Howard Hughes Medical Institute, Department of Genetics and Development, Columbia University College of Physicians and Surgeons, 701 West 168th Street, New York, NY 10032, USA
* Author for correspondence (e-mail: gs20{at}columbia.edu)
Accepted 20 August 2004
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SUMMARY |
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Key words: Drosophila, DSL-Notch signaling, Delta, Epsin/Liquid facets (Lqf), Endocytosis, Mono-ubiquitination
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Introduction |
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Although much is known about how Notch transduces signals once the receptor
undergoes the ligand-dependent S2 cleavage, the mechanism by which DSL ligands
engage Notch and trigger this cleavage are less well understood. Recent
evidence suggests that Clathrin-mediated endocytosis of DSL ligands is
required for this activating event. First, Dynamin, which functions to pinch
off invaginating coated pits to form endocytic vesicles, appears to be
required for Notch signaling prior to transmembrane (S3) cleavage of the
receptor (Seugnet et al.,
1997; Struhl and Adachi,
2000
). Second, the E3-Ubiquitin ligases Neuralized (Neur) and Mind
bomb (Mib) interact physically with Delta (Dl), promote Dl ubiquitination and
internalization, and enhance its signaling activity
(Deblandre et al., 2001
;
Lai et al., 2001
;
Pavlopoulos et al., 2001
;
Yeh et al., 2001
;
Itoh et al., 2003
;
Le Borgne and Schweisguth,
2003a
). Third, deleted or mutated forms of Dl that cannot be
efficiently endocytosed have reduced, or no, signaling activity
(Parks et al., 2000
;
Itoh et al., 2003
). Finally,
Notch signal transduction appears to be correlated with trans-endocytosis of
the Notch extracellular domain into the signal-sending cells
(Parks et al., 2000
).
It has been proposed that endocytosis of DSL ligands on signal-sending
cells that are bound to Notch on adjacent signal-receiving cells induces, via
mechanical stress, the S2 or S3 cleavage of the receptor, thus activating
signal transduction (Parks et al.,
2000). Alternatively, it has been suggested that endocytosis may
function to cluster DSL ligands, or to clear previously shed ectodomains of
Notch, to ensure robust activation of the receptor, or to package DSL ligands
into signaling exosomes (reviewed by Le
Borgne and Schweisguth, 2003b
).
To resolve the role of endocytosis in signaling by DSL ligands, we sought
to identify and manipulate endocytic proteins that are required specifically
in signaling cells for the activation of Notch in receiving cells. As we will
describe, we have found that Liquid Facets (Lqf), the sole Drosophila
Epsin (Cadavid et al., 2000),
plays just such a role, allowing us to examine the relationship between DSL
endocytosis and signaling.
Epsins are endocytic proteins that bind phosphatidylinositol
(4,5)-bisphosphate [PtdIns(4,5)P2] in the plasma membrane
as well as Clathrin, AP-2 Adapter Complex, and other accessory proteins in
coated pits (reviewed by Wendland,
2002). Epsins were initially thought to be core components of the
endocytic machinery because of the dominant-negative effects of truncated
Epsin proteins on endocytosis in mammalian cells
(Chen et al., 1998
;
Ford et al., 2002
), their
essential role in yeast endocytosis
(Wendland et al., 1999
), and
their inherent capacity to induce membrane curvature
(Ford et al., 2002
) and bind
other core components such as Clathrin and AP-2
(Chen et al., 1998
;
Owen et al., 1999
;
Rosenthal et al., 1999
;
Wendland et al., 1999
;
Drake et al., 2000
). More
recently, however, the identification of Ubiquitin-interacting motifs (UIMs)
in Epsins, as well as in other proteins involved in membrane trafficking
(Hofmann and Falquet, 2001
),
have led to the suggestion that Epsins belong to a family of cargo-selective
adapters that link mono-ubiquitinated cell-surface proteins with the endocytic
machinery (Wendland,
2002
).
Here, we report that DSL ligands must normally be endocytosed in signal-sending cells via the action of Lqf to activate Notch on the surface of signal-receiving cells. Surprisingly, however, bulk endocytosis of DSL ligands appears normal in the absence of Lqf. We resolve this apparent paradox by providing evidence that Lqf is unique amongst adapters that target mono-ubiquitinated cargo proteins for internalization, in that it allows them to enter a special endocytic pathway that DSL ligands must enter to acquire signaling activity. We also show that this requirement can be bypassed by introducing the internalization signal that normally mediates internalization and recycling of the Low Density Lipoprotein (LDL) receptor. On the basis of these results, we hypothesize that Epsin-mediated endocytosis might be required to allow DSL proteins to be recycled rather than degraded following internalization, possibly to convert them from inactive pro-ligands into active ligands.
