Browse
Submit Data
Databases
API
Help

Dataset Information

33 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Epsin-Dependent Ligand Endocytosis Activates Notch by Force.

ABSTRACT: DSL ligands activate Notch by inducing proteolytic cleavage of the receptor ectodomain, an event that requires ligand to be endocytosed in signal-sending cells by the adaptor protein Epsin. Two classes of explanation for this unusual requirement are (1) recycling models, in which the ligand must be endocytosed to be modified or repositioned before it binds Notch and (2) pulling models, in which the ligand must be endocytosed after it binds Notch to exert force that exposes an otherwise buried site for cleavage. We demonstrate in vivo that ligands that cannot enter the Epsin pathway nevertheless bind Notch but fail to activate the receptor because they cannot exert sufficient force. This argues against recycling models and in favor of pulling models. Our results also suggest that once ligand binds receptor, activation depends on a competition between Epsin-mediated ligand endocytosis, which induces cleavage, and transendocytosis of the ligand by the receptor, which aborts the incipient signal.

SUBMITTER: Langridge PD

PROVIDER: S-EPMC6219616 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Epsin-Dependent Ligand Endocytosis Activates Notch by Force.

Langridge Paul D PD Struhl Gary G

Cell 20171101 6

DSL ligands activate Notch by inducing proteolytic cleavage of the receptor ectodomain, an event that requires ligand to be endocytosed in signal-sending cells by the adaptor protein Epsin. Two classes of explanation for this unusual requirement are (1) recycling models, in which the ligand must be endocytosed to be modified or repositioned before it binds Notch and (2) pulling models, in which the ligand must be endocytosed after it binds Notch to exert force that exposes an otherwise buried si ...[more]

PMID: 29195077

Similar Datasets

Notch ligand endocytosis generates mechanical pulling force dependent on dynamin, epsins, and actin.

Project description: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 that Notch ligands require endocytosis, ubiquitylation, and epsin endocytic adaptors to activate signaling, but the exact role of ligand endocytosis 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 that the mechanical pulling force produced by endocytosis of Notch-bound ligand drives conformational changes in Notch that permit activating proteolysis.

| S-EPMC3400432 | biostudies-literature

Notch promotes dynamin-dependent endocytosis of nephrin.

Project description:Notch signaling in podocytes causes proteinuria and glomerulosclerosis in humans and rodents, but the underlying mechanism remains unknown. Here, we analyzed morphologic, molecular, and cellular events before the onset of proteinuria in newborn transgenic mice that express activated Notch in podocytes. Immunohistochemistry revealed a loss of the slit diaphragm protein nephrin exclusively in podocytes expressing activated Notch. Podocyte-specific deletion of Rbpj, which is essential for canonical Notch signaling, prevented this loss of nephrin. Overexpression of activated Notch decreased cell surface nephrin and increased cytoplasmic nephrin in transfected HEK293T cells; pharmacologic inhibition of dynamin, but not depletion of cholesterol, blocked these effects on nephrin, suggesting that Notch promotes dynamin-dependent, raft-independent endocytosis of nephrin. Supporting an association between Notch signaling and nephrin trafficking, electron microscopy revealed shortened podocyte foot processes and fewer slit diaphragms among the transgenic mice compared with controls. These data suggest that Notch signaling induces endocytosis of nephrin, thereby triggering the onset of proteinuria.

| S-EPMC3269920 | biostudies-literature

Role of epsin 1 in synaptic vesicle endocytosis.

Project description:Epsin has been suggested to act as an alternate adaptor in several endocytic pathways. Its role in synaptic vesicle recycling remains, however, unclear. Here, we examined the role of epsin in this process by using the lamprey reticulospinal synapse as a model system. We characterized a lamprey ortholog of epsin 1 and showed that it is accumulated at release sites at rest and also at clathrin-coated pits in the periactive zone during synaptic activity. Disruption of epsin interactions, by presynaptic microinjection of antibodies to either the epsin-N-terminal homology domain (ENTH) or the clathrin/AP2 binding region (CLAP), caused profound loss of vesicles in stimulated synapses. CLAP antibody-injected synapses displayed a massive accumulation of distorted coated structures, including coated vacuoles, whereas in synapses perturbed with ENTH antibodies, very few coated structures were found. In both cases coated pits on the plasma membrane showed a shift to early intermediates (shallow coated pits) and an increase in size. Moreover, in CLAP antibody-injected synapses flat clathrin-coated patches occurred on the plasma membrane. We conclude that epsin is involved in clathrin-mediated synaptic vesicle endocytosis. Our results support a model, based on in vitro studies, suggesting that epsin coordinates curvature generation with coat assembly and further indicating that epsin limits clathrin coat assembly to the size of newly formed vesicles. We propose that these functions of epsin 1 provide an additional mechanism for generation of uniformly sized synaptic vesicles.

| S-EPMC2359800 | biostudies-literature

Optical tweezers studies on Notch: single-molecule interaction strength is independent of ligand endocytosis.

