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826 Research Article
IntroductionHedgehog (Hh) is a secreted protein that patterns
and specifiescell fate in several different tissues. In Drosophila,
response toHh is mediated by Cubitus interruptus (Ci), a
transcriptionfactor with either activator or repressor functions
(Dominguezet al., 1996; Aza-Blanc et al., 1997; Methot and Basler,
1999).The processing of Ci into activator or repressor is
achievedthrough the Hh cytoplasmic complex that includes the
kinesin-like protein, Costal-2 (Cos2), the serine-threonine kinase
Fused(Fu), and Suppressor of Fused [Su(fu)] (Robbins et al.,
1997;Sisson et al., 1997; Stegman et al., 2000). Secreted Hh
bindsto its receptor Patched (Ptc). As a result, Fu and Cos2
arehyperphosphorylated (Therond et al., 1996; Robbins et al.,1997;
Nybakken et al., 2002), the cytoplasmic complexdissociates from
microtubules, and full-length Ci, Ci155,translocates into the
nucleus as a transcriptional activator(Ohlmeyer and Kalderon, 1998;
Chen et al., 1999; Wang andHolmgren, 1999). In the absence of Hh,
Ci is proteolyzed toits repressor form, Ci75. This proteolysis
involvesphosphorylation of Ci by PKA, GSK3 and CKI and the
activityof the F-box protein, Slimb (Aza-Blanc et al., 1997;
Robbinset al., 1997; Jiang and Struhl, 1998; Chen et al., 1999;
Methotand Basler, 2000).
The fate of Ci is controlled by Ptc, a twelve-passtransmembrane
protein, and Smoothened (Smo), a seven-passtransmembrane protein.
In the absence of Hh, Ptc suppressesSmo and this triggers the
proteolysis of Ci. When present, Hh
relieves the Ptc-mediated suppression of Smo, leading
tophosphorylation and stabilization of Smo, activation of Ci,
anddegradation of Ptc and the Hh ligand (Aza-Blanc et al.,
1997;Denef et al., 2000; Alcedo et al., 2000; Zhang et al.,
2004).The Smo C-terminal tail has been shown to directly bind
toCos2 (Ogden et al., 2003), and upon Hh inactivation of
Ptc,activates signaling through Cos2 and the associated
Hhcytoplasmic components (Hooper, 2003; Lum et al., 2003; Jiaet
al., 2003; Ogden et al., 2003).
Sex lethal (Sxl) functions as the master switch in
sexdetermination, controlling somatic sexual development
anddifferentiation in Drosophila. It is activated in females but
isinactive in males. These two modes of expression aremaintained
throughout the life cycle (Sanchez and Nöthiger,1983; Cline, 1984).
Sxl promotes female differentiation byregulating transformer (Boggs
et al., 1987; McKeown et al.,1987) and the dosage compensation
process (reviewed byLucchesi et al., 2005).
Previously, we showed that Sxl enhances the Hh signal
andproposed that this was the mechanism by which Sxl generatesthe
larger female body size (Horabin, 2005). In the wing discanterior
compartment, Sxl responds to the presence of Hh in aPtc-dependent
manner but Smo activity is not required (Horabinet al., 2003). Here
we show that Sxl is part of the cytoplasmicHh complex that is
tethered to the Smo carboxyl tail. Weexamined whether Ptc is also a
member of the Hh signalingcomplex, and found that Ptc and Smo can
be co-
Hedgehog acts as an organizer during development. Itssignaling
involves the receptor Patched, signal transducerSmoothened and a
cytoplasmic complex containing thetranscription factor Cubitus
interruptus tethered to theSmoothened carboxyl tail. Without
Hedgehog, Patchedrepresses Smoothened resulting in proteolysis of
Cubitusinterruptus to its repressor form. With Hedgehog,
Patchedrepression of Smoothened is relieved and Cubitusinterruptus
is activated. Sex-lethal, the master switch forsex determination in
Drosophila, has been shown toassociate with Cubitus interruptus and
the cytoplasmiccomponents of the Hedgehog signaling
pathway.Additionally, Sex-lethal responds to the presence
ofHedgehog in a Patched-dependent manner. The latterprompted us to
examine the role of Patched in signaling.We find that Cubitus
interruptus, Sex-lethal, Patched and
Smoothened co-immunoprecipitate and co-fractionate,suggesting a
large complex of both membrane andcytoplasmic components of the
Hedgehog pathway. Theentire complex is present at the plasma
membrane and theassociation of Patched changes depending on the
activationstate of the pathway; it also is not female
specific.Colocalization analyses suggest that Sex-lethal alters
theendocytic cycling of the Hedgehog components and mayaugment the
Hedgehog signal in females by decreasing theproteolytic cleavage of
Cubitus interruptus, availing moreof it for activation.