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Materials and methods |
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DlRm is identical to DlR+ except that the two Lysines
are changed to Arginine. DlUbim is identical to DlUbi+
except that the Isoleucine at position 44 is changed to Alanine. The Lysines
in the Dl stop transfer sequence were deleted for the DlLDL+,
DlLDLm, DlRm, DlUbi+ and DlUbim
chimeric proteins. For DlC and the DlR+ chimera,
both possible forms (with and without the Lysines in the stop transfer
sequence) were assayed; no difference in behavior was observed for either
protein.
Genetic material
lqf1227 (this study), lqfARI,
lqfBT (Overstreet et al.,
2003);
P[w+],lqf+(Cadavid
et al., 2000
);
hrsD28(Lloyd et al.,
2002
); Tub
1-Gal4, UAS-GFPnls
(Struhl and Greenwald, 2001
);
wgCX4, C765-Gal4, Vg-boundary-lacZ
(Zecca et al., 1996
);
nub-Gal4, UAS-y+
(Calleja et al., 1996
);
omb-lacZ, dpp-lacZ (Nellen et
al., 1996
); UAS-CD8-GFP, Tub
1-G80
(Lee and Luo, 1999
);
UAS-wg, UAS-dpp, UAS-hh (K. Basler, unpublished); UAS-fng,
UAS-Ser (Panin et al.,
1997
); UAS-Neur (Lai
et al., 2001
); Dl-lacZ, arm-lacZ, Ubi-GFP (from
Bloomington stock center).
Genotypes employed
lqf- clones
y hsp70-flp; lqf1227 FRT2A/Ubi-GFP (or
arm-lacZ) FRT2A [lacZ reporter genes on X, II or III;
mwh was used in cis with lqf1227 to mark hairs in
the adult cuticle; clones of lqfBT and
lqfARI cells were generated in the same way and gave
indistinguishable results].
lqf- clones in discs expressing UAS-Dl and/or UAS-neur
These clones were the same as above except for the added presence of the
appropriate UAS-Dl, UAS-neur, C765-Gal4 and/or nub-Gal4
transgenes. We note that Neur does not enhance the endocytosis or signaling
activity of either the DlLDL+ or DlR+ chimeric proteins,
presumably because it does not recognize their cytosolic domains as
substrates. Therefore, we could not use ectopic Neur expression to create a
sensitized background to test whether DlLDL+ or DlR+
endocytosis is compromised in lqf- cells, in contrast to
the situation with native Dl.
lqf- clones expressing markers and/or UAS-X transgenes (MARCM technique)
y hsp70-flp Tub1-Gal4 UAS-GFPnls (or
UAS-CD8-GFP)/y hsp70-flp; lqf1227
FRT2A/Tub
1-Gal80 FRT2A [UAS-X transgenes and
lacZ reporters on X, II or III; clones of lqfBT
cells (as well as control clones of lqf+ cells) that
express markers; and/or UAS-X transgenes were generated in the same
way]. Dpp, Hh and Wg signaling were assayed by, respectively,
omb-lacZ expression (Grimm and
Pflugfelder, 1996
; Nellen et
al., 1996
), dpp-lacZ and Collier expression
(Basler and Struhl, 1994
;
Vervoort et al., 1999
), and Dl
and Senseless expression (Micchelli et
al., 1997
; Nolo et al.,
2000
).
lqf-::UAS-DSL twin spots (modified MARCM technique)
y hsp70-flp Tub1-Gal4 UAS-GFPnls/y hsp70-flp; UAS-Dl (or
UASSer)/+; lqf1227 Tub
1-Gal80 FRT2A/arm-lacZ
FRT2A.
hrs-, wg- and lqf- combination clones (MARCM technique)
y hsp70-flp/y hsp70-flp UAS-HRPDl; hrsD28
(wgCX4) FRT40/ P[w+],lqf+
Tub1-Gal80 FRT40; lqf1227 C765-Gal4/lqf1227
(or +); clones of hrs- lqf- cells
expressing MycDl were obtained similarly, using a
UAS-MycDl transgene in cis with hrsD28
FRT40 in place of the UAS-HRPDl transgene in X, and
gave the same result.
Generation of clones
Clones were generated by heat shocking first or second instar larvae at
37°C for 60 minutes.
Immunofluorescent staining
Imaginal discs were fixed and stained by standard procedures (e.g.
