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First published online 6 February 2008
doi: 10.1242/dev.009530
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Section of Molecular Cell and Developmental Biology, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA.
* Author for correspondence (e-mail: jaf{at}mail.utexas.edu)
Accepted 10 January 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Notch, Delta, Epsin, Clathrin, Auxilin, liquid facets, Signaling, Eye, Drosophila, Endocytosis
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). In the signaling cells, transmembrane ligand endocytosis is
necessary for signaling (Le Borgne et al.,
2005; Le Borgne,
2006; Chitnis,
2006; Nichols et al.,
2007a). In Drosophila, ligand endocytosis and signaling
depend on Epsin (Overstreet et al.,
2004; Wang and Struhl,
2004; Wang and Struhl,
2005), an endocytic factor first identified in vertebrates that
functions in at the internalization step of endocytosis
(Chen et al., 1998).
Drosophila Epsin is encoded by the liquid facets
(lqf) gene (Cadavid et al.,
2000).
Recently, the endocytic protein Auxilin has been implicated in Notch
signaling in Drosophila (Hagedorn
et al., 2006; Eun et al.,
2007). Auxilin is an adapter for the ATPase Hsc70 (Heat shock
cognate 70); Auxilin binds Hsc70 with its J domain and also binds Clathrin
cages, which are Hsc70 substrates. Auxilin catalyzes the binding of Hsc70 to
Clathrin cages and stimulates its ATPase activity
(Lemmon, 2001;
Eisenberg and Greene, 2007).
The evidence is most solid for two direct functions of Auxilin, one in
internalization and a second in uncoating Clathrin-coated vesicles. At the
internalization step, Hsc70/Auxilin exchanges Clathrin on cages forming at the
plasma membrane, which might help to constrict vesicles prior to scission.
After scission, Hsc70/Auxilin removes the Clathrin cages, allowing vesicle
fusion with endosomes (Lemmon,
2001; Eisenberg and Greene,
2007).
Here, we show that Drosophila Auxilin is crucial in signaling cells for Delta internalization and signaling. In addition, we show that the requirement for Auxilin is, at least in part, to maintain the pool of free Clathrin. We infer that Clathrin has a key role in the signaling cells. We discuss how these observations impact current thinking on the roles of Epsin and endocytosis in the signaling cells.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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From our laboratory: w1118; w; gaux+/CyO
(Eun et al., 2007);
aux727, auxD136, auxK47,
auxD128 (Eun et al.,
2007); lqfFDD9 (FBal0104483);
UAS-aux+ (FBal0190739); UAS-GFP-Clc (FBtp0017428); and
Nts1 (FBal0012887).
Generated in this work: w; FRT42D, gaux+, ubi-GFP/CyO, YFP;
aux727/TM6B; w; Tub-aux+, ubi-GFP, FRT40A/CyO, YFP;
aux727/TM6B; w, eyFLP; FRT40A/CyO, YFP;
auxD128/TM6B; w, eyFLP; FRT42D/CyO, YFP;
auxD136/TM6B; w, eyFLP; FRT42D/CyO, YFP;
auxD136 Dl-lacZ/TM6B; w; ro-aux+/CyO;
auxK47/TM6B; w; Act5C-gal4/CyO;
aux727/TM6B; w; Act5C-gal4/CyO;
auxK47/TM6B; w; UAS-auxCBD+J/CyO;
auxD128/TM6B; w; UAS-auxK+PTEN/CyO;
auxD128/TM6B; w;
UAS-aux
J/CyO;
auxD128/TM6B; w; gauxCBD+J/CyO;
auxD128/TM6B; w; gChc+/CyO; auxK47/TM6B;
w; gChc+/CyO; auxD128/TM6B; and w; gChc+/CyO;
lqfFDD9/TM6B.
P-element constructs and transformation
Molecular biology was performed using standard procedures. Cloning enzymes
and standard oligonucleotides were from New England Biolabs and Roche, custom
oligonucleotides were from IDT, and DNA purification kits were from Qiagen.
The DNA sequences of all PCR products were verified.
ro-aux+
An aux+ cDNA in pOT2 (GH26574 from the
Drosophila Genomics Resource Center, Bloomington, IN) was purified as
an XhoI-EcoRI fragment and ligated into those sites of
pBSKSII+ (Stratagene) to generate pBS-aux+. The
EcoRI site in pBS-aux+ was changed to AscI by
ligating annealed oligonucleotides of the sequence
5'-AATTGGCGCGCC-3' into the EcoRI site. Subsequently, the
XhoI site in the resulting plasmid was changed to AscI by
ligating annealed oligonucleotides of the sequence
5'-TCGAGGCGCGCC-3' into the XhoI site. The 4.4 kb
AscI fragment containing the aux+ cDNA was ligated into the
pRo vector (Huang and
Fischer-Vize, 1996) and the correct orientation determined by
SalI digestion.
