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 13 April 2005
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SUMMARY |
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Key words: Drosophila, DSL-Notch signaling, Delta, Serrate, Mindbomb, Epsin/Liquid facets, Neuralized, Endocytosis, Ubiquitination
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Introduction |
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Here, we focus on a critical but poorly understood aspect of the mechanism
by which DSL ligands induce proteolytic processing of Notch, namely that DSL
proteins must normally be endocytosed in signal-sending cells to activate
Notch in signal-receiving cells (Parks et
al., 2000; Struhl and Adachi,
2000
; Le Borgne and
Schweisguth, 2003a
; Wang and
Struhl, 2004
). Two general classes of hypotheses have been put
forward to explain why DSL ligands must be endocytosed to activate Notch
(Le Borgne and Schweisguth,
2003a
; Wang and Struhl,
2004
). In the first class, entry of DSL ligands into coated pits
or other specializations in signal-sending cells is proposed to create
conditions essential for cleaving or shedding the ectodomain of Notch before
internalization of the ligand. Such conditions could be mechanical stress of
the intercellular bridge between the ligand and the receptor
(Parks et al., 2000
;
Struhl and Adachi, 2000
;
Wang and Struhl, 2004
),
clustering of DSL ligands in coated pits
(Le Borgne and Schweisguth,
2003a
), or the recruitment of essential accessory factors to the
same micro-environment (Wang and Struhl,
2004
). In the second class, DSL signaling activity is thought to
depend on events that take place after internalization of the ligand, such as
enzymatic processing of the ligand from an inert to an active form
(Wang and Struhl, 2004
) or
packaging into exosomes (Le Borgne and
Schweisguth, 2003a
): accordingly, signaling activity would require
recycling of the converted or repackaged ligand to the cell surface.
The endocytic adaptor protein Epsin provides a potential key to
understanding the role of endocytosis in DSL signaling. Epsins are conserved
multidomain proteins that appear likely to interact with core components of
the endocytic machinery (Clathrin, AP2, PIP2) as well as with mono-ubiquitin,
and are required in some contexts for endocytosis of mono-ubiquitinated cargo
proteins (Chen et al., 1998;
Wendland, 2002
). We have
recently shown that Drosophila Epsin, encoded by the gene liquid
facets (lqf) (Cadavid et al.,
2000
), has a remarkably specific role in normal cell signaling and
physiology: it appears to be required solely for cells to send, but not to
receive, DSL signals (Wang and Struhl,
2004
) (see also Overstreet et
al., 2004
; Tian et al.,
2004
). Furthermore, we obtained evidence that Epsin-dependent
signaling depends on ubiquitination of DSL ligands
(Wang and Struhl, 2004
). These
and related findings led us to propose: (1) that DSL ligands must normally be
ubiquitinated to be targeted for Epsin-mediated endocytosis; and (2) that the
ubiquitinated ligands must normally be internalized via the action of Epsin to
activate Notch (Wang and Struhl,
2004
).
Surprisingly, we were not able to detect any effect of the removal of Epsin
on normal, bulk endocytosis of the DSL ligand Delta (Dl)
(Wang and Struhl, 2004).
Instead, our results suggested that Epsin mediates only a small subset of the
endocytic events that internalize ubiquitinated forms of Dl. Thus, we further
proposed that it is not endocytosis, per se, that is normally essential for
DSL signaling, but rather entry of ubiquitinated DSL ligands into a select
Epsin-dependent endocytic pathway (Wang
and Struhl, 2004
).
In experiments described in this paper, we present in vivo experiments that test two predictions of this hypothesis: (1) that DSL ligands must be ubiquitinated to signal; and (2) that both ubiquitination and Epsin are required for the normal internalization of DSL ligands.