Jiang and Struhl, 1995;
Zecca et al., 1996
), using
mouse
-Dl (Developmental Studies Hybridoma Bank, DSHB), guinea pig
-Dl (Parks et al.,
2000
), guinea pig
-Hrs
(Lloyd et al., 2002
), mouse
-Wg (DSHB), mouse
-Cut (DSHB), guinea pig
-Lqf
(Overstreet et al., 2003
),
rabbit
-Col (Vervoort et al.,
1999
), rat
-Ci (DSHB), rat
-Ser
(Panin et al., 1997
), and
commercially available mouse
-Myc, rabbit
-HRP, and rabbit
anti-ßGal. Two protocols were used to stain cell surface expression (both
gave similar results): (1) living discs were incubated in Drosophila
tissue culture media with the first primary antisera at 4°C for 20
minutes, rinsed several times with ice-cold media, fixed in the absence of
detergent, and then subjected to the standard protocol using additional
primary antisera in the presence of detergent; and (2) discs were fixed and
processed for immunofluorescent staining by the standard protocol, except in
the absence of detergent until after the secondary antisera was removed.
Western blotting experiments
Proteins extracted from mature third instar wing discs carrying multiple
clones of wild-type or lqf- cells co-expressing
MYCDl and Neur (MARCM technique) were separated by SDS-PAGE
electrophoresis, and blotted to Nitrocellulose for western analysis by
standard protocol (see Struhl and Adachi,
2000). Commercially available mouse
-Myc was used to detect
MYCDl.
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Results |
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The loss of margin gene expression in lqf- clones is not cell autonomous. Instead, wild-type cells can rescue the expression of margin specific genes in adjacent lqf- cells (e.g. cut; Fig. 1D'). Similarly, we observed non-autonomous rescue of lqf- clones in the adult, where the presence of wild-type cells can rescue the ability of neighboring lqf- cells to form single bristles (Fig. 1C). In both respects, as well as in others (see supplementary material), lqf- clones resemble Dl- Ser- clones, but differ from N- clones, which show a strictly cell-autonomous loss of Notch target gene expression.
Collectively, these data establish an obligate role for Lqf in Notch signaling, and implicate Lqf in sending, rather than receiving, DSL signals.
Lqf is required in signal-sending cells to activate Notch in signal-receiving cells
To determine whether Lqf is required in signal-sending cells, we used the
MARCM technique (Lee and Luo,
1999) to generate lqf- clones that express
either Dl or Ser under Gal4 control (Materials and methods).
Notch is normally expressed in both the D and V compartments of the wing
primordium, but is modified in D cells by the action of the
glycosyltransferase Fringe (Fng) so that it responds preferentially to Dl
signaling from V cells (de Celis and Bray,
1997; Fleming et al.,
1997
; Panin et al.,
1997
; Blair, 2000
).
Ser is expressed predominantly in D compartment cells, and signals in the
opposite direction, activating unmodified Notch in V cells. Clones of cells
that express Dl under Gal4 control activate Notch strongly in adjacent
wild-type cells only when located in the D compartment, as monitored by the
expression of margin-specific genes like cut
(Fig. 2A). Conversely,
Ser-expressing clones activate Notch strongly only when located in the V
compartment (Fig. 2D). In both
cases, the levels of exogenous Dl and Ser expression are several fold higher
than the peak levels of endogenous Dl and Ser generated along the DV boundary,
and this overexpression autonomously inhibits the activation of Notch in cells
within the clones (de Celis and Bray,
1997
; Micchelli et al.,
1997
) (Fig.
2A,D).
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Lqf is not required in signal-receiving cells for transduction of DSL ligands by Notch
To determine whether Lqf is required in signal-receiving cells for Notch
activation or signal transduction, we used a modification of the MRCM
technique to determine whether clones of lqf- cells can
transduce ectopic Dl or Ser signals sent by adjacent cells that are wild-type
for lqf. To do this, we generated `twin spots' comprising adjacent
daughter clones in which one clone expresses Dl or Ser under Gal4 control and
the other clone is lqf- (see Materials and methods). We
find that Dl-expressing clones located in the D compartment induce
cut expression in surrounding cells, even when the responding cells
belong to adjacent lqf- clones
(Fig. 2C). The same is true for
Ser-expressing clones in the V compartment
(Fig. 2F). Thus, wild-type
cells that express ectopic Dl or Ser can activate Notch in adjacent
lqf- cells, indicating that Lqf is not essential in
signal-receiving cells to transduce DSL ligands. In separate experiments, we
confirmed that lqf- cells can transduce normal levels of
endogenous Dl sent by neighboring wild-type cells by assaying Cut expression
in clones of lqf- cells that ectopically express Fng in
the V compartment (see supplementary material).
The results of both sets of experiments lead us to conclude that Lqf is only required in signal-sending cells, and not in signal-receiving cells, to activate the Notch transduction pathway.
Lqf is not required for sending or receiving Decapentaplegic, Wingless or Hedgehog signals, or for normal cell growth, proliferation or intermixing
To assess whether Lqf might be required more generally for generating,
modulating or transducing extracellular signals, we have tested whether
lqf- cells are capable of sending and receiving three
other extracellular signals, each representing a different family of secreted
ligands: Decapentaplegic (Dpp), Wingless (Wg) and Hedgehog (Hh). As in the
case of DSL ligands, we generated clones of lqf- cells
that ectopically expressed each of these ligands, or which were located in
regions where these signals are normally transduced, and assayed for target
gene expression. For each ligand, we were unable to detect any change in
signaling activity, in either sending or receiving cells, as a result of
abolishing lqf function (Fig.
