text:
Drosophila Epsin's role in Notch ligand cells requires three Epsin protein functions: the lipid binding function of the ENTH domain, a single Ubiquitin interaction motif, and a subset of the C-terminal protein binding modules.
ABSTRACT Epsin is an endocytic protein that binds Clathrin, the plasma membrane, Ubiquitin, and also a variety of other endocytic proteins through well-characterized motifs. Although Epsin is a general endocytic factor, genetic analysis in Drosophila and mice revealed that Epsin is essential specifically for internalization of ubiquitinated transmembrane ligands of the Notch receptor, a process required for Notch activation. Epsin's mechanism of function is complex and context-dependent. Consequently, how Epsin promotes ligand endocytosis and thus Notch signaling is unclear, as is why Notch signaling is uniquely dependent on Epsin. Here, by generating Drosophila lines containing transgenes that express a variety of different Epsin deletion and substitution variants, we tested each of the five protein or lipid interaction modules for a role in Notch activation by each of the two ligands, Serrate and Delta. There are five main results of this work that impact present thinking about the role of Epsin in ligand cells. First, we discovered that deletion or mutation of both UIMs destroyed Epsin's function in Notch signaling and had a greater negative impact on Epsin activity than removal of any other module type. Second, only one of Epsin's two UIMs was essential. Third, the lipid-binding function of the ENTH domain was required only for maximal Epsin activity. Fourth, although the C-terminal Epsin modules that interact with Clathrin, the adapter protein complex AP-2, or endocytic accessory proteins were necessary collectively for Epsin activity, their functions were highly redundant; most unexpected was the finding that Epsin's Clathrin binding motifs were dispensable. Finally, we found that signaling from either ligand, Serrate or Delta, required the same Epsin modules. All of these observations are consistent with a model where Epsin's essential function in ligand cells is to link ubiquitinated Notch ligands to Clathrin-coated vesicles through other Clathrin adapter proteins. We propose that Epsin's specificity for Notch signaling simply reflects its unique ability to interact with the plasma membrane, Ubiquitin, and proteins that bind Clathrin.
- Mirko Messa, Ruben Fernandez-Busnadiego, Elizabeth Wen Sun, Hong Chen, Heather Czapla, Kristie Wrasman, Yumei Wu, Genevieve Ko, Theodora Ross, Beverly Wendland, Pietro De Camilli[Show abstract] [Hide abstract]
ABSTRACT: Epsin is an evolutionarily conserved endocytic clathrin adaptor whose most critical function(s) in clathrin coat dynamics remain(s) elusive. To elucidate such function(s), we generated embryonic fibroblasts from conditional epsin triple KO mice. Triple KO cells displayed a dramatic cell division defect. Additionally, a robust impairment in clathrin-mediated endocytosis was observed, with an accumulation of early and U-shaped pits. This defect correlated with a perturbation of the coupling between the clathrin coat and the actin cytoskeleton, which we confirmed in a cell-free assay of endocytosis. Our results indicate that a key evolutionary conserved function of epsin, in addition to other roles that include as we show here a low affinity interaction with SNAREs, is to help generate the force that leads to invagination and then fission of clathrin-coated pits.eLife Sciences 08/2014; · 8.52 Impact Factor - SourceAvailable from: europepmc.org[Show abstract] [Hide abstract]
ABSTRACT: Low density lipoprotein receptor (LDLR) internalization clears cholesterol-laden LDL particles from circulation in humans. Defects in clathrin-dependent LDLR endocytosis promote elevated serum cholesterol levels and can lead to atherosclerosis. However, our understanding of the mechanisms that control LDLR uptake remains incomplete. To identify factors critical to LDLR uptake, we pursued a genome-wide RNAi screen using Caenorhabditis elegans LRP-1/megalin as a model for LDLR transport. In doing so, we discovered an unanticipated requirement for the clathrin-binding endocytic adaptor epsin1 in LDLR endocytosis. Epsin1 depletion reduced LDLR internalization rates in mammalian cells, similar to that observed following clathrin depletion. Genetic and biochemical analyses of epsin in C. elegans and mammalian cells uncovered a requirement for the ubiquitin-interaction motif (UIM) as critical for receptor transport. As the epsin UIM promotes the internalization of some ubiquitinated receptors, we predicted LDLR ubiquitination as necessary for endocytosis. However, engineered ubiquitination-impaired LDLR mutants showed modest internalization defects that were further enhanced with epsin1 depletion, demonstrating epsin1-mediated LDLR endocytosis is independent of receptor ubiquitination. Finally, we provide evidence that epsin1-mediated LDLR uptake occurs independently of either of the two documented internalization motifs (FxNPxY or HIC) encoded within the LDLR cytoplasmic tail, indicating an additional internalization mechanism for LDLR.Molecular biology of the cell 12/2012; · 5.98 Impact Factor - SourceAvailable from: Julien Moretti[Show abstract] [Hide abstract]
ABSTRACT: The very conserved Notch pathway is used iteratively during development and adulthood to regulate cell fates. Notch activation relies on interactions between neighboring cells, through the binding of Notch receptors to their ligands, both transmembrane molecules. This inter-cellular contact initiates a cascade of events eventually transforming the cell surface receptor into a nuclear factor acting on the transcription of specific target genes. This review highlights how the various processes undergone by Notch receptors and ligands that regulate the pathway are linked to ubiquitination events.International Journal of Molecular Sciences 01/2013; 14(3):6359-81. · 2.34 Impact Factor
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Drosophila Epsin's role in Notch ligand cells requires three
Epsin protein functions: the lipid binding function of the ENTH
domain, a single Ubiquitin interaction motif, and a subset of the
C-terminal protein binding modules
Xuanhua Xie, Bomsoo Cho, and Janice A. Fischer*
Section of Molecular Cell and Developmental Biology, Institute for Cell and Molecular Biology,
The University of Texas at Austin, 1 University Station A4800, Austin, Texas, USA 78712
Epsin is an endocytic protein that binds Clathrin, the plasma membrane, Ubiquitin, and also a
variety of other endocytic proteins through well-characterized motifs. Although Epsin is a general
endocytic factor, genetic analysis in Drosophila and mice revealed that Epsin is essential
specifically for internalization of ubiquitinated transmembrane ligands of the Notch receptor, a
process required for Notch activation. Epsin's mechanism of function is complex and context-
dependent. Consequently, how Epsin promotes ligand endocytosis and thus Notch signaling is
unclear, as is why Notch signaling is uniquely dependent on Epsin. Here, by generating
Drosophila lines containing transgenes that express a variety of different Epsin deletion and
substitution variants, we tested each of the five protein or lipid interaction modules for a role in
Notch activation by each of the two ligands, Serrate and Delta. There are five main results of this
work that impact present thinking about the role of Epsin in ligand cells. First, we discovered that
deletion or mutation of both UIMs destroyed Epsin's function in Notch signaling and had a greater
negative impact on Epsin activity than removal of any other module type. Second, only one of
Epsin's two UIMs was essential. Third, the lipid-binding function of the ENTH domain was
required only for maximal Epsin activity. Fourth, although the C-terminal Epsin modules that
interact with Clathrin, the adapter protein complex AP-2, or endocytic accessory proteins were
necessary collectively for Epsin activity, their functions were highly redundant; most unexpected
was the finding that Epsin's Clathrin binding motifs were dispensable. Finally, we found that
signaling from either ligand, Serrate or Delta, required the same Epsin modules. All of these
observations are consistent with a model where Epsin's essential function in ligand cells is to link
ubiquitinated Notch ligands to Clathrin-coated vesicles through other Clathrin adapter proteins.
We propose that Epsin's specificity for Notch signaling simply reflects its unique ability to
interact with the plasma membrane, Ubiquitin, and proteins that bind Clathrin.
Epsin; Notch signaling; endocytosis; Drosophila; Clathrin; Ubiquitin interaction motif
© 2012 Elsevier Inc. All rights reserved.
*corresponding author: Janice Fischer, firstname.lastname@example.com, phone: 512-471-5831, fax: 512-232-3432, address for courier mail: The
University of Texas at Austin, Moffett Molecular Biology Building, Room 1.312AA, 2500 Speedway, Austin, Texas 78712.
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Dev Biol. Author manuscript; available in PMC 2013 March 15.
Published in final edited form as:
Dev Biol. 2012 March 15; 363(2): 399–412. doi:10.1016/j.ydbio.2012.01.004.
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Epsin is a multi-modular endocytic protein present in metazoans and yeast (Chen et al.,
1998; Wendland et al., 1999). Genetic studies in Drosophila, nematodes, and mice revealed
that Epsin is required specifically for Notch signaling by ligand cells, and probably for all
Notch signaling events (Overstreet et al., 2004; Wang and Struhl, 2004; Tian et al., 2004;
Chen et al., 2009).
Epsin has a structured N-terminus, called the Epsin-N-terminal homology (ENTH) domain
(Kay et al., 1998; Rosenthal et al., 1999; De Camilli et al., 2001) (Fig. 1A). The ENTH
domain binds PIP2 (phosphatidylinositol 4,5-bisphosphate) and also inserts into the plasma
membrane and induces membrane curvature (Itoh et al., 2001; Ford et al., 2002; Aguilar et
al., 2003). The yeast Epsin (Ent1) ENTH domain also binds Cdc42 GTPase-activating
protein (GAP), which may enable Ent1 to coordinate cell polarity with endocytosis (Aguilar
et al., 2006a).
The unstructured C-terminal region of Epsin contains four different protein-protein
interaction motifs in varying numbers in different animal species (Kay et al., 1998; De
Camilli et al., 2001) (Fig. 1A). There are two Epsin isoforms in Drosophila, produced by
alternate splicing of the liquid facets (lqf) gene pre-mRNA (Fig. 1B) (Cadavid et al., 2000).
Each Lqf isoform has two Ubiquitin interaction motifs (UIMs) (Hofmann and Falquet, 2001;
Polo et al., 2002; Shih et al., 2002; Oldham et al., 2002; Miller et al., 2003; Klapsiz et al.,
2002), two Clathrin binding motifs (CBMs) (Aguilar et al., 2003; Drake et al., 2000), seven
DPW motifs that bind the AP-2 endocytic adapter complex (Owen et al., 1999), and two
NPF motifs that bind EH-domain-containing endocytic factors such as Eps15 (Aguilar et al.,
2003; Salcini et al., 1997; Paoluzi et al., 1998).