To test the first prediction, we have sought to block ubiquitination of DSL
ligands by removing the relevant ubiquitin ligase(s). Previous work had
demonstrated that two RING-domain-containing E3 ubiquitin ligases, Neuralized
(Neur) in Drosophila and Mind bomb (Mib) in zebrafish promote DSL
endocytosis and signaling (Deblandre et
al., 2001; Lai et al.,
2001
; Pavlopoulos et al.,
2001
; Yeh et al.,
2001
; Itoh et al.,
2003
), implicating them in the normal pathway of Notch activation
by DSL ligands and suggesting that they perform homologous functions in their
respective organisms (Le Borgne and
Schweisguth, 2003a
). However, Neur is normally expressed and
required in only a subset of DSL signaling cells in Drosophila
(Boulianne et al., 1991
;
Yeh et al., 2000
;
Lai and Rubin, 2001
),
indicating either that ubiquitination of DSL ligands is not essential for
activating Notch in all signaling contexts, or that other ubiquitin ligase(s)
may be responsible for ubiquitinating DSL ligands in cells that do not express
Neur.
The Drosophila genome contains an ortholog of the zebrafish
mib gene, dmib (Itoh et
al., 2003), raising the possibility that dmib encodes one
such alternative ligase. Here, using newly obtained loss-of-function mutations
of dmib, we demonstrate an absolute requirement for Dmib in sending,
but not receiving, DSL signals in cells that normally do not require or
express Neur. Furthermore, we find that we can bypass the requirement for Dmib
either by ectopically expressing Neur in these cells, or by expressing a
chimeric DSL ligand in which the cytosolic domain is replaced by a
heterologous substrate for ubiquitination. Together, these findings support
the proposal that DSL ligands must be ubiquitinated to signal.
To test the second prediction, that signaling activity of DSL ligands depends on their being endocytosed in a ubiquitin- and Epsin-dependent fashion, we asked whether removing either Dmib or Epsin impairs the normal bulk endocytosis of Dl and Ser in cells that depend on Dmib function. We show that bulk endocytosis of Ser is severely impaired in Dmib-deficient cells and partially impaired in Epsin-deficient cells, whereas that of Dl is not detectably affected in either case. We infer that DSL ligands are normally internalized via multiple, distinct endocytic pathways, only some of which depend on mono-ubiquitination and only a further subset of these which depend on Epsin. Most Ser appears to be internalized via Epsin-dependent pathways, whereas most Dl is not. Nevertheless, only those DSL ligands that are internalized via the action of Epsin activate Notch.
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Materials and methods |
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Genetic materials
The following transgenes and mutations were used: UAS-Dl,
UAS-DlR+,
lqf1227(Wang and Struhl,
2004); Tub
1-Gal4, UAS-GFPnls
(Struhl and Greenwald, 1999
;
Struhl and Greenwald, 2001
);
nub-Gal4, UAS-y+
(Calleja et al., 1996
);
UAS-CD8-GFP, Tub
1-G80
(Lee and Luo, 1999
);
UAS-Ser (Panin et al.,
1997
); UAS-neur (Lai
and Rubin, 2001
); ptcG4, UAS-lacZ, arm-lacZ (Bloomington
stock center).
dmib RNAi and overexpression constructs
To generate the UAS-dmibRNAi transgene, a dmib
cDNA fragment corresponding to nucleotides 3626-4027 of the dmib cDNA
(clone SD05267;BDGP) was generated by PCR amplification and inserted in
opposite orientations into a pUAST-RNAi intron vector
(Lee and Carthew, 2003).
UAS-dmibRNAi/ptc-Gal4 and
UAS-dmibRNAi/nub-Gal4 transheterozygous larvae were
maintained at 30°C. The UAS-dmib transgene was generated by
inserting the full-length dmib coding sequence from SD05267 into the
pUAST vector (Brand and Perrimon,
1993
).