2G,H, data not shown; see Materials and methods).
To determine whether lqf- cells behave normally in terms of their ability to grow, proliferate and interdigitate with surrounding cells, we used twin-spot analysis to compare the behavior of lqf- and Dl- Ser- clones with their wild-type, sibling clones. We find that lqf- and Dl- Ser- clones behave similarly in terms of clone size, cell density and the wiggliness of their borders, indicating that the mutant cells of both genotypes grow, divide and interdigitate normally (Fig. 2H). Thus, aside from the failure to send DSL-signals, lqf- cells appear indistinguishable from wild-type cells, suggesting a dedicated requirement for Epsin in sending DSL signals.
Lqf is not required for normal expression of DSL ligands on the cell surface
To ascertain whether Lqf might be required for DSL ligands to reach the
cell surface, we examined the abundance of endogenous Dl on the surface of
lqf- mutant cells using living disc, or non-detergent,
staining protocols (see Materials and methods). In wild-type wing discs, Dl
expression peaks along the DV compartment boundary and the presumptive wing
veins. We were unable to detect a change in the surface abundance of Dl in
clones of lqf- cells relative to neighbhoring wild-type
cells, except that large clones that abut or cross the DV boundary are
associated with reduced surface expression (data not shown). This reduction,
however, can be attributed to the loss of DSL signaling associated with the
clone [which is normally required for peak Dl expression along the DV boundary
(Micchelli et al., 1997)]; we
observe a similar reduction when the staining was performed in the presence of
detergent to detect both cytosolic and surface expression of Dl (e.g.
Fig. 4C).
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First, we replaced the missing intracellular domain of
DlC with a 21 amino acid peptide from the Low Density
Lipoprotein (LDL) receptor that contains either the wild-type internalization
signal FDNPVY, or a mutant signal, ADAAVA
(Chen et al., 1990
). The LDL
peptide contains two Lysines; these were replaced by Arginine to avoid their
serving as possible acceptors for ubiquitination. Both the wild-type
(DlLDL+) and mutant (DlLDLm) chimeric proteins were
labeled by the insertion of six copies of the Myc epitope tag in the
juxtamembrane portion of the extracellular domain (see Materials and methods).
When expressed in the wing disc, DlLDL+ shows a similar subcellular
distribution to wild-type Dl, accumulating on both the apical cell surface and
in intracellular puncta (Fig.
3A; data not shown). DlLDL+-expressing clones, like
wild-type Dl-expressing clones, can induce cut activity in
surrounding cells (Fig. 3A),
indicating that the chimeric protein has signaling activity. However, they
differ from wild-type Dl-expressing clones in that they only induce
cut when located close to the DV boundary (compare
Fig. 3A and
Fig. 2A). Hence, we infer that
DlLDL+-expressing clones have reduced signaling activity relative
to wild-type Dl-expressing clones, and require the additional boost provided
by endogenous signaling from neighboring wild-type cells to activate
cut.
|
Second, we found serendipitously that replacement of the missing cytosolic
domain of DlC with a random peptide, R+, of 50 amino acids
(DlR+; see Materials and methods) also restored normal behavior.
DlR+ accumulates in intracellular puncta as well as on the apical
cell surface; in addition it activates Notch in neighboring cells
(Fig. 3D). The R+ peptide
contains two Lysines that might potentially serve as acceptors for
ubiquitination. Replacement of both Lysines with Arginine blocked the rescuing
activity of the R+ peptide. The mutant protein, DlRm, accumulated
predominantly only on the cell surface and lacked signaling activity
(Fig. 3F); moreover, clones of
DlRm that abutted the DV boundary interrupted signaling across the
boundary.
To assess the possibility that mono-ubiquitination of native Dl, as well as
the DlR+ chimera, might suffice to provide an internalization
signal, we replaced the missing cytosolic domain of DlC with
Ubiquitin itself, and with a corresponding mutant form of Ubiquitin in which
the Isoleucine at position 44 was mutated to Alanine, which functionally
inactivates the internalization signal
(Shih et al., 2002
). All seven
Lysine residues in each Ubiquitin domain were also replaced by Arginine to
avoid additional ubiquitination (Terrell
et al., 1998
). Both the resulting proteins, DlUbi+ and
DlUbim, accumulated on the cell surface, as well as in
intracellular puncta (Fig.