The mechanism of Epsin function appears to be complex and context-dependent. Given
Epsin's modular structure, the simplest imaginable role for Epsin would be as a Clathrin
adapter for transmembrane cargo that use Ubiquitin as an endocytosis signal (Aguilar et al.,
2003). Epsin might link transmembrane cargo with Clathrin cages either directly, or
indirectly through AP-2 (Aguilar and Wendland, 2006b). However, there is evidence that the
function of Epsin may be much more complex than that. For one, while the results of some
studies suggest that Epsin functions in Clathrin-dependent endocytosis (Chen et al., 1998;
Hawryluk et al., 2006), other results suggest that Epsin functions also in Clathrin-
independent membrane internalization (Sigismund et al., 2005). In addition, the ENTH
domain of yeast Epsin (Ent1) has a function separate from its role in endocytosis. The Ent1
ENTH domain alone is sufficient for the essential function of the protein in yeast, which is
not endocytosis, but the regulation of actin cytoskeleton dynamics (Aguilar et al., 2006a).
Finally, opposing roles for Epsin's UIMs and CBMs have been proposed. The UIMs are
required for Epsin ubiquitination (Hofmann and Falquet, 2001; Polo et al., 2002; Shih et al.,
2002; Oldham et al., 2002; Miller et al., 203; Klapsiz et al., 2002) and Epsin ubiquitination
inhibits Epsin function (Chen et al., 2002). Also, in vertebrate cell culture it has been
reported that Clathrin binding through the CBMs antagonizes UIM binding to Ubiquitin
(Chen and De Camilli, 2005).
Epsin is required in Notch ligand (signaling) cells for receptor activation in adjacent signal
receiving cells (Overstreet et al., 2004; Wang and Struhl, 2004; Tian et al., 2004). The Notch
receptor and its ligands (Delta and Serrate, in Drosophila) are transmembrane proteins.
Receptor activation leads ultimately to cleavage of an intracellular receptor fragment that
enters the nucleus and acts as a transcription factor (reviewed in Bray, 2006). There is
compelling evidence that ligand cells must internalize ligand in order to signal (reviewed in:
Le Borgne et al., 2005; Nichols et al., 2007a, 2007b; Weinmaster and Fischer, 2011). There
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is also persuasive evidence that Epsin's function in ligand cells is to promote Clathrin-
dependent ligand endocytosis. For one, lqf interacts genetically with a variety of genes that
encode endocytosis proteins, including Clathrin (Cadavid et al., 2000; Hagedorn et al., 2006,
Eun et al., 2007; Eun et al., 2008, Banks et al., 2011). Second, Notch ligands sometimes
accumulate abnormally at the plasma membrane in the absence of Epsin (Overstreet et al.,
2004; Wang and Struhl, 2004, 2005; Eun et al., 2008). Finally, ubiquitination of Notch
ligands is required for signaling (reviewed in Weinmaster and Fischer, 2011) and promotes
ligand endocytosis that depends on Epsin (Wang and Struhl, 2004). As genetic evidence
indicates a positive role for Clathrin in Notch signaling and Epsin has both UIMs and
CBMs, Epsin is implicated as the link between ligands and Clathrin-coated vesicles. Yet,
this simple model has not been tested experimentally.
Notch signaling is unique in its requirement for both ligand endocytosis and Epsin,
suggesting that Epsin might play a particular role in a special form of endocytosis. Ligand
internalization by the signaling cells is most likely required to exert a pulling force on Notch
that activates the receptor (reviewed in: Le Borgne et al., 2005; Nichols et al., 2007a, 2007b;
Weinmaster and Fischer, 2011). It has been speculated that generation of the pulling force
may require a special endocytic structure whose formation depends on Epsin's ability to
organize actin at the plasma membrane (see Weinmaster and Fischer, 2011). Ligand
transcytosis (recycling) is also required for Notch signaling in some cell types (reviewed in
Weinmaster and Fischer, 2011). In this process, Epsin may define a special Clathrin-
mediated endocytic pathway, distinct from the typical AP-2-dependent pathway, that routes
ligand into distinct endosomes (see Wang and Struhl, 2004). Remarkably, either the ENTH
domain of Drosophila Epsin, which has both endocytic and actin organizing functions, or
the remaining C-terminal endocytic modules, provides significant Epsin activity when
overexpressed (Overstreet et al., 2003). Thus, it is unclear which function of Epsin – the
actin regulatory or endocytic function of the ENTH domain, or the endocytic function of C-
terminus – or all of them – are important for Notch signaling under physiological conditions.
The specific requirement for Epsin in Notch ligand cells means that Epsin is somehow
intrinsic to the mechanism by which ligand cells activate Notch receptors in adjacent cells.
An important step forward in understanding the role of Epsin in Notch ligand cells is to
determine which Epsin modules are required for this process in particular. Here, by
generating a variety of transgenes that express amino acid deletion or substitution variants of
Epsin in transformed flies, we tested each Epsin module type to determine whether or not it
was needed for Notch signaling. The results suggest that in its role in Notch ligand cells,
Epsin is divided into three necessary functional regions: the lipid binding function of the
ENTH domain, a single UIM, and the C-terminus. The results are most consistent with a
model where Epsin recognizes ubiquitinated ligands at the plasma membrane, and links
ligands to Clathrin indirectly through interactions with other endocytic proteins, including
the conventional AP-2 adapter. We propose that Epsin's apparent specificity for Notch
signaling is not because Epsin generates a special endocytic environment, but because Epsin
is unique in its ability to interact with the plasma membrane, ubiquitinated transmembrane
cargo, and a variety of other proteins present in Clathrin-coated vesicles.
Materials and Methods
Drosophila strains
The following mutant alleles of lqf, maintained in our laboratory, were used: lqfFDD9
(FBal0104483), lqfARI (FBal0104485), lqfL71 (FBal0147029). Chromosomes used: hs-flp
tub-gal4 UAS-ngfp on X chr. (G. Struhl); m∂0.5-lacZ on chr. 2 (S. Bray); FRT80B lqfL71 (G.
Struhl); tub-gal80 FRT80B, FRT82B tub-gal80 (Bloomington); FRT82B SerRX106 (Y.N.
Jan); FRT82B Dlrev (N. Baker); FRT82B Dlrev SerRX82 (N. Baker).
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Molecular Biology
Enzymes were from Promega Biotech, New England BioLabs, and Boehringer Mannheim.
Herculase polymerase (Stratagene) was used for PCR. DNA sequences of all PCR
amplification products were verified. Automated fluorometric DNA sequencing was
performed in the DNA analysis facility of the Institute for Cell and Molecular Biology
(ICMB) at UT Austin.
Generation of Epsin deletion P element and PhiC31 integrase transgenes and
Deletion mutants (except for ΔUIM and ΔUIM2) were generated by a PCR-based method
(Fig. S1). This PCR method was also used to fuse GFP in-frame to the Epsin C-terminus
(Fig. S1). A list of primers used for each construct, complete construction details, and the
amino acid content of each Epsin variant are in Supplemental Text. All constructs were
ligated ultimately as Not I – Xho I fragments into pCaSpeR4 (Thummel and Pirrotta, 1992)
or into an attB vector that we constructed called pCaSper4-attB. Complete details of the
vector construction and a map are in Supplemental Text. P element transformation was by
Genetic Services (Sudbury, MA) or Genetivision (Houston, TX) and PhiC31 integrase
transformation (into site VK37 at polytene band 22A3) was by Genetivision.
Analysis of eyes and wings
Adult external eyes were photographed with an Olympus SZX12 microscope equipped with
a SPOT idea (Diagnostic Instruments) camera. Plastic sectioning of adult eyes was as
described (Tomlinson and Ready, 1987). Eye sections were photographed with a Zeiss
Axioplan equipped with an Axiocam Hrc. For immunostaining, eye discs in Figs. S2 and S3
were fixed in PEMS and antibody incubations and washes were in PBST (Fischer-Vize et
al., 1992). The antibodies used in Figs. S2 and S3 were: (A, B) guinea pig anti-Lqf (1:1000)
(Chen et al., 2002) and 488-donkey anti-guinea pig (1:200) (Jackson Laboratories), rat anti-
Chc (1:100) (Wingen et al., 2009) and 647-goat anti-rat (1:200; Molecular Probes); (C, D)
chicken anti-GFP (1:1000) (AbCam) and 488-goat anti-chicken (1:800) (Jackson
Laboratories); anti-Chc as in (A, B). The eye discs in Fig. 3 and all wing discs were
immunostained as follows. Discs were fixed in PEMS buffer with 1.0% NP-40 for 15 min.
Antibody treatment was as described previously (Lim et al., 2007) with modifications. Fixed
discs were blocked for 2 hrs. at 4°C in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5%
NP-40 and 5 mg/ml BSA, and then incubated in primary antibody diluted in blocking
solution overnight at 4°C. Discs were washed in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl
and 0.5% NP-40 3X for 5 min., and incubated with secondary antibodies in washing solution
for 2 hrs. at room temperature, and then washed 3X for 5 min. Primary antibodies were:
mouse monoclonal anti-Cut at 1:100 from (Developmental Studies Hybridoma Bank
(DSHB)), mouse monoclonal anti-β-galactosidase at 1:50 from the DSHB, rabbit anti-Svp at
1:100 (Ryan et al. 2005). Secondary antibodies (1:200; Molecular Probes) were: Alexa568-
anti-rabbit, Alexa568-anti-mouse, Alexa568-anti-guinea pig, Alexa633-anti-mouse, Cy5-anti-
rabbit. Phalloidin treatment of eye and wing discs (568-phalloidin, Invitrogen) was as
described (Chen et al., 2002). Eye and wing discs were mounted in Vectashield (Vector) and
photographed with a Leica TCSSP2 or SP2AOBS confocal microscope. Images were
processed with Adobe Photoshop CS3. MARCM clones (Lee and Luo, 1999) were
generated by heat shocking first or second instar larvae at 37°C for 60 min.