Supplementary material available online
athttp://jcs.biologists.org/cgi/content/full/120/5/826/DC1
Key words: Hedgehog, Sex-lethal, Endocycling, Patched,
Drosophila
Summary
A large complex containing Patched and Smoothenedinitiates
Hedgehog signaling in DrosophilaSabrina L. Walthall1, Michelle
Moses1 and Jamila I. Horabin2,*1Department of Biochemistry and
Molecular Genetics, University of Alabama at Birmingham,
Birmingham, AL 35294, USA2Department of Biomedical Sciences,
Florida State University, Tallahassee, FL 32306, USA*Author for
correspondence (e-mail: [email protected])
Accepted 21 December 2006Journal of Cell Science 120, 826-837
Published by The Company of Biologists
2007doi:10.1242/jcs.03382
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827Large Hh signaling complex
immunoprecipitated from embryonic extracts. This
associationcould be enhanced or disrupted, depending on the
activation stateof Hh signaling. Immunoprecipitation and
colocalization studiessuggest a large signaling complex at the
plasma membrane thatincludes both membrane proteins and the
cytoplasmiccomponents. This complex is endocytosed in a
dynamin-dependent manner. Our data, together with that of others,
suggestan altered model for passage of the Hh components through
theendocytic pathway. Colocalization of Ptc, Smo, Ci and Sxl
insalivary gland cells when signaling is on versus off suggests
that,in females, Sxl may decrease the proteolytic cleavage of Ci
byaltering its endocytic cycling with the Hh membranecomponents,
thereby enhancing the Hh signal.
ResultsSxl associates with Smo in the Hh cytoplasmic complexAs
Sxl co-immunoprecipitates with the cytoplasmic Hhcomponents Cos2,
Fu and Ci, and studies show that Smophysically interacts with the
Cos2-Fu complex via its C-terminal tail (Jia et al., 2003; Lum et
al., 2003; Ogden et al.,2003; Ruel et al., 2003), we investigated
whether Sxl wouldalso co-immunoprecipitate Smo. Wild-type embryonic
extractsshow that Sxl immunoprecipitated Smo protein (~5.2%) andthe
converse is also true; Smo immunoprecipitated Sxl (~2.7%;Fig. 1A).
By comparison, full-length Ci (Ci-155)immunoprecipitated ~4.7% of
Sxl and Smo; Cos2 ~5.1% ofSxl and ~3.5% of Smo. These
immunoprecipitation (IP) values
are in the range of or are slightly moreefficient than those
seen by others(Ogden et al., 2003) [see Fig. 5 of Jia etal. (Jia et
al., 2003)] and indicate that Sxlis present in all the known
Hhcytoplasmic complexes described to date.
Ptc is an integral member of the Hhsignaling complexAlthough Sxl
resembles Ci in itsassociation with the Hh signalingcomponents,
removing Smo activity hasno effect on Sxl nuclear entry in the
wingdisc. It is Ptc which promotes nuclearentry of Sxl in a
Hh-dependent manner(Horabin et al., 2003). This dependenceon Ptc
suggested that an associationbetween Sxl and Ptc might exist.
Toinvestigate this possibility, we determinedwhether Ptc was also
present with the Hh
Fig. 1. A large complex involving Ptc, Smoand Sxl in Drosophila
embryos. (A) Ci,Cos2, Ptc, Smo and Sxl immunoprecipitatesfrom
wild-type 0- to 12-hour embryonicextracts probed for Sxl, Ptc and
Smo. Inputlane (I) is 5% of extract used. The tablegives the
percentage of proteinimmunoprecipitated from an average of twoor
more experiments. (B) Fractionation ofcytoplasmic extract from
wild-type embryosanalyzed by western blot using the antibodiesin A
as well as anti-Fu. Arrows at top showthe elution position of the
given size marker.Several of the Hh components arephosphorylated
(asterisk) and migrate asdoublets (Cos2 and Fu); Smo is
alsophosphorylated and does not migrate as adiscrete band. ‘F’
represents total extractfrom adult females used as a marker for
eachprotein. Three arbitrary complex types ofchanging Hh components
(Complex A-C)can be described. (C) Immunoprecipitationof fractions
from each complex type withantibodies to known Hh components
showthat Ptc and other Hh components, as well asSxl are associated.
(D) Controls forimmunoprecipitates of Hh complexcomponents using
antibodies to BicD, Dlgand Fz.
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signaling components in immunoprecipitates from
wild-typeDrosophila embryo extracts. Sxl immunoprecipitated
asignificant amount of Ptc (~10.7%, Fig. 1A) and inversely
Ptcimmunoprecipitated ~3.1% of Sxl. As Sxl is in a complex withthe
other Hh components, we determined whether Ptc waspresent in
immunoprecipitates of full-length Ci, Smo and Cos2.We obtained IP
values of ~6.8, ~2% and ~1.6%, respectively,for Ptc. Although the
cause of the differences between theseproteins is not clear (there
might be different complexes or thestability of each component
within the complex may bedifferent), the fact that Sxl, Ci and Smo
can co-immunoprecipitate Ptc suggests that Ptc is part of the
Hhsignaling complex. As a negative control, we
probedimmunoprecipitates with antibodies for proteins
withsimilarities to those in the Hh signaling pathway (Fig.