3G,H; data not shown). However, far fewer puncta were found in
DlUbim-expressing cells than in DlUbi+-expressing cells,
and only DlUbi+ was able to signal to neighboring cells
(Fig. 3G,H). These data
indicate that mono-ubiquitination is sufficient for Dl endocytosis and
signalling, and suggest that at least one of the Lysines in the R+ peptide
serves as Ubiquitin acceptor, allowing the protein to be internalized and to
signal. We note that the DlUbi+ protein appears to have only weak
signaling activity relative to Dl or DlR+, as we could only detect
induction of vg boundary-specific expression, but not cut or
wg expression (Fig.
3G; data not shown).
We conclude: (1) that the cytosolic domain of Dl is essential for its endocytosis; (2) that mono-ubiquitination is sufficient for Dl internalization; and (3) that Dl endocytosis is essential for signaling activity.
Lqf is not required for bulk endocytosis of Dl
Given that Epsin has been implicated in endocytosis,
lqf- cells may fail to send DSL signals because they are
generally impaired for endocytosis. However, Dpp, Hh and Wg signaling, Wg
internalization, and cell growth and proliferation are not adversely affected
by the absence of Lqf (Fig.
2G,H, Fig. 4D,E;
data not shown) suggesting that endocytosis is not significantly impaired
overall.
Alternatively, Lqf might be required specifically for the endocytosis of
DSL ligands. To assess this possibility, we first examined the effects of
lqf- clones in the developing retina, where they have been
reported to cause abnormally high levels of Dl on the cell surface, consistent
with impaired Dl endocytosis (Overstreet
et al., 2003). We find that such clones do indeed cause elevated
surface expression of Dl, but we also observed that endogenous Dl
transcription (as assayed using a Dl-lacZ reporter gene), is strongly
upregulated in the mutant cells (Fig.
4A), apparently as a consequence of the lack of Notch signaling
(Baonza and Freeman, 2001
).
Furthermore, we can detect Dl staining in intracellular puncta in such
lqf- eye disc clones
(Fig. 4B). Thus, the elevated
surface accumulation of Dl observed in lqf- eye clones can
be ascribed to elevated Dl expression in the mutant cells, and may not reflect
impaired Dl endocytosis.
Second, we generated lqf- clones in wing discs expressing uniformly high levels of exogenous Dl under Gal4 control, and compared Dl staining in lqf- cells and their wild-type neighbors. Under this condition, the level of Dl expression does not vary between wild-type and lqf- cells, simplifying analysis. We were unable to detect any difference in the subcellular distribution of Dl between lqf- and adjacent wild-type cells. In both cases, Dl was localized predominantly at the cell surface, as well as in similar numbers of intracellular puncta, many of which co-localize with the endosomal protein Hrs (Fig. 5A,A'; data not shown). We obtained the same result in separate experiments in which we assayed the subcellular distribution of endogenous Dl only (i.e. in the absence of overexpressed Dl; Fig. 4C; data not shown).
Third, we reasoned that if the Dl-positive puncta in
lqf- clones are indeed endocytic, the appearance of such
puncta should change in the absence of hrs activity, which interferes
with the maturation of early into late endosomes, and causes the formation of
abnormal endosomal structures (Lloyd et
al., 2002). To test this, we generated both
hrs- and hrs- lqf- clones.
We find that endogenous Dl accumulates in abnormally large puncta in both
types of clones, and similar results were obtained when these clones express
exogenous Dl under Gal4 control (data not shown). We note that the block in
endosomal maturation caused by the removal of Hrs does not interfere with
signaling by Dl; nor does it alter the requirement for Lqf. Clones of
hrs- cells that express exogenous Dl induce Cut expression
in surrounding cells, whereas corresponding hrs-
lqf- clones do not (data not shown).
To determine unequivocally whether the abnormal puncta that accumulate Dl in hrs- and hrs- lqf- cells are indeed endosomal, we made use of the finding that Wg secreted from prospective wing margin cells accumulates in similar, abnormally large puncta in hrs- cells positioned at a distance from the secreting cells (E. S. Seto and H. J. Bellen, personal communication). We obtained the same result in double mutant hrs- wg- cells (data not shown), establishing that the accumulation of Wg in these puncta serves as an in vivo marker for endocytosis. We then examined Wg and Dl staining in triple mutant hrs- wg- lqf- clones that express an HRP-tagged form of Dl under Gal4 control (see Materials and methods). In this case, as in corresponding hrs- wg- double mutant clones, we observe co-localization of Wg and Dl in large intracellular puncta (Fig. 4D,E).
Thus, bulk endocytosis of both endogenous and overexpressed Dl appear normal in lqf- cells.
Lqf is required for a subset of Dl endocytic events
Although bulk Dl endocytosis appears unaffected by the absence of Lqf,
blockage of a relatively small, but specific, subset of Dl endocytic events
might escape detection, and this subset might be crucial for signaling
activity. To examine this possibility, we co-expressed Dl together with the E3
Ubiquitin Ligase Neuralized (Neur), under Gal4 control, to drive efficient
ubiquitination and internalization of the exogenous Dl
(Lai et al., 2001;
Pavlopoulos et al., 2001
). We
reasoned that under these conditions, even modest reductions in the rate of Dl
endocytosis might cause an abnormal persistence of Dl at the apical cell
surface.