The aux+ cDNA coding region is 3498 nt long, including start and stop codons, and it encodes a protein of 1165 amino acids (aa), including the Met initiator codon.
UASt-auxCBD+J
This construct contains nt 2071-3498 of the aux+ cDNA coding
region, corresponding to aa N691-A1165. A 1.4 kb region of the aux+
coding region was amplified by PCR (Platinum PCR Supermix, Invitrogen) using
pBS-aux+ as the template and the primer pair
5'-GGAATTCATGAACCAGGACTTGGATGATCTG-3' and
5'-GTCTAGATTACGCATTAAACATATTTTGCTG-3'. The PCR product was
subcloned into pGEM-T Easy (Promega). A 1.4 kb EcoRI-XhoI
fragment containing auxCBD+J was purified and ligated into
pUASt (Brand and
Perrimon, 1993) restricted with EcoRI and
XhoI.
UASt-auxK+PTEN
This construct contains nt 1-2103 of the aux+ cDNA coding region,
corresponding to aa M1-E701. A 2.1 kb AscI fragment containing
auxK+PTEN was generated by PCR using pBS-aux+ as
a template, the primers 5'-AAAGGCGCGCCATGGGCGAGTTCTTTAAGTCGCTC-3'
and 5'-AAAGGCGCGCCTCACTCAGGGTCTGGCAGATCATCCA-3', and Pfu Turbo
Polymerase (Stratagene). The amplified product was gel-purified, restricted
with AscI, and ligated into AscI-restricted
pUASt.
UASt-auxJ
This construct contains nt 1-3291 of the aux+ cDNA coding region,
corresponding to aa M1-D1097. A 3.3 kb fragment containing
auxJ was generated by PCR using
pBS-aux+ as template, the primers
5'-AAAGGCGCGCCATGGGCGAGTTCTTTAAGTCGCTC-3' and
5'-GGCTAGCTTAATGTCCCACAACACTGTGTGCATAG-3', and Herculase DNA
Polymerase (Stratagene). The product was ligated into pGEMT-Easy, re-isolated
as an AscI-XbaI fragment, and ligated into
pUASt restricted with those enzymes.
pgauxCBD+J
This construct is the 21 kb genomic DNA fragment in pgaux+
(Eun et al., 2007), subcloned
as an NheI fragment into the XbaI site of pCasper3
(Thummel and Pirrotta, 1992)
to generate pCasper3-gaux+, and with a deletion of 4101 bp that
includes the kinase and PTEN domains, and an addition of two aa (Leu-Glu),
corresponding to an XhoI site (5'-CTCGAG-3') just
downstream of the start codon. This was achieved by replacing an internal 10.7
kb KpnI fragment with a 6.6 kb KpnI fragment, which was
generated by joining three PCR products (Herculase), a 5' product, a
middle product, and a 3' product that generates the deletion. The 984 bp
3' product was amplified using pCasper3-gaux+ as template and
the primers 5'-ACTCGAGAACCAGGACTTGGATGATCTGCCAG-3' and
5'-GGTACCGCCTGTGGCTGCG-3', ligated into pGEM-T and re-isolated as
an XhoI-KpnI fragment and ligated into pBSII
restricted with the same enzymes. The middle fragment (2852 bp) was generated
the same way using the primers 5'-AGATATCTCCGGATGGGCAGACACGAA-3'
and 5'-ACTCGAGCATTTTGGTGGTGGCCAATGCTA-3', isolated as an
EcoRV-XhoI fragment and ligated into those two sites in
pBSII containing the 3' fragment. The 5' fragment (2789
bp) was generated the same way using the primers
5'-AGCGGCCGCGGTACCGCCCGAGCCCG-3' and
5'-TCCGGATGTTGCAAACTTTCCAA-3', isolated as a
NotI-BspEI fragment, and ligated into those two sites in
pBSII containing the 3' and middle fragments. Finally, a 6.6 kb
KpnI fragment was isolated from the resulting pBSII plasmid
and ligated into pCasper3-gaux+ restricted with KpnI.
pgChc+
A 14.3 kb AvrII-SacII fragment of BAC22H11 (BACPAC
Resources, Oakland, CA) containing Chc+ genomic DNA was ligated into
pCMC105 (a gift of C.-M. Chen and G. Struhl, Columbia University, New
York, NY) restricted with AvrII and SacII. The resulting
plasmid was restricted with AvrII, and a 4.5 kb
NheI-XbaI fragment of pAT806 (a gift of K. Basler
and G. Struhl) containing a w+ marker gene was ligated in.
