

    PubMed

    Nesti, Edmund; Everill, Brian; Morielli, Anthony D

    2004-09-01

    The voltage-gated potassium channel Kv1.2 undergoes tyrosine phosphorylation-dependent suppression of its ionic current. However, little is known about the physical mechanism behind that process. We have found that the Kv1.2 alpha-subunit protein undergoes endocytosis in response to the same stimuli that evoke suppression of Kv1.2 ionic current. The process is tyrosine phosphorylation-dependent because the same tyrosine to phenylalanine mutation in the N-terminus of Kv1.2 that confers resistance to channel suppression (Y132F) also confers resistance to channel endocytosis. Overexpression of a dominant negative form of dynamin blocked stimulus-induced Kv1.2 endocytosis and also blocked suppression of Kv1.2 ionic current. These data indicate that endocytosis of Kv1.2 from the cell surface is a key mechanism for channel suppression by tyrosine kinases.

  14. Candida albicans-epithelial interactions: dissecting the roles of active penetration, induced endocytosis and host factors on the infection process.

    PubMed

    Wächtler, Betty; Citiulo, Francesco; Jablonowski, Nadja; Förster, Stephanie; Dalle, Frederic; Schaller, Martin; Wilson, Duncan; Hube, Bernhard

    2012-01-01

    Candida albicans frequently causes superficial infections by invading and damaging epithelial cells, but may also cause systemic infections by penetrating through epithelial barriers. C. albicans is a remarkable pathogen because it can invade epithelial cells via two distinct mechanisms: induced endocytosis, analogous to facultative intracellular enteropathogenic bacteria, and active penetration, similar to plant pathogenic fungi. Here we investigated the contributions of the two invasion routes of C. albicans to epithelial invasion. Using selective cellular inhibition approaches and differential fluorescence microscopy, we demonstrate that induced endocytosis contributes considerably to the early time points of invasion, while active penetration represents the dominant epithelial invasion route. Although induced endocytosis depends mainly on Als3-E-cadherin interactions, we observed E-cadherin independent induced endocytosis. Finally, we provide evidence of a protective role for serum factors in oral infection: human serum strongly inhibited C. albicans adhesion to, invasion and damage of oral epithelial cells.

  15. Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses.

    PubMed

    Granseth, Björn; Odermatt, Benjamin; Royle, Stephen J; Lagnado, Leon

    2006-09-21

    The maintenance of synaptic transmission requires that vesicles be recycled after releasing neurotransmitter. Several modes of retrieval have been proposed to operate at small synaptic terminals of central neurons, including a fast "kiss-and-run" mechanism that releases neurotransmitter through a fusion pore. Using an improved fluorescent reporter comprising pHluorin fused to synaptophysin, we find that only a slow mode of endocytosis (tau = 15 s) operates at hippocampal synapses when vesicle fusion is triggered by a single nerve impulse or short burst. This retrieval mechanism is blocked by overexpression of the C-terminal fragment of AP180 or by knockdown of clathrin using RNAi, and it is associated with the movement of clathrin and vesicle proteins out of the synapse. These results indicate that clathrin-mediated endocytosis is the major, if not exclusive, mechanism of vesicle retrieval after physiological stimuli.

  16. An unmet actin requirement explains the mitotic inhibition of clathrin-mediated endocytosis.

    PubMed

    Kaur, Satdip; Fielding, Andrew B; Gassner, Gisela; Carter, Nicholas J; Royle, Stephen J

    2014-02-18

    Clathrin-mediated endocytosis (CME) is the major internalisation route for many different receptor types in mammalian cells. CME is shut down during early mitosis, but the mechanism of this inhibition is unclear. In this study, we show that the mitotic shutdown is due to an unmet requirement for actin in CME. In mitotic cells, membrane tension is increased and this invokes a requirement for the actin cytoskeleton to assist the CME machinery to overcome the increased load. However, the actin cytoskeleton is engaged in the formation of a rigid cortex in mitotic cells and is therefore unavailable for deployment. We demonstrate that CME can be 'restarted' in mitotic cells despite high membrane tension, by allowing actin to engage in endocytosis. Mitotic phosphorylation of endocytic proteins is maintained in mitotic cells with restored CME, indicating that direct phosphorylation of the CME machinery does not account for shutdown. DOI: http://dx.doi.org/10.7554/eLife.00829.001.

  17. Endocytosis of Cytotoxic Granules Is Essential for Multiple Killing of Target Cells by T Lymphocytes.

    PubMed

    Chang, Hsin-Fang; Bzeih, Hawraa; Schirra, Claudia; Chitirala, Praneeth; Halimani, Mahantappa; Cordat, Emmanuelle; Krause, Elmar; Rettig, Jens; Pattu, Varsha

    2016-09-15

    CTLs are serial killers that kill multiple target cells via exocytosis of cytotoxic granules (CGs). CG exocytosis is tightly regulated and has been investigated in great detail; however, whether CG proteins are endocytosed following exocytosis and contribute to serial killing remains unknown. By using primary CTLs derived from a knock-in mouse of the CG membrane protein Synaptobrevin2, we show that CGs are endocytosed in a clathrin- and dynamin-dependent manner. Following acidification, endocytosed CGs are recycled through early and late, but not recycling endosomes. CGs are refilled with granzyme B at the late endosome stage and polarize to subsequent synapses formed between the CTL and new target cells. Importantly, inhibiting CG endocytosis in CTLs results in a significant reduction of their cytotoxic activity. Thus, our data demonstrate that continuous endocytosis of CG membrane proteins is a prerequisite for efficient serial killing of CTLs and identify key events in this process.

  18. Chirality-Induced Budding: A Raft-Mediated Mechanism for Endocytosis and Morphology of Caveolae?

    PubMed Central

    Sarasij, R. C.; Mayor, Satyajit; Rao, Madan

    2007-01-01

    The formation of transport carriers (spherical vesicles and tubules) involves membrane budding, growth, and ultimately fission. We propose a mechanism of membrane budding, wherein the tilt and chirality of constituent molecules, confined to a patch of area A, induces buds of ∼50–100 nm that are comparable to vesicles involved in endocytosis. Because such chiral and tilted lipid molecules are likely to exist in “rafts”, we suggest the involvement of this mechanism in generating membrane buds in the clathrin and dynamin-independent, raft-component mediated endocytosis of glycosylphosphatidylinositol-anchored proteins. We argue that caveolae, permanent cell surface structures with characteristic morphology and enriched in raft constituents, are also likely to be formed by this mechanism. Thus, molecular chirality and tilt, and its expression over large spatial scales may be a common organizing principle in membrane budding of transport carriers. PMID:17237196

  19. Sequential pulses of apical epithelial secretion and endocytosis drive airway maturation in Drosophila.

    PubMed

    Tsarouhas, Vasilios; Senti, Kirsten-André; Jayaram, Satish Arcot; Tiklová, Katarína; Hemphälä, Johanna; Adler, Jeremy; Samakovlis, Christos

    2007-08-01

    The development of air-filled respiratory organs is crucial for survival at birth. We used a combination of live imaging and genetic analysis to dissect respiratory organ maturation in the embryonic Drosophila trachea. We found that tracheal tube maturation entails three precise epithelial transitions. Initially, a secretion burst deposits proteins into the lumen. Solid luminal material is then rapidly cleared from the tubes, and shortly thereafter liquid is removed. To elucidate the cellular mechanisms behind these transitions, we identified gas-filling-deficient mutants showing narrow or protein-clogged tubes. These mutations either disrupt endoplasmatic reticulum-to-Golgi vesicle transport or endocytosis. First, Sar1 is required for protein secretion, luminal matrix assembly, and diametric tube expansion. Subsequently, a sharp pulse of Rab5-dependent endocytic activity rapidly internalizes and clears luminal contents. The coordination of luminal matrix secretion and endocytosis may be a general mechanism in tubular organ morphogenesis and maturation.

  20. Exo-endocytosis and closing of the fission pore during endocytosis in single pituitary nerve terminals internally perfused with high calcium concentrations.

    PubMed Central

    Rosenboom, H; Lindau, M

    1994-01-01

    An increase in free Ca2+ triggers exocytosis in pituitary nerve terminals leading to an increase in membrane area and membrane capacitance. When Ca2+ is increased by step depolarization, an instantaneous capacitance increase during the first 80 ms is followed by a slow increase extending over several seconds. We measured capacitance changes associated with exocytosis and endocytosis in single pituitary nerve terminals internally perfused with high Ca2+. At 50 microM Ca2+ the capacitance increased by up to 2%/s, similar to the slow phase observed during depolarization. Our results indicate that at the site of fusion very high Ca2+ is required. Following exocytosis, large downward capacitance steps were measured, reflecting endocytosis of large vacuoles. These events were not abrupt but reflected a gradual decrease of fission pore conductance from 8 nS to < 40 pS during 500 ms, revealing the dynamics of individual fission pore closures. Above 300 pS, narrowing of the endocytotic fission pore was approximately 10 times slower than the previously reported expansion of the exocytotic fusion pore. The transition between 300 pS and 0 pS took approximately 200 ms, whereas it has been reported that the exocytotic fusion pore measured in mast cells opens from 0 to 280 pS in < 100 microseconds. The time course of closing of the fission pore may be explained by an exponential decrease in pore diameter occurring at a constant rate. PMID:8202480

  1. Activity-Dependent Changes in Gene Expression in Schizophrenia Human-Induced Pluripotent Stem Cell Neurons.

    PubMed

    Roussos, Panos; Guennewig, Boris; Kaczorowski, Dominik C; Barry, Guy; Brennand, Kristen J

    2016-11-01

    Schizophrenia candidate genes participate in common molecular pathways that are regulated by activity-dependent changes in neurons. One important next step is to further our understanding on the role of activity-dependent changes of gene expression in the etiopathogenesis of schizophrenia. To examine whether neuronal activity-dependent changes of gene expression are dysregulated in schizophrenia. Neurons differentiated from human-induced pluripotent stem cells derived from 4 individuals with schizophrenia and 4 unaffected control individuals were depolarized using potassium chloride. RNA was extracted followed by genome-wide profiling of the transcriptome. Neurons were planted on June 21, 2013, and harvested on August 2, 2013. We performed differential expression analysis and gene coexpression analysis to identify activity-dependent or disease-specific changes of the transcriptome. Gene expression differences were assessed with linear models. Furthermore, we used gene set analyses to identify coexpressed modules that are enriched for schizophrenia risk genes. We identified 1669 genes that were significantly different in schizophrenia-associated vs control human-induced pluripotent stem cell-derived neurons and 1199 genes that are altered in these cells in response to depolarization (linear models at false discovery rate ≤0.05). The effect of activity-dependent changes of gene expression in schizophrenia-associated neurons (59 significant genes at false discovery rate ≤0.05) was attenuated compared with control samples (594 significant genes at false discovery rate ≤0.05). Using gene coexpression analysis, we identified 2 modules (turquoise and brown) that were associated with diagnosis status and 2 modules (yellow and green) that were associated with depolarization at a false discovery rate of ≤0.05. For 3 of the 4 modules, we found enrichment with schizophrenia-associated variants: brown (χ2 = 20.68; P = .002), turquoise (χ2 = 12.95; P

  2. Differences between the endocytosis of horseradish peroxidase and its conjugate with wheat germ agglutinin by cultured fibroblasts.

    PubMed

    Stieber, A; Gonatas, J O; Gonatas, N K

    1984-04-01

    A covalent conjugate of wheat germ agglutinin (WGA) with horseradish peroxidase (HRP) was used for a morphologic study of its adsorptive endocytosis by cultured human fibroblasts. Initial binding at 4 degrees C of the conjugate was observed over the entire plasma membrane, including "coated" and smooth pits. Endocytosis of HRP and the WGA-HRP conjugate was observed in lysosomes, but only the conjugate was seen in a cisterna of the Golgi apparatus (GERL), and in adjacent coated vesicles.

  3. The endocytosis and signaling of the γδ T cell coreceptor WC1 are regulated by a dileucine motif.

    PubMed

    Hsu, Haoting; Baldwin, Cynthia L; Telfer, Janice C

    2015-03-01

    WC1 proteins, which are specifically expressed by bovine γδ T cells from a gene array containing 13 members, are part of the scavenger receptor cysteine-rich family. WC1 cytoplasmic domains contains multiple tyrosines, one of which is required to be phosphorylated for TCR coreceptor activity, and a dileucine endocytosis motif. Like the TCR coreceptor CD4, WC1 is endocytosed in response to PMA. Because WC1 endocytosis may play a role in the activation of γδ T cells, we examined WC1 endocytosis in the adherent cell 293T and Jurkat T cell lines using a fusion protein of extracellular CD4 and the transmembrane and cytoplasmic domain of WC1. Individual mutation of the two leucine residues of the endocytic dileucine motif in the WC1 cytoplasmic domain significantly reduced PMA-induced endocytosis in both cell types and enhanced IL-2 production stimulated by cocross-linking of CD3/TCR and CD4/WC1 in Jurkat cells, suggesting that the sustained membrane coligation of CD3/TCR with WC1 caused by a decrease in endocytosis increases T cell activation. Mutation of two serines upstream of the endocytic dileucine motif affected endocytosis only in adherent 293T cells. Although the two upstream serines were not required for WC1 endocytosis in Jurkat cells, the pan-protein kinase C inhibitor Gö6983 blocked endocytosis of CD4/WC1, and mutation of the upstream serines in WC1 inhibited IL-2 production stimulated by cocross-linking of CD3/TCR and CD4/WC1. These studies provide insights into the signaling of WC1 gene arrays that are present in most mammals and play critical roles in γδ T cell responses to bacterial pathogens.

  4. Oligophrenin-1 Connects Exocytotic Fusion to Compensatory Endocytosis in Neuroendocrine Cells.

    PubMed

    Houy, Sébastien; Estay-Ahumada, Catherine; Croisé, Pauline; Calco, Valérie; Haeberlé, Anne-Marie; Bailly, Yannick; Billuart, Pierre; Vitale, Nicolas; Bader, Marie-France; Ory, Stéphane; Gasman, Stéphane

    2015-08-05

    Oligophrenin-1 (OPHN1) is a protein with multiple domains including a Rho family GTPase-activating (Rho-GAP) domain, and a Bin-Amphiphysin-Rvs (BAR) domain. Involved in X-linked intellectual disability, OPHN1 has been reported to control several synaptic functions, including synaptic plasticity, synaptic vesicle trafficking, and endocytosis. In neuroendocrine cells, hormones and neuropeptides stored in large dense core vesicles (secretory granules) are released through calcium-regulated exocytosis, a process that is tightly coupled to compensatory endocytosis, allowing secretory granule recycling. We show here that OPHN1 is expressed and mainly localized at the plasma membrane and in the cytosol in chromaffin cells from adrenal medulla. Using carbon fiber amperometry, we found that exocytosis is impaired at the late stage of membrane fusion in Ophn1 knock-out mice and OPHN1-silenced bovine chromaffin cells. Experiments performed with ectopically expressed OPHN1 mutants indicate that OPHN1 requires its Rho-GAP domain to control fusion pore dynamics. On the other hand, compensatory endocytosis assessed by measuring dopamine-β-hydroxylase (secretory granule membrane) internalization is severely inhibited in Ophn1 knock-out chromaffin cells. This inhibitory effect is mimicked by the expression of a truncated OPHN1 mutant lacking the BAR domain, demonstrating that the BAR domain implicates OPHN1 in granule membrane recapture after exocytosis. These findings reveal for the first time that OPHN1 is a bifunctional protein that is able, through distinct mechanisms, to regulate and most likely link exocytosis to compensatory endocytosis in chromaffin cells.

  5. Polarized endocytosis and transcytosis in the hypothalamic tanycytes of the rat.

    PubMed

    Peruzzo, Bruno; Pastor, Francisco Efrén; Blázquez, Juan Luis; Amat, Pedro; Rodríguez, Esteban Martín

    2004-08-01

    Four types of tanycytes can be distinguished in the rat hypothalamus: alpha(1) and alpha(2) tanycytes establish an anatomical link between the ventricular cerebrospinal fluid (CSF) and the arcuate nucleus, whereas beta(1) and beta2 tanycytes establish a link between CSF and portal blood. Endocytosis and transcytosis in these cells have been investigated by (1) immunocytochemistry with antibodies against molecular markers of the endocytotic and transcytotic pathways; (2) the administration of wheat germ agglutinin (WGA) into the ventricular or subarachnoidal CSF and following its internalisation by and its routing through tanycytes. The four populations of tanycytes show marked differences concerning the expression and subcellular location of proteins involved in endocytosis and transcytosis, such as clathrin, caveolin-1, Rab4 and ARF6. Thus, beta1,2 tanycytes express caveolin-1 at the ventricular cell pole and at their terminals contacting the portal capillaries, whereas alpha1,2 tanycytes do not, suggesting that caveolae-dependant endocytosis does not occur in the latter and that, in beta1,2 tanycytes, it may occur at both cell poles. In beta1,2 tanycytes, clathrin is only expressed at the ventricular cell pole indicating that clathrin-dependant endocytosis operates for compounds present in the ventricular CSF and not for those exposed to the terminals. This agrees with the property of beta1,2 tanycytes of internalising WGA through the ventricular cell pole but not through the terminals. The subcellular distribution in beta1,2 tanycytes of WGA and of the proteins clathrin and Rab4 indicates that part of the internalised WGA follows the degradative pathway and part is sorted to a transcytotic pathway and that the transcytotic and the secretory pathways might intersect. Copyright 2004 Springer-Verlag

  6. Endocytosis regulates membrane localization and function of the fusogen EFF-1.

    PubMed

    Smurova, Ksenia; Podbilewicz, Benjamin

    2016-07-28

    Cell fusion is essential for sexual reproduction and formation of muscles, bones, and placenta. Two families of cell fusion proteins (Syncytins and FFs) have been identified in eukaryotes. Syncytins have been shown to form the giant syncytial trophoblasts in the placenta. The FFs are essential to fuse cells in the skin, reproductive, excretory, digestive and nervous systems in nematodes. EFF-1 (Epithelial Fusion Failure 1), a member of the FF family, is a type I membrane glycoprotein that is essential for most cell fusions in C. elegans. The crystal structure of EFF-1 ectodomain reveals striking structural similarity to class II fusion glycoproteins from enveloped viruses (e.g. dengue and rubella) that mediate virus to cell fusion. We found EFF-1 to be present on the plasma membrane and in RAB-5-positive early endosomes, with EFF-1 recycling between these 2 cell compartments. Only when EFF-1 proteins transiently arrive to the surfaces of 2 adjacent cells do they dynamically interact in trans and mediate membrane fusion. EFF-1 is continuously internalized by receptor-mediated endocytosis via the activity of 2 small GTPases: RAB-5 and Dynamin. Here we propose a model that explains how EFF-1 endocytosis together with interactions in trans can control cell-cell fusion. Kontani et al. showed that vacuolar ATPase (vATPase) mutations result in EFF-1-dependent hyperfusion. (1) We propose that vATPase is required for normal degradation of EFF-1. Failure to degrade EFF-1 results in delayed hyperfusion and mislocalization to organelles that appear to be recycling endosomes. EFF-1 is also required to fuse neurons as part of the repair mechanism following injury and to prune dendrites. We speculate that EFF-1 may regulate neuronal tree like structures via endocytosis. Thus, endocytosis of cell-cell fusion proteins functions to prevent merging of cells and to sculpt organs and neurons.

  7. Aspergillus nidulans Ambient pH Signaling Does Not Require Endocytosis

    PubMed Central

    Lucena-Agell, Daniel; Galindo, Antonio; Arst, Herbert N.