1D).Bicuadal-D (Bic-D), a kinesin-like protein, Discs-large (Dlg),
amember of the guanylate kinase family, Frizzled (Fz), the
Wntreceptor a seven-pass transmembrane protein homologous toSmo,
and evenskipped, an active repressor of transcription (datanot
shown) were tested. The immunoprecipitates were positivefor the Hh
component, but not for any of the controls.
To additionally show that Ptc is a part of the Hedgehogsignaling
complex, we fractionated cytoplasmic extracts fromwild-type embryos
(Fig. 1B). Three distinct populations can bedescribed. In
population A we found that Sxl cofractionateswith Ptc, Cos2, and Ci
(trace amounts of Fu were alsodetected). This population was
relatively large (>700 kDa as
Journal of Cell Science 120 (5)
noted by the size standards), suggesting the presence
ofadditional proteins or from trimerization of Ptc (Lu et
al.,2006). Population B, contained Ci in the first few fractions,
andhad Ptc, Smo, Fu, Cos2 and Su(Fu) [Su(Fu) not shown] alongwith
Sxl. Population B is also relatively large (centered around669 kDa)
and suggests that the Hh signaling complex cancontain both Sxl and
Ci, or only Sxl, as in population A. Inpopulation C, which had
little Sxl, Ptc is still present alongwith the other Hh signaling
components. These complexeswere smaller than 440 kDa, so they are
unlikely to have severalof the Hh components together, and in the
fractions of thesmallest size, probably exist as monomers.
Populations A, B and C were tested for co-IP of Sxl and Ptcwith
Cos2, Ci, Sxl or Smo for which they were positive (Fig.1C),
supporting the contention that Ptc and other Hh signalingmembers
exist in a complex. Analysis by native gelelectrophoresis also
supported this conclusion; at least twoextremely large complexes
that contain the various Hhsignaling proteins, including Ptc and
Smo, were detected (seesupplementary material Fig. S1A,B). These
large complexesentered the stacking gel but failed to enter the
resolving gel.The negative controls Eve (see supplementary material
Fig.S1B) and Dlg (not shown) were in the resolving gel alone. Fzwas
detected in the stacking as well as the resolving gel (notshown)
but as demonstrated above it did not co-immunoprecipitate with the
Hh components, suggesting that itexists in a large complex of its
own.
Pathway activation changes associationof Ptc with Hh complex
componentsWe next examined whether the activation stateof the
pathway affected the association of Ptcwith the other Hh
components. The pathwaywas activated using the Ptc1130X variant,
whichhas the last 156 amino acids of the cytoplasmictail deleted.
Ptc1130X is a dominant-negativeallele and leads to Ci activation
independentlyof the Hh morphogen, inducing targets whichrequire
high Hh levels for activation (Johnsonet al., 2000). Embryos
expressing this and thevariants that follow were generated using
theUAS-GAL4 system; homozygotes of theubiquitous daughterless-GAL4
(da-GAL4)driver were crossed to a line homozygous forthe UAS-Ptc
construct. Western blots showedthat the prevailing Ptc is the
variant form, whichfor Ptc1130X migrated a little faster than
theendogenous protein.
Immunoprecipitates of Sxl, Ci, Cos2 andSmo were probed with
antibodies for Sxl, Ptc
Fig. 2. Ptc association within the Hh complexresponds to
activation state of the pathway. Ci,Cos2, Smo and Sxl
immunoprecipitated fromembryos expressing a different Ptc
variantfollowed by western blot analysis for Sxl, Ptc andSmo.
Efficiency of IP is relative to the 5% extractin the input lane
(I). (A) Embryos expressingPtc1130X. (B) Embryos expressing
PtcD584N.(C) Embryos overexpressing wild-type Ptc.(D) Embryos
expressing Ptc�Loop2.
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and Smo and each IP yielded ~5-6% of the tested proteins
(Fig.2A). The exception was Smo which immunoprecipitated ~9%of Sxl.
Compared with wild-type embryos, the amount of
Ptcimmunoprecipitated increased regardless of the IP protein,
andthe amount of Sxl in complexes that contain Smo alsoincreased
from ~3% to ~9%. These data demonstrated that theactivity state of
the pathway alters the association of Ptc withthe other Hh
components. Removal of the last 156 amino acidsappears to not only
hinder the ability of Ptc to inhibit Smo, italso ‘locked’ Ptc into
the complex.
Intermediate activation of the pathway had a different
effect.PtcD584N has an aspartic acid changed to asparagine at
position584 in the sterol sensing domain (SSD), is also a
dominantnegative and activates the pathway, although not as
strongly asPtc1130X (Johnson et al., 2002). The amount of
Ptcimmunoprecipitated (~1%) by Sxl and Ci appeared to
decreasecompared with the wild type (Fig. 2B), Smo
alsoimmunoprecipitated slightly less Ptc whereas
Cos2immunoprecipitated ~4% of Ptc, elevated from the wild
type(~2%). The amounts of Sxl and Smo that were associated withCi
did not change substantially and remained close to ~5%.