Project description:Notch signaling controls diverse cellular processes critical to development and disease. Cell surface ligands bind Notch on neighboring cells but 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.

| S-EPMC3376165 | biostudies-literature

Dynamin-dependent amino acid endocytosis activates mechanistic target of rapamycin complex 1 (mTORC1).

Project description:The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of protein synthesis and potential target for modifying cellular metabolism in various conditions, including cancer and aging. mTORC1 activity is tightly regulated by the availability of extracellular amino acids, and previous studies have revealed that amino acids in the extracellular fluid are transported to the lysosomal lumen. There, amino acids induce recruitment of cytoplasmic mTORC1 to the lysosome by the Rag GTPases, followed by mTORC1 activation by the small GTPase Ras homolog enriched in brain (Rheb). However, how the extracellular amino acids reach the lysosomal lumen and activate mTORC1 remains unclear. Here, we show that amino acid uptake by dynamin-dependent endocytosis plays a critical role in mTORC1 activation. We found that mTORC1 is inactivated when endocytosis is inhibited by overexpression of a dominant-negative form of dynamin 2 or by pharmacological inhibition of dynamin or clathrin. Consistently, the recruitment of mTORC1 to the lysosome was suppressed by the dynamin inhibition. The activity and lysosomal recruitment of mTORC1 were rescued by increasing intracellular amino acids via cycloheximide exposure or by Rag overexpression, indicating that amino acid deprivation is the main cause of mTORC1 inactivation via the dynamin inhibition. We further show that endocytosis inhibition does not induce autophagy even though mTORC1 inactivation is known to strongly induce autophagy. These findings open new perspectives for the use of endocytosis inhibitors as potential agents that can effectively inhibit nutrient utilization and shut down the upstream signals that activate mTORC1.

| S-EPMC5672031 | biostudies-literature

Embryonic arrest at midgestation and disruption of Notch signaling produced by the absence of both epsin 1 and epsin 2 in mice.

Project description:Epsins are endocytic adaptors with putative functions in general aspects of clathrin-mediated endocytosis as well as in the internalization of specific membrane proteins. We have now tested the role of the ubiquitously expressed epsin genes, Epn1 and Epn2, by a genetic approach in mice. While either gene is dispensable for life, their combined inactivation results in embryonic lethality at E9.5-E10, i.e., at the beginning of organogenesis. Consistent with studies in Drosophila, where epsin endocytic function was linked to Notch activation, developmental defects observed in epsin 1/2 double knockout (DKO) embryos recapitulated those produced by a global impairment of Notch signaling. Accordingly, expression of Notch primary target genes was severely reduced in DKO embryos. However, housekeeping forms of clathrin-mediated endocytosis were not impaired in cells derived from these embryos. These findings support a role of epsin as a specialized endocytic adaptor, with a critical role in the activation of Notch signaling in mammals.

| S-EPMC2728981 | biostudies-literature

Interaction between Epsin/Yap180 adaptors and the scaffolds Ede1/Pan1 is required for endocytosis.

Project description:The spatial and temporal regulation of the interactions among the approximately 60 proteins required for endocytosis is under active investigation in many laboratories. We have identified the interaction between monomeric clathrin adaptors and endocytic scaffold proteins as a critical prerequisite for the recruitment and/or spatiotemporal dynamics of endocytic proteins at early and late stages of internalization. Quadruple deletion yeast cells (DeltaDeltaDeltaDelta) lacking four putative adaptors, Ent1/2 and Yap1801/2 (homologues of epsin and AP180/CALM proteins), with a plasmid encoding Ent1 or Yap1802 mutants, have defects in endocytosis and growth at 37 degrees C. Live-cell imaging revealed that the dynamics of the early- and late-acting scaffold proteins Ede1 and Pan1, respectively, depend upon adaptor interactions mediated by adaptor asparagine-proline-phenylalanine motifs binding to scaffold Eps15 homology domains. These results suggest that adaptor/scaffold interactions regulate transitions from early to late events and that clathrin adaptor/scaffold protein interaction is essential for clathrin-mediated endocytosis.

| S-EPMC2441688 | biostudies-literature

Notch-1 activates estrogen receptor-alpha-dependent transcription via IKKalpha in breast cancer cells.