    2015-01-01

    Aspergillus nidulans (Pal) ambient pH signaling takes place in cortical structures containing components of the ESCRT pathway, which are hijacked by the alkaline pH-activated, ubiquitin-modified version of the arrestin-like protein PalF and taken to the plasma membrane. There, ESCRTs scaffold the assembly of dedicated Pal proteins acting downstream. The molecular details of this pathway, which results in the two-step proteolytic processing of the transcription factor PacC, have received considerable attention due to the key role that it plays in fungal pathogenicity. While current evidence strongly indicates that the pH signaling role of ESCRT complexes is limited to plasma membrane-associated structures where PacC proteolysis would take place, the localization of the PalB protease, which almost certainly catalyzes the first and only pH-regulated proteolytic step, had not been investigated. In view of ESCRT participation, this formally leaves open the possibility that PalB activation requires endocytic internalization. As endocytosis is essential for hyphal growth, nonlethal endocytic mutations are predicted to cause an incomplete block. We used a SynA internalization assay to measure the extent to which any given mutation prevents endocytosis. We show that none of the tested mutations impairing endocytosis to different degrees, including slaB1, conditionally causing a complete block, have any effect on the activation of the pathway. We further show that PalB, like PalA and PalC, localizes to cortical structures in an alkaline pH-dependent manner. Therefore, signaling through the Pal pathway does not involve endocytosis. PMID:25841020

  8. Secreted PCSK9 downregulates low density lipoprotein receptor through receptor-mediated endocytosis.

    PubMed

    Qian, Yue-Wei; Schmidt, Robert J; Zhang, Youyan; Chu, Shaoyou; Lin, Aimin; Wang, He; Wang, Xiliang; Beyer, Thomas P; Bensch, William R; Li, Weiming; Ehsani, Mariam E; Lu, Deshun; Konrad, Robert J; Eacho, Patrick I; Moller, David E; Karathanasis, Sotirios K; Cao, Guoqing

    2007-07-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels. The mechanisms of this action, however, remain to be defined. We show here that recombinant human PCSK9 expressed in HEK293 cells was readily secreted into the medium, with the prosegment associated with the C-terminal domain. Secreted PCSK9 mediated cell surface LDLR degradation in a concentration- and time-dependent manner when added to HEK293 cells. Accordingly, cellular LDL uptake was significantly reduced as well. When infused directly into C57B6 mice, purified human PCSK9 substantially reduced hepatic LDLR protein levels and resulted in increased plasma LDL cholesterol. When added to culture medium, fluorescently labeled PCSK9 was endocytosed and displayed endosomal-lysosomal intracellular localization in HepG2 cells, as was demonstrated by colocalization with DiI-LDL. PCSK9 endocytosis was mediated by LDLR as LDLR deficiency (hepatocytes from LDLR null mice), or RNA interference-mediated knockdown of LDLR markedly reduced PCSK9 endocytosis. In addition, RNA interference knockdown of the autosomal recessive hypercholesterolemia (ARH) gene product also significantly reduced PCSK9 endocytosis. Biochemical analysis revealed that the LDLR extracellular domain interacted directly with secreted PCSK9; thus, overexpression of the LDLR extracellular domain was able to attenuate the reduction of cell surface LDLR levels by secreted PCSK9. Together, these results reveal that secreted PCSK9 retains biological activity, is able to bind directly to the LDLR extracellular domain, and undergoes LDLR-ARH-mediated endocytosis, leading to accelerated intracellular degradation of the LDLR.

  9. Dynamics of clathrin-mediated endocytosis and its requirement for organelle biogenesis in Dictyostelium.

    PubMed

    Macro, Laura; Jaiswal, Jyoti K; Simon, Sanford M

    2012-12-01

    The protein clathrin mediates one of the major pathways of endocytosis from the extracellular milieu and plasma membrane. In single-cell eukaryotes, such as Saccharomyces cerevisiae, the gene encoding clathrin is not an essential gene, raising the question of whether clathrin conveys specific advantages for multicellularity. Furthermore, in contrast to mammalian cells, endocytosis in S. cerevisiae is not dependent on either clathrin or adaptor protein 2 (AP2), an endocytic adaptor molecule. In this study, we investigated the requirement for components of clathrin-mediated endocytosis (CME) in another unicellular organism, the amoeba Dictyostelium. We identified a heterotetrameric AP2 complex in Dictyostelium that is similar to that which is found in higher eukaryotes. By simultaneously imaging fluorescently tagged clathrin and AP2, we found that, similar to higher eukaryotes, these proteins colocalized to membrane puncta that move into the cell together. In addition, the contractile vacuole marker protein, dajumin-green fluorescent protein (GFP), is trafficked via the cell membrane and internalized by CME in a clathrin-dependent, AP2-independent mechanism. This pathway is distinct from other endocytic mechanisms in Dictyostelium. Our finding that CME is required for the internalization of contractile vacuole proteins from the cell membrane explains the contractile vacuole biogenesis defect in Dictyostelium cells lacking clathrin. Our results also suggest that the machinery for CME and its role in organelle maintenance appeared early during eukaryotic evolution. We hypothesize that dependence of endocytosis on specific components of the CME pathway evolved later, as demonstrated by internalization independent of AP2 function.

  10. Pitstop 2 is a potent inhibitor of clathrin-independent endocytosis.

    PubMed

    Dutta, Dipannita; Williamson, Chad D; Cole, Nelson B; Donaldson, Julie G

    2012-01-01

    Clathrin independent endocytosis (CIE) is a form of endocytosis present in all cells that mediates the entry of nutrients, macromolecules and membrane proteins into cells. When compared to clathrin-dependent endocytosis (CDE), however, much less is known about the machinery involved in forming CIE endosomes. One way to distinguish CIE from CDE has been to deplete cells of coat proteins involved in CDE such as clathrin or the dynamin GTPase, leading to a block of CDE but not CIE. A drawback of such genetic manipulations is that depletion of proteins important for mediating CDE over a period of days can have complex indirect effects on cellular function. The identification of chemical compounds that specifically and rapidly block CDE or CIE would facilitate the determination of whether a process involved CDE or CIE. To date, all of those compounds have targeted CDE. Dynasore and the dynoles specifically target and block dynamin activity thus inhibiting CDE but not most forms of CIE. Recently, a new compound called pitstop 2 was identified as an inhibitor of the interaction of amphiphysin with the amino terminal domain of clathrin, and shown to inhibit CDE in cells. Here we show that pitstop 2 is also a potent inhibitor of CIE. The effects of pitstop 2 are not restricted to inhibition of clathrin since knockdown of clathrin fails to rescue the inhibition of endocytosis of CIE proteins by the drug. Thus pitstop 2 has additional cellular targets besides the amino terminal domain of clathrin and thus cannot be used to distinguish CIE from CDE.

  11. Dynamics of clathrin-mediated endocytosis and its requirement for organelle biogenesis in Dictyostelium

    PubMed Central

    Macro, Laura; Jaiswal, Jyoti K.; Simon, Sanford M.

    2012-01-01

    Summary The protein clathrin mediates one of the major pathways of endocytosis from the extracellular milieu and plasma membrane. In single-cell eukaryotes, such as Saccharomyces cerevisiae, the gene encoding clathrin is not an essential gene, raising the question of whether clathrin conveys specific advantages for multicellularity. Furthermore, in contrast to mammalian cells, endocytosis in S. cerevisiae is not dependent on either clathrin or adaptor protein 2 (AP2), an endocytic adaptor molecule. In this study, we investigated the requirement for components of clathrin-mediated endocytosis (CME) in another unicellular organism, the amoeba Dictyostelium. We identified a heterotetrameric AP2 complex in Dictyostelium that is similar to that which is found in higher eukaryotes. By simultaneously imaging fluorescently tagged clathrin and AP2, we found that, similar to higher eukaryotes, these proteins colocalized to membrane puncta that move into the cell together. In addition, the contractile vacuole marker protein, dajumin-green fluorescent protein (GFP), is trafficked via the cell membrane and internalized by CME in a clathrin-dependent, AP2-independent mechanism. This pathway is distinct from other endocytic mechanisms in Dictyostelium. Our finding that CME is required for the internalization of contractile vacuole proteins from the cell membrane explains the contractile vacuole biogenesis defect in Dictyostelium cells lacking clathrin. Our results also suggest that the machinery for CME and its role in organelle maintenance appeared early during eukaryotic evolution. We hypothesize that dependence of endocytosis on specific components of the CME pathway evolved later, as demonstrated by internalization independent of AP2 function. PMID:22992464

  12. HIV-1 Vpu Promotes Release and Prevents Endocytosis of Nascent Retrovirus Particles from the Plasma Membrane

    PubMed Central

    Neil, Stuart J. D; Eastman, Scott W; Jouvenet, Nolwenn; Bieniasz, Paul D

    2006-01-01

    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. PMID:16699598

  13. Chloride transporters and receptor-mediated endocytosis in the renal proximal tubule

    PubMed Central

    Devuyst, Olivier; Luciani, Alessandro

    2015-01-01

    Abstract The epithelial cells lining the proximal tubules of the kidney reabsorb a large amount of filtered ions and solutes owing to receptor-mediated endocytosis and polarized transport systems that reflect final cell differentiation. Dedifferentiation of proximal tubule cells and dysfunction of receptor-mediated endocytosis characterize Dent’s disease, a rare disorder caused by inactivating mutations in the CLCN5 gene that encodes the endosomal chloride–proton exchanger, ClC-5. The disease is characterized by a massive urinary loss of solutes (renal Fanconi syndrome), with severe metabolic complications and progressive renal failure. Investigations of mutations affecting the gating of ClC-5 revealed that the proximal tubule dysfunction may occur despite normal endosomal acidification. In addition to defective endocytosis, proximal tubule cells lacking ClC-5 show a trafficking defect in apical receptors and transporters, as well as lysosomal dysfunction and typical features of dedifferentiation, proliferation and oxidative stress. A similar but milder defect is observed in mouse models with defective CFTR, a chloride channel that is also expressed in the endosomes of proximal tubule cells. These data suggest a major role for endosomal chloride transport in the maintenance of epithelial differentiation and reabsorption capacity of the renal proximal tubule. Key points The reabsorptive activity of renal proximal tubule cells is mediated by receptor-mediated endocytosis and polarized transport systems that reflect final cell differentiation. Loss-of-function mutations of the endosomal chloride–proton exchanger ClC-5 (Dent’s disease) cause a major trafficking defect in proximal tubule cells, associated with lysosomal dysfunction, oxidative stress and dedifferentiation/proliferation. A similar but milder defect is associated with mutations in CFTR (cystic fibrosis transmembrane conductance regulator). Vesicular chloride transport appears to be important for

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  15. Analysis of Occludin Trafficking, Demonstrating Continuous Endocytosis, Degradation, Recycling and Biosynthetic Secretory Trafficking

    PubMed Central

    Fletcher, Sarah J.; Iqbal, Mudassar; Jabbari, Sara; Stekel, Dov; Rappoport, Joshua Z.

    2014-01-01

    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. PMID:25422932

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

    PubMed

    Fletcher, Sarah J; Iqbal, Mudassar; Jabbari, Sara; Stekel, Dov; Rappoport, Joshua Z

    2014-01-01

    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.

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

    PubMed

    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.

  18. Discovery of new cargo proteins that enter cells through clathrin-independent endocytosis.

    PubMed

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

    2009-05-01

    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 associated with endosomal membranes carrying CIE cargo and expression of an active form of Arf6 leads to the generation of vacuolar structures that trap CIE cargo immediately after endocytosis, blocking the convergence with transferrin-containing endosomes. We isolated these trapped vacuolar structures and analyzed their protein composition by mass spectrometry. Here we identify and validate six new endogenous cargo proteins (CD44, CD55, CD98, CD147, Glut1, and ICAM1) that use CIE to enter cells. CD55 and Glut1 appear to closely parallel the trafficking of MHCI, merging with transferrin endosomes before entering the recycling tubules. In contrast, CD44, CD98, and CD147 appear to directly enter the recycling tubules and by-pass the merge with EEA1-positive, transferrin-containing endosomes. This divergent itinerary suggests that sorting may occur along this CIE pathway. Furthermore, the identification of new cargo proteins will assist others studying CIE in different cell types and tissues.

  19. Endocytosis of the tachykinin neuropeptide, neurokinin B, in astrocytes and its role in cellular copper uptake.

    PubMed

    Shahzad, Reeha; Jones, Mark R; Viles, John H; Jones, Christopher E

    2016-09-01

    The tachykinin neuropeptide, neurokinin B (NKB), belongs to a family of peptides having diverse roles in the brain. NKB, along with several other tachykinins, has been identified as a copper-binding peptide, however the physiological relevance of the binding is unclear. Previously, NKB was shown to limit the ability of copper to enter astrocytes and disrupt calcium homeostasis and it was thought that the peptide was sequestering the metal extracellularly. Here we use a fluorescein-labelled NKB peptide (F-NKB) to show that NKB is not retained extracellularly, but is endocytosed within 10-20min after addition to the cell media. The endocytosis is not inhibited when NKB is delivered as a copper-complex, [Cu(II)(F-NKB)2]. Endocytosis of NKB can increase intracellular copper. Comparison to cells cultured in copper-free buffer indicated that apo-NKB can facilitate uptake of copper found in normal culture media. To achieve this NKB must compete with a variety of copper proteins, and we show that NKB can successfully compete with copper-binding peptides derived from the prion protein, itself associated with Cu(II) and Zn(II) metabolism. We suggest a mechanism of receptor mediated endocytosis to account for the observations. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Clathrin light chains’ role in selective endocytosis influences antibody isotype switching

    PubMed Central

    Wu, Shuang; Majeed, Sophia R.; Evans, Timothy M.; Camus, Marine D.; Wong, Nicole M. L.; Schollmeier, Yvette; Park, Minjong; Muppidi, Jagan R.; Reboldi, Andrea; Parham, Peter; Cyster, Jason G.; Brodsky, Frances M.

    2016-01-01

    Clathrin, a cytosolic protein composed of heavy and light chain subunits, assembles into a vesicle coat, controlling receptor-mediated endocytosis. To establish clathrin light chain (CLC) function in vivo, we engineered mice lacking CLCa, the major CLC isoform in B lymphocytes, generating animals with CLC-deficient B cells. In CLCa-null mice, the germinal centers have fewer B cells, and they are enriched for IgA-producing cells. This enhanced switch to IgA production in the absence of CLCa was attributable to increased transforming growth factor β receptor 2 (TGFβR2) signaling resulting from defective endocytosis. Internalization of C-X-C chemokine receptor 4 (CXCR4), but not CXCR5, was affected in CLCa-null B cells, and CLC depletion from cell lines affected endocytosis of the δ-opioid receptor, but not the β2-adrenergic receptor, defining a role for CLCs in the uptake of a subset of signaling receptors. This instance of clathrin subunit deletion in vertebrates demonstrates that CLCs contribute to clathrin’s role in vivo by influencing cargo selectivity, a function previously assigned exclusively to adaptor molecules. PMID:27540116

  1. The Trypanosome Exocyst: A Conserved Structure Revealing a New Role in Endocytosis

    PubMed Central

    Boehm, Cordula M.; Obado, Samson; Gadelha, Catarina; Kaupisch, Alexandra; Manna, Paul T.; Gould, Gwyn W.; Rout, Michael P.; Field, Mark C.

    2017-01-01

    Membrane transport is an essential component of pathogenesis for most infectious organisms. In African trypanosomes, transport to and from the plasma membrane is closely coupled to immune evasion and antigenic variation. In mammals and fungi an octameric exocyst complex mediates late steps in exocytosis, but comparative genomics suggested that trypanosomes retain only six canonical subunits, implying mechanistic divergence. We directly determined the composition of the Trypanosoma brucei exocyst by affinity isolation and demonstrate that the parasite complex is nonameric, retaining all eight canonical subunits (albeit highly divergent at the sequence level) plus a novel essential subunit, Exo99. Exo99 and Sec15 knockdowns have remarkably similar phenotypes in terms of viability and impact on morphology and trafficking pathways. Significantly, both Sec15 and Exo99 have a clear function in endocytosis, and global proteomic analysis indicates an important role in maintaining the surface proteome. Taken together these data indicate additional exocyst functions in trypanosomes, which likely include endocytosis, recycling and control of surface composition. Knockdowns in HeLa cells suggest that the role in endocytosis is shared with metazoan cells. We conclude that, whilst the trypanosome exocyst has novel components, overall functionality appears conserved, and suggest that the unique subunit may provide therapeutic opportunities. PMID:28114397

  2. Specific Sterols Required for the Internalization Step of Endocytosis in Yeast

    PubMed Central

    Munn, Alan L.; Heese-Peck, Antje; Stevenson, Brian J.; Pichler, Harald; Riezman, Howard

    1999-01-01

    Sterols are major components of the plasma membrane, but their functions in this membrane are not well understood. We isolated a mutant defective in the internalization step of endocytosis in a gene (ERG2) encoding a C-8 sterol isomerase that acts in the late part of the ergosterol biosynthetic pathway. In the absence of Erg2p, yeast cells accumulate sterols structurally different from ergosterol, which is the major sterol in wild-type yeast. To investigate the structural requirements of ergosterol for endocytosis in more detail, several erg mutants (erg2Δ, erg6Δ, and erg2Δerg6Δ) were made. Analysis of fluid phase and receptor-mediated endocytosis indicates that changes in the sterol composition lead to a defect in the internalization step. Vesicle formation and fusion along the secretory pathway were not strongly affected in the ergΔ mutants. The severity of the endocytic defect correlates with changes in sterol structure and with the abundance of specific sterols in the ergΔ mutants. Desaturation of the B ring of the sterol molecules is important for the internalization step. A single desaturation at C-8,9 was not sufficient to support internalization at 37°C whereas two double bonds, either at C-5,6 and C-7,8 or at C-5,6 and C-8,9, allowed internalization. PMID:10564282

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

    SciTech Connect

    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.

  4. Endocytosis of heat-denatured albumin by cultured rat Kupffer cells

    SciTech Connect

    Brouwer, A.; Knook, D.L.

    1982-10-01

    Purified Kupffer cells were obtained by centrifugal elutriation of sinusoidal cells isolated by pronase treatment of the rat liver. The endocytosis of radioactively labeled heat-aggregated colloidal albumin (CA /sup 125/I) was investigated in maintenance cultures of the purified Kupffer cells. The endocytic capacity of the cells was studied during 4 days of culture. Maximum uptake was observed after 24 hr of culture, with a gradual decline during the following days. When the uptake was measured after incubation with increasing concentrations of CA /sup 125/I, a saturation effect was observed. This finding and the observed high rate of uptake are strong indications that receptor sites on the cell membrane are involved in the mechanism of endocytosis. The uptake of CA /sup 125/I by Kupffer cells was inhibited by the metabolic inhibitors fluoride and antimycin A, indicating that endocytosis of CA /sup 125/I is dependent on energy derived from both glycolysis and mitochondrial respiration. The mechanism of internalization may also require the action of microfilaments as well as intact microtubules, since both cytochalasin B and colchicine inhibited the uptake of CA /sup 125/I. The intracellular degradation of CA /sup 125/I by Kupffer cells was strongly inhibited by chloroquine but not by colchicine. The degradation of ingested CA /sup 125/I occurred within the Kupffer cell lysosomes.

  5. Virion endocytosis is a major target for murid herpesvirus-4 neutralization

    PubMed Central

    Glauser, Daniel L.; Gillet, Laurent

    2012-01-01

    Herpesviruses consistently transmit from immunocompetent carriers, implying that their neutralization is hard to achieve. Murid herpesvirus-4 (MuHV-4) exploits host IgG Fc receptors to bypass blocks to cell binding, and pH-dependent protein conformation changes to unveil its fusion machinery only after endocytosis. Nevertheless, neutralization remains possible by targeting the virion glycoprotein H (gH)–gL heterodimer, and the neutralizing antibody responses of MuHV-4 carriers are improved by boosting with recombinant gH–gL. We analysed here how gH–gL-directed neutralization works. The MuHV-4 gH–gL binds to heparan sulfate. However, most gH–gL-specific neutralizing antibodies did not block this interaction; neither did they act directly on fusion. Instead, they blocked virion endocytosis and transport to the late endosomes, where membrane fusion normally occurs. The poor endocytosis of gH–gL-neutralized virions was recapitulated precisely by virions genetically lacking gL. Therefore, driving virion uptake appears to be an important function of gH–gL that provides a major target for antibody-mediated neutralization. PMID:22377583

  6. Cbl ubiquitination of p85 is essential for Epo-induced EpoR endocytosis.

    PubMed

    Bulut, Gamze B; Sulahian, Rita; Yao, Huiyu; Huang, Lily Jun-shen

    2013-12-05

    Erythropoietin (Epo) binding to the Epo receptor (EpoR) elicits downstream signaling that is essential for red blood cell production. One important negative regulatory mechanism to terminate Epo signaling is Epo-induced EpoR endocytosis and degradation. Defects in this mechanism play a key role in the overproduction of erythrocytes in primary familial and congenital polycythemia (PFCP). Here we have identified a novel mechanism mediating Epo-dependent EpoR internalization. Epo induces Cbl-dependent ubiquitination of the p85 regulatory subunit of PI3K, which binds to phosphotyrosines on EpoR. Ubiquitination allows p85 to interact with the endocytic protein epsin-1, thereby driving EpoR endocytosis. Knockdown of Cbl, expression of its dominant negative forms, or expression of an epsin-1 mutant devoid of ubiquitin-interacting motifs all compromise Epo-induced EpoR internalization. Mutated EpoRs mimicking those from PFCP patients cannot bind p85, co-localize with epsin-1, or internalize on Epo stimulation and exhibit Epo hypersensitivity. Similarly, knockdown of Cbl also causes Epo hypersensitivity in primary erythroid progenitors. Restoring p85 binding to PFCP receptors rescues Epo-induced epsin-1 co-localization and EpoR internalization and normalizes Epo hypersensitivity. Our results uncover a novel Cbl/p85/epsin-1 pathway in EpoR endocytosis and show that defects in this pathway contribute to excessive Epo signaling and erythroid hyperproliferation in PFCP.