These data suggest that complexes that contain full-lengthCi
with Sxl or Smo remain unaltered when the pathway isactivated or
‘on’. However, Smo was able to more effectivelyimmunoprecipitate
Sxl than in the wild-type condition (from~3% to ~5%), suggesting
that the association of Sxl with Smowithin the complex may be
enhanced while the association ofPtc is disrupted.
Although Ptc1130X and PtcD584N were overexpressed, eachdisplayed
different interactions with the other components,suggesting that
overexpression per se does not determineassociation. Both activate
the Hh pathway; however, with respectto protein turnover, PtcD584N
more closely resembles wild-typeprotein bound to ligand (Martin et
al., 2001; Strutt et al., 2001;Lu et al., 2006). This suggests that
the association of Ptc withinthe complex is disrupted when the
pathway is turned on.
Association of Ptc with the Hh complex is enhancedwhen the
pathway is offFor the ‘off’ state, wild-type Ptc or Ptc�Loop2 was
expressed inembryos. Overexpression of wild-type Ptc switches the
systemoff as there is not sufficient endogenous Hh. Ptc�Loop2
alsosuppresses pathway activation; it is unable to bind Hh as
itlacks the extracellular loop between transmembrane segmentsseven
and eight necessary for Hh binding. Embryosoverexpressing wild-type
Ptc showed full-length Ci, Cos2,Smo as well as Sxl
co-immunoprecipitated with significantamounts of Ptc (10% or
greater, Fig. 2C). The Hh componentsalso immunoprecipitated
significant amounts of Sxl (~8% orgreater), suggesting that Sxl
remains a part of the complexwhen the pathway is off. Association
of Ptc with Smo and thecytoplasmic Hh components appeared to be
enhanced when thepathway is off. Compared with the wild type, all
thecomponents tested showed substantial increases in the amountsof
Ptc that co-immunoprecipitated with them. Similar resultswere
obtained when the pathway was off through Ptc�Loop2
expression (Fig. 2D).The data taken together, suggest a large
complex that
consists of Ptc, Smo and the cytoplasmic Hh components (andSxl
in females). The similarity in the IP profile of wild-typeembryos
to that of embryos expressing PtcD584N rather than
either variant that turns the system off, also suggests that
wild-type embryos reflect primarily an activated state.
Presence of Ptc in the Hh complex is not dependent onSxlAs
embryonic extracts do not distinguish between males andfemales, it
could be argued that Sxl accounts for the IP of Ptcwith the other
Hh components. Wild-type male and female fliesshowed that females
do indeed give a greater yield of Ptc intheir Ci and Cos2
immunoprecipitates (Fig. 3G), however,males also showed Ptc,
indicating that its presence is not sexspecific.
If the Hh cytoplasmic complex directly interacts with Ptc,
itshould be able to do so in the absence of Smo. To demonstratethat
Ci can associate with the plasma membrane when Ptc isthe lone Hh
membrane component, we removed Smo in malesalivary gland cells
using the SmoD16 deletion allele. As thelevels of Ci are greatly
reduced under these conditions, amutation in PKA was introduced to
increase the levels of Ci.Clones of cells mutant for SmoD16 and PKA
showed that 15%of the Ptc and 14% of the Ci colocalize at the
plasmamembrane and the vesicular network (Fig. 3A-F). Althoughthis
was a little lower than we observed in the wild-typecondition (see
below) and might suggest a more stable complexexists when both Hh
membrane proteins are present, thecolocalization is consistent with
the idea that the Hhcytoplasmic complex is associated with Ptc.
Overexpression of Ptc and its variants titrates Sxl out ofthe
nucleusAs Sxl depends on Ptc to respond to Hh in wing discs, we
Fig. 3. Association of Ptc with the Hh complex is independent
ofSxl. (A-F) Male salivary gland cells with the SmoD16 deletion
allele(and PKAH2). Ci and Ptc colocalize at the plasma membrane
andvesicular network (C and F colocalized pixels only). (G) Adult
maleand female extracts treated with anti-Ci and Cos2 show that
Cos2immunoprecipitates ~16% of the Ptc in males, ~22% in females;
Ciimmunoprecipitates ~1% of the Ptc in males and ~16% in
females.
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reasoned that Sxl might associate directly with Ptc.
Thisprompted us to determine whether overexpression of Ptc
wouldaffect the localization of Sxl (Fig. 4A-F), because, unlike
mostof the other Hh components that reside in the cytoplasm, Sxlin
embryos is primarily nuclear (Fig. 4A’).
Overexpression of Ptc1130X, PtcC (deletion of the N-terminusup
to the seventh transmembrane region) and Ptc13 titrated Sxlout of
the nucleus (Fig. 4D’,E’; Ptc13 not shown). Wild-typePtc and
Ptc�Loop2 gave intermediate effects: Sxl was detectedin both
cytoplasm and nucleus (Fig. 4B’,F’), whereas PtcD584N
did not significantly alter the nuclear localization of Sxl
(Fig.4C’). Both dominant negatives, Ptc1130X and PtcD584N, result
inthe activation of Ci; however, only Ptc1130X sequesters Sxl
inembryos and facilitates its nuclear import in wing discs
(ourunpublished results), correlating these two properties of
Ptc.