Tub-aux+
An 
Tub84B (Tub) promoter fragment was excised from
pKB700 (a gift of K. Basler and G. Struhl) as a 2.6 kb
NotI-KpnI fragment and ligated into pBSKSII
restricted with the same enzymes. The NotI site in the resulting
plasmid was changed to AvrII by inserting the linker
5'-GGCCCCTAGG-3'. The Tub promoter was excised as an
AvrII-KpnI(3' overhang removed) fragment. A 4.4 kb
EcoRI-XhoI fragment containing the aux+ cDNA was
excised from clone GH26574 and ligated into pBSKSII
restricted with the same enzymes. The XhoI site of the resulting
plasmid was changed to NheI using the linker
5'-TCGAGCTAGC-3', the EcoRI site was changed to
EcoRV using the linker 5'-AATTCGATATCG-3', and the
NotI site was changed to AvrII using the linker
5'-GGCCCCTAGG-3'. The Tub promoter fragment was ligated
into the resulting plasmid restricted with EcoRV and AvrII,
and an AvrII-NheI fragment containing Tub-aux+ was
excised. A 1.2 kb SpeI-XbaI fragment containing
transcription termination signals was excised from pAT806 and ligated
into pCasper4 (Thummel and
Pirrotta, 1992) restricted with SpeI. A plasmid with the
termination signal in the appropriate orientation was isolated, restricted
with SpeI, and the Tub-aux+ ligated in.
Transformation
P-element transformation of w1118 flies was
performed according to standard methods in our laboratory or by Genetic
Services (Sudbury, MA).
Auxilin antibodies
Antibodies were generated to Auxilin amino acids Q702-A1165. The
corresponding DNA sequence was amplified by PCR using primers
5'-GGATCCCAGGTGACACCTCGGTTCTGCG-3' and
5'-GAATTCCGCATTAAACATATTTTGCTGCGTG-3', with plasmid
GH26574 as a template, and subcloned into pGEMT. A
BamHI-EcoRI fragment containing the aux coding
sequences was ligated into pRSET (Invitrogen) restricted with the same
enzymes, which added six His codons at the beginning of the open reading
frame. Protein expression from pRSET-aux was in BL21-CodonPlus-RIL cells
(Invitrogen) induced with 0.1 mM IPTG. The 6xHis-Aux protein was purified from
sonicated cells using HisBind (Novagen) and used to raise antibodies in rats
(Pocono Rabbit Farm and Laboratory, Canadensis, PA). Prior to use for
immunostaining, the antisera were preadsorbed with fixed w1118
Drosophila embryos.
Analysis of eyes and wings
Third instar larval eye discs were fixed in PEMS and antibody incubations
and washes were in PBST (see Fischer-Vize
et al., 1992). Primary antibodies used were: mAb323 (1:2) obtained
from Sarah Bray (University of Cambridge, UK); mAb202 (1:10) from
Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA; rat anti-Elav
(9:1) from DSHB; and anti-Aux (1:500). Secondary antibodies (Molecular Probes)
were AlexaFluor 568 anti-mouse and AlexaFluor 633 anti-rat (1:300). AlexaFluor
568-phalloidin (Molecular Probes) was dried and resuspended in PBST at 0.1
unit/l. Eye discs were mounted in Vectashield (Vector) and viewed with a Leica
TCSSP2 confocal microscope. Plastic sectioning of adult eyes was as described
(Tomlinson and Ready, 1987),
and wings were dehydrated in 70% ethanol and mounted in DPX (Fluka). Wing and
eye sections were photographed with a Zeiss Axioplan and Axiocam HRc. Images
were processed with Adobe Photoshop.
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RESULTS |
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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).] The
nonsense mutant auxD136 is likely to be null because the
truncated protein it may produce would lack the J domain
(Fig. 1A), which we show below
is necessary for Auxilin function. aux727 and
auxD128 are also likely null alleles because of their
early nonsense mutations (Fig.
1A), and because their phenotypes in trans to each other or
auxD136 are indistinguishable. Also, we show below that
the mutant eye phenotype of either aux- genotype is identical to
lqf or Delta null mutants. Finally, in eye discs
immunolabeled with a polyclonal antibody to the C-terminal 464 amino acids of
Auxilin, whereas adjacent aux+ cells showed strong signals, clones of
aux727/auxD128
(Fig. 1B,B') cells showed
no signal above background. Nevertheless, there could be some truncated and
functional Auxilin protein produced by aux727 or
auxD128 that escaped detection. In
aux727 or auxD128 homozygous larvae,
mutant aux mRNA accumulates to levels similar to those seen in wild
type (data not shown), and thus through reinitiation of translation downstream
of the nonsense mutation, aux727 or
auxD128 potentially could produce truncated Auxilin
protein. As we show that the C-terminal region of Auxilin containing the
Clathrin-binding and J domains is sufficient for function, truncated Auxilin
protein so produced could be functional. We could not test whether or not
these alleles produce truncated protein undetectable by immunolabeling because
the Auxilin antibody we generated does not detect Auxilin protein on blots of
Drosophila wild-type protein extracts.