  7. CFTR and defective endocytosis: new insights in the renal phenotype of cystic fibrosis.

    PubMed

    Jouret, François; Devuyst, Olivier

    2009-04-01

    Inactivation of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) causes cystic fibrosis (CF). Although CFTR is expressed in the kidney, no overwhelming renal phenotype is associated with CF. Recent studies have shown that the level of CFTR mRNA in mouse kidney approaches that found in lung. CFTR is particularly abundant in the apical area of proximal tubule cells, where it co-distributes with the Cl(-)/H(+) exchanger ClC-5 and Rab5a in endosomes. The biological relevance of CFTR in proximal tubule endocytosis has been tested in CF mouse models and CF patients. Mice lacking CFTR show a defective receptor-mediated endocytosis, as evidenced by impaired uptake of (125)I-beta(2)-microglobulin, a decreased expression of the cubilin receptor in the kidney, and a significant excretion of cubilin and its low-molecular-weight ligands into the urine. Low-molecular-weight proteinuria (and particularly transferrinuria) is similarly detected in CF patients in comparison with normal controls or patients with chronic lung inflammation. These studies suggest that the functional loss of CFTR impairs the handling of low-molecular-weight proteins by the kidney, supporting a role of CFTR in receptor-mediated endocytosis in proximal tubule cells. The selective proteinuria should be integrated in the pathophysiology of multi-systemic complications increasingly observed in CF patients.

  8. Secretion and Endocytosis in Pollen Tubes: Models of Tip Growth in the Spot Light.

    PubMed

    Grebnev, Gleb; Ntefidou, Maria; Kost, Benedikt

    2017-01-01

    Pollen tube tip growth is a widely used model ideally suited to study cellular processes underlying polarized cell expansion. Local secretion supplying material for plasma membrane (PM) and cell wall extension is essential for this process. Cell wall biogenesis requires fusion of secretory vesicles with the PM at an about 10× higher rate than PM extension. Excess material is therefore incorporated into the PM, which needs to be reinternalized through endocytosis. The classical model of tip growth proposes that exocytosis occurs at the apex and that newly incorporated PM material is transported to adjacent lateral regions, where excess material is endocytically recycled. This model was recently challenged based on studies indicating that lateral exocytosis may be balanced by apical endocytosis. This review provides an overview of published data pertaining to exocytosis, endocytosis and vesicular trafficking in pollen tubes. Its key aim is to present classical and alternative models of tip growth in the light of available experimental data. By necessity, the review focusses on pollen tubes of angiosperm models (Nicotiana tabacum, Arabidopsis, Lilium longiflorum), which have been studied far more extensively and grow much faster than structurally strikingly different gymnosperm pollen tubes. Only major transport pathways are considered, which substantially contribute to the mass-flow of membrane material at the pollen tube tip. Growth oscillation, which may be displayed in particular by fast-growing pollen tubes, are not discussed as their influence on the spatial organization of apical membrane traffic is not understood.

  9. AP-2-complex-mediated endocytosis of Drosophila Crumbs regulates polarity by antagonizing Stardust.

    PubMed

    Lin, Ya-Huei; Currinn, Heather; Pocha, Shirin Meher; Rothnie, Alice; Wassmer, Thomas; Knust, Elisabeth

    2015-12-15

    Maintenance of epithelial polarity depends on the correct localization and levels of polarity determinants. The evolutionarily conserved transmembrane protein Crumbs is crucial for the size and identity of the apical membrane, yet little is known about the molecular mechanisms controlling the amount of Crumbs at the surface. Here, we show that Crumbs levels on the apical membrane depend on a well-balanced state of endocytosis and stabilization. The adaptor protein 2 (AP-2) complex binds to a motif in the cytoplasmic tail of Crumbs that overlaps with the binding site of Stardust, a protein known to stabilize Crumbs on the surface. Preventing endocytosis by mutation of AP-2 causes expansion of the Crumbs-positive plasma membrane domain and polarity defects, which can be partially rescued by removing one copy of crumbs. Strikingly, knocking down both AP-2 and Stardust leads to the retention of Crumbs on the membrane. This study provides evidence for a molecular mechanism, based on stabilization and endocytosis, to adjust surface levels of Crumbs, which are essential for maintaining epithelial polarity.

  10. Iminochromene inhibitors of dynamins I and II GTPase activity and endocytosis.

    PubMed

    Hill, Timothy A; Mariana, Anna; Gordon, Christopher P; Odell, Luke R; Robertson, Mark J; McGeachie, Andrew B; Chau, Ngoc; Daniel, James A; Gorgani, Nick N; Robinson, Phillip J; McCluskey, Adam

    2010-05-27

    Herein we report the synthesis of discrete iminochromene ("iminodyn") libraries (14-38) as potential inhibitors of dynamin GTPase. Thirteen iminodyns were active (IC(50) values of 260 nM to 100 microM), with N,N-(ethane-1,2-diyl)bis(7,8-dihydroxy-2-iminochromene-3-carboxamide) (17), N,N-(ethane-1,2-diyl)bis(7,8-dihydroxy-2-iminochromene-3-carboxamide) (22), and N,N-(ethane-1,2-diyl)bis(7,8-dihydroxy-2-iminochromene-3-carboxamide) (23) (IC(50) values of 330 +/- 70, 450 +/- 50, and 260 +/- 80 nM, respectively) being the most potent. Five of the most potent iminodyns all inhibited dynamins I and II approximately equally. Iminodyn-22 displayed uncompetitive inhibition with respect to GTP. Selected iminodyns were evaluated for their ability to block receptor mediated endocytosis (RME, mediated by dynamin II) and synaptic vesicle endocytosis (SVE, mediated by dynamin I), with 17 showing no activity while 22 returned RME and SVE IC(50) values of 10.7 +/- 4.5 and 99.5 +/- 1.7 microM, respectively. The iminodyns reported herein represent a new chemical class of the first nanomolar potent dynamin inhibitors that are also effective endocytosis inhibitors.

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

    PubMed Central

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

    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 catalysis itself. Following an approach based on permease structural modeling, mutagenesis, and kinetic parameter analysis, we obtained evidence that substrate-induced endocytosis requires transition of the permease to a conformational state preceding substrate release into the cell. Furthermore, this transient conformation must be stable enough, and thus sufficiently populated, for the permease to undergo efficient downregulation. Additional observations, including the constitutive downregulation of two active Gap1 mutants altered in cytosolic regions, support the model that the substrate-induced conformational transition inducing endocytosis involves remodeling of cytosolic regions of the permeases, thereby promoting their recognition by arrestin-like adaptors of the Rsp5 ubiquitin ligase. Similar mechanisms might control many other plasma membrane transporters according to the external concentrations of their substrates. PMID:25266656

  12. Nedd4-1 binds and ubiquitylates activated FGFR1 to control its endocytosis and function.

    PubMed

    Persaud, Avinash; Alberts, Philipp; Hayes, Madeline; Guettler, Sebastian; Clarke, Ian; Sicheri, Frank; Dirks, Peter; Ciruna, Brian; Rotin, Daniela

    2011-07-15

    Fibroblast growth factor receptor 1 (FGFR1) has critical roles in cellular proliferation and differentiation during animal development and adult homeostasis. Here, we show that human Nedd4 (Nedd4-1), an E3 ubiquitin ligase comprised of a C2 domain, 4 WW domains, and a Hect domain, regulates endocytosis and signalling of FGFR1. Nedd4-1 binds directly to and ubiquitylates activated FGFR1, by interacting primarily via its WW3 domain with a novel non-canonical sequence (non-PY motif) on FGFR1. Deletion of this recognition motif (FGFR1-Δ6) abolishes Nedd4-1 binding and receptor ubiquitylation, and impairs endocytosis of activated receptor, as also observed upon Nedd4-1 knockdown. Accordingly, FGFR1-Δ6, or Nedd4-1 knockdown, exhibits sustained FGF-dependent receptor Tyr phosphorylation and downstream signalling (activation of FRS2α, Akt, Erk1/2, and PLCγ). Expression of FGFR1-Δ6 in human embryonic neural stem cells strongly promotes FGF2-dependent neuronal differentiation. Furthermore, expression of this FGFR1-Δ6 mutant in zebrafish embryos disrupts anterior neuronal patterning (head development), consistent with excessive FGFR1 signalling. These results identify Nedd4-1 as a key regulator of FGFR1 endocytosis and signalling during neuronal differentiation and embryonic development.

  13. Nedd4-1 binds and ubiquitylates activated FGFR1 to control its endocytosis and function

    PubMed Central

    Persaud, Avinash; Alberts, Philipp; Hayes, Madeline; Guettler, Sebastian; Clarke, Ian; Sicheri, Frank; Dirks, Peter; Ciruna, Brian; Rotin, Daniela

    2011-01-01

    Fibroblast growth factor receptor 1 (FGFR1) has critical roles in cellular proliferation and differentiation during animal development and adult homeostasis. Here, we show that human Nedd4 (Nedd4-1), an E3 ubiquitin ligase comprised of a C2 domain, 4 WW domains, and a Hect domain, regulates endocytosis and signalling of FGFR1. Nedd4-1 binds directly to and ubiquitylates activated FGFR1, by interacting primarily via its WW3 domain with a novel non-canonical sequence (non-PY motif) on FGFR1. Deletion of this recognition motif (FGFR1-Δ6) abolishes Nedd4-1 binding and receptor ubiquitylation, and impairs endocytosis of activated receptor, as also observed upon Nedd4-1 knockdown. Accordingly, FGFR1-Δ6, or Nedd4-1 knockdown, exhibits sustained FGF-dependent receptor Tyr phosphorylation and downstream signalling (activation of FRS2α, Akt, Erk1/2, and PLCγ). Expression of FGFR1-Δ6 in human embryonic neural stem cells strongly promotes FGF2-dependent neuronal differentiation. Furthermore, expression of this FGFR1-Δ6 mutant in zebrafish embryos disrupts anterior neuronal patterning (head development), consistent with excessive FGFR1 signalling. These results identify Nedd4-1 as a key regulator of FGFR1 endocytosis and signalling during neuronal differentiation and embryonic development. PMID:21765395

  14. L-type calcium channels in exocytosis and endocytosis of chromaffin cells.

    PubMed

    Nanclares, Carmen; Baraibar, Andrés M; Gandía, Luis

    2017-09-02

    The coexistence of different subtypes of voltage-dependent calcium channels (VDCC) within the same chromaffin cell (CC) and the marked interspecies variability in the proportion of VDCC subtypes that are present in the plasmalemma of the CCs raises the question on their roles in controlling different physiological functions. Particularly relevant seems to be the role of VDCCs in the regulation of the exocytotic neurotransmitter release process, and its tightly coupled membrane retrieval (endocytosis) process since both are Ca(2+)-dependent processes. This review is focused on the role of Ca(2+) influx through L-type VDCC in the regulation of these two processes. It is currently accepted that the different VDCC subtypes (i.e., T, L, N, P/Q, R) contribute to exocytosis proportionally to their density of expression and gating properties. However, the pattern of stimulation defines a preferential role of the different subtypes of VDCC on exocytosis and endocytosis. Thus, L-type channels seem to control catecholamine release induced by prolonged stimuli while fast exocytosis in response to short square depolarizing pulses or action potentials is mediated by Ca(2+) entering CCs through P/Q channels. The pattern of stimulation also influences the endocytotic process, and thus, electrophysiological data suggest the sustained Ca(2+) entry through slow-inactivating L-type channels could be responsible for the activation of fast endocytosis.

  15. The giardial ENTH protein participates in lysosomal protein trafficking and endocytosis.

    PubMed

    Feliziani, Constanza; Zamponi, Nahuel; Gottig, Natalia; Rópolo, Andrea S; Lanfredi-Rangel, Adriana; Touz, Maria C

    2015-03-01

    In the protozoa parasite Giardia lamblia, endocytosis and lysosomal protein trafficking are vital parasite-specific processes that involve the action of the adaptor complexes AP-1 and AP-2 and clathrin. In this work, we have identified a single gene in Giardia encoding a protein containing an ENTH domain that defines monomeric adaptor proteins of the epsin family. This domain is present in the epsin or epsin-related (epsinR) adaptor proteins, which are implicated in endocytosis and Golgi-to-endosome protein trafficking, respectively, in other eukaryotic cells. We found that GlENTHp (for G. lamblia ENTH protein) localized in the cytosol, strongly interacted with PI3,4,5P3, was associated with the alpha subunit of AP-2, clathrin and ubiquitin and was involved in receptor-mediated endocytosis. It also bonded PI4P, the gamma subunit of AP-1 and was implicated in ER-to-PV trafficking. Alteration of the GlENTHp function severely affected trophozoite growth showing an unusual accumulation of dense material in the lysosome-like peripheral vacuoles (PVs), indicating that GlENTHp might be implicated in the maintenance of PV homeostasis. In this study, we showed evidence suggesting that GlENTHp might function as a monomeric adaptor protein supporting the findings of other group indicating that GlENTHp might be placed at the beginning of the ENTH family.

  16. ArfB links protein lipidation and endocytosis to polarized growth of Aspergillus nidulans

    PubMed Central

    Lee, Soo Chan

    2008-01-01

    Aspergillus nidulans undergoes polarized hyphal growth during the majority of its life cycle. Regulatory mechanisms for hyphal polarity have been intensively investigated in a variety of filamentous fungi. Two important cellular processes, which have received recent attention, include protein myristoylation and endocytosis. It is clear that protein myristoylation is essential for polarity establishment because germinating A. nidulans conidia lost polarity in the presence of cerulenin, a lipid metabolism inhibitor and in an N-myristoyl transferase mutant background. Only 41 predicted proteins encoded by A. nidulans posses an N-myristoylation motif, one of which is ADP ribosylation factor B (ArfB). Disruption of ArfB leads to failure of polarity establishment and maintenance during early morphogenesis and in a delay in endocytosis. Therefore, ArfB connects N-myristoylation and endocytosis to polarized growth. Exocytotic vesicle trafficking through the Spitzenkörper may also require Arf proteins in their role in vesicle formation. Taken together, ArfB is one of the important key components for the fungal hyphal growth. PMID:19704790

  17. Clostridium difficile Toxin A Undergoes Clathrin-Independent, PACSIN2-Dependent Endocytosis

    PubMed Central

    Kenworthy, Anne K.

    2016-01-01

    Clostridium difficile infection affects a significant number of hospitalized patients in the United States. Two homologous exotoxins, TcdA and TcdB, are the major virulence factors in C. difficile pathogenesis. The toxins are glucosyltransferases that inactivate Rho family-GTPases to disrupt host cellular function and cause fluid secretion, inflammation, and cell death. Toxicity depends on receptor binding and subsequent endocytosis. TcdB has been shown to enter cells by clathrin-dependent endocytosis, but the mechanism of TcdA uptake is still unclear. Here, we utilize a combination of RNAi-based knockdown, pharmacological inhibition, and cell imaging approaches to investigate the endocytic mechanism(s) that contribute to TcdA uptake and subsequent cytopathic and cytotoxic effects. We show that TcdA uptake and cellular intoxication is dynamin-dependent but does not involve clathrin- or caveolae-mediated endocytosis. Confocal microscopy using fluorescently labeled TcdA shows significant colocalization of the toxin with PACSIN2-positive structures in cells during entry. Disruption of PACSIN2 function by RNAi-based knockdown approaches inhibits TcdA uptake and toxin-induced downstream effects in cells indicating that TcdA entry is PACSIN2-dependent. We conclude that TcdA and TcdB utilize distinct endocytic mechanisms to intoxicate host cells. PMID:27942025

  18. Clathrin light chains' role in selective endocytosis influences antibody isotype switching.

    PubMed

    Wu, Shuang; Majeed, Sophia R; Evans, Timothy M; Camus, Marine D; Wong, Nicole M L; Schollmeier, Yvette; Park, Minjong; Muppidi, Jagan R; Reboldi, Andrea; Parham, Peter; Cyster, Jason G; Brodsky, Frances M

    2016-08-30

    Clathrin, a cytosolic protein composed of heavy and light chain subunits, assembles into a vesicle coat, controlling receptor-mediated endocytosis. To establish clathrin light chain (CLC) function in vivo, we engineered mice lacking CLCa, the major CLC isoform in B lymphocytes, generating animals with CLC-deficient B cells. In CLCa-null mice, the germinal centers have fewer B cells, and they are enriched for IgA-producing cells. This enhanced switch to IgA production in the absence of CLCa was attributable to increased transforming growth factor β receptor 2 (TGFβR2) signaling resulting from defective endocytosis. Internalization of C-X-C chemokine receptor 4 (CXCR4), but not CXCR5, was affected in CLCa-null B cells, and CLC depletion from cell lines affected endocytosis of the δ-opioid receptor, but not the β2-adrenergic receptor, defining a role for CLCs in the uptake of a subset of signaling receptors. This instance of clathrin subunit deletion in vertebrates demonstrates that CLCs contribute to clathrin's role in vivo by influencing cargo selectivity, a function previously assigned exclusively to adaptor molecules.

  19. Internalisation of the bleomycin molecules responsible for bleomycin toxicity: a receptor-mediated endocytosis mechanism.

    PubMed

    Pron, G; Mahrour, N; Orlowski, S; Tounekti, O; Poddevin, B; Belehradek, J; Mir, L M

    1999-01-01

    Bleomycin (BLM) does not diffuse through the plasma membrane but nevertheless displays cytotoxic activity due to DNA break generation. The aim of the study was to describe the mechanism of BLM internalisation. We previously provided evidence for the existence of BLM-binding sites at the surface of DC-3F Chinese hamster fibroblasts, as well as of their involvement in BLM cytotoxicity on DC-3F cells and related BLM-resistant sublines. Here we report that A253 human cells and their BLM-resistant subline C-10E also possessed a membrane protein of ca. 250 kDa specifically binding BLM. Part of this C-10E cell resistance could be explained by a decrease in the number of BLM-binding sites exposed at the cell surface with respect to A253 cells. The comparison between A253 and DC-3F cells exposing a similar number of BLM-binding sites revealed that the faster the fluid phase endocytosis, the greater the cell sensitivity to BLM. Moreover, the experimental modification of endocytotic vesicle size showed that BLM cytotoxicity was directly correlated with the flux of plasma membrane area engulfed during endocytosis rather than with the fluid phase volume incorporated. Thus, BLM would be internalised by a receptor-mediated endocytosis mechanism which would first require BLM binding to its membrane receptor and then the transfer of the complex into intracellular endocytotic vesicles, followed by BLM entry into the cytosol, probably from a nonacidic compartment.