Quantitative Western analysis
Epsin-GFP variants expressed by transgenes were quantified using Western blots using anti-
GFP and anti-β-tubulin, and compared to endogenous Epsin indirectly through one copy of
FL. Eye disc protein extracts were generated and analyzed on Western blots as described
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(Chen et al., 2002), probed with guinea pig-anti-Lqf (1:1000) or mouse-anti-GFP (1:1000)
from Santa Cruz Biotechnology, and mouse mAbE7 (anti-β-tubulin from DSHB) at 1:100.
Secondary antibodies were HRP-anti-guinea pig (Jackson) at 1:20,000, HRP-goat-anti-
mouse (Sigma) at 1:2000 and HRP-anti-mouse (Santa Cruz Biotechnology) at 1:500. The
results were quantified using NIH Image J.
Analysis of lqfFDD9 mRNAs
RT-PCR (Invitrogen) of total mRNA from five lqfFDD9 third instar larvae (Qiagen RNAeasy
Mini Kit) amplified only the mutant splice form. Thus we infer that only mutant protein is
produced from the lqfFDD9 allele. The primers were 5'-ATTCCAGCAACAGCAGCCAG-3'
(in exon 6) and 5'-TGCTGACTGAAAACGGGGC-3' (in exon 7). These primers amplified
a 146 bp fragment of lqf genomic DNA, a 71 bp fragment of wild-type lqf mRNA, and an 82
bp fragment of lqfFDD9 mRNA. Both the 71 bp and 82 bp bands were detected in mRNA
from lqfFDD9/lqf+ heterozygotes.
Protein interaction experiments
The Gateway (Invitrogen) vectors pDEST15 (GST fusions) or pVP13 (MBP fusions) (Thao
et al., 2004) were used to express proteins in bacteria according to manufacturer's
instructions (Invitrogen). Details of the plasmid constructions are in Supplemental Text. The
fusion proteins were purified from BL21 Rosetta Cells (Novagen) according to the
procedure at http://wolfson.huji.ac.il/purification/TagProteinPurif/MBP_Tag_nature.html.
Proteins were quantified on Coomassie stained gels with Precision Plus Protein Unstained
Standards (Bio-Rad). GST-tagged proteins were immobilized on glutathione Sepharose 4B
(GE Healthcare) by mixing 1 ml of bacterial cell lysate with 200 ul Sepharose prewashed
with GST binding buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 10% glycerol, 0.5% NP-40;
just before use, 10µl 1M DTT and 1 tablet Protease Inhibitor Cocktail (Roche) added per 10
ml buffer). A 30 µl aliquot of the GST-fusion loaded Sepharose was used for protein
quantification as described above. The GST pull-down procedure used is modified from
Drake et al., 2000. Aliquots (30 µl) of loaded Sepharose were mixed with MBP-Epsin
proteins (the same weight as GST-fusion protein) and assay buffer (25 mM Hepes-KOH ph
7.2, 125 mM potassium acetate, 2.5 mM magnesium acetate, 5 mM EGTA, 1 mM DTT)
added to make the total volume 100 µl. After overnight incubation at 4°C with continuous
gentle mixing, the Sepharose beads were recovered by centrifugation at 10,000 g for 1 min.
The supernatant was removed and a portion of that (the "S" fraction) was mixed with SDS-
PAGE buffer. The Sepharose pellets were washed 5X with 1 ml ice-cold PBS and after
centrifugation, the final supernatants were aspirated and a portion of each pellet (the "P"
fraction) was resuspended in SDS-PAGE buffer. GST-Ub experiment: GST-Ub was 0.6 µg/
µl and MBP-Epsins were 0.2 µg/µl. The S lane on the gel was 10 µl of a dilution (10 µl
supernatant adjusted to 80 µl with SDS-PAGE buffer) and the P lane was 10 µl of the pellet
resuspended in 30 µl. GST-ChcTD experiment: GST-ChcTD was 0.1 µg/µl and MBP-
Epsins were 1 µg/µl. The S lane on the gel was 20 µl of a dilution (25 µl supernatant
adjusted to 120 µl) and the P lane was 20 µl of the pellet resuspended in 50 µl. GST-α-
AdaEar experiment: GST-α-AdaEar was 0.2 µg/µl and MBP-Epsins were 0.2 µg/µl. The S
lane on the gel was 20 µl of a dilution (5 µl supernatant adjusted to 80 µl) and the P lane was
20 µl of the pellet resuspended in 80 µl. GST-Eps15EH experiment: GST-Eps15EH was 0.4
µg/µl and MBP-Epsins were 0.4 µg/µl. The S lane on the gel was 20 µl of a dilution (25 µl
supernatant adjusted to 120 µl) and the P lane was 20 µl of the pellet resuspended in 50 µl.
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Deletion of individual Epsin module types: the UIMs are the single module type most
important to Epsin's ability to promote ligand signaling
The first aim was to generate Drosophila lines that express, at levels as close to the
endogenous Epsin (lqf) gene as possible, one of five different variants of Epsin in which all
copies (or most copies in the case of the DPWs) of a particular module type were deleted.
To this end, we first generated a 16 kilobase Drosophila genomic DNA fragment containing
a lqf gene tagged at the 3' end with gfp, and transformed flies with it using a P element
vector. (The GFP tag is mainly to facilitate quantitation of protein expression levels.) A
single copy of this transgene, called FL for "full length", complemented completely the
embryonic lethality of lqf null mutants (lqfL71/lqfARI) and the rescued adults were normal
morphologically (Fig. 2). Moreover, endogenous Epsin and Epsin-GFP from the FL
transgene each colocalized extensively with Clathrin in eye discs and were present mainly in
puncta at the apical plasma membrane (Fig. S2). Next we generated the five deletion
derivatives of FL shown in Fig. 2A. The Epsin-GFP proteins expressed by each transgene in
several different transformant lines were quantified using Western blots of eye disc protein
extracts (Materials and methods) and lines that matched normal endogenous Epsin levels
most closely were used for further analysis (Fig. 2A).
Rescue of lethality in lqf nulls and eye defects in lqf hypomorphs—First, we
tested whether or not a single copy of each transgene could substitute for the endogenous
gene as well as FL did. We found that three of the transgenes - ΔCBM, ΔDPW, and ΔNPF –
did so; each complemented lqf null mutants completely (Fig. 2). (In the ΔDPW construct,
the two clusters of 3 DPW motifs are deleted leaving a single motif which would not be
expected to bind AP-2 significantly (Owen et al., 1999)). Although ΔENTH did not rescue
the lqf nulls completely, it did retain significant Epsin activity as some viable adults eclosed
with morphological defects typical of Notch signaling mutants (including malformed eyes,
wings, and legs) (Fig. 2). In contrast, ΔUIM had almost no lqf+ activity; the developing
animals died as pupae and no escapers ever eclosed (Fig. 2). To better detect and resolve the
low levels of lqf+ gene activity in the ΔENTH and ΔUIM transgenes, we also tested how
well each of the transgenes rescued the morphological defects of lqf hypomorphs (lqfFDD9
homozygotes, Fig. 2A). lqfFDD9 homozygotes are semi-viable with typical Notch pathway
gene mutant defects, including abnormal eye morphology (Cadavid et al., 2000). Consistent
with the idea that ΔENTH retained significant lqf+ gene activity and ΔUIM did not, ΔENTH
rescued lqfFDD9 to wild-type while ΔUIM rescued the lqfFDD9 defects only slightly (Fig.
2A). The absence of UIMs or CBMs did not prevent ΔUIM nor ΔCBM from accumulating
at the plasma membrane similarly to wild-type Epsin-GFP (FL) (Figs. S2,S3). ΔENTH
protein also accumulates at the plasma membrane, but to a lesser extent than FL (Figs. S2,
S3). Therefore, deletion of the ENTH domain may lower Epsin activity at least in part by
preventing efficient localization of Epsin to the plasma membrane (see below).
Rescue of Delta-mediated Notch activation in the eye—In order to test directly
whether expression of ΔUIM in ligand cells affects Notch activation in adjacent cells, we
used the MARCM technique (Lee and Luo, 1999) to generate GFP-marked lqf− (lqf null)
cell clones in eye discs that either do or do not contain an Epsin transgene - either FL or
ΔUIM. (GFP signal from the transgenes was invisible in these and subsequent imaginal disc
clone experiments because Epsin-GFP was expressed at much lower levels than the nuclear
GFP that marks the clones.) In the eye discs, we monitored Notch activation in the R3/R4
photoreceptor cell pair using a transgene called m∂0.5-lacZ that is transcribed in response to
Notch activation (Cooper and Bray, 1999). Normally, Delta signaling by the equatorial cell
(R3) activates Notch in the polar cell (R4) and thus R4 expresses m∂0.5-lacZ (Fig. 3A–A")
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(Fanto and Mlodzik, 1999; Cooper and Bray, 1999; Tomlinson and Struhl, 1999). In mosaic
R3/R4 pairs in which R3 is lqf− and R4 is lqf+, R3's ability to signal through Delta was
impaired and R4 did not express m∂0.5-lacZ (0/36 pairs restored; Fig. 3B). In contrast, FL
largely restored the ability of R3s to activate Notch in adjacent R4s (25/31 pairs restored;
Fig. 3C,C'), while ΔUIM expression did not (2/47 pairs restored; Fig. 3D,D'). We conclude
that in Delta signaling cells, Epsin requires its UIMs to activate Notch in adjacent cells.
Rescue of Delta- or Serrate-mediated Notch activation in the wing—Both Delta
and Serrate require Epsin to signal (Wang and Struhl, 2005). We used wing discs to ask if
like Delta, the ligand Serrate requires Epsin's UIMs. Notch is activated in a stripe of cells at
the dorsal/ventral (D/V) boundary of the wing disc by Delta from the ventral side and
Serrate from the dorsal side (Fig. 4A) (reviewed by Irvine and Vogt, 1997; Irvine, 1999 and
Blair, 2000). In response to Notch activation, the transcription factor Cut is expressed at the
D/V boundary. In clones of Dl- Ser- cells (Fig. 4B,B') or lqf− cells (Fig. 4C,C') (Wang and
Struhl, 2004) that span the D/V boundary, Cut expression is absent. Expression of FL
restores Cut expression in lqf-cells (Fig. 4D,D'), but ΔUIM expression does not (Fig.