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). As cells exit the furrow, lateral inhibition results in
evenly spaced ommatidial preclusters; failure of Notch activation at this
point results in disordered photoreceptors (R-cells)
(Baker, 2002). R-cell
determination in aux- clones appeared identical to that in
Delta or lqf null clones
(Baker and Yu, 1996;
Overstreet et al., 2004). No
R-cells (Elav+) were present in the center of large aux- clones, and
aux- R-cells present at clone borders were disorganized
(Fig. 1D,D'). Another
indicator of lateral inhibition failure is increased Delta
transcription (Baker and Yu,
1998; Wang and Struhl,
2004). aux- clones showed increased expression of a
Delta-lacZ enhancer trap (Fig.
1E,E') and also increased Delta protein
(Fig. 1F,F'). It was
reported previously that Delta-lacZ is not upregulated in eye discs
(Hagedorn et al., 2006).
Perhaps the discrepancy is due to differences in the mutant aux
alleles used. A later Notch signaling event is R-cell restriction, in which
R2/5 and R3/4 signal precluster cells in the developing ommatidium to limit
the number of outer R-cells to six (Fig.
1C) (Overstreet et al.,
2004). Like Delta or lqf hypomorphs
(Parks et al., 1995;
Cadavid et al., 2000),
aux hypomorphic eyes contain ommatidia with extra outer R-cells
(Hagedorn et al., 2006;
Eun et al., 2007).
As is the case for Epsin, the requirement for Auxilin in eye patterning
appears limited to Notch signaling. Several different signaling pathways are
needed for morphogenetic furrow movement, and Epidermal growth factor receptor
signaling is essential for R-cell determination
(Lee and Treisman, 2002;
Kumar, 2002;
Nagaraj et al., 2002). These
pathways are operating in aux- clones because furrow movement inside
aux- clones resembles that in wild type
(Fig. 1F,F'), and cells
expressed Elav, a marker of R-cell determination
(Fig. 1D,D'). In
addition, neither Epsin (Overstreet et
al., 2004; Wang and Struhl,
2004) nor Auxilin is required for cell viability.
Auxilin is essential in signaling cells only
aux- cells become R-cells at clone borders, where they are
adjacent to aux+ cells that signal and impart proneural potential
(Fig. 1D,D'). This
suggests that aux- cells can receive Notch activation signals.
Indeed, aux- cells expressed Enhancer of split proteins [E(spl)], a
direct response to Notch activation
(Jennings et al., 1994), but
only when adjacent to aux+ cells at clone borders
(Fig. 1G,G'). In
addition, in aux hypomorphic eyes, expression of aux+ in
R2/5 and R3/4 by a transgene (ro-aux+) rescued the mutant phenotypes
of ommatidia with ectopic R-cells: 
25% of facets in
auxK47/auxD128 were wild-type, and the addition
of ro-aux+ increased the fraction to 95% (data not shown). The
ro expression construct is remarkably specific for R2/5 and R3/4
(Overstreet et al., 2004).
Thus, R-cell restriction fails when R2/5 and R3/4 have little aux+
activity because they cannot signal to extraneous precluster cells. We
conclude that aux- cells in the eye can receive, but not send, Notch
signals.
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;
Baker and Yu, 1998;
Overstreet et al., 2003;
Overstreet et al., 2004).
Rather, nearly all Delta detected is in endocytic vesicles that are probably
irrelevant to signaling, as they are observed in lqf- cells that do
not signal (Fig. 2C)
(Overstreet et al., 2004;
Wang and Struhl, 2004). In
lqf- clones, lateral inhibition failure results in Delta
overexpression (Baker and Yu,
1998; Wang and Struhl,
2004), which allows detection of Delta on the plasma membrane
(Fig. 2C). This is observed
also in eye discs that are hypomorphic for both lqf and heterozygous
for a neuralized (neur) mutation
(Fig. 2D)
(Overstreet et al., 2004).
neur encodes a Ubiquitin ligase required for Delta ubiquitination and
signaling (Le Borgne et al.,
2005; Le Borgne,
2006). Delta eventually clears from the membrane and is observed
again only in intracellular puncta several rows posterior to the furrow
(Overstreet et al., 2003;
Overstreet et al., 2004). A
similar phenomenon is observed with Notch temperature sensitive
(Nts) mutant discs raised to the restrictive temperature
for 6 hours (Fig. 2B)
(Baker and Yu, 1998). However,
in Nts discs, where lateral inhibition fails owing to a
failure in signal reception, Delta clears from the membrane quickly and only a
tight band of membrane-associated Delta is apparent at the furrow
(Fig. 2B)
(Baker and Yu, 1998). By
contrast, in lqf- cells, Delta remains on the membrane for a longer
time (more rows of cells) posterior to the furrow
(Fig. 2C,D)
(Overstreet et al., 2003;
Overstreet et al., 2004) and
we interpret this as inefficient endocytosis. This observation is similar to
that in the wing disc, where Delta was overexpressed using a transgene and
clones of lqf-cells accumulated more Delta on the membrane than did
adjacent wild-type cells (Wang and Struhl,
2004). Delta also persisted on the membrane in aux mutant
cells (Fig.