  20. Endocytosis and intracellular processing of BODIPY-sphingomyelin by murine CATH.a neurons☆

    PubMed Central

    Nusshold, Christoph; Uellen, Andreas; Bernhart, Eva; Hammer, Astrid; Damm, Sabine; Wintersperger, Andrea; Reicher, Helga; Hermetter, Albin; Malle, Ernst; Sattler, Wolfgang

    2013-01-01

    Neuronal sphingolipids (SL) play important roles during axonal extension, neurotrophic receptor signaling and neurotransmitter release. Many of these signaling pathways depend on the presence of specialized membrane microdomains termed lipid rafts. Sphingomyelin (SM), one of the main raft constituents, can be formed de novo or supplied from exogenous sources. The present study aimed to characterize fluorescently-labeled SL turnover in a murine neuronal cell line (CATH.a). Our results demonstrate that at 4 °C exogenously added BODIPY-SM accumulates exclusively at the plasma membrane. Treatment of cells with bacterial sphingomyelinase (SMase) and back-exchange experiments revealed that 55–67% of BODIPY-SM resides in the outer leaflet of the plasma membrane. Endocytosis of BODIPY-SM occurs via caveolae with part of internalized BODIPY-fluorescence ending up in the Golgi and the ER. Following endocytosis BODIPY-SM undergoes hydrolysis, a reaction substantially faster than BODIPY-SM synthesis from BODIPY-ceramide. RNAi demonstrated that both, acid (a)SMase and neutral (n)SMases contribute to BODIPY-SM hydrolysis. Finally, high-density lipoprotein (HDL)-associated BODIPY-SM was efficiently taken up by CATH.a cells. Our findings indicate that endocytosis of exogenous SM occurs almost exclusively via caveolin-dependent pathways, that both, a- and nSMases equally contribute to neuronal SM turnover and that HDL-like particles might represent physiological SM carriers/donors in the brain. PMID:23973266

  1. EPEC effector EspF promotes Crumbs3 endocytosis and disrupts epithelial cell polarity.

    PubMed

    Tapia, Rocio; Kralicek, Sarah E; Hecht, Gail A

    2017-06-15

    Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system to inject effector proteins into host intestinal epithelial cells causing diarrhoea. EPEC infection redistributes basolateral proteins β1-integrin and Na(+) /K(+) ATPase to the apical membrane of host cells. The Crumbs (Crb) polarity complex (Crb3/Pals1/Patj) is essential for epithelial cell polarisation and tight junction (TJ) assembly. Here, we demonstrate that EPEC displaces Crb3 and Pals1 from the apical membrane to the cytoplasm of cultured intestinal epithelial cells and colonocytes of infected mice. In vitro studies show that EspF, but not Map, alters Crb3, whereas both effectors modulate Pals1. EspF perturbs polarity formation in cyst morphogenesis assays and induces endocytosis and apical redistribution of Na(+) /K(+) ATPase. EspF binds to sorting nexin 9 (SNX9) causing membrane remodelling in host cells. Infection with ΔespF/pespFD3, a mutant strain that ablates EspF binding to SNX9, or inhibition of dynamin, attenuates Crb3 endocytosis caused by EPEC. In addition, infection with ΔespF/pespFD3 has no impact on Na(+) /K(+) ATPase endocytosis. These data support the hypothesis that EPEC perturbs apical-basal polarity in an EspF-dependent manner, which would contribute to EPEC-associated diarrhoea by disruption of TJ and altering the crucial positioning of membrane transporters involved in the absorption of ions and solutes. © 2017 John Wiley & Sons Ltd.

  2. Biocompatibility, uptake and endocytosis pathways of polystyrene nanoparticles in primary human renal epithelial cells.

    PubMed

    Monti, Daria Maria; Guarnieri, Daniela; Napolitano, Giuliana; Piccoli, Renata; Netti, Paolo; Fusco, Sabato; Arciello, Angela

    2015-01-10

    Recent years have witnessed an unprecedented growth in the number of applications—such as drug delivery, nutraceuticals and production of improved biocompatible materials—in the areas of nanoscience and nanotechnology. Engineered nanoparticles (NPs) are an important tool for the development of quite a few of these applications. Despite intense research activity, mechanisms regulating the uptake of NPs into cells are not completely defined, being the phenomenon dramatically influenced by physico-chemical properties of NPs and cell-specific differences. Since the cellular uptake of NPs is a prerequisite for their use in nanomedicine, the definition of their internalization pathway is crucial. For this reason, we used 44 nm polystyrene NPs as a model to analyze the uptake and endocytosis pathways in primary human renal cortical epithelial (HRCE) cells, which play a key role in the clearance of drugs. NPs were found not to affect the viability and cell cycle progression of HRCE cells. Distinct internalization pathways were analyzed by the use of drugs known to inhibit specific endocytosis routes. Analyses, performed by confocal microscopy in combination with quantitative spectrofluorimetric assays, indicated that NPs enter HRCE cells through multiple mechanisms, either energy-dependent (endocytosis) or energy-independent.

  3. Quantification of nanoparticle endocytosis based on double fluorescent pH-sensitive nanoparticles.

    PubMed

    Kurtz-Chalot, Andréa; Klein, Jean-Philippe; Pourchez, Jérémie; Boudard, Delphine; Bin, Valérie; Sabido, Odile; Marmuse, Laurence; Cottier, Michèle; Forest, Valérie

    2015-04-01

    Amorphous silica is a particularly interesting material because of its inertness and chemical stability. Silica nanoparticles have been recently developed for biomedical purposes but their innocuousness must be carefully investigated before clinical use. The relationship between nanoparticles physicochemical features, their uptake by cells and their biological activity represents a crucial issue, especially for the development of nanomedicine. This work aimed at adapting a method for the quantification of nanoparticle endocytosis based on pH-sensitive and double fluorescent particles. For that purpose, silica nanoparticles containing two fluorophores: FITC and pHrodo(TM) were developed, their respective fluorescence emission depends on the external pH. Indeed, FITC emits a green fluorescence at physiological pH and pHrodo(TM) emits a red fluorescence which intensity increased with acidification. Therefore, nanoparticles remained outside the cells could be clearly distinguished from nanoparticles uptaken by cells as these latter could be spotted inside cellular acidic compartments (such as phagolysosomes, micropinosomes…). Using this model, the endocytosis of 60 nm nanoparticles incubated with the RAW 264.7 macrophages was quantified using time-lapse microscopy and compared to that of 130 nm submicronic particles. The amount of internalized particles was also evaluated by fluorimetry. The biological impact of the particles was also investigated in terms of cytotoxicity, pro-inflammatory response and oxidative stress. Results clearly demonstrated that nanoparticles were more uptaken and more reactive than submicronic particles. Moreover, we validated a method of endocytosis quantification.

  4. Decoding ubiquitin sorting signals for clathrin-dependent endocytosis by CLASPs.

    PubMed

    Traub, Linton M; Lukacs, Gergely L

    2007-02-15

    Cargo selectivity is a hallmark of clathrin-mediated endocytosis. A wide range of structurally unrelated internalization signals specify the preferential clustering of transmembrane cargo into clathrin coats forming on the plasma membrane. Intriguingly, the classical endocytic adaptor AP-2 appears to recognize only a subset of these endocytic sorting signals. New data now reveal the molecular basis for recognition of other internalization signals, including post-translationally appended ubiquitin, by clathrin-coat-associated sorting proteins (CLASPs). Curiously, structurally related ubiquitin-recognition modules are shared by select CLASPs and the 26S proteasome, and recent work indicates that both display similar requirements for ubiquitin binding. During endocytosis, these modules engage oligoubiquitylated cargo in the form of polyubiquitin chains and/or multiple single ubiquitin molecules appended to different acceptor lysines. Functional separation between clathrin-mediated endocytosis and proteasome-dependent proteolysis is probably ensured by temporally regulated, local assembly of ubiquitin-tagged membrane cargo at sorting stations on the cell surface, shielding ubiquitin sorting signals from the proteasome. Thus, an expanded repertoire of CLASPs couples the process of clathrin-coat assembly with high-fidelity incorporation of assorted, cargo-specific sorting signals.

  5. The redox sensor TXNL1 plays a regulatory role in fluid phase endocytosis.

    PubMed

    Felberbaum-Corti, Michela; Morel, Etienne; Cavalli, Valeria; Vilbois, Francis; Gruenberg, Jean

    2007-11-07

    Small GTPases of the Rab family can cycle between a GTP- and a GDP-bound state and also between membrane and cytosol. The latter cycle is mediated by the Guanine Nucleotide Dissociation Inhibitor GDI, which can selectively extract GDP-bound Rab proteins from donor membranes, and then reload them on target membranes. In previous studies, we found that capture of the small GTPase Rab5, a key regulator of endocytic membrane traffic, by GDI is stimulated by oxidative stress via p38MAPK, resulting in increased fluid phase endocytosis. When purifying the GDI stimulating activity we found that that it copurified with a high MW protein complex, which included p38MAPK. Here we report the identification and characterization of another component of this complex as the thioredoxin-like protein TXNL1. Our observations indicate that TXNL1 play a selective role in the regulation of fluid phase endocytosis, by controlling GDI capacity to capture Rab5. Oxidants, which are known to cause cellular damage, can also trigger signaling pathways, in particular via members of the thioredoxin family. We propose that TXNL1 acts as an effector of oxidants or a redox sensor by converting redox changes into changes of GDI capacity to capture Rab5, which in turn modulates fluid phase endocytosis.

  6. The Redox Sensor TXNL1 Plays a Regulatory Role in Fluid Phase Endocytosis

    PubMed Central

    Felberbaum-Corti, Michela; Morel, Etienne; Cavalli, Valeria; Vilbois, Francis; Gruenberg, Jean

    2007-01-01

    Background Small GTPases of the Rab family can cycle between a GTP- and a GDP-bound state and also between membrane and cytosol. The latter cycle is mediated by the Guanine Nucleotide Dissociation Inhibitor GDI, which can selectively extract GDP-bound Rab proteins from donor membranes, and then reload them on target membranes. In previous studies, we found that capture of the small GTPase Rab5, a key regulator of endocytic membrane traffic, by GDI is stimulated by oxidative stress via p38MAPK, resulting in increased fluid phase endocytosis. Methodology/Principal Findings When purifying the GDI stimulating activity we found that that it copurified with a high MW protein complex, which included p38MAPK. Here we report the identification and characterization of another component of this complex as the thioredoxin-like protein TXNL1. Our observations indicate that TXNL1 play a selective role in the regulation of fluid phase endocytosis, by controlling GDI capacity to capture Rab5. Conclusions/Significance Oxidants, which are known to cause cellular damage, can also trigger signaling pathways, in particular via members of the thioredoxin family. We propose that TXNL1 acts as an effector of oxidants or a redox sensor by converting redox changes into changes of GDI capacity to capture Rab5, which in turn modulates fluid phase endocytosis. PMID:17987124

  7. Study of serum interaction with a cationic nanoparticle: Implications for in vitro endocytosis, cytotoxicity and genotoxicity.

    PubMed

    Merhi, Maysaloun; Dombu, Christophe Youta; Brient, Alizée; Chang, Jiang; Platel, Anne; Le Curieux, Frank; Marzin, Daniel; Nesslany, Fabrice; Betbeder, Didier

    2012-02-14

    We used well-characterized and positively charged nanoparticles (NP(+)) to investigate the importance of cell culture conditions, specifically the presence of serum and proteins, on NP(+) physicochemical characteristics, and the consequences for their endocytosis and genotoxicity in bronchial epithelial cells (16HBE14o-). NP(+) surface charge was significantly reduced, proportionally to NP(+)/serum and NP(+)/BSA ratios, while NP(+) size was not modified. Microscopy studies showed high endocytosis of NP(+) in 16HBE14o-, and serum/proteins impaired this internalization in a dose-dependent manner. Toxicity studies showed no cytotoxicity, even for very high doses of NP(+). No genotoxicity was observed with classic comet assay while primary oxidative DNA damage was observed when using the lesion-specific repair enzyme, formamidopyrimidine DNA-glycosylase (FPG). The micronucleus test showed NP(+) genotoxicity only for very high doses that cannot be attained in vivo. The low toxicity of these NP(+) might be explained by their high exocytosis from 16HBE14o- cells. Our results confirm the importance of serum and proteins on nanoparticles endocytosis and genotoxicity.

  8. DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur.

    PubMed

    Nichols, James T; Miyamoto, Alison; Olsen, Samantha L; D'Souza, Brendan; Yao, Christine; Weinmaster, Gerry

    2007-02-12

    Cleavage of Notch by furin is required to generate a mature, cell surface heterodimeric receptor that can be proteolytically activated to release its intracellular domain, which functions in signal transduction. Current models propose that ligand binding to heterodimeric Notch (hNotch) induces a disintegrin and metalloprotease (ADAM) proteolytic release of the Notch extracellular domain (NECD), which is subsequently shed and/or endocytosed by DSL ligand cells. We provide evidence for NECD release and internalization by DSL ligand cells, which, surprisingly, did not require ADAM activity. However, losses in either hNotch formation or ligand endocytosis significantly decreased NECD transfer to DSL ligand cells, as well as signaling in Notch cells. Because endocytosis-defective ligands bind hNotch, but do not dissociate it, additional forces beyond those produced through ligand binding must function to disrupt the intramolecular interactions that keep hNotch intact and inactive. Based on our findings, we propose that mechanical forces generated during DSL ligand endocytosis function to physically dissociate hNotch, and that dissociation is a necessary step in Notch activation.

  9. DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur

    PubMed Central

    Nichols, James T.; Miyamoto, Alison; Olsen, Samantha L.; D'Souza, Brendan; Yao, Christine; Weinmaster, Gerry

    2007-01-01

    Cleavage of Notch by furin is required to generate a mature, cell surface heterodimeric receptor that can be proteolytically activated to release its intracellular domain, which functions in signal transduction. Current models propose that ligand binding to heterodimeric Notch (hNotch) induces a disintegrin and metalloprotease (ADAM) proteolytic release of the Notch extracellular domain (NECD), which is subsequently shed and/or endocytosed by DSL ligand cells. We provide evidence for NECD release and internalization by DSL ligand cells, which, surprisingly, did not require ADAM activity. However, losses in either hNotch formation or ligand endocytosis significantly decreased NECD transfer to DSL ligand cells, as well as signaling in Notch cells. Because endocytosis-defective ligands bind hNotch, but do not dissociate it, additional forces beyond those produced through ligand binding must function to disrupt the intramolecular interactions that keep hNotch intact and inactive. Based on our findings, we propose that mechanical forces generated during DSL ligand endocytosis function to physically dissociate hNotch, and that dissociation is a necessary step in Notch activation. PMID:17296795

  10. Protein kinase C induces endocytosis of the sodium taurocholate cotransporting polypeptide.

    PubMed

    Stross, Claudia; Helmer, Angelika; Weissenberger, Katrin; Görg, Boris; Keitel, Verena; Häussinger, Dieter; Kubitz, Ralf

    2010-08-01

    Bile salts influence signaling and metabolic pathways. In hepatocytes, the sodium taurocholate cotransporting polypeptide (Ntcp) is a major determinant of intracellular bile salt levels. Short-term downregulation of Ntcp is not well characterized to date. FLAG and enhanced green fluorescent protein (EGFP) tags were cloned to the extra- and intracellular termini of Ntcp. Endocytosis of Ntcp in transfected HepG2 cells was visualized by fluorescence of EGFP, and membrane surface expression of Ntcp was quantified by flow cytometry with fluorochrome-labeled FLAG antibodies. Activation of protein kinase C (PKC) by phorbolester or thymeleatoxin an activator of Ca(2+)-dependent conventional PKCs (cPKCs), induced endocytosis of Ntcp, whereas the Na(+)-K(+)-ATPase remained in the plasma membrane. The PKC inhibitor BIM I and the cPKC-selective inhibitor Gö6976 abolished PMA-induced endocytosis. Because of this internalization, cell surface expression of Ntcp was reduced by 36 +/- 7%, bile salt uptake was decreased by 25%, and taurolithocholate sulfate-induced cell toxicity was prevented. In conclusion, Ca(2+)-dependent PKCs induce vesicular retrieval of Ntcp, thereby reducing bile salt uptake. This mechanism may protect hepatocytes from toxic intracellular bile salt concentrations.

  11. Secretion and Endocytosis in Pollen Tubes: Models of Tip Growth in the Spot Light

    PubMed Central

    Grebnev, Gleb; Ntefidou, Maria; Kost, Benedikt

    2017-01-01

    Pollen tube tip growth is a widely used model ideally suited to study cellular processes underlying polarized cell expansion. Local secretion supplying material for plasma membrane (PM) and cell wall extension is essential for this process. Cell wall biogenesis requires fusion of secretory vesicles with the PM at an about 10× higher rate than PM extension. Excess material is therefore incorporated into the PM, which needs to be reinternalized through endocytosis. The classical model of tip growth proposes that exocytosis occurs at the apex and that newly incorporated PM material is transported to adjacent lateral regions, where excess material is endocytically recycled. This model was recently challenged based on studies indicating that lateral exocytosis may be balanced by apical endocytosis. This review provides an overview of published data pertaining to exocytosis, endocytosis and vesicular trafficking in pollen tubes. Its key aim is to present classical and alternative models of tip growth in the light of available experimental data. By necessity, the review focusses on pollen tubes of angiosperm models (Nicotiana tabacum, Arabidopsis, Lilium longiflorum), which have been studied far more extensively and grow much faster than structurally strikingly different gymnosperm pollen tubes. Only major transport pathways are considered, which substantially contribute to the mass-flow of membrane material at the pollen tube tip. Growth oscillation, which may be displayed in particular by fast-growing pollen tubes, are not discussed as their influence on the spatial organization of apical membrane traffic is not understood. PMID:28224002

  12. ENTH and ANTH domain proteins participate in AP2-independent clathrin-mediated endocytosis

    PubMed Central

    Manna, Paul T.; Gadelha, Catarina; Puttick, Amy E.; Field, Mark C.

    2015-01-01

    ABSTRACT Clathrin-mediated endocytosis (CME) is a major route of entry into eukaryotic cells. A core of evolutionarily ancient genes encodes many components of this system but much of our mechanistic understanding of CME is derived from a phylogenetically narrow sampling of a few model organisms. In the parasite Trypanosoma brucei, which is distantly related to the better characterised animals and fungi, exceptionally fast endocytic turnover aids its evasion of the host immune system. Although clathrin is absolutely essential for this process, the adaptor protein complex 2 (AP2) has been secondarily lost, suggesting mechanistic divergence. Here, we characterise two phosphoinositide-binding monomeric clathrin adaptors, T. brucei (Tb)EpsinR and TbCALM, which in trypanosomes are represented by single genes, unlike the expansions present in animals and fungi. Depletion of these gene products reveals essential, but partially redundant, activities in CME. Ultrastructural analysis of TbCALM and TbEpsinR double-knockdown cells demonstrated severe defects to clathrin-coated pit formation and morphology associated with a dramatic inhibition of endocytosis. Depletion of TbCALM alone, however, produced a distinct lysosomal segregation phenotype, indicating an additional non-redundant role for this protein. Therefore, TbEpsinR and TbCALM represent ancient phosphoinositide-binding proteins with distinct and vital roles in AP2-independent endocytosis. PMID:25908855

  13. A dynamin-actin interaction is required for vesicle scission during endocytosis in yeast.

    PubMed

    Palmer, Sarah E; Smaczynska-de Rooij, Iwona I; Marklew, Christopher J; Allwood, Ellen G; Mishra, Ritu; Johnson, Simeon; Goldberg, Martin W; Ayscough, Kathryn R

    2015-03-30

    Actin is critical for endocytosis in yeast cells, and also in mammalian cells under tension. However, questions remain as to how force generated through actin polymerization is transmitted to the plasma membrane to drive invagination and scission. Here, we reveal that the yeast dynamin Vps1 binds and bundles filamentous actin. Mutational analysis of Vps1 in a helix of the stalk domain identifies a mutant RR457-458EE that binds actin more weakly. In vivo analysis of Vps1 function demonstrates that the mutation disrupts endocytosis but not other functions of Vps1 such as vacuolar trafficking or peroxisome fission. The mutant Vps1 is stably expressed in cells and co-localizes with the endocytic reporters Abp1 and the amphiphysin Rvs167. Detailed analysis of individual endocytic patch behavior indicates that the mutation causes aberrant movements in later stages of endocytosis, consistent with a scission defect. Ultrastructural analysis of yeast cells using electron microscopy reveals a significant increase in invagination depth, further supporting a role for the Vps1-actin interaction during scission. In vitro analysis of the mutant protein demonstrates that--like wild-type Vps1--it is able to form oligomeric rings, but, critically, it has lost its ability to bundle actin filaments into higher-order structures. A model is proposed in which actin filaments bind Vps1 during invagination, and this interaction is important to transduce the force of actin polymerization to the membrane to drive successful scission.