4G,G'). We conclude that Epsin's UIMs are required for signaling by Serrate as well as by
We also used wing discs to ask if Delta and Serrate have the same requirements for other
Epsin modules. In lqf− clones that span the D/V boundary we saw Cut expression restored
to apparently normal levels by ΔENTH, ΔCBM, ΔDPW, or ΔNPF (Fig. 4E–F' and data not
shown). As this result may be observed sometimes in clones that are only Dl- or only Ser-
(Micchelli et al., 1997), to determine if in the clones both Delta and Serrate were signaling,
we analyzed dorsal and ventral clones that abut the D/V border. Clones generated after the
first larval instar stage do not cross the D/V midline (Garcia-Bellido et al., 1976). Ventral
Dl- clones that abut the D/V border can receive but not send signals and thus Cut is
expressed inside the clone but not in adjacent midline cells (Fig. 5A; Glittenberg et al.,
2006). In contrast, dorsal Dl-clones can send and receive signals (Fig. 5A; Glittenberg et al.,
2006). Conversely, dorsal Ser- clones can receive but not send, while ventral Ser- clones can
both send and receive (Figs. 5A, S4). We found that each of the four transgenes (ΔENTH,
ΔCBM, ΔDPW, or ΔNPF) restored the ability of lqf− cells to signal from either the dorsal or
ventral side (Fig. 5B-G and data not shown). We conclude that in the context of an
otherwise intact Epsin protein, neither Delta nor Serrate require the ENTH domain, the
CBMs, the DPWs, nor the NPF motifs of Epsin to signal.
Differences in the assays—The embryonic lethality of lqf null mutants as well as the
defects later in development are probably due entirely to the failure of Notch signaling, and
thus the transgene complementation test is a reasonable assay for Epsin activity in the Notch
pathway. This assumption is based on several observations. First, analysis of lqf null clones
in developing eye and wing imaginal discs indicates a specific failure of Notch signaling
(Overstreet et al., 2004; Wang and Struhl, 2004, 2005). Second, Notch signaling is essential
during Drosophila embryonic development, and consequently homozygous null mutants in
core Notch pathway genes die during embryogenesis (Poulson, 1937; Lehmann et al., 1983;
Nusslein-Volhard et al.,1984; Fleming et al., 1990). Likewise, mice with knock-out
mutations in both Epsin genes (epsin 1 and epsin 2) show typical Notch signaling defects
throughout development, including embryogenesis (Chen et al., 2009). Finally, different
mutant alleles of lqf affect viability and Notch-like morphology to a similar degree (Cadavid
et al., 2000; Overstreet et al., 2003). Similarly, we always observed a correspondence
between the ability of a transgene to rescue the lethality of lqf null mutants and Notch
signaling defects later in development in either lqf null mutants or lqf hypomorphs (Fig. 2B
and see below).
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Although the general results were similar in all assays, there were two subtle differences in
the results in the wing as compared with the eye and early development assays. First, while
ΔUIM retains some residual activity that rescues embryonic lethality and eye morphology
slightly, it has no apparent activity in the Cut expression assay. Second, while ΔENTH
rescues the lethality and eye morphology defects in lqf null mutants not quite completely,
ΔENTH-expressing ligand cells activated Cut apparently normally in the wing. Most likely
these discrepancies reflect differences in the sensitivities of the different assays in terms of
how much Epsin activity is required to activate Notch, and/or in terms of how much Notch
activation is required to observe a wild-type signal output. In addition, the levels of
expression of the Epsin variants were tested in eye discs. It is possible that for some
transgenes, wing disc expression levels may not correlate with the expression levels in eye
discs (see Discussion).
Taken together, the results of these experiments indicate that Epsin's UIMs are vital to
Epsin's ability to enable Delta or Serrate signaling. In contrast, the ENTH domain is
required only for maximal Epsin activity, and in an otherwise intact Epsin protein, the
CBMs, the DPW motifs, and the NPF motifs are each unnecessary.
Either UIM is sufficient for Epsin function in ligand signaling
Epsin has two UIMs (Fig. 6A) and we were curious to know whether or not both of Epsin's
UIMs are required for ligand cell signaling. To test this, we first generated a lqf-gfp
transgene in the context of the 16 kb genomic DNA used above with a deletion of UIM2
(ΔUIM2). In transformed flies, ΔUIM2 rescued the lethality of lqf− null mutants and the
viable flies were almost completely wild-type morphologically (Fig. 6B,C). Likewise, in
ΔUIM2-expressing lqf− null clones in the wing disc, Notch is activated by both Delta and
Serrate (Fig. 6D). We conclude from these results that only a single UIM is essential. To test
if the single UIM could be UIM2 instead of UIM1, we first generated a transgene identical
to ΔUIM2, except that three UIM1 consensus Glutamic acid codons were changed to
Alanine codons (UIM1EEE/AAAΔUIM2; Fig. 6B). Mutation of the N-terminal acidic UIM
residues has been shown to eliminate the ability of UIM peptides to bind Ubiquitin in vitro
(Fisher et al. 2003). UIM1EEE/AAAΔUIM2 had only a very small amount of Epsin activity in
the lqf− phenotypic rescue assays (Fig. 6B), and likewise in lqf-wing disc clones spanning
the D/V boundary, UIM1EEE/AAAΔUIM2 did not promote Cut activation (Fig. 6D). Thus, the
UIM1 mutations rendered UIM1EEE/AAAΔUIM2 essentially inactive. Next, we added back to
UIM1EEE/AAAΔUIM2 the UIM2 sequences to generate UIM1EEE/AAA (Fig. 6B). We found
that UIM1EEE/AAA had significant activity, just below that of ΔUIM2 (Fig. 6B).
UIM1EEE/AAA rescued lqf null mutants to nearly wild-type (Fig. 6B,C) and in about half the
dorsal or ventral lqf-wing disc clones assayed, UIM1EEE/AAA restored the ability of Serrate
or Delta, respectively, to signal (Fig. 6D). We conclude that only one UIM is necessary for
very nearly full Epsin activity in ligand cells, and that either UIM1 or UIM2 is sufficient.
The CBMs, DPWs and NPFs are redundant with each other
Above we discovered that Epsin's function in Notch ligand cells is independent of its
CBMs, DPW motifs, or NPF motifs in an otherwise intact protein. To determine whether the
C-terminal modules are required at all, we generated a lqf-gfp transgene containing only the
ENTH domain and the UIMs (ENTH-UIM). ENTH-UIM has no ability to rescue the mutant
phenotypes of lqf- mutants (Fig. 7A,C). As we have shown that each of the C-terminal
module types is dispensable, the C-terminal module types must be redundant for Epsin's
role in ligand signaling. To explore the nature of the redundancy, we generated two
additional lqf-gfp transgenes, one with deletions of the CBMs and one DPW cluster (ΔCBM–
ΔDPW1), and the other with the CBMs and both DPW clusters deleted (ΔCBM–ΔDPW).
ΔCBM–ΔDPW1 rescues lqf null mutants to wild-type, and ΔCBM–ΔDPW has just slightly
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less activity in that the null eye has a small number of mutant facets (Fig. 7A,B). Similarly,
in the wing, each transgene restores the ability of Serrate and Delta to signal (Fig. 7C). This
result indicates that Epsin functions nearly normally in ligand cells with only one of the C-
terminal module types intact – the NPFs.
We wondered whether like the NPFs, the DPW motifs or the CBMs would support Epsin
function independent of the other two module types. Instead of testing this with additional
deletion variants, we attempted to generate "minimal Epsin-GFP" genes in the context of the
16kb lqf genomic DNA fragment, containing only the ENTH domain, UIM1, and four
copies of either the CBMs (4XCBM), DPW clusters (4XDPW), or NPF motifs (4XNPF). We
took this approach in order to address whether or not there are unknown motifs in the C-
terminus that may be contributing to Epsin function. We detected no Epsin-GFP protein in
flies transformed with 4xCBM or 4XDPW (data not shown). In contrast, the 4XNPF protein
was expressed at high levels and the transgene rescued lqf− mutant phenotypes significantly
(about as well as ΔENTH does) (Fig. 7A,B). However, 4XNPF-expressing clones abutting
the D/V wing boundary did not activate Cut detectably (Fig. 7C). This was surprising as
ΔENTH activity is similar to 4XNPF in the eye, and ΔENTH supports Cut activation (Fig.
5E). A likely possibility is that the transgene is expressed at somewhat lower levels in the
wing than in the eye or in early development, and that this expression level is beneath the
threshold for detectable Cut expression. Alternatively, lower levels of NPF binding proteins
in the wing than in the eye could account for the difference in 4XNPF activity (see
We conclude that there is functional redundancy among the C-terminal modules, and that
the NPF motifs alone were sufficient to provide a significant portion of the function of this
region. Additional experiments are needed to determine whether the CBMs or DPW motifs
alone would provide as much Epsin function in ligand cells as the NPFs do.
Deletion of UIMs, CBMs, DPWs or NPFs interferes with Epsin binding in vitro to Ubiquitin,
Clathrin, α-Adaptin, and Eps15, respectively
Protein-protein interactions between Epsin's various modules (UIMs, CBMs, DPWs, and
NPFs) have been characterized previously in a variety of different contexts (see
Introduction). Nevertheless, we wanted to test whether the deletions of modules were indeed
disrupting the ability of the particular Drosophila Epsin variants we generated to interact
with the expected Drosophila proteins. To this end, we generated GST fusion proteins in
bacteria with Ubiquitin (GST-Ub), the Clathrin heavy chain "terminal domain" (GST-
ChcTD), the EH domain (NPF-binding region) of Eps15 (GST-Eps15EH), and the "ear
domain" of α-Adaptin, the AP-2 subunit that binds Epsin (GST-α-AdaEar). Purified GST
fusion proteins were tested in GST "pull-down" experiments for interaction with wild-type
Drosophila Epsin (FL) or Epsin deletion derivatives, all without GFP tags, purified in
bacteria as MBP fusions. As a control for artifactual results, Epsin and each derivative were
also tested under similar conditions for binding to GST alone and none of the proteins bound
GST (data not shown).
First, we found that under conditions in which FL bound to GST-Ub, ΔUIM2 (Fig. 2) bound
to GST-Ub also, but ΔUIM (Fig. 2) lost the ability to interact with GST-Ub (Fig. 8A). We
conclude that the UIMs are required not only for Epsin function but also for Epsin to bind
Ub in vitro. In addition, there is a correlation between Epsin's ability to bind Ub in vitro and
its ability to function in vivo; UIM1, which is sufficient for Epsin function without UIM2 in
an otherwise intact protein, is also sufficient for Ub binding in vitro.