2E-E'',F-F''). Also, as in lqf- clones
(Overstreet et al., 2004;
Wang and Struhl, 2004), Delta
puncta were present in aux- cells
(Fig. 2F,G). We conclude that
like Epsin, Auxilin is required for some, but not all, Delta internalization.
Similarly, in yeast and vertebrate cells, aux mutations result in
decreased endocytosis (Pishvaee et al.,
2000; Umeda et al.,
2000).
An Auxilin fragment including the Clathrin-binding domain and J domain is sufficient for Notch signaling
Drosophila Auxilin, like vertebrate auxilin 2 or GAK (cyclin
G-associated kinase), has N-terminal kinase and PTEN (phosphatase and
tensin-related) domains (Fig.
3), the functions of which are unclear
(Umeda et al., 2000;
Lemmon, 2001;
Korolchuk and Banting, 2002;
Lee et al., 2006;
Massol et al., 2006;
Eisenberg and Greene, 2007).
The auxilins of yeast and C. elegans, which lack kinase and PTEN
domains, uncoat clathrin-coated vesicles (CCVs) in vitro and in vivo
(Gall et al., 2000;
Pishvaee et al., 2000;
Greener et al., 2001), and the
Clathrin-binding and J domains of GAK are sufficient for uncoating in vivo
(Holstein et al., 1996;
Greener et al., 2000). The
presence of the kinase and PTEN domains in Drosophila Auxilin
suggests that the function of Auxilin in Notch signaling might not be limited
to Clathrin uncoating or exchange.
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J had no activity
(Fig. 3A).
Act5C>auxilinCBD+J might overexpress
AuxilinCBD+J relative to endogenous Auxilin. Because genomic
transgenes are likely to be expressed at physiological levels, we also
generated a transgene (gauxCBD+J) that expresses
AuxilinCBD+J in the context of a genomic DNA construct,
gaux+ (Eun et al.,
2007). A single copy of either gaux+ or
gauxCBD+J complemented all aux- phenotypes
(Fig. 3A). These results
suggest that the ability of AuxilinCBD+J to complement
aux- is unlikely to require its overexpression. We conclude that the J domain is necessary and a C-terminal Auxilin fragment including the Clathrin-binding and J domains is sufficient for function in Notch signaling. Thus, the role of Auxilin is likely to depend on its function as a link between Hsc70 and Clathrin cages. Consistent with functions in endocytic vesicle internalization and uncoating, Auxilin was detected apically in cytoplasmic puncta near the plasma membrane (Fig. 3B).
The Notch pathway phenotypes of aux mutants are partially suppressed by an extra copy of the Chc+ gene
In aux mutants, it would be expected that the levels of free
Clathrin are limiting owing to inefficient uncoating of CCVs. In yeast and
C. elegans, free clathrin is depleted in aux mutants
(Gall et al., 2000;
Pishvaee et al., 2000).
Consistent with Clathrin depletion in Drosophila aux mutants, we
found that a loss-of-function mutation in the Clathrin heavy chain
gene, Chc1, is a strong dominant enhancer of aux
hypomorphs: Chc1/+ flies appeared normal, and
auxK47/auxD128 adult flies were viable with
Notch-like phenotypes, but Chc1/+;
auxK47/auxD128 adults could not be obtained. It was
reported previously that expression of GFP-Clathrin light chain (Clc) enhances
aux phenotypes (Hagedorn et al.,
2006), and this result was repeatable in our hands. We do not
think, however, that this genetic interaction contradicts the results with
Chc mutants. Rather, as GFP-Clc does not colocalize with Chc in the
eye disc (data not shown), we propose that GFP-Clc acts as a dominant
negative. There are no Clc mutants available to test for
complementation by GFP-Clc.
We also tested whether an additional copy of the Chc+ gene
(PChc+) suppresses the Notch-like phenotypes of
aux- hypomorphs. We found that PChc+ suppresses the wing
phenotype completely (Fig.
4A,B) and the eye phenotype significantly
(Fig. 4C-F). Similarly, in
C. elegans, aux(RNAi) mutant phenotypes are suppressed by
overexpression of clathrin heavy chain
(Greener et al., 2001). Taken
together, these results suggest that Notch pathway phenotypes of aux
mutants are due at least in part to Clathrin depletion. Clathrin is likely to
be needed in some manner for Delta internalization. Indeed, we have shown that
Delta endocytosis is inefficient in aux mutants. Thus, the
requirement for Auxilin does not necessarily infer a requirement in the
signaling cells for trafficking of Delta-containing endosomes downstream of
internalization, as proposed previously
(Hagedorn et al., 2006).