  14. Unconventional EGF-induced ERK1/2-mediated Kv1.3 endocytosis.

    PubMed

    Martínez-Mármol, Ramón; Comes, Núria; Styrczewska, Katarzyna; Pérez-Verdaguer, Mireia; Vicente, Rubén; Pujadas, Lluís; Soriano, Eduardo; Sorkin, Alexander; Felipe, Antonio

    2016-04-01

    The potassium channel Kv1.3 plays roles in immunity, neuronal development and sensory discrimination. Regulation of Kv1.3 by kinase signaling has been studied. In this context, EGF binds to specific receptors (EGFR) and triggers tyrosine kinase-dependent signaling, which down-regulates Kv1.3 currents. We show that Kv1.3 undergoes EGF-dependent endocytosis. This EGF-mediated mechanism is relevant because is involved in adult neural stem cell fate determination. We demonstrated that changes in Kv1.3 subcellular distribution upon EGFR activation were due to Kv1.3 clathrin-dependent endocytosis, which targets the Kv1.3 channels to the lysosomal degradative pathway. Interestingly, our results further revealed that relevant tyrosines and other interacting motifs, such as PDZ and SH3 domains, were not involved in the EGF-dependent Kv1.3 internalization. However, a new, and yet undescribed mechanism, of ERK1/2-mediated threonine phosphorylation is crucial for the EGF-mediated Kv1.3 endocytosis. Our results demonstrate that EGF triggers the down-regulation of Kv1.3 activity and its expression at the cell surface, which is important for the development and migration of adult neural progenitors.

  15. Endocytosis of fluorescent cyclodextrins by intestinal Caco-2 cells and its role in paclitaxel drug delivery.

    PubMed

    Réti-Nagy, Katalin; Malanga, Milo; Fenyvesi, Éva; Szente, Lajos; Vámosi, György; Váradi, Judit; Bácskay, Ildikó; Fehér, Pálma; Ujhelyi, Zoltán; Róka, Eszter; Vecsernyés, Miklós; Balogh, György; Vasvári, Gábor; Fenyvesi, Ferenc

    2015-12-30

    Cyclodextrins are widely used excipients in pharmaceutical formulations. They are mainly utilized as solubilizers and absorption enhancers, but recent results revealed their effects on cell membranes and pharmacological barriers. In addition to the growing knowledge on their interaction with plasma membranes, it was confirmed that cyclodextrins are able to enter cells by endocytosis. The number of the tested cyclodextrins was limited, and the role of this mechanism in drug absorption and delivery is not known. Our aim was to examine the endocytosis of fluorescently labeled hydroxypropyl-β-cyclodextrin, random methyl-β-cyclodextrin and soluble β-cyclodextrin polymer, and the cellular uptake of the fluorescent paclitaxel derivative-random methyl-β-cyclodextrin complex. The studied cyclodextrin derivatives were able to enter Caco-2 intestinal cells and localized in vesicles in the cytoplasm, while their permeability was very limited through Caco-2 monolayers. We demonstrated for the first time that the fluorescent paclitaxel derivative and rhodamine-labeled random methyl-β-cyclodextrin were detected in the same intracellular vesicles after treating cells with their inclusion complex. These results indicate that the endocytosis of cyclodextrin complexes can contribute to drug absorption processes.

  16. Regulation of cargo-selective endocytosis by dynamin 2 GTPase-activating protein girdin.

    PubMed

    Weng, Liang; Enomoto, Atsushi; Miyoshi, Hiroshi; Takahashi, Kiyofumi; Asai, Naoya; Morone, Nobuhiro; Jiang, Ping; An, Jian; Kato, Takuya; Kuroda, Keisuke; Watanabe, Takashi; Asai, Masato; Ishida-Takagishi, Maki; Murakumo, Yoshiki; Nakashima, Hideki; Kaibuchi, Kozo; Takahashi, Masahide

    2014-09-17

    In clathrin-mediated endocytosis (CME), specificity and selectivity for cargoes are thought to be tightly regulated by cargo-specific adaptors for distinct cellular functions. Here, we show that the actin-binding protein girdin is a regulator of cargo-selective CME. Girdin interacts with dynamin 2, a GTPase that excises endocytic vesicles from the plasma membrane, and functions as its GTPase-activating protein. Interestingly, girdin depletion leads to the defect in clathrin-coated pit formation in the center of cells. Also, we find that girdin differentially interacts with some cargoes, which competitively prevents girdin from interacting with dynamin 2 and confers the cargo selectivity for CME. Therefore, girdin regulates transferrin and E-cadherin endocytosis in the center of cells and their subsequent polarized intracellular localization, but has no effect on integrin and epidermal growth factor receptor endocytosis that occurs at the cell periphery. Our results reveal that girdin regulates selective CME via a mechanism involving dynamin 2, but not by operating as a cargo-specific adaptor.

  17. Cadherin-11 endocytosis through binding to clathrin promotes cadherin-11-mediated migration in prostate cancer cells.

    PubMed

    Satcher, Robert L; Pan, Tianhong; Bilen, Mehmet A; Li, Xiaoxia; Lee, Yu-Chen; Ortiz, Angelica; Kowalczyk, Andrew P; Yu-Lee, Li-Yuan; Lin, Sue-Hwa

    2015-12-15

    Cadherin-11 (Cad11) cell adhesion molecule plays a role in prostate cancer cell migration. Because disassembly of adhesion complexes through endocytosis of adhesion proteins has been shown to play a role in cell migration, we examined whether Cad11 endocytosis plays a role in Cad11-mediated migration. The mechanism by which Cad11 is internalized is unknown. Using a GST pulldown assay, we found that clathrin binds to the Cad11 cytoplasmic domain but not to that of E-cadherin. Using deletion analysis, we identified a unique sequence motif, VFEEE, in the Cad11 membrane proximal region (amino acid residues 11-15) that binds to clathrin. Endocytosis assays using K(+)-depletion buffer showed that Cad11 internalization is clathrin dependent. Proximity ligation assays showed that Cad11 colocalizes with clathrin, and immunofluorescence assays showed that Cad11 localizes in vesicles that stain for the early endosomal marker Rab5. Deletion of the VFEEE sequence from the Cad11 cytoplasmic domain (Cad11-cla-Δ5) leads to inhibition of Cad11 internalization and reduces Cad11-mediated cell migration in C4-2B and PC3-mm2 prostate cancer cells. These observations suggest that clathrin-mediated internalization of Cad11 regulates surface trafficking of Cad11 and that dynamic turnover of Cad11 regulates the migratory function of Cad11 in prostate cancer cells.

  18. Target shape dependence in a simple model of receptor-mediated endocytosis and phagocytosis.

    PubMed

    Richards, David M; Endres, Robert G

    2016-05-31

    Phagocytosis and receptor-mediated endocytosis are vitally important particle uptake mechanisms in many cell types, ranging from single-cell organisms to immune cells. In both processes, engulfment by the cell depends critically on both particle shape and orientation. However, most previous theoretical work has focused only on spherical particles and hence disregards the wide-ranging particle shapes occurring in nature, such as those of bacteria. Here, by implementing a simple model in one and two dimensions, we compare and contrast receptor-mediated endocytosis and phagocytosis for a range of biologically relevant shapes, including spheres, ellipsoids, capped cylinders, and hourglasses. We find a whole range of different engulfment behaviors with some ellipsoids engulfing faster than spheres, and that phagocytosis is able to engulf a greater range of target shapes than other types of endocytosis. Further, the 2D model can explain why some nonspherical particles engulf fastest (not at all) when presented to the membrane tip-first (lying flat). Our work reveals how some bacteria may avoid being internalized simply because of their shape, and suggests shapes for optimal drug delivery.

  19. Inhibitors of endocytosis prevent Wnt/Wingless signalling by reducing the level of basal β-catenin/Armadillo

    PubMed Central

    Gagliardi, Maria; Hernandez, Ana; McGough, Ian J.; Vincent, Jean-Paul

    2014-01-01

    ABSTRACT A key step in the canonical Wnt signalling pathway is the inhibition of GSK3β, which results in the accumulation of nuclear β-catenin (also known as CTNNB1), and hence regulation of target genes. Evidence suggests that endocytosis is required for signalling, yet its role and the molecular understanding remains unclear. A recent and controversial model suggests that endocytosis contributes to Wnt signalling by causing the sequestration of the ligand–receptor complex, including LRP6 and GSK3 to multivesicular bodies (MVBs), thus preventing GSK3β from accessing β-catenin. Here, we use specific inhibitors (Dynasore and Dyngo-4a) to confirm the essential role of endocytosis in Wnt/Wingless signalling in human and Drosophila cells. However, we find no evidence that, in Drosophila cells or wing imaginal discs, LRP6/Arrow traffics to MVBs or that MVBs are required for Wnt/Wingless signalling. Moreover, we show that activation of signalling through chemical blockade of GSK3β is prevented by endocytosis inhibitors, suggesting that endocytosis impacts on Wnt/Wingless signalling downstream of the ligand–receptor complex. We propose that, through an unknown mechanism, endocytosis boosts the resting pool of β-catenin upon which GSK3β normally acts. PMID:25236598

  20. μ2-Dependent endocytosis of N-cadherin is regulated by β-catenin to facilitate neurite outgrowth.

    PubMed

    Chen, Yi-Ting; Tai, Chin-Yin

    2017-02-22

    Circuit formation in the brain requires neurite outgrowth throughout development to establish synaptic contacts with target cells. Active endocytosis of several adhesion molecules facilitates the dynamic exchange of these molecules at the surface and promotes neurite outgrowth in developing neurons. The endocytosis of N-cadherin, a calcium-dependent adhesion molecule, has been implicated in the regulation of neurite outgrowth, but the mechanism remains unclear. Here, we identified that a fraction of N-cadherin internalizes through clathrin-mediated endocytosis (CME). Two tyrosine-based motifs in the cytoplasmic domain of N-cadherin recognized by the μ2 subunit of the AP-2 adaptor complex are responsible for CME of N-cadherin. Moreover, β-catenin, a core component of the N-cadherin adhesion complex, inhibits N-cadherin endocytosis by masking the 2 tyrosine-based motifs. Removal of β-catenin facilitates μ2 binding to N-cadherin, thereby increasing clathrin-mediated N-cadherin endocytosis and neurite outgrowth without affecting the steady-state level of surface N-cadherin. These results identify and characterize the mechanism controlling N-cadherin endocytosis through β-catenin-regulated μ2 binding to modulate neurite outgrowth.

  1. Inhibitors of endocytosis prevent Wnt/Wingless signalling by reducing the level of basal β-catenin/Armadillo.

    PubMed

    Gagliardi, Maria; Hernandez, Ana; McGough, Ian J; Vincent, Jean-Paul

    2014-11-15

    A key step in the canonical Wnt signalling pathway is the inhibition of GSK3β, which results in the accumulation of nuclear β-catenin (also known as CTNNB1), and hence regulation of target genes. Evidence suggests that endocytosis is required for signalling, yet its role and the molecular understanding remains unclear. A recent and controversial model suggests that endocytosis contributes to Wnt signalling by causing the sequestration of the ligand-receptor complex, including LRP6 and GSK3 to multivesicular bodies (MVBs), thus preventing GSK3β from accessing β-catenin. Here, we use specific inhibitors (Dynasore and Dyngo-4a) to confirm the essential role of endocytosis in Wnt/Wingless signalling in human and Drosophila cells. However, we find no evidence that, in Drosophila cells or wing imaginal discs, LRP6/Arrow traffics to MVBs or that MVBs are required for Wnt/Wingless signalling. Moreover, we show that activation of signalling through chemical blockade of GSK3β is prevented by endocytosis inhibitors, suggesting that endocytosis impacts on Wnt/Wingless signalling downstream of the ligand-receptor complex. We propose that, through an unknown mechanism, endocytosis boosts the resting pool of β-catenin upon which GSK3β normally acts.

  2. Tyrosine-based signal mediates LRP6 receptor endocytosis and desensitization of Wnt/β-catenin pathway signaling.

    PubMed

    Liu, Chia-Chen; Kanekiyo, Takahisa; Roth, Barbara; Bu, Guojun

    2014-10-03

    Wnt/β-catenin signaling orchestrates a number of critical events including cell growth, differentiation, and cell survival during development. Misregulation of this pathway leads to various human diseases, specifically cancers. Endocytosis and phosphorylation of the LDL receptor-related protein 6 (LRP6), an essential co-receptor for Wnt/β-catenin signaling, play a vital role in mediating Wnt/β-catenin signal transduction. However, its regulatory mechanism is not fully understood. In this study, we define the mechanisms by which LRP6 endocytic trafficking regulates Wnt/β-catenin signaling activation. We show that LRP6 mutant with defective tyrosine-based signal in its cytoplasmic tail has an increased cell surface distribution and decreased endocytosis rate. These changes in LRP6 endocytosis coincide with an increased distribution to caveolae, increased phosphorylation, and enhanced Wnt/β-catenin signaling. We further demonstrate that treatment of Wnt3a ligands or blocking the clathrin-mediated endocytosis of LRP6 leads to a redistribution of wild-type receptor to lipid rafts. The LRP6 tyrosine mutant also exhibited an increase in signaling activation in response to Wnt3a stimulation when compared with wild-type LRP6, and this activation is suppressed when caveolae-mediated endocytosis is blocked. Our results reveal molecular mechanisms by which LRP6 endocytosis routes regulate its phosphorylation and the strength of Wnt/β-catenin signaling, and have implications on how this pathway can be modulated in human diseases.

  3. Bulking agents in sludge composting

    SciTech Connect

    De Bertoldi, M.; Citernesi, U.; Griselli, M.

    1980-01-01

    Composting is one of the most effective ways of disposing of sludge in agriculture. Three bulking agents were studied: (1) the organic fraction of solid wastes, (2) solid agricultural and forestry waste (straw, maize cobs, sawdust, cork, pine cones, etc.), and (3) recyclable inert substrates (polystyrene or polyethylene balls, porous clay balls, etc.). The sole purpose of the inert bulking agent is to aid in the aeration and drying of the composting material.

  4. Activity-dependent PSA expression regulates inhibitory maturation and onset of critical period plasticity.

    PubMed

    Di Cristo, Graziella; Chattopadhyaya, Bidisha; Kuhlman, Sandra J; Fu, Yu; Bélanger, Marie-Claude; Wu, Cai Zhi; Rutishauser, Urs; Maffei, Lamberto; Huang, Z Josh

    2007-12-01

    Functional maturation of GABAergic innervation in the developing visual cortex is regulated by neural activity and sensory inputs and in turn influences the critical period of ocular dominance plasticity. Here we show that polysialic acid (PSA), presented by the neural cell adhesion molecule, has a role in the maturation of GABAergic innervation and ocular dominance plasticity. Concentrations of PSA significantly decline shortly after eye opening in the adolescent mouse visual cortex; this decline is hindered by visual deprivation. The developmental and activity-dependent regulation of PSA expression is inversely correlated with the maturation of GABAergic innervation. Premature removal of PSA in visual cortex results in precocious maturation of perisomatic innervation by basket interneurons, enhanced inhibitory synaptic transmission, and earlier onset of ocular dominance plasticity. The developmental and activity-dependent decline of PSA expression therefore regulates the timing of the maturation of GABAergic inhibition and the onset of ocular dominance plasticity.

  5. Activity-dependent signal changes in neurons by fiber-coupled microscopy

    NASA Astrophysics Data System (ADS)

    Sakurai, Takashi; Koida, Kowa

    2014-03-01

    To study neuronal functions in brain, we developed a higher resolution type fiber-coupled microscope (FCM), and measured the activity-dependent fluorescence intensity of the excitable cells over time. FCM was constructed by combining a fluorescence microscope with the high density type of fiber bundle, which consisted of 1.5 x 104 unit fiber in the assemble less than 0.5 mm tip. The spatial resolution was calculated to be 2.4 mm with the 5 mm focal depth. The activity-dependent Ca signals were detectable in each cell of either the pancreatic spheroids or the brain slices. The present FCM is very promising for detailed studies with the live imaging of signal molecules in the body at a single cell level.

  6. Activity-dependent regulation of genes implicated in X-linked non-specific mental retardation.

    PubMed

    Boda, B; Mas, C; Muller, D

    2002-01-01

    X-linked forms of non-specific mental retardation are complex disorders, for which mutations in several genes have recently been identified. These include OPHN1, GDI1, PAK3, IL1RAPL, TM4SF2, FMR2 and RSK2. To investigate the mechanisms through which alterations of these gene products could result in cognitive impairment, we analyzed their expression using quantitative PCR technique in two in vitro models of activity-dependent gene regulation: kainate-induced seizures and long-term synaptic potentiation (LTP). We found that the level of expression of four genes, PAK3, IL1RAPL, RSK2 and TM4SF2, was significantly up-regulated following kainate treatment. Furthermore we observed a significant increase in mRNA levels of PAK3 and IL1RAPL following LTP induction. These results suggest a possible role for these four genes in activity-dependent brain plasticity.

  7. Activity-dependent signaling: influence on plasticity in circuits controlling fear-related behavior

    PubMed Central

    Hill, Julia L; Martinowich, Keri

    2015-01-01

    Fear regulation is impaired in anxiety and trauma-related disorders. Patients experience heightened fear expression and reduced ability to extinguish fear memories. Because fear regulation is abnormal in these disorders and extinction recapitulates current treatment strategies, understanding the underlying mechanisms is vital for developing new treatments. This is critical because although extinction-based exposure therapy is a mainstay of treatment, relapse is common. We examine recent findings describing changes in network activity and functional connectivity within limbic circuits during fear regulation, and explore how activity-dependent signaling contributes to the neural activity patterns that control fear and anxiety. We review the role of the prototypical activity-dependent molecule, brain-derived neurotrophic factor (BDNF), whose signaling has been critically linked to regulation of fear behavior. PMID:26485574

  8. Dynamin2, Clathrin, and Lipid Rafts Mediate Endocytosis of the Apical Na/K/2Cl Cotransporter NKCC2 in Thick Ascending Limbs*

    PubMed Central

    Ares, Gustavo R.; Ortiz, Pablo A.

    2012-01-01

    Steady-state surface levels of the apical Na/K/2Cl cotransporter NKCC2 regulate NaCl reabsorption by epithelial cells of the renal thick ascending limb (THAL). We reported that constitutive endocytosis of NKCC2 controls NaCl absorption in native THALs; however, the pathways involved in NKCC2 endocytosis are unknown. We hypothesized that NKCC2 endocytosis at the apical surface depends on dynamin-2 and clathrin. Measurements of steady-state surface NKCC2 and the rate of NKCC2 endocytosis in freshly isolated rat THALs showed that inhibition of endogenous dynamin-2 with dynasore blunted NKCC2 endocytosis by 56 ± 11% and increased steady-state surface NKCC2 by 67 ± 27% (p < 0.05). Expression of the dominant negative Dyn2K44A in THALs slowed the rate of NKCC2 endocytosis by 38 ± 8% and increased steady-state surface NKCC2 by 37 ± 8%, without changing total NKCC2 expression. Inhibition of clathrin-mediated endocytosis with chlorpromazine blunted NKCC2 endocytosis by 54 ± 6%, while preventing clathrin from interacting with synaptojanin also blunted NKCC2 endocytosis by 52 ± 5%. Disruption of lipid rafts blunted NKCC2 endocytosis by 39 ± 4% and silencing caveolin-1 by 29 ± 4%. Simultaneous inhibition of clathrin- and lipid raft-mediated endocytosis completely blocked NKCC2 internalization. We concluded that dynamin-2, clathrin, and lipid rafts mediate NKCC2 endocytosis and maintain steady-state apical surface NKCC2 in native THALs. These are the first data identifying the endocytic pathway for apical NKCC2 endocytosis. PMID:22977238

  9. Modeling activity-dependent changes of axonal spike conduction in primary afferent C-nociceptors

    PubMed Central

    Tigerholm, Jenny; Petersson, Marcus E.; Obreja, Otilia; Lampert, Angelika; Carr, Richard; Schmelz, Martin

    2013-01-01

    Action potential initiation and conduction along peripheral axons is a dynamic process that displays pronounced activity dependence. In patients with neuropathic pain, differences in the modulation of axonal conduction velocity by activity suggest that this property may provide insight into some of the pathomechanisms. To date, direct recordings of axonal membrane potential have been hampered by the small diameter of the fibers. We have therefore adopted an alternative approach to examine the basis of activity-dependent changes in axonal conduction by constructing a comprehensive mathematical model of human cutaneous C-fibers. Our model reproduced axonal spike propagation at a velocity of 0.69 m/s commensurate with recordings from human C-nociceptors. Activity-dependent slowing (ADS) of axonal propagation velocity was adequately simulated by the model. Interestingly, the property most readily associated with ADS was an increase in the concentration of intra-axonal sodium. This affected the driving potential of sodium currents, thereby producing latency changes comparable to those observed for experimental ADS. The model also adequately reproduced post-action potential excitability changes (i.e., recovery cycles) observed in vivo. We performed a series of control experiments replicating blockade of particular ion channels as well as changing temperature and extracellular ion concentrations. In the absence of direct experimental approaches, the model allows specific hypotheses to be formulated regarding the mechanisms underlying activity-dependent changes in C-fiber conduction. Because ADS might functionally act as a negative feedback to limit trains of nociceptor activity, we envisage that identifying its mechanisms may also direct efforts aimed at alleviating neuronal hyperexcitability in pain patients. PMID:24371290