Next, we found that GST-ChcTD pulled down FL, but pulled down neither ΔCBM nor
ΔCBMΔDPW (Figs. 2, 8B). Thus, deletion of the CBMs hampered the ability of Epsin to
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bind Clathrin in vitro. Similarly, we found that deletion of the NPF motifs (ΔNPF, Fig. 2)
prevented Epsin from interacting in vitro with the EH domain of Eps15 (Fig. 8C), and
deletion of the two clusters of DPW motifs (ΔDPW, Fig. 2) weakened the in vitro
interaction between α-AdaEar and Epsin (Fig. 8D). We conclude that at least in vitro,
deletion of the characterized protein binding motifs is indeed interfering with the ability of
Epsin to bind the proteins predicted.
The lipid-binding function of the ENTH domain, but not the Cdc42 GAP interaction
function, is required for Epsin's role in ligand cells
We found above that the ENTH domain, while not essential for Epsin function in ligand
cells, is required for maximal Epsin activity (Fig. 2). Two functions have been proposed for
the ENTH domain. First, the ENTH domain brings Epsin to the plasma membrane through
interactions with PIP2 (Itoh et al., 2001; Aguilar et al., 2003). Plasma membrane binding of
the ENTH domain promotes endocytosis in two ways: it brings the other Epsin modules to
the membrane and also induces membrane curvature (Ford et al., 2002). Second, the ENTH
domain binds Cdc42 GAP and thus regulates actin dynamics and cell polarity (Aguilar et al.,
2006a). The observation that ΔENTH protein localizes to the plasma membrane less well
than wild-type Epsin (Fig. S3) is consistent with either function, as ENTH domain/Cdc42
GAP interactions are expected to occur at the plasma membrane (Aguilar et al., 2006a).
Expression in yeast of either full-length Drosophila Epsin or the ENTH domain only rescues
the lethality of yeast mutants lacking Epsin (ent1Δent2Δ) (Overstreet et al., 2003). As the
lethality of yeast Epsin mutants is due to the loss of the ENTH domain's ability to polarize
actin via Cdc42 regulation (Aguilar et al., 2006a), we reason that the ENTH domains of
Drosophila and yeast Epsin likely interact with Cdc42 GAP the same way. Distinct ENTH
domain amino acids have been identified that are specific either for plasma membrane or
Cdc42 GAP binding (Itoh et al., 2001; Aguilar et al., 2006a). To determine which of the two
functions of the ENTH domain is required for maximal Epsin activity in ligand cells, we
generated lqf-gfp transgenes that generate Epsins with mutations that alter amino acids key
to one function or the other.
We first generated ENTHT/D and ENTHFTVF/RDAA transgenes which produce Epsin variants
with alterations in amino acids required for Cdc42 GAP interaction. Each of them functions
as well as the FL (normal Epsin) transgene (Fig. 9A,B). Thus, we conclude that the actin
organizing function is not essential to Epsin's role in Notch ligand cells. By default, the
ENTH domain's role in PIP2 binding is implicated as its function in Notch ligand cells. We
tested this directly by generating transgenes that express Epsin variants with alterations in
amino acids needed for PIP2 binding. The transgene ENTHRWRK/AAAA produces an Epsin
with four key amino acid changes (Fig. 9A). ENTHRWRK/AAAA provides only a barely
detectable amount of Epsin activity, but the protein accumulates only to a small fraction of
wild-type Epsin levels (Fig. 9A). Because the low levels of ENTHRWRK/AAAA protein make
these results difficult to interpret, we generated ENTHR/A, which contains a mutation that
changes only one key residue to Alanine (Fig. 9A). ENTHR/A protein accumulates to normal
levels and although it retains more activity than ΔENTH (see Fig. 1B), its activity is
noticeably below wild-type (Fig. 9A,B). As ENTHR/A protein differs from wild-type Epsin
(FL) by only one amino acid and yet has less activity than wild-type Epsin, this result
supports the idea that the ENTH domain is required for its PIP2 binding function.
Epsin is a complex multi-modular protein that functions differently in different contexts. In
C. elegans, Drosophila, and mice, Epsin is needed specifically in Notch ligand cells (Tian et
al., 2004; Overstreet et al., 2004; Wang and Struhl, 2004; Chen et al., 2009). The structure/
function analysis of Epsin we performed here shows that modules of Epsin associated with
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the internalization step of endocytosis – the lipid binding function of the ENTH domain and
the C- terminal modules that bind proteins present in Clathrin-coated vesicles – are required
for Epsin's function in Notch ligand cells. In addition, we have shown that a UIM is
necessary. We discuss below how these results lead to a specific model for Epsin function in
ligand cells.
The actin organizing function of the ENTH domain is not essential for Epsin function in
Notch ligand cells
The dispensability of the Cdc42 GAP binding function of the ENTH domain suggests that in
ligand cells the primary role of Drosophila Epsin, unlike yeast Ent1, is not regulation of
actin dynamics. The other known function of the ENTH domain is the endocytic function,
and the results suggest that the ability of the ENTH domain to interact with PIP2 explains
why it is needed for maximal Epsin function in Notch ligand cells. These observations are
consistent with the lack of typical Notch signaling defects in Drosophila cdc42 mutants
(Genova et al., 2000). In contrast, flies with mutations in genes for either of two actin
regulators, the Arp2/3 complex and WASp, do have notal bristle defects indicative of Notch
signaling failure (Rajan et al., 2009). The results we present here are not in conflict with the
findings of Ragan et al. (2009). The notal bristle phenotype described is not due to failure of
the Epsin-dependent endocytosis of ligand that activates Notch in all cell types, but instead
to failure of ligand transcytosis required in only some cell types to relocalize ligand prior to
signaling (see Weinmaster and Fischer, 2011). The absence of the Arp2/3 complex or WASp
in mutants inhibits signaling by blocking traffic of endocytosed Delta to apical microvilli of
sensory organ precursors (Rajan et al., 2009). Whether or not Delta transcytosis in sensory
organ precursors also depends on Epsin is unknown. If Epsin is involved, it may be
interesting to use the Epsin variant transgenes we generated to determine whether or not the
Cdc42 GAP interaction function of the ENTH domain is required.
A UIM is critical to Epsin activity in Notch ligand cells, and a single UIM is sufficient
There are two types of UIMs: single-sided UIMs that bind one Ubiquitin, and double-sided
UIMs that bind two Ubiquitins simultaneously (Hirano et al., 2006). As the affinity between
a UIM and Ubiquitin is low, successful interaction between a mono-ubiquitinated protein
and a UIM-containing protein is thought to require either one double-sided UIM, or two
single-sided UIMs (Hawryluk et al., 2006; Hirano et al., 2006; Barriere et al., 2006;
Madshus, 2006). Epsins have single-sided UIMs, and so the observation that only one
single-sided UIM is required for Drosophila Epsin function in Notch signaling is
unexpected. The simplest explanation is that Notch ligands use multiple mono-Ubiquitins or
Ubiquitin chains as a signal for Epsin-mediated internalization (Traub and Lukacs, 2007;
Heuss et al., 2008). Two distinct Lysine residues in the intracellular domains of both Delta
and Serrate have been implicated as important for the function of each ligand. In the case of
Serrate, simultaneous mutation of both of these Lysines results in a Serrate ligand that can
neither activate Notch nor be endocytosed in wing discs (Glittenberg et al., 2006). These
observations identify two particular Lysines as candidates for the critical Ub attachments,
but do not distinguish whether one or both Lysines are required. In the case of Delta, single
mutation of either of two specific Lysines results in accumulation of Delta at the cell surface
of eye discs and failure to signal (Parks et al., 2006). Although Delta is thought to be mono-
ubiquitinated (Deblandre et al., 2001), these results suggest the possibility that Delta is
multiply mono-ubiquitinated. An alternative explanation for Epsin's ability to promote
ligand endocytosis with a single UIM is that mono-ubiquitinated ligands cluster to generate
an environment where multiple Ubiquitins attract Epsin to ligand at the plasma membrane
(LeBorgne and Schweisguth, 2003).
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Epsin function in Notch ligand cells is not as a monomeric Clathrin adapter
There is compelling evidence that in somatic cells, Notch ligand endocytosis associated with
signaling is Clathrin-dependent. First, there are exceedingly strong genetic interactions
between the Clathrin heavy chain (Chc) gene and lqf, the gene for Epsin. Flies with only one
Chc+ gene copy are wild-type, but this condition is lethal in homozygotes for a normally
viable hypomorphic allele of lqf (Cadavid et al., 2000). Second, the Clathrin-coated vesicle
uncoating protein Auxilin is, like Epsin, required specifically for Notch signaling in
Drosophila and in ligand cells (hagedorn et al., 2006; Eun et al., 2007; Eun et al., 2008;
Kandachar et al., 2008). Given the clear involvement of Clathrin and the lack of strong
genetic interaction between α-Adaptin (the gene for an AP-2 subunit) and lqf (Cadavid et al.,
2000), the simplest model for Epsin function in Notch signaling was as an adapter protein
that links Clathrin and the plasma membrane, independent of AP-2. This model predicted
that direct interaction between Epsin and Clathrin would be necessary, and thus the most
surprising result of this work is that deletion of the CBMs had no detectable effect on Epsin
activity. The dispensability of the CBMs rules out models where Epsin acts as a monomeric
Clathrin adapter that links ligand to Clathrin cages.
In the Drosophila female germline, Notch signaling requires Epsin but neither Clathrin nor
Auxilin (Windler and Bilder, 2010; Banks et al., 2011). Although this is surprising, Epsin
has been shown to function in Clathrin-independent internalization of ubiquitinated
transmembrane cargos in vertebrate cell culture (Sigismund et al., 2005). Epsin must
therefore function differently in Notch signaling in the female germline than in somatic
cells. We speculate that the ENTH domain and UIMs may be required in germline cells to
guide the ubiquitinated proteins into an endocytic vesicle (see Aguilar and Wendland, 2005).