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). As Epsin has both Ubiquitin- and Clathrin-binding motifs,
and also binds the plasma membrane, the simplest scenario imaginable for
Clathrin and Epsin function in Delta internalization is for Epsin to act as a
Clathrin adapter that recognizes ubiquitinated Delta, and brings Clathrin to
the membrane for CCV formation (Wendland,
2002). However, in light of evidence that Epsin-dependent
endocytosis of ubiquitinated transmembrane proteins such as Delta may not
occur through formation of CCVs (Chen and
De Camilli, 2005; Sigismund et
al., 2005; Aguilar and
Wendland, 2005; Hawryluk et
al., 2006; Barriere et al.,
2006; Madshus,
2006), it became unclear how to interpret the Chc/lqf
genetic interaction. The results presented here point to a crucial role for
Clathrin in Notch signaling cells. One intriguing possibility is that Delta
internalization depends on Clathrin not because Delta is endocytosed in CCVs,
but because Clathrin is a positive regulator of Epsin function. More
experiments are required to test this idea.
Why do tissues that lack Epsin or Auxilin display Delta-like
phenotypes, rather than phenotypes indicating failure of many signaling
pathways or even cell death? One possibility is that the apparent specificity
of both Epsin and Auxilin might simply reflect the usual redundancy of
endocytic protein functions, and an unusual dependence of Notch signaling on
efficient endocytosis. Alternatively, a special function of Epsin might be
crucial to Notch signaling cells. Two kinds of models have been proposed to
explain why Notch signaling requires ligand endocytosis by the signaling cells
(Le Borgne et al., 2005;
Le Borgne, 2006;
Chitnis, 2006;
Nichols et al., 2007a). One
idea (the `pulling model') is that after receptor binding, ligand endocytosis
generates mechanical forces that result in cleavage of the Notch intracellular
domain (Notch activation), either by exposing the proteolytic cleavage site on
the Notch extracellular domain, or by causing the heterodimeric Notch receptor
to dissociate (Parks et al.,
2000; Nichols et al.,
2007b). Alternatively, ligand internalization prior to receptor
binding might be required to process the ligand endosomally, and recycle it
back to the plasma membrane in an activated form (the `recycling model')
(Le Borgne and Schweisguth,
2003; Wang and Struhl,
2004; Wang and Struhl,
2005; Emery et al.,
2005; Jafar-Nejad et al.,
2005). Epsin might generate an environment particularly conducive
to either pulling or recycling, and Auxilin might be required specifically by
Notch signaling cells because it activates Epsin, perhaps by providing free
Clathrin. Alternatively, Auxilin might be needed to provide free Clathrin
because Delta is internalized through CCVs. In this case, if Auxilin is
required in Notch signaling solely to provide free Clathrin, the implication
would be that efficient CCV uncoating is not important for generating uncoated
Delta-containing vesicles per se, which are prerequisite for travel through an
endosomal recycling pathway. Further understanding of the role of Auxilin in
Notch signaling cells might be key to understanding the role of ligand
endocytosis.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Aguilar, R. C. and Wendland, B. (2005).
Endocytosis of membrane receptors: two pathways are better than one.
Proc. Natl. Acad. Sci. USA
102,2679
-2680.
Baker, N. E. (2002). Notch and the patterning
of ommatidial founder cells in the developing Drosophila eye. In
Drosophila Eye Development (ed. K. Moses), pp.35
-58. Berlin: Springer-Verlag.
Baker, N. E. and Yu, S.-Y. (1996). Proneural
function of neurogenic genes in the developing Drosophila eye.
Curr. Biol. 7,122
-132.[CrossRef]
Baker, N. E. and Yu, S.-Y. (1998). The
R8-photoreceptor equivalence group in Drosophila: fate choice precedes
regulated Delta transcription and is independent of Notch
gene dose. Mech. Dev.
74, 3-14.[CrossRef][Medline]
Barriere, H., Nemes, C., Lechardeue, D., Kahn-Mohammed, M.,
Fruh, K. and Lukacs, G. L. (2006). Molecular basis of
oligoubiquitin-dependent internalization of membrane proteins in mammalian
cells. Traffic 7,282
-297.[CrossRef][Medline]
Bazinet, C., Katzen, A. L., Morgan, M., Mahowald, A. P. and
Lemmon, S. K. (1993). The Drosophila clathrin heavy
chain gene: clathrin function is essential in a multicellular organism.
Genetics 134,1119
-1134.[Abstract]
Brand, A. H. and Perrimon, N. (1993). Targeted
gene expression as a means of altering cell fates and generating dominant
phenotypes. Development
118,401
-415.[Abstract]
Bray, S. J. (2006). Notch signalling: a simple
pathway becomes complex. Nat. Rev. Mol. Cell Biol.
7, 678-689.[CrossRef][Medline]
Cadavid, A. L. M., Ginzel, A. and Fischer, J. A.
(2000). The function of the Drosophila Fat facets
deubiquitinating enzyme in limiting photoreceptor cell number is intimately
associated with endocytosis. Development
127,1727
-1736.[Abstract]
Chen, H. and De Camilli, P. (2005). The
association of epsin with ubiquitinated cargo along the endocytic pathway is
negatively regulated by its interaction with clathrin. Proc. Natl.