Genotypes employed
Clones of dmib-, lqf-, or wild-type
cells marked by GFP and/or y+ and expressing
UAS-X transgenes (MARCM technique) y hsp70-flp Tub1-Gal4
UAS-GFPnls/y hsp70-flp; UAS-y/+; [dmib-,
lqf- or wild type] FRT2A/Tub
1-Gal80 FRT2A,
UAS-X. dmib-=dmibL53, dmibL70
or dmibEY09780; UAS-X was provided either on II
in trans to UAS-y, or on III, in trans to Tub
1-Gal80
FRT2A.
Clones of dmib- or lqf- cells in discs expressing UAS-Dl or UAS-Ser in all prospective wing cells under nub-G4 control y hsp70-flp; nub-Gal4/UAS-Dl (or UAS-Ser); dmib- (or lqf-) FRT2A/arm-lacZ FRT2A. 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 as described previously
(Wang and Struhl, 2004), using
mouse
-Dl (Developmental Studies Hybridoma Bank, DSHB), Ginuea pig
-Dl (Parks et al.,
2000
), Ginuea pig
-Hrs
(Lloyd et al., 2002
); mouse
-Wg (DSHB), mouse
-Cut (DSHB), rat
-Ser
(Panin et al., 1997
), and
rabbit
-ßGal (Cappel). To monitor cell surface accumulation of
Ser, living discs were incubated for 20-30 minutes at room temperature with
rat
-Ser antisera in Drosophila tissue culture media, and then
rinsed and fixed in the absence of detergent, prior to executing the standard
staining protocol. We could not distinguish any difference in Ser staining
associated with the apical cell surface in such living discs, compared with
that in discs fixed in detergent prior to incubation with rat
-Ser, as
in our standard protocol.
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Results |
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Clones of dmib- cells are associated with large wing notches and thickened veins (Fig. 1D), both hallmark phenotypes associated with loss of DSL-Notch signaling. However, the formation of bristles on the mesonotum is only weakly affected. Both macro and microchaetes develop normally, except that the density of microchaetes is somewhat higher than normal and some macrochaetes are occasionally duplicated (Fig. 1B; data not shown). Nevertheless, each individual bristle organ appears to be morphologically normal. Thus, although we can detect evidence that the process of SOP segregation is compromised, albeit modestly, the subsequent, Neur-dependent segregations of cell types comprising each bristle sense organ appear unaffected.
|
Dmib is required in signal-sending cells to activate Notch in signal-receiving cells
To determine unequivocally whether Dmib is required in signal-sending cells
to activate Notch in signal-receiving cells, we used the MARCM technique
(Lee and Luo, 1999) to
generate clones of dmib- cells that ectopically express
high levels of either Dl or Ser under Gal4 control. Normally, clones
that ectopically express Dl but are otherwise wild type, induce Cut expression
in adjacent, non-expressing cells only when located in the dorsal compartment,
whereas clones of ectopic Ser-expressing cells induce Cut expression, but only
when located in the ventral compartment
(Fig. 3A,C)
(Fleming et al., 1997
;
Panin et al., 1997
). By
contrast, clones of dmib- cells that ectopically express
either Dl or Ser fail to induce Cut expression, no matter where they are
located. Furthermore, Dl and Ser expressing dmib- clones
that abut or cross the D-V boundary block normal Cut expression on both sides
(Fig. 3B'-B''',
D'-D''').
|
We conclude that in the context of inductive Notch signaling across the D-V compartment boundary, Dmib is selectively required for sending, but not receiving, DSL signals.