However, it is not clear how any of the characterized modules within Epsin's C-terminus
might be involved in Clathrin-independent endocytosis (Mayor and Pagano, 2007). It would
be of interest to use the transgenes we have generated to determine which motifs are
required in the female germline. Additional experiments could potentially identify unknown
C-terminal interaction motifs used in Clathrinin-dependent endocytosis.
Does Epsin function in the same way in the embryo, eye, and wing?
We began the experiments with the assumption that Epsin functions through the same
mechanism in all signaling contexts, and thus we expected the same Epsin modules would
be required for Epsin function in all contexts. Epsin appears to be required in every Notch
signaling event and thus could be regarded as a core component of Notch signaling. It
therefore seems reasonable to expect that Epsin would function in the same manner in all
tissues. As described above, the female germline is apparently an exception. Nevertheless, in
the three assays we used for Epsin activity – rescue of lethality and eye morphology defects
due to lqf mutations and rescue of the ability of lqf null cells to activate Cut expression in
cells at the D/V boundary in the wing disc – we detected only subtle differences between the
eye and the wing in the activity of two Epsin variants, ΔENTH and ΔUIM. (The only major
difference was with the highly artificial Epsin variant, 4XNPF.) Despite these differences,
we think that Epsin likely functions the same way in the eye and wing, as well as during
embryogenesis. For one, the differences in activity we observed may be explained easily
without invoking different mechanisms for Epsin in the eye and wing (see Results).
Importantly, we never observed even one case where modules were essential in one context
(embryogenesis, eye, or wing development) and dispensable in another one. In fact, it is
possible to observe all-or-none differences in requirements for Epsin modules. We have
discovered that Epsin has a function outside of Notch ligand cells and we did find modules
that are dispensable completely in this context yet absolutely essential for Epsin's function
in ligand cells (B.C., X.X., and J.A.F., manuscript in preparation).
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A model for Epsin function in Notch ligand cells
Notch ligands require ubiquitination and (usually) Clathrin-dependent endocytosis, and
formation of Clathrin-coated vesicles requires adapter proteins that link the plasma
membrane with Clathrin (Owen et al., 2004). The absolute necessity of at least one UIM and
the observation that the lipid-binding function of the ENTH domain plays a role in ligand
cells suggests that Epsin indeed binds ubiquitinated Notch ligands at the plasma membrane.
However, as an Epsin derivative lacking CBMs functions as well as wild-type Epsin in
ligand cells, the essential role of Epsin in Notch signaling cannot be as a monomeric
Clathrin adapter that links Clathrin directly to ligand at the plasma membrane. As any pair
of the three types of modules is sufficient for Epsin function (CBMs+DPWs, CBMs+NPFs,
or DPWs+ NPFs), Epsin must be able to support Notch activation by linking ligand to
Clathrin in a variety of different ways (Fig. 10). We speculate that Eps15 is involved
because of the three EH-domain proteins in Drosophila (Eps15, Dap160, Past1), none have
Clathrin binding motifs, and Eps15 is the only one with motifs for a known Clathrin-binding
protein (AP-2) (Koh et al., 2007; Olswang-Kutz et al., 2008). From analysis of mutant
phenotypes and genetic interaction studies, there is no evidence for the involvement of
Eps15 nor AP-2 in Notch signaling (Gonzalez-Gaitan and Jackle, 1997; Koh et al., 2007;
Windler and Bilder, 2010). The results presented here suggest that Eps15 and AP-2 may
play redundant roles in the presence of intact Epsin and this idea could be tested with
additional genetic experiments. In light of the evidence indicating a requirement for Clathrin
in ligand cells (outside of the germline), the results suggest that Epsin is required absolutely
for Notch signaling not because it generates a special endocytic environment, but simply
because it is the only UIM-containing endocytic protein with the appropriate complement of
interaction modules to target ubiquitinated cargo to Clathrin-coated vesicles.
Epsin requires only one UIM for its function in Notch ligand cells.
Epsin requires the plasma membrane binding function of the ENTH domain for
maximal activity in Notch ligand cells.
Epsin requires its C-terminal modules collectively for its role in Notch ligand
cells, but the roles of the different modules are highly redundant
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Ben Doolan helped to generate the ΔUIM constructs. We thank M. Behr for his gift of Drosophila Clathrin heavy
chain antibody, R. Cripps for anti-Svp and S. Sung for plasmids. For fly strains, we thank Y. N. Jan, N. Baker, G.
Struhl, K. Irvine, T. Klein, and S. Bray. Special thanks to E. Lafer and Y. Zhuo for their generosity in supplying
materials and advice regarding Clathrin/Epsin interactions, and to Ji-Hoon Lee for help with the in vitro
experiments. We also thank P. Macdonald for use of his confocal microscope, and acknowledge the DNA Analysis
facility at UT Austin for DNA sequencing. This work was supported by grant R01-HD36080 to J. A. F. from the
USA National Institutes of Health, Institute of Child Health and Human Development.
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Drake MT, Downs MA, Traub LM. Epsin binds to Clathrin by associating directly with the Clathrin-
terminal domain. J. Biol. Chem. 2000; 275:6479–6489. [PubMed: 10692452]
Eun SH, Lea K, Overstreet E, Stevens S, Lee J-H, Fischer JA. Identification of genes that interact with
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Eun SH, Banks SML, Fischer JA. Auxilin is essential for Delta signaling. Development. 2008;
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Fanto M, Mlodzik M. Asymmetric activation specifies photoreceptors R3 and R4 and planar polarity
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ISLAMIC STATE: US RELEASES IMAGES OF OIL REFINERY AIRSTRIKES IN SYRIA-The US military has released footage and still photos of its air strikes on oil refineries controlled by Islamic State (IS) militants in eastern Syria.
Media Center
Monthly Feature
And the days dwindle down to a precious few, the lyrics of September Song go on. But first we must trace the history of September. The month comes from an old Roman word 'septem' meaning seven. How come we now commonly call it the ninth month. Well, the ancient Roman calendar began with March. As previously explained, the calendar was reformed by Rome by adding two new months (January and February) and altering the numbering of the old months. Thus September came to be called as the ninth month.
Although not connected with September being referred to as the ninth month, a further reform was made - the Gregorian calendar. It was introduced by Pope Gregory XIII and that is how the calendar got its name. It was made for accuracy - to change a full year to 11 minutes less than 365.25 days. Many countries didn't switch over immediately to the Gregorian calendar perhaps because it was made by a Roman Catholic pope but these nations have migrated to the Gregorian which is now widely in use.
Read more: The Days Grow Short When You Reach SeptemberThough there appears to be no official word on the move to Loyola, we presume that it was done in September and in this segment we present two articles saying hello to Loyola, then the new site of Ateneo. We feature the articles of John Anthony Lazo and Ted Montemayor.
Read more: Welcome LoyolaThe Philippines takes the cake as the country that celebrates the longest Christmas season. With parols (Filipino for Christmas lanterns) popping up and Christmas carols heard as early as September, and lasting perhaps until either Epiphany or January 9, the feast of the Black Nazarene, the Philippines easily earns that distinction.
For the Filipino the parol is as traditional as the Christmas tree is to the Westerner. Many Filipinos abroad have bought parols from the Philippines to adorn their homes or, if handy, made them. These star-shaped lanterns represent the star of Bethlehem that guided the magi to the baby Jesus.
Simple parols can be constructed with ten sticks, paper and paste. Present-day parols are made from a variety of materials such as cellophane, glass and rope or are so complicated and well-done that many Filipinos prefer to buy them from the Philippines.
Hereunder we feature one of the "Thoughts and Stories" which we feel supports this theory.
Read more: The Longest Christmas SeasonAbout Us
ADM5054 is in many ways an organization that is wondrous strange and unique. For one, by an accident of timing, it is limited to men. Secondly, its membership can no longer grow, instead it has been diminishing and will continue to diminish until in the fullness of time it will altogether disappear. Furthermore, a member cannot refuse to join ADM5054 because that has already happened. Neither can he decide to leave the organization because that too will simply happen. At this time there is nothing he can do about either...
Read More...There is an obvious connection between Ateneo's high school class of 1950 and college class of 1954. Clearly, from HS50 came the nucleus of College 54. But is this the only connection?
A kinship exists not only between the two graduating classes but also among their members. Their unique bond transcends school ties and must have been forged even before they came to Ateneo.
They were just grade school boys when the Japanese bombed Pearl Harbor, an event that embroiled the Philippines in World War II,...
In 1946 following the end of WWII, Ateneo, "bloodied, but unbowed", reopened its Padre Faura gates. With its buildings lying in ruins, the school erected renovated Quonset huts behind the rubble to serve as classrooms, a chapel and living quarters for the Jesuits. When Ateneo relocated to Loyola Heights in January 1952, the Padre Faura campus gave way to commercial buildings over the years. Now, the campus HS50 knows is gone without a trace and lives only in the memories of those who were there. In time, those memories...
Read More...The boys who would later be College 54 have the distinction of being among the first batch of students to move from their humble preserves in Padre Faura to the grandeur in Loyola Heights. For many, sadness mingled with anxiety as they bade farewell to Padre Faura and said hello to their new campus. Their student lives were to be spent mostly in Bellarmine Hall. Here was where they sat down for lectures, recitations, quizzes and tests and where they had anxious as well as triumphant moments. It was more than 50 years...
Read More...

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text: We arrived in Greymouth after dark so this was the only motel we found with a reasonably priced vacancy for one night. The asian man on reception said he didn't eat out so couldn't advise where we could go for dinner. The room was spacious but needed some TLC - the hose cover to the shower was unravelling and there... More


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Weymouth New Testament (WNT) | New International Version (NIV) |
6 and said, "If you are God's Son, throw yourself down; for it is written, "`To His angels He will give orders concerning thee, and on their hands they shall bear thee up, lest at any moment thou shouldst strike thy foot against a stone.'" | 6 "If you are the Son of God," he said, "throw yourself down. For it is written: " 'He will command his angels concerning you, and they will lift you up in their hands, so that you will not strike your foot against a stone.' " |
The Weymouth New Testament is in the public domain. (Weymouth New Testament - Holy Bible ) | Scripture quoted by permission. Quotations designated (NIV) are from THE HOLY BIBLE: NEW INTERNATIONAL VERSION®. NIV®. Copyright © 1973, 1978, 1984 by Biblica. All rights reserved worldwide. (New International Version Bible Online) |


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Current mayor of Hillsboro, Roger Vance, talks to volunteer John Witmer at the Old Stone School voting location. Anna Harris, Times-Mirror
Singer, songwriter Sloan Wainwright will take the stage at 8 p.m. June 6 at the Old Stone School in Hillsboro. Courtesy Photo
Photo courtesy/Loudoun County Bed & Breakfast Guild The green bedroom at Fieldstone Farm in Hillsboro. Fieldstone Farm is one of 15 bed and breakfasts or cottages featured in the 5th annual Bed & Breakfast Open House.