  10. Histone methyltransferase Ash1L mediates activity-dependent repression of neurexin-1α

    PubMed Central

    Zhu, Τao; Liang, Chen; Li, Dongdong; Tian, Miaomiao; Liu, Sanxiong; Gao, Guanjun; Guan, Ji-Song

    2016-01-01

    Activity-dependent transcription is critical for the regulation of long-term synaptic plasticity and plastic rewiring in the brain. Here, we report that the transcription of neurexin1α (nrxn1α), a presynaptic adhesion molecule for synaptic formation, is regulated by transient neuronal activation. We showed that 10 minutes of firing at 50 Hz in neurons repressed the expression of nrxn1α for 24 hours in a primary cortical neuron culture through a transcriptional repression mechanism. By performing a screening assay using a synthetic zinc finger protein (ZFP) to pull down the proteins enriched near the nrxn1α promoter region in vivo, we identified that Ash1L, a histone methyltransferase, is enriched in the nrxn1α promoter. Neuronal activity triggered binding of Ash1L to the promoter and enriched the histone marker H3K36me2 at the nrxn1α promoter region. Knockout of Ash1L in mice completely abolished the activity-dependent repression of nrxn1α. Taken together, our results reveal that a novel process of activity-dependent transcriptional repression exists in neurons and that Ash1L mediates the long-term repression of nrxn1α, thus implicating an important role for epigenetic modification in brain functioning. PMID:27229316

  11. Kinesin-1–syntaphilin coupling mediates activity-dependent regulation of axonal mitochondrial transport

    PubMed Central

    Chen, Yanmin

    2013-01-01

    Axonal mitochondria are recruited to synaptic terminals in response to neuronal activity, but the mechanisms underlying activity-dependent regulation of mitochondrial transport are largely unknown. In this paper, using genetic mouse model combined with live imaging, we demonstrate that syntaphilin (SNPH) mediates the activity-dependent immobilization of axonal mitochondria through binding to KIF5. In vitro analysis showed that the KIF5–SNPH coupling inhibited the motor adenosine triphosphatase. Neuronal activity further recruited SNPH to axonal mitochondria. This motor-docking interplay was induced by Ca2+ and synaptic activity and was necessary to establish an appropriate balance between motile and stationary axonal mitochondria. Deleting snph abolished the activity-dependent immobilization of axonal mitochondria. We propose an “Engine-Switch and Brake” model, in which SNPH acts both as an engine off switch by sensing mitochondrial Rho guanosine triphosphatase-Ca2+ and as a brake by anchoring mitochondria to the microtubule track. Altogether, our study provides new mechanistic insight into the molecular interplay between motor and docking proteins, which arrests axonal mitochondrial transport in response to changes in neuronal activity. PMID:23857772

  12. Evidence for evolutionary divergence of activity-dependent gene expression in developing neurons

    PubMed Central

    Qiu, Jing; McQueen, Jamie; Bilican, Bilada; Dando, Owen; Magnani, Dario; Punovuori, Karolina; Selvaraj, Bhuvaneish T; Livesey, Matthew; Haghi, Ghazal; Heron, Samuel; Burr, Karen; Patani, Rickie; Rajan, Rinku; Sheppard, Olivia; Kind, Peter C; Simpson, T Ian; Tybulewicz, Victor LJ; Wyllie, David JA; Fisher, Elizabeth MC; Lowell, Sally; Chandran, Siddharthan; Hardingham, Giles E

    2016-01-01

    Evolutionary differences in gene regulation between humans and lower mammalian experimental systems are incompletely understood, a potential translational obstacle that is challenging to surmount in neurons, where primary tissue availability is poor. Rodent-based studies show that activity-dependent transcriptional programs mediate myriad functions in neuronal development, but the extent of their conservation in human neurons is unknown. We compared activity-dependent transcriptional responses in developing human stem cell-derived cortical neurons with those induced in developing primary- or stem cell-derived mouse cortical neurons. While activity-dependent gene-responsiveness showed little dependence on developmental stage or origin (primary tissue vs. stem cell), notable species-dependent differences were observed. Moreover, differential species-specific gene ortholog regulation was recapitulated in aneuploid mouse neurons carrying human chromosome-21, implicating promoter/enhancer sequence divergence as a factor, including human-specific activity-responsive AP-1 sites. These findings support the use of human neuronal systems for probing transcriptional responses to physiological stimuli or indeed pharmaceutical agents. DOI: http://dx.doi.org/10.7554/eLife.20337.001 PMID:27692071

  13. Activity-Dependent Callosal Axon Projections in Neonatal Mouse Cerebral Cortex

    PubMed Central

    Tagawa, Yoshiaki; Hirano, Tomoo

    2012-01-01

    Callosal axon projections are among the major long-range axonal projections in the mammalian brain. They are formed during the prenatal and early postnatal periods in the mouse, and their development relies on both activity-independent and -dependent mechanisms. In this paper, we review recent findings about the roles of neuronal activity in callosal axon projections. In addition to the well-documented role of sensory-driven neuronal activity, recent studies using in utero electroporation demonstrated an essential role of spontaneous neuronal activity generated in neonatal cortical circuits. Both presynaptic and postsynaptic neuronal activities are critically involved in the axon development. Studies have begun to reveal intracellular signaling pathway which works downstream of neuronal activity. We also review several distinct patterns of neuronal activity observed in the developing cerebral cortex, which might play roles in activity-dependent circuit construction. Such neuronal activity during the neonatal period can be disrupted by genetic factors, such as mutations in ion channels. It has been speculated that abnormal activity caused by such factors may affect activity-dependent circuit construction, leading to some developmental disorders. We discuss a possibility that genetic mutation in ion channels may impair callosal axon projections through an activity-dependent mechanism. PMID:23213574

  14. Genome-wide identification and characterization of functional neuronal activity-dependent enhancers

    PubMed Central

    Malik, Athar N.; Vierbuchen, Thomas; Hemberg, Martin; Rubin, Alex A.; Ling, Emi; Couch, Cameron H.; Stroud, Hume; Spiegel, Ivo; Farh, Kyle Kai-How; Harmin, David A.; Greenberg, Michael E.

    2015-01-01

    SUMMARY Experience-dependent gene transcription is required for nervous system development and function. However, the DNA regulatory elements that control this program of gene expression are not well defined. Here we characterize the enhancers that function across the genome to mediate activity-dependent transcription in mouse cortical neurons. We find that the subset of enhancers enriched for monomethylation of histone H3 lysine 4 (H3K4me1) and binding of the transcriptional co-activator CREBBP (CBP) that shows increased acetylation of histone H3 lysine 27 (H3K27ac) upon membrane depolarization of cortical neurons functions to regulate activity-dependent transcription. A subset of these enhancers appears to require binding of FOS, which previously was thought to bind primarily to promoters. These findings suggest that FOS functions at enhancers to control activity-dependent gene programs that are critical for nervous system function and provide a resource of functional cis-regulatory elements that may give insight into the genetic variants that contribute to brain development and disease. PMID:25195102

  15. Chronic Mild Stress Modulates Activity-Dependent Transcription of BDNF in Rat Hippocampal Slices.

    PubMed

    Molteni, Raffaella; Rossetti, Andrea C; Savino, Elisa; Racagni, Giorgio; Calabrese, Francesca

    2016-01-01

    Although activity-dependent transcription represents a crucial mechanism for long-lasting experience-dependent changes in the hippocampus, limited data exist on its contribution to pathological conditions. We aim to investigate the influence of chronic stress on the activity-dependent transcription of brain-derived neurotrophic factor (BDNF). The ex vivo methodology of acute stimulation of hippocampal slices obtained from rats exposed to chronic mild stress (CMS) was used to evaluate whether the adverse experience may alter activity-dependent BDNF gene expression. CMS reduces BDNF expression and that acute depolarization significantly upregulates total BDNF mRNA levels only in control animals, showing that CMS exposure may alter BDNF transcription under basal conditions and during neuronal activation. Moreover, while the basal effect of CMS on total BDNF reflects parallel modulations of all the transcripts examined, isoform-specific changes were found after depolarization. This different effect was also observed in the activation of intracellular signaling pathways related to the neurotrophin. In conclusion, our study discloses a functional alteration of BDNF transcription as a consequence of stress. Being the activity-regulated transcription a critical process in synaptic and neuronal plasticity, the different regulation of individual BDNF promoters may contribute to long-lasting changes, which are fundamental for the vulnerability of the hippocampus to stress-related diseases.

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

    NASA Astrophysics Data System (ADS)

    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 bx<1 , it is subcritical. For stock prices we find bx=1 within statistical uncertainty, for all x , consistent with an “efficient 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.

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

    PubMed

    Martin, Elliot; Shreim, Amer; Paczuski, Maya

    2010-01-01

    We define an activity-dependent branching ratio that allows comparison of different time series X(t). The branching ratio b(x) is defined as b(x)=E[xi(x)/x]. The random variable xi(x) is the value of the next signal given that the previous one is equal to x, so xi(x)=[X(t+1) | X(t)=x]. If b(x)>1, the process is on average supercritical when the signal is equal to x, while if b(x)<1, it is subcritical. For stock prices we find b(x)=1 within statistical uncertainty, for all x, consistent with an "efficient market hypothesis." For stock volumes, solar x-ray flux intensities, and the Bak-Tang-Wiesenfeld (BTW) sandpile model, b(x) 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 b(x) approximately equal 1, which we interpret as an indicator of critical behavior. This is true despite different underlying probability distributions for X(t) and for xi(x). For the BTW model the distribution of xi(x) 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 b(x) is close to one disappears once bulk dissipation is introduced in the BTW model-supporting our hypothesis that it is an indicator of criticality.

  18. Oestrogen promotes KCNQ1 potassium channel endocytosis and postendocytic trafficking in colonic epithelium

    PubMed Central

    Rapetti-Mauss, Raphael; O'Mahony, Fiona; Sepulveda, Francisco V; Urbach, Valerie; Harvey, Brian J

    2013-01-01

    The cAMP-regulated potassium channel KCNQ1:KCNE3 plays an essential role in transepithelial Cl− secretion. Recycling of K+ across the basolateral membrane provides the driving force necessary to maintain apical Cl− secretion. The steroid hormone oestrogen (17β-oestradiol; E2), produces a female-specific antisecretory response in rat distal colon through the inhibition of the KCNQ1:KCNE3 channel. It has previously been shown that rapid inhibition of the channel conductance results from E2-induced uncoupling of the KCNE3 regulatory subunit from the KCNQ1 channel pore complex. The purpose of this study was to determine the mechanism required for sustained inhibition of the channel function. We found that E2 plays a role in regulation of KCNQ1 cell membrane abundance by endocytosis. Ussing chamber experiments have shown that E2 inhibits both Cl− secretion and KCNQ1 current in a colonic cell line, HT29cl.19A, when cultured as a confluent epithelium. Following E2 treatment, KCNQ1 was retrieved from the plasma membrane by a clathrin-mediated endocytosis, which involved the association between KCNQ1 and the clathrin adaptor, AP-2. Following endocytosis, KCNQ1 was accumulated in early endosomes. Following E2-induced endocytosis, rather than being degraded, KCNQ1 was recycled by a biphasic mechanism involving Rab4 and Rab11. Protein kinase Cδ and AMP-dependent kinase were rapidly phosphorylated in response to E2 on their activating phosphorylation sites, Ser643 and Thr172, respectively (as previously shown). Both kinases are necessary for the E2-induced endocytosis, because E2 failed to induce KCNQ1 internalization following pretreatment with specific inhibitors of both protein kinase Cδ and AMP-dependent kinase. The ubiquitin ligase Nedd4.2 binds KCNQ1 in response to E2 to induce channel internalization. This study has provided the first demonstration of hormonal regulation of KCNQ1 trafficking. In conclusion, we propose that internalization of KCNQ1 is a key event

  19. Temperature dependence of fluid phase endocytosis coincides with membrane properties of pig platelets.

    PubMed

    Wolkers, Willem F; Looper, Sheri A; Fontanilla, Ray A; Tsvetkova, Nelly M; Tablin, Fern; Crowe, John H

    2003-06-10

    In previous studies we have shown that platelets take up low molecular weight molecules from the medium by fluid phase endocytosis, a phenomenon that we previously have used to load trehalose into human platelets, after which we have successfully freeze-dried them. We now extend those findings to a species to be used in animal trials of freeze-dried platelets:pigs. Further, we report results of studies aimed at elucidating the mechanism of the uptake. Temperature dependence of fluid-phase endocytosis was determined in pig platelets, using lucifer yellow carbohydrazide (LY) as a marker. A biphasic curve of marker uptake versus temperature was obtained. The activation energy was significantly higher above 22 degrees C (18.7+/-1.8 kcal/mol) than below that critical temperature (7.5+/-1.5 kcal/mol). The activation energy of fluid phase endocytosis in human platelets was 24.1+/-1.6 kcal/mol above 15 degrees C. In order to establish a correlation between the effect of temperature on fluid phase endocytosis and the membrane physical state, Fourier transform infrared spectroscopy (FTIR) and fluorescence anisotropy experiments were conducted. FTIR studies showed that pig platelets exhibit a main membrane phase transition at approximately 12 degrees C, and two smaller transitions at 26 and 37 degrees C. Anisotropy experiments performed with 1,6 diphenyl-1,3,5 hexatriene (DPH) complemented FTIR results and showed a major transition at 8 degrees C and smaller transitions at 26 and 35 degrees C. In order to investigate the relative roles of known participants in fluid phase endocytosis, the effects of several chemical inhibitors were investigated. LY uptake was unaffected by colchicine, methylamine, and amiloride. However, disruption of specific microdomains in the membrane (rafts) by methyl-beta-cyclodextrin reduced uptake of LY by 35%. Treatment with cytochalasin B, which inhibits actin polymerization, reduced the uptake by 25%. We conclude that the inflection point in the LY

  20. Modelling of bulk superconductor magnetization

    NASA Astrophysics Data System (ADS)

    Ainslie, M. D.; Fujishiro, H.

    2015-05-01

    This paper presents a topical review of the current state of the art in modelling the magnetization of bulk superconductors, including both (RE)BCO (where RE = rare earth or Y) and MgB2 materials. Such modelling is a powerful tool to understand the physical mechanisms of their magnetization, to assist in interpretation of experimental results, and to predict the performance of practical bulk superconductor-based devices, which is particularly important as many superconducting applications head towards the commercialization stage of their development in the coming years. In addition to the analytical and numerical techniques currently used by researchers for modelling such materials, the commonly used practical techniques to magnetize bulk superconductors are summarized with a particular focus on pulsed field magnetization (PFM), which is promising as a compact, mobile and relatively inexpensive magnetizing technique. A number of numerical models developed to analyse the issues related to PFM and optimise the technique are described in detail, including understanding the dynamics of the magnetic flux penetration and the influence of material inhomogeneities, thermal properties, pulse duration, magnitude and shape, and the shape of the magnetization coil(s). The effect of externally applied magnetic fields in different configurations on the attenuation of the trapped field is also discussed. A number of novel and hybrid bulk superconductor structures are described, including improved thermal conductivity structures and ferromagnet-superconductor structures, which have been designed to overcome some of the issues related to bulk superconductors and their magnetization and enhance the intrinsic properties of bulk superconductors acting as trapped field magnets. Finally, the use of hollow bulk cylinders/tubes for shielding is analysed.

  1. Mechanisms of Toll-like Receptor 4 Endocytosis Reveal a Common Immune-Evasion Strategy Used by Pathogenic and Commensal Bacteria.

    PubMed

    Tan, Yunhao; Zanoni, Ivan; Cullen, Thomas W; Goodman, Andrew L; Kagan, Jonathan C

    2015-11-17

    Microbe-induced receptor trafficking has emerged as an essential means to promote innate immune signal transduction. Upon detection of bacterial lipopolysaccharides (LPS), CD14 induces an inflammatory endocytosis pathway that delivers Toll-like receptor 4 (TLR4) to endosomes. Although several regulators of CD14-dependent TLR4 endocytosis have been identified, the cargo-selection mechanism during this process remains unknown. We reveal that, in contrast to classic cytosolic interactions that promoted the endocytosis of transmembrane receptors, TLR4 was selected as cargo for inflammatory endocytosis entirely through extracellular interactions. Mechanistically, the extracellular protein MD-2 bound to and dimerized TLR4 in order to promote this endocytic event. Our analysis of LPS variants from human pathogens and gut commensals revealed a common mechanism by which bacteria prevent inflammatory endocytosis. We suggest that evasion of CD14-dependent endocytosis is an attribute that transcends the concept of pathogenesis and might be a fundamental feature of bacteria that inhabit eukaryotic hosts.

  2. Study on the endocytosis and the internalization mechanism of aminosilane-coated Fe3O4 nanoparticles in vitro.

    PubMed

    Ma, Yong-Jie; Gu, Hong-Chen

    2007-11-01

    In this study, the endocytosis and the internalization mechanism of aminosilane-coated Fe(3)O(4) nanoparticles into human lung cancer cell line SPC-A1 was studied compared with human lung cell line WI-38 in vitro. The particle endocytosis behavior was studied by using Transmission Electron Microscope (TEM) and Coupled Plasma-Atomic Emission Spectrometry (ICP-AES). It was found that aminosilane-coated Fe(3)O(4) nanoparticles could be greatly taken up by SPC-A1 human cancer cells (202 pg iron/cell) but not by WI-38 human lung cells (13 pg iron/cell). The particles could be retained in SPC-A1 cells over a number of generations in vitro. Different endocytosis was observed by TEM after SPC-A1 cells were treated with different temperature or with/without Cytochalasin B (Inhibitor of phagocytosis) at 37 degrees C. No nanoparticles were taken up by SPC-A1 after the endocytosis inhibited in low temperature. Restoring the endocytosis activity at 37 degrees C, the process of nanoparticles from coated pit to endosomes and lysosomes was observed by TEM. Endocytosis activity was effectively inhibited by the presence of Cytochalasin B at 37 degrees C, while a lot of nanoparticles were uptaken to the cytoplasm of SPC-A1 cells in the control group. Our results suggest that the process of endocytosis of aminosilane-coated Fe(3)O(4) nanoparticles can efficiently takes place in lung cancer cells and nanoparticles can be kept in cancer cells for generations. Phagocytosis may be involved in the internalization process of aminosilane-coated Fe(3)O(4) nanoparticles.

  3. Prevention of export of anoxia/reoxygenation injury from ischemic to nonischemic cardiomyocytes via inhibition of endocytosis.

    PubMed

    Khaidakov, Magomed; Mercanti, Federico; Wang, Xianwei; Ding, Zufeng; Dai, Yao; Romeo, Francesco; Sawamura, Tatsuya; Mehta, Jawahar L

    2014-06-15

    Myocardial infarct size is determined by the death of nonischemic border zone cardiomyocytes caused by export of injury signals from the infarct zone. The countermeasures to limit infarct size, therefore, should be aimed at nonselective blockade of most, if not all, injury signals from entering nonischemic cells. To test whether inhibition of endocytosis might limit infarct size, HL-1 cardiomyocytes were subjected to anoxia (6 h) and reoxygenation (1 h). Anoxic and reoxygenated cells showed a multifold increase in mitochondrial ROS production accompanied with upregulation of scavenger receptors lectin-like oxidized low-density lipoprotein receptor-1 and CD36 and stimulation of stress signals, including NADPH oxidase subunit p22(phox), SOD2, and beclin-1. Incubation of healthy cardiomyocytes in media from anoxic and reoxygenated cells (conditioned media) resulted in qualitatively similar responses, including increase in the generation of mitochondrial ROS, p22(phox), SOD2, and beclin-1. Anoxia and reoxygenation caused collapse of clathrin-mediated endocytosis and stimulation of macropinocytosis, whereas in cultures exposed to conditioned media, the activity of endocytosis was uniformly higher. Conditioned media also significantly aggravated cytotoxic effects of TNF-α and angiotensin II, and suppression of endocytosis reversed these trends, resulting in an overall increase of metabolic activity. Moreover, inhibition of endocytosis prevented binding of oxidized cellular fragments with greater efficiency than targeted neutralization of the scavenger receptor lectin-like oxidized low-density lipoprotein receptor-1. Many of the observations in HL-1 cardiomyocytes were confirmed in primary cardiomyocyte cultures. Our data suggest that endocytosis is upregulated in border zone cardiomyocytes, and inhibition of endocytosis may be an effective approach to prevent export of injury signals from the infarct zone.