A chimeric form of Delta with heterologous sites for ubiquitination bypasses the requirement for Dmib
The ability of cells to send DSL signals across the D-V compartment
boundary also depends on Epsin [encoded by the liquid facets
(lqf) gene (Cadavid et al.,
2000)]: like clones of dmib- cells, clones of
lqf- cells in the wing primordium can receive, but not
send, DSL signals that specify the border cell fate
(Wang and Struhl, 2004
). Epsin
is thought to target mono-ubiquitinated cargo proteins for endocytosis
(Hofmann and Falquet, 2001
;
Wendland, 2002
); hence,
dmib- wing cells may not be able to send DSL signals
because they normally depend on Dmib to ubiquitinate Dl and Ser and thereby
target them for Epsin-mediated endocytosis. To test this, we assayed whether
Dmib is required for signaling by a chimeric form of Dl, DlR+,
which appears to contain one or more heterologous sites for
ubiquitination.
|
We generated clones of dmib- cells that express
DlR+. Such clones induce Cut in surrounding cells
(Fig. 3H'), just like
clones of wild-type cells that express DlR+
(Fig. 3G), but in contrast to
clones of lqf- cells that express DlR+
(Wang and Struhl, 2004).
Hence, we infer that expression of the chimeric DlR+ protein
bypasses the requirement for Dmib because it is ubiquitinated by some other
ubiquitin ligase, thereby providing the necessary signal for Epsin-mediated
internalization and signaling.
Ectopic expression of Neur bypasses the requirement for Dmib
Although dmib and lqf are both essential for wing cells
to send DSL signals, the requirement for Dmib is context dependent, whereas
that for lqf appears general. Specifically, dmib has only a
modest role during the establishment of bristle SOPs, and little or no role in
the segregation of their descendents, both contexts in which DSL signaling
depends on the action of Neuralized (Neur)
(Lai and Rubin, 2001). Hence,
Neur and Dmib might constitute functionally related ligases that normally
ubiquitinate DSL proteins in different signaling contexts.
To test this, we first asked whether ectopic expression of Neur, which is
normally not expressed during wing development except in SOPs and their lineal
descendents, can rescue the ability of dmib- cells to send
DSL signals that specify Cut-expressing border cells and wing veins. We find
that ectopic Neur expression does indeed rescue the ability of clones of
dmib- cells to signal in both contexts, as indicated by
the rescue of Cut expression on both sides of the D-V boundary in the wing
disc (Fig. 4A-H), as well as by
the rescue of the wing notching and vein thickening phenotypes that would
otherwise result from the loss of dmib activity (data not shown).
Second, we asked whether co-expression of Neur can rescue the ability of
clones of dmib- cells that ectopically express Dl to
activate Cut. As shown in Fig.
4I-L, we find that this is also the case. In fact, clones of
dmib- cells that ectopically co-express Neur and Dl
exhibit enhanced signaling activity in that they activate Cut expression even
in ventral compartment cells (Fig.
4K); clones of otherwise wild-type cells that ectopically
co-express Neur and Dl similarly activate Cut in ventral, as well as dorsal,
cells (Pavlopoulos et al.,
2001). Thus, ectopic Neur activity can substitute functionally for
the absence of Dmib activity, suggesting that both proteins can execute a
common ubiquitin ligase activity necessary for DSL signaling.
Dmib is required for normal bulk endocytosis of Ser but not Dl
Previously, we found that Epsin is essential for cells to send DSL signals.
However, we were not able to detect any effect of removing Epsin on the bulk
endocytosis of Dl in otherwise wild-type cells
(Wang and Struhl, 2004).
Hence, we hypothesized that if Dl must enter a select endocytic pathway
mediated by Epsin to acquire signaling activity, this pathway would constitute
a relatively small subset of the available pathways by which it can be
internalized. Similar results were also obtained with the chimeric
DlR+ ligand: like native Dl, DlR+ is strictly dependent
on Epsin for signaling activity; however we could not detect any difference in
bulk endocytosis of DlR+ protein in clones of
lqf- cells. Hence, even if one considers only those
pathways that target ubiquitinated Dl for endocytosis, only a small subset
might be Epsin-dependent and responsible for conferring signaling
activity.
To determine the extent to which Dl and Ser internalization might depend on Dmib activity, we examined the subcellular distribution of both ligands in clones of dmib- cells. As we describe below, we observe a pronounced and abnormal cell surface accumulation of Ser in dmib- cells, but can not detect a change in Dl.