Times-Mirror Staff Photo/Beverly Denny Former Secretary of State Madeleine Albright was the special guest speaker at Woodgrove High School's Class of 2012 awards ceremony June 1.


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What's new in this version:
- Fixed a help menu bug


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Owners Manual
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Get up to $400 cash back, with mail-in rebate, on select Maytag appliances. Ex. • Purchase a Dishwasher - $40 Off • Purchase a Range - $40 Off • Purchase a Refrigerator - $40 Off • Purchase a Wall Oven - $40 Off • Purchase a Washer - $40 Off • Purchase any 3 Dishwasher, Cooktop, OTR, Range, Refrigerator, Wall Oven - $100 Bonus • Purchase any 4 Dishwasher, Cooktop, OTR, Range, Refrigerator, Wall Oven - $200 Bonus Only one model per category qualifies. All purchases must be on one sales slip. $40 rebate reward is only for Dishwasher, Range, Refrigerator, Wall Oven, and Washer. Washers do not qualify for kitchen bonus. Rebate form must be postmarked by November 14, 2014. It is the sole responsibility of the customer to download, complete and mail in the rebate form and all required documentations by the given postmark date.
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Continental Carbonic Products, Inc.
We are located 1 block east of I 35 North, off of Manana, on the northwest side of Dallas to the east of the Dallas/Fortworth International Airport.
The Dallas location serves central Texas including the cities of Fort Worth, Waco, Greenville, Longview, Tyler, Plano, Wichita Falls, Garland and Arlington.
Monday - Friday: 7:00 a.m. - 4:00 p.m. or call after hours for 24-hour service
Special Halloween Hours!
Saturday, October 25th, 2014 8:00 a.m. - 3:00 p.m.


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James Paxton: 'You Do The Attacking Or You're Going To Be Attacked'
February 26, 2014 12:08 PM
Steve Sandmeyer
Steve Sandmeyer hosts 1090 The Fan's local show. Sandmeyer, a Seattle...
Read More
(1090 The Fan) — Seattle Mariners lefty James Paxton joined the Steve Sandmeyer Show on Tuesday, February 25th from Peoria, Arizona.
Paxton, 25, saw his first Major League action in September of the 2013 season and in 24 innings of work he held a 1.50 ERA and a WHIP of 0.917. The Richmond, B.C. native figures to anchor the starting rotation in 2014.
Listen to what he expects out of the upcoming season below:
(TM and © Copyright 2014 CBS Radio Inc. and its relevant subsidiaries. CBS RADIO and EYE Logo TM and Copyright 2014 CBS Broadcasting Inc. Used under license. All Rights Reserved. This material may not be published, broadcast, rewritten, or redistributed.)


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Black and White Photography by Yusuke Yamatani
Photography // Wednesday, February 05, 2014
We have loved the photography of Japan's Yusuke Yamatani for years, and seeing that he will open his new solo exhibition at Yuka Tsuruno Gallery in Tokyo on February 12, we thought it would be a good idea to see some of the black and white photos from the artist.
Ceramics, Not Wood, From Christopher David White
Juxtapoz // Wednesday, February 05, 2014
Yes, this sculpture by Christopher David White looks like wood, and yes, it's actually ceramic, and yes, we are still not convinced but we will take the artist's word for it. The piece, Cycles of Decay, was made to resemble old wood, and explained by White, "Often, the process of change occurs at an almost unnoticeable rate...
Jean-Pierre Seguin's Toy Soldier Portraits
Juxtapoz // Wednesday, February 05, 2014
Montreal-based artist Jean-Pierre Seguin uses thousands of painted toy soldiers to construct portraits and scenes. Seguin's process involves studying a source image from a distance and then painting each individual soldier (sometimes over 15,000) a different color.
The Works of Francesco Lo Castro
Juxtapoz // Wednesday, February 05, 2014
The multicolor paintings of Florida-based artist Francesco Lo Castro recently caught our attention. Using oils, acrylics, spray paint, silkscreen as well as layered epoxy resin and gold leaf, hes created a technicolor word of geometrics.
The Work of Monir Farmanfarmaian
Juxtapoz // Wednesday, February 05, 2014
http://synapticstimuli.com/Born in Tehran in 1924, Monir Shahroudy Farmanfarmaian is at the forefront of contemporary Iranian art. Her work illustrates a commitment to traditional Persian techniques and patterns, combining mirror mosaics, Islamic geometric patterns and...
"Control Room" by Roxy Paine
Juxtapoz // Wednesday, February 05, 2014
Artist Roxy Paine takes the concept of a diorama to a new level, creating life-size recreations of fast-food restaurants or a control room. Payne is inspired by environments that are meant to be activated by human interaction. This installation, titled Control Room, is hand-carved from birch wood, formed with steal, and finished with automotive paint.
Paintings by Brett Williams
Juxtapoz // Wednesday, February 05, 2014
We are enjoying these paintings by South African artist Brett Williams. Brett is a self-taught artist who is also color blind and currently based in Cape Town.
"Time For Change" by Waone of Interesni Kazki in India
Street Art // Tuesday, February 04, 2014
Waone of the surrealist muralist duo, Interesni Kazk,i has been trekking through India and occasionally leaving one of his otherworldly pieces behind. The one is on the side of a small weathered building and is entitled "Time for Change." It's located in Kairalinagar, Varkala, Kerala, India.
Louise Zhang's Neon Viscera
Illustration // Tuesday, February 04, 2014
Louise Zhang's brilliantly psychedelic abstractions are mind-numbing meditations on the vagaries of color. Great fields of purples and pinks gradate into intestinal tracts of neon viscerae, the cosmos and the microscopic all coming to mind in a candy-sweet cacophony.
Ben Eine "Heartfelt" @ Judith Charles Gallery, NYC
Juxtapoz // Tuesday, February 04, 2014
Ben Eine, who is famed for traveling the world with his signature style of block letter street art proclamations and is in the personal collection of the White House, will open a new solo show, "Heartfelt," at Judith Charles Gallery in NYC this week, February 5, 2014. The British artist will also be presenting a series of limited edition prints to coincide with the new series of paintings.


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It hasn't been an easy couple of months for RIM, makers of the BlackBerry. The Waterloo, Canada-based company has lost key board members as well as significant smartphone market share to Apple and Android phone makers. But today the smartphone maker is announcing more details on the next version of its phones, which it hopes will help it regain momentum.
BlackBerry OS 10 is the next version of its operating system, and today the company released the software to developers and app makers at its BlackBerry World 2012 conference in Orlando.
"There is tremendous interest, anticipation and momentum building toward the launch of BlackBerry 10 devices, and today we're extremely excited to release the BlackBerry 10 developer beta tools for general use," Christopher Smith, RIM's vice president for handheld application platform and tools, said in a statement.
RIM is giving those developers at the conference a BlackBerry 10 phone — called the Dev Alpha developer testing device – to use to develop apps for the new software. RIM also previewed the BlackBerry 10 software, which has a drastically different interface and design than previous versions of the smartphone software. The interface lets you swipe between apps and has a new and improved touchscreen keyboard.
"We're making incredible progress on BlackBerry 10," RIM CEO Thorsten Heins said in his keynote at the conference. "You can see the detail that we are putting into this platform," he added.
While consumers won't be able to buy this phone, the device gives us an idea of what these future BlackBerrys might look like. Straying from the traditional BlackBerry form factor, the phone doesn't have a hardware keyboard but a big 4.2-inch, 1280×768-resolution touchscreen.
Heins also announced that the first BlackBerry 10 devices would will be out later this year. He didn't provide a concrete date, but it has been rumored for an October launch.


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Grammy Awards 2013: Ellen Gets A Good Look At Katy Perry's... Assets (PHOTOS)
DeGeneres gets a full view of the pop star's ample cleavage at the award show.
Here's looking at you, Katy Perry!
Ellen DeGeneres got a grand view of the pop star's ample assets as the duo — along with the talk show's wife Portia de Rossi — before taking their seats at the 2013 Grammy Awards in Los Angeles on Sunday night.
With de Rossi giggling beside her, DeGeneres jokingly showed off her shocked expression with Perry's cleavage — who donned a green Gucci frock — front and center.
After the trio's hilarious encounter, DeGeneres joined Beyonce to introduce Justin Timberlake's much-awaited comeback performance — but not without cracking a joke.
"Why, thank you. I am a talk show host, a best-selling author, and a selfless humanitarian," she quipped as Bey praised JT.
Meanwhile, Katy was nominated for Best Pop Solo Performance for "Wide Awake, but lost out to Adele's live version of "Set Fire to the Rain."Tell us: What has been your favorite moment of the Grammys so far? Sound off in the comments, below.


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2:23 pm
Wed January 15, 2014
Special Election Bodes Ill For Arkansas Health Plan
A Republican takeover of a northeast Arkansas state Senate seat dims the prospects for continuing the state's compromise Medicaid expansion plan and could be a troubling early sign for Democrats who had hoped to rebound from recent losses at the polls.
Republican John Cooper's win in the special election Tuesday for the seat vacated by Democratic Sen. Paul Bookout over ethics violations gives Republicans a 22-13 majority in the Senate and takes away a key vote for the "private option" Medicaid expansion lawmakers approved last year.
Cooper called his win a clear sign that voters don't support the private option.
Democratic Gov. Mike Beebe said the results make passing the measure more difficult when the legislative session begins Feb. 10, but remained hopeful lawmakers will continue the program.
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Name: Teressa
Location: Alabama
Date: Nov. 25, 2011
My sister and I stayed here last week and it will be were I stay whenever I return to New Orleans.The staff and botel are exceptioal.When ever we had a question the staff were there to help .The hotel and exceptional staff are everything that is New Orleans.I would recommend the Hotel Provencial to anyone who wants to feel the warmth of New Orleans.