  4. Role of endocytosis in localization and maintenance of the spatial markers for bud-site selection in yeast.

    PubMed

    Tuo, Shanshan; Nakashima, Kenichi; Pringle, John R

    2013-01-01

    The yeast Saccharomyces cerevisiae normally selects bud sites (and hence axes of cell polarization) in one of two distinct patterns, the axial pattern of haploid cells and the bipolar pattern of diploid cells. These patterns depend on distinct sets of cortical-marker proteins that transmit positional information through a common signaling pathway based on a Ras-type GTPase. It has been reported previously that various proteins of the endocytic pathway may be involved in determining the bipolar pattern but not the axial pattern. To explore this question systematically, we constructed and analyzed congenic haploid and diploid deletion mutants for 14 genes encoding proteins that are involved in endocytosis. The mutants displayed a wide range of severities in their overall endocytosis defects, as judged by their growth rates and abilities to take up the lipophilic dye FM 4-64. Consistent with the previous reports, none of the mutants displayed a significant defect in axial budding, but they displayed defects in bipolar budding that were roughly correlated with the severities of their overall endocytosis defects. Both the details of the mutant budding patterns and direct examination of GFP-tagged marker proteins suggested that both initial formation and maintenance of the normally persistent bipolar marks depend on endocytosis, as well as polarized exocytosis, in actively growing cells. Interestingly, maintenance of the bipolar marks in non-growing cells did not appear to require normal levels of endocytosis. In some cases, there was a striking lack of correlation between the overall severities of the general-endocytosis defect and the bud-site selection defect, suggesting that various endocytosis proteins may differ in their importance for the uptake of various plasma-membrane targets.

  5. Looking for a bulk point

    DOE PAGES

    Maldacena, Juan; Simmons-Duffin, David; Zhiboedov, Alexander

    2017-01-03

    Here, 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 also 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 thesemore » locations. Finally, we prove this statement in 1+1 dimensions by CFT methods.« less

  6. Looking for a bulk point

    NASA Astrophysics Data System (ADS)

    Maldacena, Juan; Simmons-Duffin, David; Zhiboedov, Alexander

    2017-01-01

    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.

  7. 19 CFR 149.4 - Bulk and break bulk cargo.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Importers must still report 24 hours in advance of loading any containerized or non-qualifying break bulk... this chapter that a cargo declaration be filed with Customs and Border Protection (CBP) 24 hours before... Border Protection (CBP) 24 hours before such cargo is laden aboard the vessel at the foreign port,...

  8. Activity-Dependent Human Brain Coding/Noncoding Gene Regulatory Networks

    PubMed Central

    Lipovich, Leonard; Dachet, Fabien; Cai, Juan; Bagla, Shruti; Balan, Karina; Jia, Hui; Loeb, Jeffrey A.

    2012-01-01

    While most gene transcription yields RNA transcripts that code for proteins, a sizable proportion of the genome generates RNA transcripts that do not code for proteins, but may have important regulatory functions. The brain-derived neurotrophic factor (BDNF) gene, a key regulator of neuronal activity, is overlapped by a primate-specific, antisense long noncoding RNA (lncRNA) called BDNFOS. We demonstrate reciprocal patterns of BDNF and BDNFOS transcription in highly active regions of human neocortex removed as a treatment for intractable seizures. A genome-wide analysis of activity-dependent coding and noncoding human transcription using a custom lncRNA microarray identified 1288 differentially expressed lncRNAs, of which 26 had expression profiles that matched activity-dependent coding genes and an additional 8 were adjacent to or overlapping with differentially expressed protein-coding genes. The functions of most of these protein-coding partner genes, such as ARC, include long-term potentiation, synaptic activity, and memory. The nuclear lncRNAs NEAT1, MALAT1, and RPPH1, composing an RNAse P-dependent lncRNA-maturation pathway, were also upregulated. As a means to replicate human neuronal activity, repeated depolarization of SY5Y cells resulted in sustained CREB activation and produced an inverse pattern of BDNF-BDNFOS co-expression that was not achieved with a single depolarization. RNAi-mediated knockdown of BDNFOS in human SY5Y cells increased BDNF expression, suggesting that BDNFOS directly downregulates BDNF. Temporal expression patterns of other lncRNA-messenger RNA pairs validated the effect of chronic neuronal activity on the transcriptome and implied various lncRNA regulatory mechanisms. lncRNAs, some of which are unique to primates, thus appear to have potentially important regulatory roles in activity-dependent human brain plasticity. PMID:22960213

  9. Activity-dependent transmission and integration control the timescales of auditory processing at an inhibitory synapse.

    PubMed

    Ammer, Julian J; Siveke, Ida; Felmy, Felix

    2015-06-15

    To capture the context of sensory information, neural networks must process input signals across multiple timescales. In the auditory system, a prominent change in temporal processing takes place at an inhibitory GABAergic synapse in the dorsal nucleus of the lateral lemniscus (DNLL). At this synapse, inhibition outlasts the stimulus by tens of milliseconds, such that it suppresses responses to lagging sounds, and is therefore implicated in echo suppression. Here, we untangle the cellular basis of this inhibition. We demonstrate with in vivo whole-cell patch-clamp recordings in Mongolian gerbils that the duration of inhibition increases with sound intensity. Activity-dependent spillover and asynchronous release translate the high presynaptic firing rates found in vivo into a prolonged synaptic output in acute slice recordings. A key mechanism controlling the inhibitory time course is the passive integration of the hyperpolarizing inhibitory conductance. This prolongation depends on the synaptic conductance amplitude. Computational modeling shows that this prolongation is a general mechanism and relies on a non-linear effect caused by synaptic conductance saturation when approaching the GABA reversal potential. The resulting hyperpolarization generates an efficient activity-dependent suppression of action potentials without affecting the threshold or gain of the input-output function. Taken together, the GABAergic inhibition in the DNLL is adjusted to the physiologically relevant duration by passive integration of inhibition with activity-dependent synaptic kinetics. This change in processing timescale combined with the reciprocal connectivity between the DNLLs implements a mechanism to suppress the distracting localization cues of echoes and helps to localize the initial sound source reliably.

  10. Selectivity of commonly used inhibitors of clathrin-mediated and caveolae-dependent endocytosis of G protein-coupled receptors.

    PubMed

    Guo, Shuohan; Zhang, Xiaohan; Zheng, Mei; Zhang, Xiaowei; Min, Chengchun; Wang, Zengtao; Cheon, Seung Hoon; Oak, Min-Ho; Nah, Seung-Yeol; Kim, Kyeong-Man

    2015-10-01

    Among the multiple G protein-coupled receptor (GPCR) endocytic pathways, clathrin-mediated endocytosis (CME) and caveolar endocytosis are more extensively characterized than other endocytic pathways. A number of endocytic inhibitors have been used to block CME; however, systemic studies to determine the selectivity of these inhibitors are needed. Clathrin heavy chain or caveolin1-knockdown cells have been employed to determine the specificity of various chemical and molecular biological tools for CME and caveolar endocytosis. Sucrose, concanavalin A, and dominant negative mutants of dynamin blocked other endocytic pathways, in addition to CME. In particular, concanavalin A nonspecifically interfered with the signaling of several GPCRs tested in the study. Decreased pH, monodansylcadaverine, and dominant negative mutants of epsin were more specific for CME than other treatments were. A recently introduced CME inhibitor, Pitstop2™, showed only marginal selectivity for CME and interfered with receptor expression on the cell surface. Blockade of receptor endocytosis by epsin mutants and knockdown of the clathrin heavy chain enhanced the β2AR-mediated ERK activation. Overall, our studies show that previous experimental results should be interpreted with discretion if they included the use of endocytic inhibitors that were previously thought to be CME-selective. In addition, our study shows that endocytosis of β2 adrenoceptor through clathrin-mediated pathway has negative effects on ERK activation.

  11. A dileucine motif is involved in plasma membrane expression and endocytosis of rat sodium taurocholate cotransporting polypeptide (Ntcp).

    PubMed

    Stross, Claudia; Kluge, Stefanie; Weissenberger, Katrin; Winands, Elisabeth; Häussinger, Dieter; Kubitz, Ralf

    2013-11-15

    The sodium taurocholate cotransporting polypeptide (Ntcp) is the major uptake transporter for bile salts into liver parenchymal cells, and PKC-mediated endocytosis was shown to regulate the number of Ntcp molecules at the plasma membrane. In this study, mechanisms of Ntcp internalization were analyzed by flow cytometry, immunofluorescence, and Western blot analyses in HepG2 cells. PKC activation induced endocytosis of Ntcp from the plasma membrane by ~30%. Endocytosis of Ntcp was clathrin dependent and was followed by lysosomal degradation. A dileucine motif located in the third intracellular loop of Ntcp was essential for endocytosis but also for processing and plasma membrane targeting, suggesting a dual function of this motif for intracellular trafficking of Ntcp. Mutation of two of five potential phosphorylation sites surrounding the dileucine motif (Thr225 and Ser226) inhibited PKC-mediated endocytosis. In conclusion, we could identify a motif, which is critical for Ntcp plasma membrane localization. Endocytic retrieval protects hepatocytes from elevated bile salt concentrations and is of special interest, because NTCP has been identified as a receptor for the hepatitis B and D virus.

  12. Silencing megalin and cubilin genes inhibits myeloma light chain endocytosis and ameliorates toxicity in human renal proximal tubule epithelial cells.

    PubMed

    Li, Min; Balamuthusamy, Saravanan; Simon, Eric E; Batuman, Vecihi

    2008-07-01

    Using target-specific short interfering (si) RNAs, we silenced the tandem endocytic receptors megalin and cubilin genes in cultured human renal proximal tubule epithelial cells. Transfection by siRNA resulted in up to 90% suppression of both megalin and cubilin protein and mRNA expression. In HK-2 cells exposed to kappa-light chain for up to 24 h, light chain endocytosis was reduced in either megalin- or cubilin-silenced cells markedly but incompletely. Simultaneous silencing of both the cubilin and megalin genes, however, resulted in near-complete inhibition of light chain endocytosis, as determined by measuring kappa-light chain protein concentration in cell cytoplasm and by flow cytometry using FITC-labeled kappa-light chain. In these cells, light chain-induced cytokine responses (interleukin-6 and monocyte chemoattractant protein-1) and epithelial-to-mesenchymal transition as well as the associated cellular and morphological alterations were also markedly suppressed. The results demonstrate that light chain endocytosis is predominantly mediated by the megalin-cubilin tandem endocytic receptor and identify endocytosis as a key step in light chain cytotoxicity. Blocking light chain endocytosis prevents its nephrotoxic effects on human kidney proximal tubule cells.

  13. Blocking endocytosis in Drosophila's circadian pacemaker neurons interferes with the endogenous clock in a PDF-dependent way.

    PubMed

    Wülbeck, Corinna; Grieshaber, Eva; Helfrich-Förster, Charlotte

    2009-10-01

    The neuropeptide pigment-dispersing factor (PDF) plays an essential role in the circadian clock of the fruit fly Drosophila melanogaster, but many details of PDF signaling in the clock network are still unknown. We tried to interfere with PDF signaling by blocking the GTPase Shibire in PDF neurons. Shibire is an ortholog of the mammalian Dynamins and is essential for endocytosis of clathrin-coated vesicles at the plasma membrane. Such endocytosis is used for neurotransmitter reuptake by presynaptic neurons, which is a prerequisite of synaptic vesicle recycling, and receptor-mediated endocytosis in the postsynaptic neuron, which leads to signal termination. By blocking Shibire function via overexpression of a dominant negative mutant form of Shibire in PDF neurons, we slowed down the behavioral rhythm by 3 h. This effect was absent in PDF receptor null mutants, indicating that we interfered with PDF receptor-mediated endocytosis. Because we obtained similar behavioral phenotypes by increasing the PDF level in regions close to PDF neurons, we conclude that blocking Shibire did prolong PDF signaling in the neurons that respond to PDF. Obviously, terminating the PDF signaling via receptor-mediated endocytosis is a crucial step in determining the period of behavioral rhythms.

  14. Receptor-mediated endocytosis of albumin by kidney proximal tubule cells is regulated by phosphatidylinositide 3-kinase.

    PubMed Central

    Brunskill, N J; Stuart, J; Tobin, A B; Walls, J; Nahorski, S

    1998-01-01

    Receptor-mediated endocytosis of albumin is an important function of the kidney proximal tubule epithelium. We have measured endocytosis of [125I]-albumin in opossum kidney cells and examined the regulation of this process by phosphatidylinositide 3-kinase (PI 3-kinase). Albumin endocytosis was inhibited by both wortmannin (IC50 6.9 nM) and LY294002 (IC50 6.5 microM) at concentrations that suggested the involvement of PI 3-kinase in its regulation. Recycling rates were unaffected. We transfected OK cells with either a wild-type p85 subunit of PI 3-kinase, or a dominant negative form of the p85 subunit (Deltap85) using the LacSwitch expression system. Transfects were screened by immunoblotting with anti-PI 3-kinase antibodies. Under basal conditions, transfects demonstrated no expression of p85 or Deltap85, but expression was briskly induced by treatment of the cells with IPTG (EC50 13.7 microM). Inhibition of PI 3-kinase activity by Deltap85 was confirmed by in vitro kinase assay of anti-phosphotyrosine immunoprecipitates from transfected cells stimulated with insulin. Expression of Deltap85 resulted in marked inhibition of albumin endocytosis, predominantly as a result of reduction of the Vmax of the transport process. Expression of p85 had no significant effect on albumin uptake. The results demonstrate that PI 3-kinase regulates an early step in the receptor-mediated endocytosis of albumin by kidney proximal tubular cells. PMID:9593770

  15. Notch down-regulation by endocytosis is essential for pigment cell determination and survival in the Drosophila retina.

    PubMed

    Peralta, Susana; Gómez, Yolanda; González-Gaitán, Marcos A; Moya, Fernando; Vinós, Javier

    2009-01-01

    The clathrin heavy chain is a fundamental element in endocytosis and therefore, in the internalization of several cell-surface receptors through which cells interact with their environment. Here we show that the only non-lethal mutant allele of the clathrin heavy chain identified to date in metazoans, the Drosophila Chc(4), involves the substitution of a residue at the knee region of the molecule that impairs clathrin-dependent endocytosis. We have investigated the consequences of this endocytic defect in Drosophila retinal development and found that it produces an inhibition of programmed cell death in the retinal lattice, followed by widespread death of interommatidial pigment cells once retinal development has been completed. Through genetic interactions and transgenic analyses, we show that Chc(4) phenotypes are caused by a Notch receptor gain-of-function, providing a dramatic example of the importance of Notch down-regulation by endocytosis. An increase in Notch signaling is also observed in Drosophila wings in response to the mutant clathrin, suggesting that Notch levels are controlled by clathrin-dependent endocytosis. We discuss the implications of these findings for current models on eye-development and for the role of endocytosis in Notch signaling.

  16. Adaptor Protein Complex 2–Mediated Endocytosis Is Crucial for Male Reproductive Organ Development in Arabidopsis[W

    PubMed Central

    Kim, Soo Youn; Xu, Zheng-Yi; Song, Kyungyoung; Kim, Dae Heon; Kang, Hyangju; Reichardt, Ilka; Sohn, Eun Ju; Friml, Jiří; Juergens, Gerd; Hwang, Inhwan

    2013-01-01

    Fertilization in flowering plants requires the temporal and spatial coordination of many developmental processes, including pollen production, anther dehiscence, ovule production, and pollen tube elongation. However, it remains elusive as to how this coordination occurs during reproduction. Here, we present evidence that endocytosis, involving heterotetrameric adaptor protein complex 2 (AP-2), plays a crucial role in fertilization. An Arabidopsis thaliana mutant ap2m displays multiple defects in pollen production and viability, as well as elongation of staminal filaments and pollen tubes, all of which are pivotal processes needed for fertilization. Of these abnormalities, the defects in elongation of staminal filaments and pollen tubes were partially rescued by exogenous auxin. Moreover, DR5rev:GFP (for green fluorescent protein) expression was greatly reduced in filaments and anthers in ap2m mutant plants. At the cellular level, ap2m mutants displayed defects in both endocytosis of N-(3-triethylammonium-propyl)-4-(4-diethylaminophenylhexatrienyl) pyridinium dibromide, a lypophilic dye used as an endocytosis marker, and polar localization of auxin-efflux carrier PIN FORMED2 (PIN2) in the stamen filaments. Moreover, these defects were phenocopied by treatment with Tyrphostin A23, an inhibitor of endocytosis. Based on these results, we propose that AP-2–dependent endocytosis plays a crucial role in coordinating the multiple developmental aspects of male reproductive organs by modulating cellular auxin level through the regulation of the amount and polarity of PINs. PMID:23975898

  17. DJ-1 associates with lipid rafts by palmitoylation and regulates lipid rafts-dependent endocytosis in astrocytes.

    PubMed

    Kim, Kwang Soo; Kim, Jin Soo; Park, Ji-Young; Suh, Young Ho; Jou, Ilo; Joe, Eun-Hye; Park, Sang Myun

    2013-12-01

    Parkinson's disease (PD) is the second most common progressive neurodegenerative disease. Several genes have been associated with familial type PD, providing tremendous insights into the pathogenesis of PD. Gathering evidence supports the view that these gene products may operate through common molecular pathways. Recent reports suggest that many PD-associated gene products, such as α-synuclein, LRRK2, parkin and PINK1, associate with lipid rafts and lipid rafts may be associated with neurodegeneration. Here, we observed that DJ-1 protein also associated with lipid rafts. Palmitoylation of three cysteine residues (C46/53/106) and C-terminal region of DJ-1 were required for this association. Lipopolysaccharide (LPS) induced the localization of DJ-1 into lipid rafts in astrocytes. The LPS-TLR4 signaling was more augmented in DJ-1 knock-out astrocytes by the impairment of TLR4 endocytosis. Furthermore, lipid rafts-dependent endocytosis including the endocytosis of CD14, which play a major role in regulating TLR4 endocytosis was also impaired, but clathrin-dependent endocytosis was not. This study provides a novel function of DJ-1 in lipid rafts, which may contribute the pathogenesis of PD. Moreover, it also provides the possibility that many PD-related proteins may operate through common molecular pathways in lipid rafts.

  18. Srv2/CAP is required for polarized actin cable assembly and patch internalization during clathrin-mediated endocytosis.

    PubMed

    Toshima, Junko Y; Horikomi, Chika; Okada, Asuka; Hatori, Makiko N; Nagano, Makoto; Masuda, Atsushi; Yamamoto, Wataru; Siekhaus, Daria Elisabeth; Toshima, Jiro

    2016-01-15

    The dynamic assembly and disassembly of actin filaments is essential for the formation and transport of vesicles during endocytosis. In yeast, two types of actin structures, namely cortical patches and cytoplasmic cables, play a direct role in endocytosis, but how their interaction is regulated remains unclear. Here, we show that Srv2/CAP, an evolutionarily conserved actin regulator, is required for efficient endocytosis owing to its role in the formation of the actin patches that aid initial vesicle invagination and of the actin cables that these move along. Deletion of the SRV2 gene resulted in the appearance of aberrant fragmented actin cables that frequently moved past actin patches, the sites of endocytosis. We find that the C-terminal CARP domain of Srv2p is vitally important for the proper assembly of actin patches and cables; we also demonstrate that the N-terminal helical folded domain of Srv2 is required for its localization to actin patches, specifically to the ADP-actin rich region through an interaction with cofilin. These results demonstrate the in vivo roles of Srv2p in the regulation of the actin cytoskeleton during clathrin-mediated endocytosis.