In mature wing discs, Dl protein is expressed at low level throughout the
wing primordium, but accumulates at higher level in two stripes flanking the
D-V boundary and along the `pro-veins' that will give rise to the longitudinal
veins of the wing (Fig. 2E). Ser protein shows a similar expression pattern, except that its expression is
much weaker in the ventral compartment
(Fig. 2A). Within cells, Dl
appears to accumulate primarily on the apical cell surface, but is also
readily detected in intracellular puncta. By contrast, we find that the
majority of Ser appears to be localized in intracellular puncta
(Fig. 2A'), raising the
possibility that it might normally be cleared from the apical cell surface
more efficiently than Dl. For both Dl and Ser, we observe significant
co-staining of labelled cytosolic puncta with an antisera directed against
Hepatocyte growth factor-regulated tyronine kinase substrate (Hrs), an
endosomal marker protein (Lloyd et al.,
2002), indicating that at least some of these puncta are endosomal
[data not shown; confirmed for Dl by independent experiments
(Wang and Struhl, 2004
)].
In general, the distribution of endogenous Dl appears unchanged in clones
of dmib- cells relative to neighboring wild-type cells
(Fig. 2E', E''). However, as described previously for clones of lqf- cells
(Wang and Struhl, 2004),
dmib- clones that abut or cross the D-V boundary show
reduced expression of Dl and clones in pro-vein regions show increased
expression; both these effects can be attributed to alterations in Dl
transcription resulting from the loss of DSL-Notch signaling. In general, we
could not detect a change in the subcellular distribution of Dl in
dmib- cells relative to neighboring wild-type cells,
although a small enhancement of staining at the apical cell surface was
occasionally apparent in pro-vein regions; this difference could be due to
either enhanced transcription of Dl or an abnormal surface
accumulation of Dl protein.
By contrast, endogenous Ser protein accumulates apically to abnormally high
levels in clones of dmib- cells
(Fig. 2A',A''). We
obtained similar results whether or not the discs were fixed in the presence
of detergent prior to incubation with Ser antisera (Materials and
methods; data not shown); hence, it appears that Ser accumulates abnormally on
the apical cell surface of dmib- cells. In addition, the
number of intracellular Ser-positive puncta sometimes appears to be
significantly reduced in dmib- cells relative to
surrounding wild-type cells (Fig.
2A',A''). Both effects are more obvious when the mutant
clones are located in or near pro-vein regions, where Ser
transcription is likely to be upregulated as a consequence of the loss of
DSL-Notch signaling. However, we also detect abnormally high levels of cell
surface accumulation of Ser in intervein regions where such transcriptional
upregulation is not expected to occur. Moreover, the level of surface
accumulation of Ser in dmib- cells exceeds that of their
wild-type neighbors even within provein regions. Hence, the abnormal
accumulation of Ser in dmib- cells appears to result
primarily from altered Ser trafficking and/or stability rather than from an
abnormal upregulation of Ser transcription.
|
For over-expressed Dl, we were not able to detect any difference in subcellular distribution between dmib- cells and their wild-type neighbors (Fig. 5A-C''). In both populations of cells, the over-expressed Dl protein is localized mostly at the cell surface (Fig. 5B'',C''). Hence, bulk Dl endocytosis does not appear to be affected in dmib- cells, as we previously observed for lqf- cells. By contrast, a different result was obtained for over-expressed Ser. Here, as observed for endogenous Ser in wild-type discs, we again detected a dramatic accumulation of Ser protein at the apical surface of dmib- cells (Fig. 5D-F''), consistent with a block in the clearance of Ser from the cell surface.
|
Epsin is required for normal bulk endocytosis of Ser
Our finding that normal bulk Ser endocytosis depends on Dmib activity
raises the possibility that it may also depend on Epsin. Hence, we
investigated whether Ser endocytosis is affected in lqf-
mutant cells. We first examined endogenous Ser expression in clones of
lqf- cells in otherwise wild-type wing discs. As in
dmib- cells, we observed an enhanced accumulation of Ser
on the apical cell surface (Fig.