The two Ptc dominant negatives also suggest that
pathwayactivation is not responsible for the observed change in
Sxllocalization. To confirm this, the state of Hh signaling
wasdirectly assayed by staining embryos for full-length Ci
andWingless (Wg; Fig. 4G-L). As predicted, the levels of Ci
Journal of Cell Science 120 (5)
increase when Ptc1130X, PtcD584N or Smo isexpressed, and
decrease when Ptc+ or Ptc�Loop2 isexpressed. The Wg signal was also
altered in thesebackgrounds, reflecting its dependence on
Hhsignaling. That transcriptional activation by Hhwas not involved
in changing the subcellularlocalization of Sxl in embryos, is also
supportedby the observation that overexpression of Hh itselfhad
little effect, as did the overexpression of Smo(data not shown).
Since Ptc variants that bothactivate or inhibit Hh signaling can
sequester Sxl,and the sequestration does not show segmentalrepeats,
it is more likely that the effect is causeddirectly by the
overexpression of the Ptc proteins.
As PtcC can titrate Sxl out of the nucleus, thecarboxyl half of
Ptc must provide the docking site. The 156 C-terminal residues do
not appear to be involved, however, astheir removal in Ptc1130X did
not severely compromisesequestering Sxl out of the nucleus. Between
PtcC andPtc1130X, the cytoplasmic regions remaining are the
loopbetween transmembrane segments 10 and 11 and the last ~27amino
acids before the 1130X deletion point.
Ptc and Smo colocalize with Hh componentsDepending on the
signaling condition, Ptc and Smo appearedto be in the same complex.
To determine whether theycolocalize in vivo, we examined their
distribution with full-length Ci in the large salivary gland cells
(Fig. 5A-L); Ci servesas the marker of the cytoplasmic Hh complex.
The state of Hhsignaling in wild-type salivary glands is normally
off (Zhu etal., 2003). To activate it, Hh was expressed using a
salivarygland GAL4 driver, sgs3 (Fig. 5M-X).
Optical sections were taken near the plasma membrane aswell as
deeper within the cell to include a cross section of thenucleus.
Sections near the plasma membrane sample vesicles
Fig. 4. Ptc can alter Sxl subcellular location.(A-F) Embryos
expressing different Ptc variants stainedfor Sxl (green, A-F), and
propidium iodide (PI; red)merged with Sxl (A’-F’). (A’) Sxl is
primarily nuclearin wild-type (wt) embryos. Ptc+ (B’) and Ptc�Loop2
(F’)do not completely titrate Sxl out of the nucleus giving
adiffuse image; the PI signal is yellow to orange.(C’) PtcD584N
resembles the wild type and Sxl is moredistinctly nuclear. (D’,E’)
Ptc1130X and the carboxylhalf of Ptc, PtcC, strongly titrate Sxl
out of the nucleus;the PI signal is more red. (G-L) Effects of Ptc
variantson Hh signaling in embryos reported by full-length Ci(red)
and Wg (blue) expression. Relative to wt (G), Cilevels are
increased by the expression of PtcD584N (I)and PtcC (K), decreased
by Ptc+ (H) and Ptc�Loop2 (L).The increase caused by Ptc1130X (J)
is very modest. Wgexpression is depressed by Ptc+ and Ptc�Loop2 (H’
andL’; arrowheads mark disrupted Wg stripe), elevated byPtcC (K’)
and modestly elevated by PtcD584N (I’). Thefeedback between Ci and
Wg (Lessing and Nusse,1998) appears most affected by Ptc1130X and
Wg levelsare not strongly elevated (J’). This is more evident asthe
embryos get older and the Wg levels drop. Embryosscanned at similar
settings with a 40� objective. Bars,20 �m.
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more recently budded from the plasma membrane, whereasthose
deeper in the cell sample later stages of the endocyticcycle.
Colocalization of the two Hh membrane proteins withCi was compared
between males and females, in the presenceand absence of Hh (Fig.
5, Fig. 7A1).
Several observations stand out. First, for both Ptc and Smothe
plasma membrane as well as the underlying vesicularnetwork,
appeared as sites of colocalization with Ci. Second,in the absence
of Hh the degree of colocalization of the twoHh membrane proteins
with Ci was generally a little lower infemales than males, both
near the plasma membrane anddeeper within the cell. Third, in the
presence of Hh, thesetrends were quite dramatically altered.
Females showed moreCi colocalizing with Ptc at the plasma membrane
than males,presumably reflecting a heightened response to signaling
(Ptcis the receptor). They also showed a more dramatic decrease
of Ci colocalized with Ptc deeper within the cellsthan males.
This enhanced separation of Ci from Ptcin females was mirrored by
an increased level ofcolocalization of Ci with Smo within the
cell.Combined, this suggests that in the presence of Hhfemale cells
might recruit more Ci to receive thesignal at the plasma membrane,
but as Ptc trafficstowards degradation deeper within the cells,
femalessegregate more of their Ci away from Ptc. Changeswere also
seen in the colocalization of Sxl with Ptc,Smo and Ci in the
presence of Hh (Fig. 7A2); moreof the Sxl and Ptc signal overlapped
at the plasmamembrane, and much of the Sxl appeared todissociate
from these Hh components within the cell.