Acad. Sci. USA 102,2766
-2771.
Chen, H., Fre, S., Slepnev, V. I., Capua, M. R., Takei, K.,
Butler, M. H., Di Fiore, P. P. and De Camilli, P. (1998).
epsin is an EH-domain-binding protein implicated in clathrin-mediated
endocytosis. Nature 394,793
-797.[CrossRef][Medline]
Chitnis, A. (2006). Why is Delta endocytosis
required for effective activation of Notch? Dev. Dyn.
235,886
-894.[CrossRef][Medline]
Eisenberg, E. and Greene, L. E. (2007).
Multiple roles of auxilin and Hsc70 in clathrin-mediated endocytosis.
Traffic 8,640
-646.[CrossRef][Medline]
Emery, G., Hutterer, A., Berdnik, D., Mayer, B., Wirtz-Peitz,
T., Gaitan, M. G. and Knoblich, J. A. (2005). Asymmetric
Rab11 endosomes regulate Delta recycling and specify cell fate in the
Drosophila nervous system. Cell
122,763
-773.[CrossRef][Medline]
Eun, S. H., Lea, K., Overstreet, E., Stevens, S., Lee, J.-H. and
Fischer, J. A. (2007). Identification of genes that interact
with Drosophila liquid facets. Genetics
175,116
3-1174.
Fischer-Vize, J. A., Rubin, G. M. and Lehmann, R.
(1992). The fat facets gene is required for Drosophila
eye and embryo development. Development
116,985
-1000.[Abstract]
Gall, W. E., Higginbotham, M. A., Chen, C.-Y., Ingram, M. F.,
Cyr, D. M. and Graham, T. R. (2000). The auxilin-like
phosphoprotein Swa2p is required for clathrin function in yeast.
Curr. Biol. 10,1349
-1358.[CrossRef][Medline]
Greener, T., Zhao, X., Nojima, H., Eisenberg, E. and Greene, L.
E. (2000). Role of cyclin G-associated kinase in uncoating
clathrin-coated vesicles from non-neuronal cells. J. Biol.
Chem. 275,1365
-1370.
Greener, T., Grant, B., Zhang, Y., Wu, X., Greene, L. E., Hirsh,
D. and Eisenberg, E. (2001). Caenorhabditis elegans
auxilin: a J-domain protein essential for clathrin-mediated endocytosis in
vivo. Nat. Cell Biol.
3, 215-219.[CrossRef][Medline]
Hagedorn, E. J., Bayraktar, J. L., Kandachar, V. R., Bai, T.,
Englert, D. M. and Chang, H. C. (2006). Drosophila
melanogaster auxilin regulates the internalization of Delta to control
activity of the Notch signaling pathway. J. Cell Biol.
173,443
-452.
Hawryluk, M. J., Keyel, P. A., Mishra, S. K., Watkins, S. C.,
Heuser, J. E. and Traug, L. M. (2006). Epsin is a
polyubiquitin-selective clathrin-associated sorting protein.
Traffic 7,262
-281.[CrossRef][Medline]
Holstein, S. E., Ungewickell, H. and Ungewickell, E.
(1996). Mechanism of clathrin basket dissociation: separate
functions of protein domains of the DnaJ homologue auxilin. J. Cell
Biol. 135,925
-937.
Huang, Y. and Fischer-Vize, J. A. (1996).
Undifferentiated cells in the developing Drosophila eye influence facet
assembly and require the Fat facets ubiquitin-specific protease.
Development 122,3207
-3216.[Abstract]
Jafar-Nejad, H., Andrews, H. K., Acar, M., Bayat, B.,
Wirtz-Peitz, F., Mehta, S. Q., Knoblich, J. A. and Bellen, H. J.
(2005). Sec15, a component of the exocyst, promotes Notch
signaling during the asymmetric division of Drosophila sensory organ
precursors. Dev. Cell 9,351
-363.[CrossRef][Medline]
Jennings, B., Preiss, A., Delidakis, C. and Bray, S.
(1994). The Notch signaling pathway is required for Enhancer of
split protein expression during neurogenesis in the Drosophila embryo.
Development 120,3537
-3548.[Abstract]
Korolchuk, V. I. and Banting, G. (2002). CK2
and GAK/auxilin2 are major protein kinases in clathrin-coated vesicles.
Traffic 3,428
-439.[CrossRef][Medline]
Kumar, J. (2002). The epidermal growth factor
receptor in Drosophila eye development. In Drosophila Eye
Development (ed. K. Moses), pp. 59-71.
Berlin: Springer-Verlag.
Le Borgne, R. (2006). Regulation of Notch
signalling by endocytosis and endosomal sorting. Curr. Opin. Cell
Biol. 18,213
-222.[CrossRef][Medline]
Le Borgne, R. and Schweisguth, F. (2003).