6A-A''). However, the degree of enhancement was less dramatic
than that observed for dmib- cells
(Fig. 2A',A'') making it less easy to attribute the abnormal accumulation to impaired
endocytosis, as opposed to transcriptional upregulation. To resolve this
uncertainty, we repeated the experiment in wing discs over-expressing
uniformly high levels of exogenous Ser under Gal4 control. As in clones of
dmib- cells obtained under the same conditions, we find
that Ser accumulated at dramatically higher levels along the apical surface of
lqf- cells (Fig.
6F-H'').
Thus, Ser differs from Dl in that it does not normally accumulate to high
levels on the apical cell surface, but instead appears to be cleared from the
cell surface by the actions of both Dmib and Epsin. Notably, the effects of
abolishing Epsin activity on bulk endocytosis of Ser are less severe than
those of abolishing Dmib, suggesting that Epsin mediates only a subset of the
endocytic events that internalize ubiquitinated forms of Ser. However, as is
the case for Dl, it is the subset of Epsin-mediated events that are essential
for signaling activity (Fig.
6E) (Wang and Struhl,
2004).
Over-expression of Dmib enhances endocytosis and signaling activity of DSL ligands
Ectopic expression of Neur in presumptive wing cells has previously been
shown to enhance both endocytosis and signaling activity of ectopically
expressed Dl, suggesting that under these conditions, the rate of
ubiquitination is limiting. Accordingly, over-expression of Dmib might be
expected, similarly, to enhance DSL endocytosis and signaling, and we have
found evidence that this so.
First, we find that ectopic expression of Dmib enhances endocytosis of ectopically expressed Dl and Ser. For example, when Dl is ectopically expressed along the A-P compartment boundary under the control of ptc-Gal4, it is localized predominantly at the apical cell surface, while Ser shows only a modest cell surface accumulation and is found, instead, mostly in intracellular vesicles (Fig. 7A-A''',C-C'''). However, when coexpressed with exogenous Dmib, the subcellular distributions of both Dl and Ser shift towards localization in intracellular puncta at the expense of accumulation at the cell surface (Fig. 7B-B''',D-D''', data not shown). Second, we observe that uniform over-expression of Dmib in the wing primordium under nub-Gal4 control suppresses vein formation (Fig. 1F). This phenotype is reciprocal to the vein-thickening phenotype caused by loss of dmib, lqf, or Dl activity but similar to that caused by ectopic activation of Notch. Hence, the level of endogenous Dmib activity in wing cells appears to limit both the strength of DSL-Notch signaling, as well as the rate of internalization of DSL ligands.
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Discussion |
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Distinct roles for Epsin and the ubiquitin ligases Dmib and Neur in sending DSL signals
To date, two E3 ubiquitin ligases have been implicated in DSL signaling:
zebrafish Mind bomb (Mib) and Drosophila Neuralized (Neur). Both
proteins have been shown to promote DSL ubiquitination, endocytosis and
signaling (Deblandre et al.,
2001; Lai et al.,
2001
; Pavlopoulos et al.,
2001
; Yeh et al.,
2001
; Itoh et al.,
2003
). Moreover, loss-of-function mutations in zebrafish
mib and Drosophila neur cause essentially the same hallmark
phenotype exemplifying a failure of DSL-signaling in their respective
organisms; namely, a dramatic hyperplasia of the embryonic nervous system at
the expense of the epidermis (Lehmann et
al., 1983
; Jiang et al.,
1996
; Schier et al.,
1996
). These observations have led to the suggestion that
zebrafish Mib and Drosophila Neur are functional homologs
(Le Borgne and Schweisguth,
2003a
). Yet, the two proteins show only limited sequence homology;
moreover they appear to be members of distinct Mib and Neur ubiquitin ligase
families, each having true orthologs in both vertebrate and invertebrate
genomes. As a consequence, the relative roles of Mib and Neur are not known in
any animal system and this uncertainty complicates the use of mutations in
these genes to assay the role of ubiquitination in DSL endocytosis and
signaling.