Although this colocalization approach is limited –the images are
not a perfect sampling of specificsubcellular compartments – it did
show Cicolocalization with Ptc, besides the expectedcolocalization
of Ci with Smo. Additionally, thedifferences between the sexes are
reliable as they aregenerated with similarly treated samples.
Blocking the first step in endocytosis increasescolocalization
of Hh componentsThe wild-type data support the idea that Ptc, Smo
andCi are together at the plasma membrane. SinceDynamin [Shibire
(Shi)] is required for the pinchingof clathrin-coated pits from the
plasma membrane(van der Bliek and Meyerowitz, 1991), reducing
Shiactivity should inhibit the first step of endocytosis
and‘freeze’ the Hh components there. Consistent with
thisprediction, expressing the ShiK44A dominant negativeessentially
doubled the colocalization of Ci with Ptcin sections near the
plasma membrane in both sexes[from 20% to 41% in males, 15% to 33%
in females(Fig. 6G-L, Fig. 7B1)]. Ci and Ptc colocalization isalso
increased in sections deeper within the cell. Thecolocalization of
Ci and Smo near the plasmamembrane did not change as dramatically,
and wasaffected even less within the cell (Fig. 6A-F, Fig.7B1,C1).
Sxl showed the same trends as Ci in itschanges in colocalization
with Ptc and Smo,particularly at the plasma membrane (Fig.
7B1,B2).
Besides affecting the proteins at the plasmamembrane, inhibiting
the first step of endocytosis
decreased the sexual dimorphism in the Ci and Ptccolocalization,
and eliminated it for Ci and Smo. This suggeststhat the sexual
difference is primarily a function of endocyticcycling, occurring
after the events at the plasma membrane.
Sxl dissociates from Ptc early in endocytosisBlocking
endocytosis at the first step appears to increase thecolocalization
of Ci, and Sxl with Smo and Ptc. To confirm this,IPs using extracts
from embryos expressing the dominantnegative ShiK44A were
performed. Relative to the wild type, thedominant negative Shi
increased the amount of Ptc complexedwith some of the Hh
components. Ci and Sxlimmunoprecipitated ~8.4% and ~8.7% of the
Ptc, which issimilar to the wild type. However, Smo and
Cos2immunoprecipitated ~4.9 and 4% of the Ptc, an increase from~2%
for both of them (Fig. 8B). The amount of Sxl
Fig. 5. Colocalization of Ptc and Smo, with Ci and Sxl.
Wild-type female andmale salivary gland cells stained for Ci and
Ptc or Ci and Smo (and Sxl infemales) in the presence and absence
of Hh. Female glands in the absence ofHh stained for Sxl, Smo and
Ci (A-C) or Sxl, Ptc and Ci (G-I). Male glands inthe absence of Hh
signal stained for Smo and Ci (D,E) or Ptc and Ci (J,K).Female
glands expressing Hh stained for Sxl, Smo and Ci (M-O) or Sxl,
Ptcand Ci (S-U). Male glands expressing Hh stained for Smo and Ci
(P,Q) or Ptcand Ci (V,W). ‘Col.’ panels
(A’-C’,F,G’-I’,L,M’-O’,R,S’-U’,X) show only thepixels that are
common between two proteins. Note the extensive colocalizationclose
to the plasma membrane in most panels.
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immunoprecipitated by Smo and Ci was also elevated.This is in
keeping with the idea that blocking the firststep of endocytosis
traps Ptc and the entire Hhsignaling complex at the plasma membrane
and thatPtc segregates from Smo and Cos2 as it progressesthrough
the endocytic pathway.
From clathrin-coated pits, budding vesicles fusewith endosomes
in a process that is mediated by thesmall Rab5 GTPase. Inhibiting
the endocytic pathwayat this stage using a dominant negative
Rab5S43N
variant (Stenmark et al., 1994; Entchev et al., 2000),showed the
amount of Ptc immunoprecipitated by Sxlis considerably less (~3.9%,
Fig. 8B) than for the Shidominant negative. The amount of Ptc in
the Smo andCos2 IP appeared to elevate a little (from ~4 and 4.9%to
~7.2 and 7.3%, respectively), while Ci levelsappeared to drop (from
~8.4 to 5.2%). The co-IP ofSxl with Ci and Smo also decreased
relative to the Shidominant negative condition. This suggests that
theassociation of both Sxl and Ci with Ptc is weakened,while Ptc
continues to progress with Smo and Cos2through the endocytic
pathway. Sxl also appeared toreduce its association with Smo and Ci
but itsassociation with Cos2 was not significantly altered.
Rab7 is a GTPase essential for transportingendocytic cargo from
the early to late endosomes andlysosomes (Vitelli et al., 1997).
Embryos expressing adominant gain-of-function Rab7 (Rab7Q67L)
(Entchevet al., 2000) also showed lower Sxl and Ptc
association(~2.9%, similar to the Rab5 dominant negative anddown
from ~8.7% in the Shi dominant negative).However, the degree of
association between Ptc withCi, Smo or Cos2 was not substantially
different fromthe Shi dominant negative condition (Fig. 8B).