Unequal segregation of Neuralized biases Notch activation during asymmetric
cell division. Dev. Cell
5, 139-148.[CrossRef][Medline]
Le Borgne, R., Bardin, A. and Schweisguth, F.
(2005). The roles of receptor and ligand endocytosis in
regulating Notch signaling. Development
132,1751
-1762.
Lee, D.-W., Wu, X., Eisenberg, E. and Greene, L. E.
(2006). Recruitment dynamics of GAK and auxilin to
clathrin-coated pits during endocytosis. J. Cell Sci.
119,3502
-3512.
Lee, J. D. and Treisman, J. E. (2002).
Regulators of the morphogenetic furrow. In Drosophila Eye
Development (ed. K. Moses), pp. 21-58.
Berlin: Springer-Verlag.
Lemmon, S. K. (2001). Clathrin uncoating:
auxilin comes to life. Curr. Biol.
11,R49
-R52.[CrossRef][Medline]
Madshus, I. H. (2006). Ubiquitin binding in
endocytosis - how tight should it be and where does it happen?
Traffic 7,258
-261.[CrossRef][Medline]
Massol, R. H., Boll, W., Griffin, A. M. and Kirchhausen, T.
(2006). A burst of auxilin recruitment determines the onset of
clathrin-coated vesicle uncoating. Proc. Natl. Acad. Sci.
USA 103,10265
-10270.
Nagaraj, R., Canon, J. and Banerjee, U. (2002).
Cell fate specification in the Drosophila eye. In Drosophila eye
development (ed. K. Moses), pp. 73-88.
Berlin: Springer-Verlag.
Nichols, J. T., Miyamoto, A. and Weinmaster, G.
(2007a). Notch signaling-constantly on the move.
Traffic 8,959
-969.[CrossRef][Medline]
Nichols, J. T., Miyamoto, A., Olsen, S. L., D'Souza, B., Yao, C.
and Weinmaster, G. (2007b). DSL ligand endocytosis physically
dissociates Notch1 heterodimers before activating proteolysis can occur.
J. Cell Biol. 176,445
-458.
Overstreet, E., Chen, X., Wendland, B. and Fischer, J. A.
(2003). Either part of a Drosophila epsin protein, divided after
the ENTH domain, functions in endocytosis of Delta in the developing eye.
Curr. Biol. 13,854
-860.[CrossRef][Medline]
Overstreet, E., Fitch, E. and Fischer, J. A.
(2004). Fat facets and Liquid facets promote Delta endocytosis
and Delta signaling in the signaling cells.
Development 131,5355
-5366.
Parks, A. L., Turner, F. R. and Muskavitch, M. A. T.
(1995). Relationships between complex Delta expression and the
specification of retinal cell fates during Drosophila eye development.
Mech. Dev. 50,201
-216.[CrossRef][Medline]
Parks, A. L., Klueg, K. M., Stout, J. R. and Muskavitch, M.
A. (2000). Ligand endocytosis drives receptor dissociation
and activation in the Notch pathway. Development
127,1373
-1385.[Abstract]
Pishvaee, B., Costaguta, G., Yeung, B. G., Ryazantsev, S.,
Greener, T., Greene, L. E., Eisenberg, E., McCaffery, J. M. and Payne, G.
S. (2000). A yeast DNA J protein required for uncoating of
clathrin-coated vesicles in vivo. Nat. Cell Biol.
2, 958-963.[CrossRef][Medline]
Sigismund, S., Woelk, T., Puri, C., Maspero, E., Tacchetti, C.,
Transidico, P., Di Fiore, P. P. and Polo, S. (2005).
Clathrin-independent endocytosis of ubiquitinated cargos. Proc.
Natl. Acad. Sci. USA 102,2760
-2765.
Thummel, C. S. and Pirrotta, V. (1992).
Technical notes: new pCaSpeR P element vectors. Drosoph. Inf.
Serv. 71,150
.
Tomlinson, A. and Ready, D. F. (1987). Neuronal
differentiation in the Drosophila ommatidium. Dev.
Biol. 120,366
-376.[CrossRef][Medline]
Umeda, A., Meyerholz, A. and Ungewickell, E.
(2000). Identification of the universal cofactor (auxilin 2) in
clathrin coat dissociation. Eur. J. Cell Biol.
79,336
-342.[CrossRef][Medline]
Wang, W. and Struhl, G. (2004). Drosophila
epsin mediates a select endocytic pathway the DSL ligands must enter to
activate Notch. Development
131,5367
-5380.
Wang, W. and Struhl, G. (2005). Distinct roles
for Mind bomb, Neuralized and epsin in mediating DSL endocytosis and signaling
in Drosophila. Development
132,2883
-2894.
Wendland, B. (2002). Epsins: adaptors in
endocytosis? Nat. Rev. Mol. Cell Biol.
3, 971-977.[CrossRef][Medline]
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