|
|
We note that if Mib and Neur ligases have overlapping molecular functions in all animal systems, as we find them to have in Drosophila, there is no compelling reason why they would need to be deployed in the same way in different animal species. Instead, any given DSL-signaling process might depend on Mib in one animal system but on Neur in another, as appears to be the case for neurogenesis in zebrafish and Drosophila.
In contrast to the selective requirement for Dmib and Neur in overlapping
subsets of DSL signaling contexts, Epsin is required for most or all DSL
signaling events (Overstreet et al.,
2004; Tian et al.,
2004
; Wang and Struhl,
2004
). This difference is expected if ubiquitination of DSL
ligands by either Dmib or Neur is normally a prerequisite for Epsin-mediated
endocytosis, and hence for signaling activity.
Do Dmib and Neur directly bind and ubiquitinate DSL ligands and thereby
confer signaling activity by targeting them for Epsin-mediated endocytosis?
Although ectopic Dmib and Neur activity are both associated with enhanced DSL
ubiquitination, endocytosis and signaling, there is still no compelling
evidence that either ligase directly binds and ubiquitinates DSL proteins, or
that Dmib/Neur-dependent ubiquitination of DSL ligands confers signalng
activity. However, we show here that the obligate requirement for Dmib for
Dl-signaling by wing cells can be bypassed by replacing the cytosolic domain
of Dl with a random peptide, R+, that may serve as the substrate
for ubiquitination by an unrelated ubiquitin ligase
(Wang and Struhl, 2004). This
result provides in vivo evidence that Dmib/Neur-dependent ubiquitination of
DSL ligands is normally essential to confer signaling activity. Moreover, the
failure of the chimeric DlR+ ligand to bypass the requirment for
Epsin (Wang and Struhl, 2004
),
supports the interpretation that ubiquitination of DSL ligands confers
signaling activity because it targets them for Epsin-mediated endocytosis.
Bulk endoctyosis of Ser, but not Dl, depends on Dmib and Epsin
During wing development, Ser and Dl both serve as unidirectional signals
that specify the `border' cell fate in cells across the D-V compartment
boundary, and we find that both are equally dependent on Dmib and Epsin
function for signaling activity. However, we find the two ligands differ in
the extent to which they accumulate on the cell surface, and to which they are
cleared from the surface as a consequence of Dmib and Epsin activity.
Specifically, we find that most Ser accumulates in cytosolic puncta rather
than on the cell surface, whereas the reverse is the case for Dl. Furthermore,
removing either Dmib or Epsin activity results in a dramatic and abnormal
retention of Ser on the cell surface, whereas it has no detectable effect on
the surface accumulation of Dl. Similar results for Dmib were also obtained by
Le Borgne et al. (Le Borgne et al.,
2005). Thus, it appears that most Ser is efficiently cleared from
the cell surface by the actions of Dmib and Epsin, whereas most Dl remains on
the cell surface, irrespective of Dmib and Epsin activity. This unexpected
difference provides two insights.
First, in our previous analysis of the role of Epsin, we focused almost
exclusively on Dl endocytosis and signaling
(Wang and Struhl, 2004) and
failed to obtain direct evidence that Epsin is required for normal DSL
endocytosis, despite the obligate role for Epsin in sending both Dl and Ser
signals. Instead, we could only detect such a requirement in experiments in
which we abnormally enhanced surface clearance of over-expressed Dl by
ectopically co-expressing Neur, or infer it from experiments in which we
bypassed the requirement for Epsin by replacing the cytosolic domain of Dl
with the well-characterized endocytic recycling signal from the mammalian low
density lipoprotein (LDL) receptor. By contrast, the different endocytic
behavior of Ser has now allowed us to obtain direct evidence that Dmib and
Epsin are both required for normal DSL endocytosis.