TheRab7 gain of function increased the Ci and Ptcassociation
compared with the Rab5 dominantnegative, as well as the interaction
of Sxl with Smoand Ci (Fig. 8A). Consistent with this
observedinhibition in sorting of Hh components,overexpression of
wild-type Rab7, which increasesoverall Rab7 activity, also impairs
the motility ofendosomes (Lebrand et al., 2002).
In summary, these data suggest that Sxl and Cibegin to segregate
from Ptc relatively early, prior tothe stage requiring Rab5. Sxl
also decreases itsassociation with Ci and Smo. The colocalization
analyses (Fig.7A2) also suggest that within the cell, Sxl has low
levels ofcolocalization with Ptc, Smo and Ci when signaling is on:
thestate embryos more strongly reflect. Overactive Rab7 reversesthe
segregation of Sxl from Ci and Smo, but not from Ptcsuggesting that
Sxl and Ptc separate earlier in endocytosis. Ciappears to separate
from Ptc a little later than Sxl, whereassegregation of Smo from
Ptc must occur beyond the stageregulated by Rab5. Overactive Rab7
prevents Smo and Ptcsegregation, however, as they have opposite
endocytic fatesthey would have to separate prior to sorting to the
lysosome(Denef et al., 2000; Incardona et al., 2002; Zhu et al.,
2003).
Detecting segregation of the Hh components and Sxlduring
endocytosisThe sexually dimorphic colocalization of Ci with Ptc and
Smo
Journal of Cell Science 120 (5)
presumably reflects the effects of Sxl. Sxl appears to
dissociatefrom some of the Hh components relatively early in
theendocytic cycle. We therefore compared the colocalization ofPtc
and Smo with Ci (and Sxl in females) in males and femalesexpressing
a Rab5 dominant negative and a Rab7 gain-of-function variant.
Inhibiting the endocytic cycle at the Rab5 stage showed adrop in
the level of Ci and Ptc that colocalized at the plasmamembrane in
both sexes compared with the Shi dominantnegative (41% to 29%
males, 33% to 22% in females; Fig. 6R’,Fig. 7B1). These values are
still higher than the wild type, andsince the Shi dominant negative
presumably reflects the stateof the components as they begin
endocytosis at the plasmamembrane, suggest that sorting of the
proteins is still inprogress. Colocalization of Ci with Smo showed
a smallerdecrease relative to Shi, in both sexes (33% to 27% in
males,
Fig. 6. Colocalization analyses of Hh components when
endocytosis is blocked.Salivary gland cells expressing the dominant
negative form of Shi, ShiK44A, orRab5, Rab5SN stained for Ci and
Ptc or Ci and Smo (and Sxl in females).Female glands expressing
ShiK44A stained for Sxl, Smo and Ci (A-C) or Sxl, Ptcand Ci (G-I).
Male glands expressing ShiK44A stained for Smo and Ci (D,E) orPtc
and Ci (J,K). ‘Col.’ panels (A’-C’,F,G’-I’,L) show only pixels
common tothe indicated proteins. (M-O,S-U) Female glands expressing
Rab5SN stained forSxl, Smo and Ci (M-O) or Sxl, Ptc and Ci (S-U).
‘Col.’ panels (M’-O’,P’-R’)show only pixels common to both
proteins. All sections are from near theplasma membrane.
Percentages of all colocalizations are given in Fig. 7.
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33% to 29% in females, Fig. 6O’, Fig. 7B2). Within the
cells,these trends were upheld for Ptc, but not for Smo,
whichremained almost unchanged. These data suggest that when
thepathway is off (the ground state of salivary glands), Ptc
andfull-length Ci segregate from each other by the Rab5 stage;
butthe segregation of Ci and Smo does not change considerably.
Progression from early to late endosomes involvesdisplacement of
Rab5 by Rab7 (Rink et al., 2005). Thedominant gain-of-function Rab7
(Rab7Q67L) (Entchev et al.,2000) might be predicted to accelerate
Rab7 function andcompromise sorting of components in Rab5 positive
vesicles.
We found that the colocalization values resembled the
Shidominant negative and also the Rab5 dominant negativecondition
(Fig. 7B1-2,C1-2, images not shown), but not thewild type
suggesting a reversal in sorting events. In general,the changes in
Smo and Ci colocalization were smaller thanfor Ptc with Ci. What
stands out most clearly is that femalesshowed an elevation over
males of Ci and Ptc colocalization atthe plasma membrane, and Ci
and Smo colocalization withinthe cell. These differences indicate
that the sorting effects onthe Hh proteins caused by the Rab7 gain
of function do notoccur similarly in males and females.
Fig. 7. Colocalization percentages of Ci and Sxl with Ptc and
Smo in both sexes. (A) Colocalization of full-length Ci with the
two Hhmembrane components, Ptc and Smo (1), as well as Sxl (2), in
wild-type salivary glands in the absence (–Hh) and presence of Hh
(+Hh) at theplasma membrane (PM) and within the cell.