Wing discs that express uniformly high levels of Dl under Gal4 control accumulate high levels of Dl on the apical cell surface. However, in discs that co-express high levels of both Dl and Neur, this surface accumulation is strongly reduced and Dl accumulates instead in an abnormally large number of intracellular puncta. Clones of lqf- cells generated in such co-expressing discs do not appear to alter the number or general appearance of these Dl-positive puncta, many of which co-localize with Hrs (Fig. 5B',B''). However, they do affect the level of Dl staining associated with the apical cell surface (as visualized in discs processed either with, or without, detergent). Such lqf- clones show residual surface staining of Dl, in contrast to neighboring wild-type cells where surface-associated staining is depleted (Fig. 5B). We infer that lqf- cells cannot endocytose Dl as efficiently as their wild-type neighbors, accounting for why we detect a difference under sensitized conditions in which the rate of surface clearance appears to be limiting.
Significantly, the residual staining of Dl on the surface of
lqf- cells that overexpress Neur and Dl correlates with
the failure of these cells to signal. We find that clones of
lqf- cells that overexpress Neur and Dl fail to activate
cut in neighboring cells (Fig.
5C), even though clones of otherwise wild-type cells that
overexpress Neur and Dl show enhanced Dl signaling
(Pavlopoulos et al., 2001).
Hence, it appears that the impairment in Dl endoctyosis we detect in
lqf- clones in this sensitized background correlates with
an absolute block in signaling activity.
Evidence that Lqf is required for signaling by mono-ubiquitinated forms of Dl
The cytosolic domain of DSL ligands contains multiple Lysines at least some
of which serve as acceptors for Ubiquitin
(Deblandre et al., 2001;
Itoh et al., 2003
). Lqf
contains two Ubiquitin Interacting Motifs (UIMs)
(Hofmann and Falquet, 2001
).
Hence, mono-ubiquitination of DSL ligands might allow Lqf to target them for a
special subset of endocytic events that are required for signaling activity.
By contrast, bulk endocytosis of DSL ligands mediated by interactions with
other Ubiquitin-binding adaptor proteins might not suffice to confer signaling
activity. To test this hypothesis, we investigated whether the signaling
activity of the DlR+ protein depends on Lqf activity.
Endocytosis and signaling activity of DlR+ depends on the presence of at least one of the two Lysines in the R+ peptide comprising the cytosolic domain (Fig. 3D,F). We find that clones of lqf- cells that express DlR+ fail to induce cut expression in adjacent wing disc cells (Fig. 3E). However, DlR+ protein in these lqf- clones accumulates both on the apical surface and in intracellular puncta (Fig. 3E). Moreover, we could not detect any difference in the punctate, cytosolic accumulation of DlR+ between lqf- and wild-type cells in wing discs that generally overexpress DlR+ (data not shown). Both results indicate that bulk endocytosis of DlR+ is not significantly altered in the absence of Lqf. Because substitution of both Lysines by Arginine blocks internalization and signaling activity of DlRm (Fig. 3F), we infer that DlR+ is targeted for internalization solely by ubiquitination at one or both of these Lysines. Hence, we suggest that other Ubiquitin-interacting proteins aside from Lqf can target mono-ubiquitinated cargo proteins, such as DlR+ or endogenous Dl, for internalization. However, only Lqf appears able to direct endocytosis of these proteins in a way that allows DSL ligands to signal.
Dl proteins carrying the LDL internalization signal bypass the requirement for Lqf
Both endocytosis and signaling activity of DlLDL+ depends on the
FDNPVY internalization signal (Fig.
3A,C). However, unlike either native Dl or DlR+, we
find that clones of lqf- cells expressing
DlLDL+ can induce cut expression in adjacent wild-type
cells (Fig. 3B), indicating
that the presence of the LDL internalization signal in the chimeric
DlLDL+ protein bypasses the requirement for Lqf. As observed for
clones of wild-type cells overexpressing DlLDL+, the `rescued'
lqf- clones only induced cut when located close
to the DV boundary. Nevertheless, their ability to signal, albeit weakly,
contrasts with that of lqf- clones that overexpress native
Dl, native Dl plus Neur, or DlR+, all of which are devoid of
signaling activity. Hence, we conclude that the FDNPVY signal directs
internalization of DlLDL+ in a manner that permits the protein to
acquire signaling activity even in the absence of Lqf activity.