Project description:Approximately 80% of breast cancers express the estrogen receptor-alpha (ERalpha) and are treated with anti-estrogens. Resistance to these agents is a major cause of mortality. We have shown that estrogen inhibits Notch, whereas anti-estrogens or estrogen withdrawal activate Notch signaling. Combined inhibition of Notch and estrogen signaling has synergistic effects in ERalpha-positive breast cancer models. However, the mechanisms whereby Notch-1 promotes the growth of ERalpha-positive breast cancer cells are unknown. Here, we demonstrate that Notch-1 increases the transcription of ERalpha-responsive genes in the presence or absence of estrogen via a novel chromatin crosstalk mechanism. Our data support a model in which Notch-1 can activate the transcription of ERalpha-target genes via IKKalpha-dependent cooperative chromatin recruitment of Notch-CSL-MAML1 transcriptional complexes (NTC) and ERalpha, which promotes the recruitment of p300. CSL binding elements frequently occur in close proximity to estrogen-responsive elements (EREs) in the human and mouse genomes. Our observations suggest that a hitherto unknown Notch-1/ERalpha chromatin crosstalk mediates Notch signaling effects in ERalpha-positive breast cancer cells and contributes to regulate the transcriptional functions of ERalpha itself.

| S-EPMC4976641 | biostudies-literature

Epsin 1 is a cargo-specific adaptor for the clathrin-mediated endocytosis of the influenza virus.

Project description:During clathrin-mediated endocytosis, adaptor proteins recognize specific internalization signals on cargo receptors, either recruiting cargos into clathrin-coated pits (CCPs) or initiating clathrin-coat assembly around the cargo molecules. Here, we identify epsin 1, a clathrin-, ubiquitin-, and phospholipid-interacting protein, as a cargo-specific adaptor for influenza virus entry through the clathrin-mediated pathway. Using live-cell imaging to monitor the entry of individual virus particles, we observed recruitment of epsin 1 to the binding sites of influenza viruses in synchrony with the assembly of CCPs. Epsin 1 knockdown by siRNA significantly inhibited the clathrin-mediated endocytosis of the influenza virus and caused the majority of the virus particles to enter through a clathrin-independent pathway. The same treatment did not affect the entry of several classical ligands for clathrin-mediated endocytosis, including transferrin, LDL, and EGF. Overexpression of the dominant-negative epsin 1 mutant lacking the ubiquitin-interaction motifs nearly completely blocked the clathrin-mediated entry of the influenza virus without affecting transferrin uptake. These results suggest that epsin 1 functions as a cargo-specific adaptor for the clathrin-mediated entry of the influenza virus.

| S-EPMC2504482 | biostudies-literature

Complimentary action of structured and unstructured domains of epsin supports clathrin-mediated endocytosis at high tension.

Project description:Membrane tension plays an inhibitory role in clathrin-mediated endocytosis (CME) by impeding the transition of flat plasma membrane to hemispherical clathrin-coated structures (CCSs). Membrane tension also impedes the transition of hemispherical domes to omega-shaped CCSs. However, CME is not completely halted in cells under high tension conditions. Here we find that epsin, a membrane bending protein which inserts its N-terminus H0 helix into lipid bilayer, supports flat-to-dome transition of a CCS and stabilizes its curvature at high tension. This discovery is supported by molecular dynamic simulation of the epsin N-terminal homology (ENTH) domain that becomes more structured when embedded in a lipid bilayer. In addition, epsin has an intrinsically disordered protein (IDP) C-terminus domain which induces membrane curvature via steric repulsion. Insertion of H0 helix into lipid bilayer is not sufficient for stable epsin recruitment. Epsin's binding to adaptor protein 2 and clathrin is critical for epsin's association with CCSs under high tension conditions, supporting the importance of multivalent interactions in CCSs. Together, our results support a model where the ENTH and unstructured IDP region of epsin have complementary roles to ensure CME initiation and CCS maturation are unimpeded under high tension environments.

| S-EPMC7722716 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data