  19. Chronic insulin effects on insulin signalling and GLUT4 endocytosis are reversed by metformin.

    PubMed Central

    Pryor, P R; Liu, S C; Clark, A E; Yang, J; Holman, G D; Tosh, D

    2000-01-01

    Decreases in insulin-responsive glucose transport and associated levels of cell surface GLUT4 occur in rat adipocytes maintained in culture for 20 h under hyperinsulinaemic and hyperglycaemic conditions. We have investigated whether this defect is due to reduced signalling from the insulin receptor, GLUT4 expression or impaired GLUT4 trafficking. The effects of chronic insulin treatment on glucose transport and GLUT4 trafficking were ameliorated by inclusion of metformin in the culture medium. In comparison with the ic insulin treatment attenuated changes in signalling processes leading to glucose transport. These included insulin receptor tyrosine phosphorylation, phosphoinositide 3-kinase activity and Akt activity, which were all reduced by 60-70%. Inclusion of metformin in the culture medium prevented the effects of the chronic insulin treatment on these signalling processes. In comparison with cells maintained in culture without insulin, the total expression of GLUT4 protein was not significantly altered by chronic insulin treatment, although the level of GLUT1 expression was increased. Trafficking rate constants for wortmannin-induced cell-surface loss of GLUT4 and GLUT1 were assessed by 2-N-4-(1-azi-2, 2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannose-4-yloxy)-2-propyla min e (ATB-BMPA) photolabelling. In comparison with cells acutely treated with insulin, chronic insulin treatment resulted in a doubling of the rate constants for GLUT4 endocytosis. These results suggest that the GLUT4 endocytosis process is very sensitive to the perturbations in signalling that occur under hyperinsulinaemic and hyperglycaemic conditions, and that the resulting elevation of endocytosis accounts for the reduced levels of net GLUT4 translocation observed. PMID:10794717

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

    PubMed

    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.

  1. Involvement of BLT1 endocytosis and Yes kinase activation in leukotriene B4-induced neutrophil degranulation.

    PubMed

    Gaudreault, Eric; Thompson, Charles; Stankova, Jana; Rola-Pleszczynski, Marek

    2005-03-15

    One of the important biological activities of human neutrophils is degranulation, which can be induced by leukotriene B4 (LTB4). Here we investigated the intracellular signaling events involved in neutrophil degranulation mediated by the high affinity LTB4 receptor, BLT1. Peripheral blood neutrophils as well as the promyeloid PLB-985 cell line, stably transfected with BLT1 cDNA and differentiated into a neutrophil-like cell phenotype, were used throughout this study. LTB4-induced enzyme release was inhibited by 50-80% when cells were pretreated with the pharmacological inhibitors of endocytosis sucrose, Con A and NH4Cl. In addition, transient transfection with a dominant negative form of dynamin (K44A) resulted in approximately 70% inhibition of ligand-induced degranulation. Pretreating neutrophils or BLT1-expressing PLB-985 cells with the Src family kinase inhibitor PP1 resulted in a 30-60% inhibition in BLT1-mediated degranulation. Yes kinase, but not c-Src, Fgr, Hck, or Lyn, was found to exhibit up-regulated kinase activity after LTB4 stimulation. Moreover, BLT1 endocytosis was found to be necessary for Yes kinase activation in neutrophils. LTB4-induced degranulation was also sensitive to inhibition of PI3K. In contrast, it was not affected by inhibition of the mitogen-activated protein kinase MEK kinase, the Janus kinases, or the receptor tyrosine kinase epidermal growth factor receptor or platelet-derived growth factor receptor. Taken together, our results suggest an essential role for BLT1 endocytosis and Yes kinase activation in LTB4-mediated degranulation of human neutrophils.

  2. Role of Endothelial Cell Septin 7 in the Endocytosis of Candida albicans

    PubMed Central

    Phan, Quynh T.; Eng, David K.; Mostowy, Serge; Park, Hyunsook; Cossart, Pascale; Filler, Scott G.

    2013-01-01

    ABSTRACT Candida albicans invades endothelial cells by binding to N-cadherin and other cell surface receptors. This binding induces rearrangement of endothelial cell actin microfilaments, which results in the formation of pseudopods that surround the organism and pull it into the endothelial cell. Here, we investigated the role of endothelial cell septin 7 (SEPT7) in the endocytosis of C. albicans hyphae. Using confocal microscopy, we determined that SEPT7 accumulated with N-cadherin and actin microfilaments around C. albicans as it was endocytosed by endothelial cells. Affinity purification studies indicated that a complex containing N-cadherin and SEPT7 was recruited by C. albicans and that formation of this complex around C. albicans was mediated by the fungal Als3 and Ssa1 invasins. Knockdown of N-cadherin by small interfering RNA (siRNA) reduced recruitment of SEPT7 to C. albicans, suggesting that N-cadherin functions as a link between SEPT7 and the fungus. Also, depolymerization of actin microfilaments with cytochalasin D decreased the association between SEPT7 and N-cadherin and inhibited recruitment of both SEPT7 and N-cadherin to C. albicans, indicating the necessity of an intact cytoskeleton in the functional interaction between SEPT7 and N-cadherin. Importantly, knockdown of SEPT7 decreased accumulation of N-cadherin around C. albicans in intact endothelial cells and reduced binding of N-cadherin to this organism, as revealed by the affinity purification assay. Furthermore, SEPT7 knockdown significantly inhibited the endocytosis of C. albicans. Therefore, in response to C. albicans infection, SEPT7 forms a complex with endothelial cell N-cadherin, is required for normal accumulation of N-cadherin around C. albicans hyphae, and is necessary for maximal endocytosis of the organism. PMID:24345743

  3. High affinity nanobodies against the Trypanosome brucei VSG are potent trypanolytic agents that block endocytosis.

    PubMed

    Stijlemans, Benoît; Caljon, Guy; Natesan, Senthil Kumar A; Saerens, Dirk; Conrath, Katja; Pérez-Morga, David; Skepper, Jeremy N; Nikolaou, Alexandros; Brys, Lea; Pays, Etienne; Magez, Stefan; Field, Mark C; De Baetselier, Patrick; Muyldermans, Serge

    2011-06-01

    The African trypanosome Trypanosoma brucei, which persists within the bloodstream of the mammalian host, has evolved potent mechanisms for immune evasion. Specifically, antigenic variation of the variant-specific surface glycoprotein (VSG) and a highly active endocytosis and recycling of the surface coat efficiently delay killing mediated by anti-VSG antibodies. Consequently, conventional VSG-specific intact immunoglobulins are non-trypanocidal in the absence of complement. In sharp contrast, monovalent antigen-binding fragments, including 15 kDa nanobodies (Nb) derived from camelid heavy-chain antibodies (HCAbs) recognizing variant-specific VSG epitopes, efficiently lyse trypanosomes both in vitro and in vivo. This Nb-mediated lysis is preceded by very rapid immobilisation of the parasites, massive enlargement of the flagellar pocket and major blockade of endocytosis. This is accompanied by severe metabolic perturbations reflected by reduced intracellular ATP-levels and loss of mitochondrial membrane potential, culminating in cell death. Modification of anti-VSG Nbs through site-directed mutagenesis and by reconstitution into HCAbs, combined with unveiling of trypanolytic activity from intact immunoglobulins by papain proteolysis, demonstrates that the trypanolytic activity of Nbs and Fabs requires low molecular weight, monovalency and high affinity. We propose that the generation of low molecular weight VSG-specific trypanolytic nanobodies that impede endocytosis offers a new opportunity for developing novel trypanosomiasis therapeutics. In addition, these data suggest that the antigen-binding domain of an anti-microbial antibody harbours biological functionality that is latent in the intact immunoglobulin and is revealed only upon release of the antigen-binding fragment.

  4. Folic acid-capped PEGylated magnetic nanoparticles enter cancer cells mostly via clathrin-dependent endocytosis.

    PubMed

    Allard-Vannier, Emilie; Hervé-Aubert, Katel; Kaaki, Karine; Blondy, Thibaut; Shebanova, Anastasia; Shaitan, Konstantin V; Ignatova, Anastasia A; Saboungi, Marie-Louise; Feofanov, Alexey V; Chourpa, Igor

    2017-06-01

    This work is focused on mechanisms of uptake in cancer cells of rationally designed, covalently assembled nanoparticles, made of superparamagnetic iron oxide nanoparticles (SPIONs), fluorophores (doxorubicin or Nile Blue), polyethylene glycol (PEG) and folic acid (FA), referred hereinafter as SFP-FA. SFP-FA were characterized by DLS, zetametry and fluorescence spectroscopy. The SFP-FA uptake in cancer cells was monitored using fluorescence-based methods like fluorescence-assisted cell sorting, CLSM with single-photon and two-photon excitation. The SFP-FA endocytosis was also analyzed with electron microscopy approaches: TEM, HAADF-STEM and EELS. The SFP-FA have zeta potential below -6mW and stable hydrodynamic diameter close to 100nm in aqueous suspensions of pH range from 5 to 8. They contain ca. 109 PEG-FA, 480 PEG-OCH3 and 22-27 fluorophore molecules per SPION. The fluorophores protected under the PEG shell allows a reliable detection of intracellular NPs. SFP-FA readily enter into all the cancer cell lines studied and accumulate in lysosomes, mostly via clathrin-dependent endocytosis, whatever the FR status on the cells. The present study highlights the advantages of rational design of nanosystems as well as the possible involvement of direct molecular interactions of PEG and FA with cellular membranes, not limited to FA-FR recognition, in the mechanisms of their endocytosis. Composition, magnetic and optical properties of the SFP-FA as well their ability to enter cancer cells are promising for their applications in cancer theranosis. Combination of complementary analytical approaches is relevant to understand the nanoparticles behavior in suspension and in contact with cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Monomeric IgG Is Neuroprotective via Enhancing Microglial Recycling Endocytosis and TNF-α

    PubMed Central

    Hulse, Raymond E.; Swenson, Wade G.; Kunkler, Phillip E.; White, David M.; Kraig, Richard P.

    2009-01-01

    In brain, monomeric immunoglobin G (IgG) is regarded as quiescent and only poised to initiate potentially injurious inflammatory reactions via immune complex formation associated with phagocytosis and tumor necrosis factor α (TNF-α) production in response to disease. Using rat hippocampal slice and microglial cultures, here we show instead that physiological levels (i.e., 0.2−20 μg/ml) of monomeric IgG unassociated with disease triggered benign low-level proinflammatory signaling that was neuroprotective against CA1 area excitotoxicity and followed a U-shaped or hormetic dose–response. The data indicate that physiological IgG levels activated micro-glia by enhancing recycling endocytosis plus TNF-α release from these cells to produce the neuroprotection. Minocycline, known for its anti-inflammatory and neuroprotective effects when given after disease onset, abrogated IgG-mediated neuroprotection and related microglial effects when given before injury. In contrast, E-prostanoid receptor subtype 2 (EP2) activation, which served as an exemplary paracrine stimulus like the one expected from neuronal activity, amplified IgG-mediated increased microglial recycling endocytosis and TNF-α production. Furthermore, like monomeric IgG these EP2 related effects took days to be effective, suggesting both were adaptive anabolic effects consistent with those seen from other long-term preconditioning stimuli requiring de novo protein synthesis. The data provide the first evidence that brain monomeric IgG at physiological levels can have signaling function via enhanced recycling endocytosis/TNF-α production from microglia unassociated with disease and that these IgG-mediated changes may be a means by which paracrine signaling from neuronal activity influences microglia to evoke neuroprotection. The data provide further support that low-level proinflammatory neural immune signaling unassociated with disease enhances brain function. PMID:19020014

  6. Endocytosis of a maltose permease is induced when amylolytic enzyme production is repressed in Aspergillus oryzae.

    PubMed

    Hiramoto, Tetsuya; Tanaka, Mizuki; Ichikawa, Takanori; Matsuura, Yuka; Hasegawa-Shiro, Sachiko; Shintani, Takahiro; Gomi, Katsuya

    2015-09-01

    In the filamentous fungus Aspergillus oryzae, amylolytic enzyme production is induced by the presence of maltose. Previously, we identified a putative maltose permease (MalP) gene in the maltose-utilizing cluster of A. oryzae. malP disruption causes a significant decrease in α-amylase activity and maltose consumption, indicating that MalP is a maltose transporter required for amylolytic enzyme production in A. oryzae. Although the expression of amylase genes and malP is repressed by the presence of glucose, the effect of glucose on the abundance of functional MalP is unknown. In this study, we examined the effect of glucose and other carbon sources on the subcellular localization of green fluorescence protein (GFP)-tagged MalP. After glucose addition, GFP-MalP at the plasma membrane was internalized and delivered to the vacuole. This glucose-induced internalization of GFP-MalP was inhibited by treatment with latrunculin B, an inhibitor of actin polymerization. Furthermore, GFP-MalP internalization was inhibited by repressing the HECT ubiquitin ligase HulA (ortholog of yeast Rsp5). These results suggest that MalP is transported to the vacuole by endocytosis in the presence of glucose. Besides glucose, mannose and 2-deoxyglucose also induced the endocytosis of GFP-MalP and amylolytic enzyme production was inhibited by the addition of these sugars. However, neither the subcellular localization of GFP-MalP nor amylolytic enzyme production was influenced by the addition of xylose or 3-O-methylglucose. These results imply that MalP endocytosis is induced when amylolytic enzyme production is repressed.

  7. Endocytosis and Vacuolar Degradation of the Yeast Cell Surface Glucose Sensors Rgt2 and Snf3*

    PubMed Central

    Roy, Adhiraj; Kim, Jeong-Ho

    2014-01-01

    Sensing and signaling the presence of extracellular glucose is crucial for the yeast Saccharomyces cerevisiae because of its fermentative metabolism, characterized by high glucose flux through glycolysis. The yeast senses glucose through the cell surface glucose sensors Rgt2 and Snf3, which serve as glucose receptors that generate the signal for induction of genes involved in glucose uptake and metabolism. Rgt2 and Snf3 detect high and low glucose concentrations, respectively, perhaps because of their different affinities for glucose. Here, we provide evidence that cell surface levels of glucose sensors are regulated by ubiquitination and degradation. The glucose sensors are removed from the plasma membrane through endocytosis and targeted to the vacuole for degradation upon glucose depletion. The turnover of the glucose sensors is inhibited in endocytosis defective mutants, and the sensor proteins with a mutation at their putative ubiquitin-acceptor lysine residues are resistant to degradation. Of note, the low affinity glucose sensor Rgt2 remains stable only in high glucose grown cells, and the high affinity glucose sensor Snf3 is stable only in cells grown in low glucose. In addition, constitutively active, signaling forms of glucose sensors do not undergo endocytosis, whereas signaling defective sensors are constitutively targeted for degradation, suggesting that the stability of the glucose sensors may be associated with their ability to sense glucose. Therefore, our findings demonstrate that the amount of glucose available dictates the cell surface levels of the glucose sensors and that the regulation of glucose sensors by glucose concentration may enable yeast cells to maintain glucose sensing activity at the cell surface over a wide range of glucose concentrations. PMID:24451370

  8. REL - English Bulk Data Input.

    ERIC Educational Resources Information Center

    Bigelow, Richard Henry

    A bulk data input processor which is available for the Rapidly Extensible Language (REL) English versions is described. In REL English versions, statements that declare names of data items and their interrelationships normally are lines from a terminal or cards in a batch input stream. These statements provide a convenient means of declaring some…

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

    SciTech Connect

    Diederich, Sandra; Thiel, Lena; Maisner, Andrea

    2008-06-05

    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.

  10. Entry of Bombyx mori nucleopolyhedrovirus into BmN cells by cholesterol-dependent macropinocytic endocytosis.

    PubMed

    Huang, Jinshan; Hao, Bifang; Cheng, Chen; Liang, Fei; Shen, Xingjia; Cheng, Xiaowen

    2014-10-10

    Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious viral pathogen of silkworm, and no drug or specific protection against BmNPV infection is available at present time. Although functions of most BmNPV genes were depicted in recent years, knowledge on the mechanism of BmNPV entry into insect cells is still limited. Here BmNPV cell entry mechanism is investigated by different endocytic inhibitor application and subcellular analysis. Results indicated that BmNPV enters BmN cells by clathrin-independent macropinocytic endocytosis, which is mediated by cholesterol in a dose-dependent manner, and cholesterol replenishment rescued the BmNPV infection partially.

  11. Inactivation of clathrin heavy chain inhibits synaptic recycling but allows bulk membrane uptake

    PubMed Central

    Kasprowicz, Jaroslaw; Kuenen, Sabine; Miskiewicz, Katarzyna; Habets, Ron L.P.; Smitz, Liesbet; Verstreken, Patrik

    2008-01-01

    Synaptic vesicle reformation depends on clathrin, an abundant protein that polymerizes around newly forming vesicles. However, how clathrin is involved in synaptic recycling in vivo remains unresolved. We test clathrin function during synaptic endocytosis using clathrin heavy chain (chc) mutants combined with chc photoinactivation to circumvent early embryonic lethality associated with chc mutations in multicellular organisms. Acute inactivation of chc at stimulated synapses leads to substantial membrane internalization visualized by live dye uptake and electron microscopy. However, chc-inactivated membrane cannot recycle and participate in vesicle release, resulting in a dramatic defect in neurotransmission maintenance during intense synaptic activity. Furthermore, inactivation of chc in the context of other endocytic mutations results in membrane uptake. Our data not only indicate that chc is critical for synaptic vesicle recycling but they also show that in the absence of the protein, bulk retrieval mediates massive synaptic membrane internalization. PMID:18762582

  12. Reduced Activity-Dependent Protein Levels in a Mouse Model of the Fragile X Premutation

    PubMed Central

    von Leden, Ramona E.; Curley, Lindsey C.; Greenberg, Gian D.; Hunsaker, Michael R.; Willemsen, Rob; Berman, Robert F.

    2014-01-01

    Environmental enrichment results in increased levels of Fmrp in brain and increased dendritic complexity. The present experiment evaluated activity-dependent increases in Fmrp levels in the motor cortex in response to training on a skilled forelimb reaching task in the CGG KI mouse model of the fragile X premutation. Fmrp, Arc, and c-Fos protein levels were quantified by Western blot in the contralateral motor cortex of mice following training to reach for sucrose pellets with a non-preferred paw and compared to levels in the ipsilateral motor cortex. After training, all mice showed increases in Fmrp, Arc, and c-Fos protein levels in the contralateral compared to the ipsilateral hemisphere; however, the increase in CGG KI mice was less than wildtype mice. Increases in Fmrp and Arc proteins scaled with learning, whereas this relationship was not observed with the c-Fos levels. These data suggest the possibility that reduced levels of activity-dependent proteins associated with synaptic plasticity such as Fmrp and Arc may contribute to the neurocognitive phenotype reported in the CGG KI mice and the fragile X premutation. PMID:24462720

  13. Evidence supporting the existence of an activity-dependent astrocyte-neuron lactate shuttle.

    PubMed

    Pellerin, L; Pellegri, G; Bittar, P G; Charnay, Y; Bouras, C; Martin, J L; Stella, N; Magistretti, P J

    1998-01-01

    Mounting evidence from in vitro experiments indicates that lactate is an efficient energy substrate for neurons and that it may significantly contribute to maintain synaptic transmission, particularly during periods of intense activity. Since lactate does not cross the blood-brain barrier easily, blood-borne lactate cannot be a significant source. In vitro studies by several laboratories indicate that astrocytes release large amounts of lactate. In 1994, we proposed a mechanism whereby lactate could be produced by astrocytes in an activity-dependent, glutamate-mediated manner. Over the last 2 years we have obtained further evidence supporting the notion that a transfer of lactate from astrocytes to neurons might indeed take place. In this article, we first review data showing the presence of mRNA encoding for two monocarboxylate transporters, MCT1 and MCT2, in the adult mouse brain. Second, by using monoclonal antibodies selectively directed against the two distinct lactate dehydrogenase isoforms, LDH1 and LDH5, a specific cellular distribution between neurons and astrocytes is revealed which suggests that a population of astrocytes is a lactate 'source' while neurons may be a lactate 'sink'. Third, we provide biochemical evidence that lactate is interchangeable with glucose to support oxidative metabolism in cortical neurons. This set of data is consistent with the existence of an activity-dependent astrocyte-neuron lactate shuttle for the supply of energy substrates to neurons.

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

    PubMed Central

    Li, Hui; 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 a series of permanently charged capsaicinoids and found that one, cap-ET, efficaciously evoked TRPV1-dependent entry of Ca2+ or the large cationic dye YO-PRO-1 comparably to capsaicin, but far smaller electrical currents. Cap-ET–induced YO-PRO-1 transport required permeation of both the agonist and the dye through the TRPV1 pore and could be enhanced by kinase activation or oxidative covalent modification. Moreover, cap-ET reduced capsaicin-induced currents by a voltage-dependent block of the pore. A low dose of cap-ET elicited entry of permanently charged Na+ channel blockers to effectively suppress Na+ currents in sensory neurons presensitized with oxidative chemicals. These results implicate therapeutic potential of these unique TRPV1 agonists exhibiting activity-dependent ion transport but of minimal pain-producing risks. PMID:21536874