Lqf is required for Dl processing
Lqf-dependent endocytosis of DSL ligands might be accompanied by
modifications of these ligands, either as a pre-requisite for, or a
consequence of, signaling activity. To examine this possibility, we asked
whether the size of Dl protein changes as a consequence of Lqf-dependent
endocytosis.
Initially, we generated clones of wild-type and lqf- cells that express Dl tagged by the insertion of six copies of the Myc epitope in the extracellular juxtamembrane domain, and analyzed the profile of Dl peptides that retain the Myc epitope by western blotting (see Materials and methods). Under these conditions, we observed similar, complex profiles of Myc-tagged Dl peptides from both wild-type and lqf- cells corresponding to full-length Myc-Dl protein, as well as several lower molecular weight peptides (data not shown).
We then repeated this experiment using wild-type and
lqf- cells that overexpress Neur and Myc-tagged Dl, the
sensitized condition under which we can detect residual surface expression of
Myc-tagged Dl in lqf-, but not in wild-type, cells. In
this case, the profile of Myc-tagged Dl is remarkably simple. Wild-type cells
show two bands, one corresponding by size to full-length Myc-tagged Dl
(105 kDa) and the other to a Myc-tagged cleavage product of
50 kDa.
By contrast, lqf- cells show only a single band,
corresponding to full-length Myc-tagged Dl
(Fig. 6). Thus, the failure to
clear Dl from the cell surface of lqf- cells is associated
with an apparent failure in Dl processing. These results provide evidence for
a Lqf-dependent cleavage of Dl that correlates with Lqf-dependent endocytosis
and signaling activity.
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Discussion |
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We have demonstrated an absolute, cell-autonomous requirement for Lqf in generating functional DSL ligands. However, we have not been able to detect any other role for Lqf in cell-cell signaling, such as in receiving DSL ligands, or in sending, receiving, or controlling the distribution of other extracellular signals, notably Hg, Wg and Dpp. Furthermore, cells devoid of Lqf activity appear to grow, proliferate and interdigitate in a manner that is indistinguishable from cells devoid of Dl and Ser, the two DSL ligands in Drosophila. These results suggest that Epsin function in Drosophila may be essential solely for the production of active DSL ligands.
Surprisingly, we also failed to detect an effect of removing Lqf on the steady state accumulation of Dl in endocytic compartments. However, we were able to detect a modest effect on Dl internalization in a sensitized background in which we greatly enhance Dl endocytosis by overexpressing Neur, an E3-Ubiquitin ligase that ubiquitinates Dl. Strikingly, high levels of Dl accumulate in endocytic vesicles of such Neur overexpressing cells whether or not they have Lqf, but only cells that have Lqf can signal. We therefore infer that Epsin is required for a discrete and apparently small subset of the endocytic events that normally internalize DSL ligands; however, it is this subset that is crucial for generating active DSL signals.
The selective requirement for Epsin in sending DSL ligands is reminiscent
of that for the Presenilin/-secretase complex in transmembrane cleavage
and signal transduction by Notch (Struhl
and Greenwald, 1999
; Struhl
and Adachi, 2000
). Selectivity in the case of the
Presenilin/
-secretase complex does not reflect a dedicated role in
Notch proteolysis, but rather an unusual property of the Notch transduction
mechanism, namely that ectodomain shedding activates the pathway by inducing
transmembrane proteolysis (Mumm and Kopan,
2000
; Struhl and Adachi,
2000
). Similarly, selectivity in the case of Epsin may reflect an
unusual requirement for DSL ligands to signal, and not a dedicated role of
Epsin in confering their signaling activity.
It is generally thought that Epsins target cargo proteins for endocytosis
via mono-ubiquitin internalization signals
(Wendland, 2002). We replaced
the cytosolic domain of Dl with a random peptide (R+) that contains two
Lysines, and showed that the presence of at least one Lysine is essential for
both the endocytosis and signaling activity of the chimeric DlR+
ligand. To test the possibility that the presence of Lysine targets
DlR+, as well as wild-type Dl, for endocytosis by serving as an
Ubiquitin acceptor, we replaced the cytosolic domain of Dl with a non-Lysine
containing form of Ubiquitin. The resulting DlUbi+ ligand could be
endocytosed and had at least partial signaling activity, but not if the
Ubiquitin domain contained an additional mutation that blocks its ability to
be targeted for endocytosis. Finally, and critically, we demonstrated that the
signaling activity of DlR+, like that of wild-type Dl, depends on
Lqf. Collectively, these findings implicate mono-ubiquitination as the
internalization signal required to target DSL ligands for endocytosis by
Epsin.