Second, we find that even though bulk endocytosis of Ser depends on both
Dmib and Epsin activity, neither requirement appears absolute. Instead, we can
still detect the accumulation of Ser in cytosolic puncta in both Dmib- and
Epsin-deficient cells. Moreover, we can detect a difference in the abnormal
cell surface accumulation of Ser in Dmib-deficient versus Epsin-deficient
cells; significantly more Ser appears to accumulate in the absence of Dmib
than in the absence of Epsin. As diagrammed in
Fig. 8A, we infer that both Dl
and Ser are normally internalized by multiple endocytic pathways, only some of
which depend on ubiquitination of the ligand, and only a subset of these that
depends on Epsin. However, the two ligands normally utilize these pathways to
different extents, most Ser being internalized by ubiquitin- and
Epsin-dependent pathways, and most Dl being internalized by alternative
pathways. We presume that this difference reflects the presence of different
constellations of internalization signals in the two ligands, especially the
presence of signals in Delta, but not Ser, that target the great majority of
the protein for internalization pathways that do not depend on ubiquitination
or Epsin. Nevertheless, only those molecules of Ser and Dl that are targeted
by ubiquitination to enter the Epsin-dependent pathway have the capacity to
activate Notch; all other routes of entry that are normally available appear
to be non-productive in terms of signaling. These results reinforce our
previous evidence (Wang and Struhl,
2004) that endocytosis of DSL ligands, per se, is not sufficient
to confer signaling activity; instead, DSL ligands must normally be
internalized via the action of Epsin to signal.
Epsin-dependent endocytosis and DSL signaling activity
Why must DSL ligands normally be internalized by an Epsin-dependent
endocytic mechanism to activate Notch? We can distinguish two general classes
of explanation (Fig. 8B) (see
also Wang and Struhl, 2004).
In the first, Epsin confers signaling activity by regulating an early event in
DSL endocytosis that occurs before internalization. For example, Epsin might
cluster DSL ligands in a particular way or recruit them to a select subset of
coated pits or other endocytic specializations. Alternatively, Epsin-mediated
invagination of these structures might control the physical tension across the
ligand/receptor bridge linking the sending and receiving cell, creating a
sufficiently strong or special mechanical stress necessary to induce Notch
cleavage or ectodomain shedding. In the second class of models, Epsin acts by
regulating a later event in DSL endocytosis that occurs after internalization.
For example, Epsin might direct, or accompany, DSL proteins into a particular
recycling pathway that is essential to convert or repackage them into ligands
that can activate Notch upon return to the cell surface. In both cases,
internalization of DSL ligands via the other endocytic routes normally
available to them would not provide the necessary conditions, even in the
extreme case of Dl, which appears to be internalized primarily by these other
pathways.
Our present results do not distinguish between these models. However,
recent studies of Epsin-dependent endocytosis in mammalian tissue culture
cells suggest that Epsin may direct cargo proteins to different endocytic
specializations or pathways, depending on their state of ubiquitination
(Chen and De Camilli, 2005;
Sigismund et al., 2005
). They
also suggest that interactions between Epsin and components of the core
Clathrin endocytic machinery normally regulate where and how Epsin
internalizes target proteins. Both properties might govern how DSL proteins
are internalized, allowing the ligands to gain access to the select endocytic
pathway they need to enter to activate Notch.
Note added in proof
Lai et al. present complementary findings that similarly support a direct
role for Dmib in ubiquitination, internalization and signaling by DSL ligands
(Lai et al., 2005).
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ACKNOWLEDGMENTS |
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