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Americans 18-30 are public libraries' biggest fans. "And people are going to libraries not only for the Internet-enabled computers there but also for library reference books, newspapers and magazines," reports the Associated Press, citing a new study by the University of Illinois at Urbana-Champaign, and the Pew Internet & American Life Project. Pew/Internet director Lee […]
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More Web playgrounds coming
Stories about kids' virtual worlds are becoming perennial because children 6-10 appear to be a growth market. Twenty million children are expected to be virtual-world members by 2011, up from 8.2 million right now, according to eMarketer figures cited by the New York Times. This latest article paints a pretty good landscape. There's Disney's new […]
Wii-related 'parental challenges'
A California mom was "lucky" enough this past fall to walk into a toy store right after a fresh shipment of Nintendo Wii consoles had been received. So she bought one for her child as a gift, only too soon to discover some "hidden costs." "Be prepared for "post-Wii stress disorder," she wrote in the […]
Oral history online
You know how most communication, story-telling, and history used to be oral? Well, with social networking, humanity may be coming full circle. "Academic researchers are starting to [explore] the parallels between online social networks and tribal societies," the New York Times reports. "In the collective patter of profile-surfing, messaging and 'friending,' they see the resurgence […]
The social Web Petri dish
Social-networking sites are important Petri dishes. By studying the social Web, researchers are learning a lot about how people interact – not just about how they do so now and online but about human interaction in general. In fact, research in social-networking sites "may be more accurate than personal information offered elsewhere online, such as […]
Politician's profile deleted
It was Liberal Democrat Steve Webb, a British member of Parliament, whose Facebook page was deleted after someone sent in an abuse report calling it an imposter profile. Soon there was a Facebook group called "Steve Webb is Real!", CNET reports. His profile was shortly reinstated, to the satisfaction of his 2,500 Facebook friends and […]
New features afoot at MySpace
MySpace plans to be people's dashboard for navigating cyberspace, USATODAY reports – the place "where they can check in on the activities of friends, peruse email, get the latest on news and weather, and post their favorite photos and videos." To deal with the growing threat Facebook represents to MySpace, USATODAY says, the latter is […]
Controversial 'Cool Girl' game in Oz
Is it a way for "cool girl" wannabes to vent their frustrations, does it teach them to bully, or does it simply entertain? Those are the questions reportedly surrounding a new mobile-phone game in Australia that's drawing international attention. Called "Coolest Girl in School," the game – quite an anomaly because designed specifically for girls […]
Australia's very connected families
Ninety percent of Australian families with children are online, up from 7% in 2005, reports Australian IT, citing new findings from the Australian Communications and Media Authority, Three-quarters (76%) of those online families have broadband connections. The study also found that "most Australian families with children older than eight now have three televisions, three mobile […]
Oz union's Facebook profile
The Australian Workers Union is marketing itself to youth by establishing a presence in Facebook, Australian IT reports. Though union leaders say its profile will get more sophisticated, for now "users can add the 'Proud AWU Supporter' application to their profile pages to obtain the organisation's latest news feeds." Version 2.0 will let users "interact […]
The One Good Thing project
The vast majority of things that happen online are positive. Let's tell that story - together. Learn more
NetFamilyNews – by Anne Collier
- Of student digital privacy & schools demanding passwords
- Sexting & the plummeting teen pregnancy rate
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- Kids' top social media picks: Great new resource for parents
- Two 2014 anniversaries that say reams about our kids' futures
- An app for teens that promotes (& gets) positivity
Analysis & News – by Larry Magid
- ConnectSafely to host Safer Internet Day with Calif AG Kamala Harris and Facebook's Sheryl Sandberg
- Ford CEO Mark Fields on mobility, connected cars & teen safety
- ClassDojo app offers students & parents words of encouragement
- Social media still growing, with Instagram leading the way
- Facebook to carry Amber Alerts — exclusive interview with John Walsh
- Connected devices at CES raise security, privacy and safety questions
- Researcher sets the record straight on teen sexting
- Intel CEO Brian Krzanich on safety, privacy & security issues of drones, wearables and Internet of Things
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Ibiss LED Single Flood HeadPin It Houzz |
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Related Info Tools |
Constructed of die cast aluminum, the IBISS heads from TECH Lighting, are designed to celebrate what?s achievable with LED, resulting in a form that would never be possible with traditional lamping. Going beyond simply incorporating LED, the IBISS heads are beautiful in their minimalist simplicity and deliver ultimate performance.
- Pivots 330° vertically and rotates 360° horizontally
- 50,000 hour lamp life at 70%
- Single head flood
- Embedded lens to diffuse glare and minimize shadowing
- Choice of edge lit or solid edge
- Integrated LED driver, dimmable with Low Voltage Electronic Dimmer.
- Available in Satin Nickel or Antique Bronze finishes.
Choosing your connector:
- FJ- Freejack: For use with mono and multipoint Freejack LED canopies (sold separately).
- MO- Monorail: Includes a connector for use with TECH Lighting's Monorail system.
Socket terminates into a system connector. Elements ordered with a system prefix include a connector for that system.
Lightfair Innovation Awards' Design Excellence Winner.
Material(s): Die-Cast Aluminum, Glass
Dimensions: 5.2"L X 1"W
Lamp Type: LED
Bulbs: 1 X 10W LED Array (2700K or 3000K), 60 lumnes/watt(included); 600 lumens
Listing: ETL, CETL
Manufacturer Specifications: Click to download specifications.
Model(s): 700FJIBISFESL203S 700FJIBISFESL203Z 700FJIBISFESL206S 700FJIBISFESL206Z 700FJIBISFESL212S 700FJIBISFESL212Z 700FJIBISFESL218S 700FJIBISFESL218Z 700FJIBISFESL303S 700FJIBISFESL303Z 700FJIBISFESL306S 700FJIBISFESL306Z 700FJIBISFESL312S 700FJIBISFESL312Z 700FJIBISFESL318S 700FJIBISFESL318Z 700FJIBISFSSL203S 700FJIBISFSSL203Z 700FJIBISFSSL206S 700FJIBISFSSL206Z 700FJIBISFSSL212S 700FJIBISFSSL212Z 700FJIBISFSSL218S 700FJIBISFSSL218Z 700FJIBISFSSL303S 700FJIBISFSSL303Z 700FJIBISFSSL306S 700FJIBISFSSL306Z 700FJIBISFSSL312S 700FJIBISFSSL312Z 700FJIBISFSSL318S 700FJIBISFSSL318Z 700MOIBISFESL203S 700MOIBISFESL203Z 700MOIBISFESL206S 700MOIBISFESL206Z 700MOIBISFESL212S 700MOIBISFESL212Z 700MOIBISFESL218S 700MOIBISFESL218Z 700MOIBISFESL303S 700MOIBISFESL303Z 700MOIBISFESL306S 700MOIBISFESL306Z 700MOIBISFESL312S 700MOIBISFESL312Z 700MOIBISFESL318S 700MOIBISFESL318Z 700MOIBISFSSL203S 700MOIBISFSSL203Z 700MOIBISFSSL206S 700MOIBISFSSL206Z 700MOIBISFSSL212S 700MOIBISFSSL212Z 700MOIBISFSSL218S 700MOIBISFSSL218Z 700MOIBISFSSL303S 700MOIBISFSSL303Z 700MOIBISFSSL306S 700MOIBISFSSL306Z 700MOIBISFSSL312S 700MOIBISFSSL312Z 700MOIBISFSSL318S 700MOIBISFSSL318Z 700WMOIBISFESL203S 700WMOIBISFESL203Z 700WMOIBISFESL206S 700WMOIBISFESL206Z 700WMOIBISFESL212S 700WMOIBISFESL212Z 700WMOIBISFESL303S 700WMOIBISFESL303Z 700WMOIBISFESL306S 700WMOIBISFESL306Z 700WMOIBISFESL312S 700WMOIBISFESL312Z 700WMOIBISFSSL203S 700WMOIBISFSSL203Z 700WMOIBISFSSL206S 700WMOIBISFSSL206Z 700WMOIBISFSSL212S 700WMOIBISFSSL212Z 700WMOIBISFSSL303S 700WMOIBISFSSL303Z 700WMOIBISFSSL306S 700WMOIBISFSSL306Z 700WMOIBISFSSL312S 700WMOIBISFSSL312Z
TECH Lighting
TECH Lighting is North America's premier designer of architectural lighting systems. TECH's products are unique thanks to their quality and attention to detail, making TECH a great solution for both residential and commercial applications.
What began as a Chicago lighting showroom in 1983 evolved into an industry-leading manufacturer. Over the years, TECH Lighting became famous for its low-voltage track lighting systems, such as MonoRail, Kable Lite, and TwinRail.
This tradition of excellence continues with the introduction of T-trak™, winner of the Lightfair 2003 Design Excellence Award for most innovative new product. T-trak is the first-ever hand-bendable, line-voltage track lighting system. This revolutionary system features a sleek metal track that can be curved in the field to form practically any shape, while simultaneously providing powerful and versatile illumination options.
These systems are complemented by a wide range of pendants, wall sconces, bath lighting, chandeliers, ceiling fixtures, picture lights, and more. TECH's commitment to innovation, design and craftsmanship is evident in every component, from their rail and cable, to signature glass, to the smallest hardware detailing.
Four Inch Flush FreeJack CanopyPrice: $92.00+ Free Shipping
FreeJack 4 Inch Round Flush Wood CanopyPrice: $108.00+ Free Shipping
FreeJack Surface CanopyPrice: $96.00+ Free Shipping
Free shipping
on most orders
Expert advice
+ service
Best collection
of modern designs
Price guarantee
100% price match


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Meteorologists from the Philippine Atmospheric, Geophysical and Astronomical Services Administration (PAGASA) monitor and plot the direction of super typhoon Hagupit at PAGASA in suburban Manila on December 4, 2014. A super typhoon gained strength on December 4 as it tracked towards the Philippines, threatening more devastation for mostly poor communities where thousands of people have died in an annual tirade of mega storms. AFP PHOTO / Jay DIRECTOJAY DIRECTO/AFP/Getty Images
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