Significantly, bulk endocytosis of the chimeric DlR+ ligand, like that of wild-type Dl, appears to be unaffected in cells devoid of Lqf, even though signaling activity is abolished. Hence, we infer that Lqf is not the only adapter protein that can target mono-ubiquitinated substrate proteins for endocytosis. Nevertheless, Lqf appears to be unique amongst all such adapter proteins in its ability to direct internalization of mono-ubiquitinated DSL ligands in a manner that confers signaling activity. We therefore suggest that Epsin has a dedicated role in directing mono-ubiquitinated cargo proteins into a particular endocytic pathway, one that DSL ligands must enter in order to acquire signaling activity. As we detail in the next section, we suggest that Epsin might direct DSL ligands specificially into a recycling pathway.
It is notable that substitution of the cytosolic domain of Dl with a peptide carrying the FDNPVY internalization signal from the LDL receptor yields a chimeric DlLDL+ ligand that is endocytosed and has signaling activity. However, in this case, Lqf is not essential for signaling. One interpretation of this result is that mono-ubiquitinated DSL ligands are normally targeted for endocytic pathways that preclude their signaling activity, unless they are diverted from entering these pathways by association with Lqf, or by the presence of a heterologous internalization signal such as FDNPVY. In both cases, endocytosis would take place via an alternate pathway compatible with signaling activity.
Endocytosis and DSL signaling
Why must DSL ligands on the surface of signal-sending cells be endocytosed
in order to activate Notch on the surface of signal-receiving cells? We can
distinguish two general classes of explanation. In the first
(Fig. 7A), activation of Notch
is triggered by early events in the process of DSL endocytosis that occur
while the ligands are still on the cell surface, prior to the pinching off of
coated vesicles. In the second (Fig.
7B), internalization of DSL proteins is a necessary prerequisite
for endocytic recycling, which is required for subsequent signaling
activity.
|
For such internalization models to accommodate our results, it seems necessary to posit that productive interactions between DSL ligands and Notch require a special micro-environment that is associated only with a particular subclass of coated pits or other specializations (Fig. 7A). Mono-ubiquitinated cargo proteins might be excluded from such structures, unless chaperoned there by Lqf. Thus, only DSL ligands that gain entry, whether via Lqf, or by the targeting mediated by the LDL receptor signal, would be able to activate Notch on the abutting surface of the receiving cell. Furthermore, one would have to posit the existence of accessory molecules that are provided by the sending cell, sequestered in these specializations, and essential for DSL-dependent activation of Notch on the receiving cell, whether by mechanical stress, DSL clustering, or some other means.
We are directed to the second general class of explanation, in which
recycling is the key element, by the ability of the internalization signal
from the LDL receptor to bypasses the requirement for Lqf. In general,
mono-ubiquitination acts as a sorting signal in the endosomal system that
leads to delivery of membrane proteins to late endosomes and eventually
lysosomes (reviewed by Hicke and Dunn,
2003). By contrast, the FDNPVY signal is associated with rapid
recycling back to the cell surface after entry into endosomes
(Chen et al., 1990
;
Matter et al., 1993
). Hence,
Epsin-binding to mono-ubiquitinated DSL proteins during endocytosis might
allow those DSL proteins to escape degradation by altering their sorting, thus
allowing them to enter a recycling pathway. Passage through this pathway would
be essential to confer signaling activity.
Why might recycling be necessary for DSL ligands to acquire signaling activity? One possibility is that recycling allows DSL ligands to be stripped of the bound ectodomain of Notch so that they can be re-used. Multiple rounds of recycling might then enhance the level of active DSL ligands on the surface of signal-sending cells above a critical threshold necessary to activate Notch transduction in the signal-receiving cell. According to this view, one might expect that massive overexpression of DSL ligands would be able to bypass the requirement for Lqf. However, our results suggest that this is not the case: we estimate that, in our experiments, overexpressed Dl accumulates on the cell surface at levels up to tenfold higher than peak accumulation of endogenous Dl, yet is unable to rescue DSL signaling activity in cells devoid of Lqf.
Alternatively, recycling of nascent DSL proteins may be important to
convert inactive `pro-ligands' into active ligands
(Fig. 7B). Conversion might
entail recruitment of DSL proteins into signaling exosomes
(Le Borgne and Schweisguth,
2003b). However, Dl signaling appears to be unaffected in cells
devoid of Hrs, despite impairment in the maturation of early to late
endosomes, and in the formation of multi-vesicular bodies from which exosomes
might derive (Lloyd et al.,
2002
). Another possibility is that DSL proteins need to be
processed in order to be converted to active ligands, a hypothesis that is
consistent with our evidence that Lqf-dependent endocytosis of Dl correlates
with a specific proteolytic cleavage of the ligand. Lqf would be required in
this scenario to allow DSL ligands to enter a recycling pathway in which the
required processing event can occur. The only specificity one needs to invoke
in this model is that of Epsin to allow mono-ubiquitinated cargo proteins to
gain access to a recycling pathway. The conditions necessary to convert DSL
pro-ligands into active signals (e.g. low pH) might exist generally in early
endosomes or recycling endosomes.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/21/5367/DC1
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