Colocalization of the proteins at the plasma membrane (B) and
within the cell (C), in salivaryglands of the wild type or animals
expressing Shi, Rab5 or Rab7 mutant proteins. Males and females
show differences in amounts. Plasmamembrane optical sections
include region near the membrane. Sections taken within the cell
include a cross section of the nucleus. Percentagesreflect the
average of at least two, usually three separate optical
sections.
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With respect to Sxl, altering endocytosis with the Rab5dominant
negative and Rab7 gain-of-function variants,generally increased the
amounts of Sxl that colocalized withall three Hh proteins relative
to the wild type near the plasmamembrane. Sxl did show a drop in
its colocalization levels withboth Ptc and Ci with the Rab5
dominant negative, suggestinga separation of Sxl from these two
components around theRab5 stage. The Rab7 gain of function appeared
to reverse thisdissociation. Within the cell, the colocalization of
Sxl with Ptcwas enhanced by both Rab5 and Rab7 variants,
particularly theRab7 gain-of-function variant; the association of
Sxl with Smoand Ci was generally also slightly elevated.
These dynamics suggest a complex picture of componentsin
different associations, depending on their stage within
theendocytic cycle. They suggest that (1) in the
wild-typecondition, the overall colocalization of Ptc and Smo with
Ci islow, and perturbing the early stages of endocytosis tends
toincrease their (and Sxl in females) colocalization levels;
(2)females generally show lower protein colocalization levels
thanmales and perturbing the early stages of endocytosis tends
todecrease the difference; (3) males and females show variancesin
their response to sorting perturbations, particularly by theRab7
gain-of-function variant, where females show greaterfluctuations;
(4) changes in Ptc colocalization with Ci tend tobe greater than
for Ci with Smo, with Smo showing greaterchanges near the plasma
membrane than within the cell.
Journal of Cell Science 120 (5)
DiscussionWe proposed that Sxl enhances the Hh signal to
generate thelarger female body size (Horabin, 2005). Sxl is part of
the Hhcytoplasmic signaling complex (Horabin et al., 2003) as
wellas the complex attached to the Smo tail, suggesting that it
isan integral signaling member. To elucidate how Sxl enhancesthe Hh
signal, we attempted to quantify the associations of thevarious
components under different signaling conditions. Wefound that Sxl
influenced the interactions between thecomponents during
endocytosis and, more importantly, that Ptcis also a member of the
Hh signaling complex.
A large complex that contains both Hh membranecomponentsThe IP
data showed Ptc with full-length Ci, Sxl and the otherHh signaling
proteins in wild-type embryos; complexescontaining both Ptc and Smo
suggest that this associationbecomes stronger when signaling is
off. The complexcontaining Ptc is not sex specific, although female
flies givegreater yields than males.
The idea that Drosophila Ptc and Smo are in a commoncomplex runs
counter to prevailing thought. Experiments inmammalian cells
suggested that Ptc might associate with Smo(Stone et al., 1996;
Murone et al., 1999), but subsequentexperiments in S2 cells
questioned whether theseobservations applied to the Drosophila
proteins (Johnson etal., 2000). Ruel et al. (Ruel et al., 2003)
also reported that Ptcdid not immunoprecipitate with Cos2 in cl-8
cells. This reportis the first using Drosophila embryonic extracts,
and we alsofind a weak association of Ptc with both Cos2 and
Smo.However, this result changes depending on the state of
Hhsignaling.
The addition of Ptc to the Hh signaling complex was
furthercorroborated by size fractionation experiments, which
showPtc in large complexes that also contained other known
Hhcomponents. It is not clear what proteins account for the
verylarge complexes of population A in Fig. 1B, which contain
Ptc,Cos2, Sxl, Ci and trace amounts of Fu. Ptc can trimerize (Luet
al., 2006), and in vivo data suggest Ptc functions as a
trimer(Casali and Struhl, 2004) requiring its co-receptors Ihog
andBoi (Yao et al., 2006). Additionally, some of the other
Hhcomponents besides Ptc may be present as multimers, or thecomplex
may also contain unknown proteins.
Colocalization analyses also showed Ptc and Smo togetherwith Ci
at the plasma membrane and in the network of theendocytic pathway.
When endocytosis is blocked in embryosexpressing a dominant
negative Shi variant, an enhancement inco-IP of Hh complex proteins
with Ptc is observed, suggestinga large complex with all known
signaling components at theplasma membrane. Images also show an
increase in theamounts of Ptc and Smo that colocalize with Ci at
the plasmamembrane, supporting this view.
Finally, the Ptc tail must play a key role in influencing
theinteractions within the complex. Removal of the last 156
aminoacids not only hinders Ptc1130X from inhibiting Smo, it
alsostabilizes most of the interactions within the complex. It
alsoappears to affect the endocytic cycling and stability of Ptc.
Asopposed to Ptc1130X being proteolyzed, which is the normal
fatefor Ptc when Hh signaling is on, high levels of the
dominantnegative protein are detected at the plasma membrane (Zhu
etal., 2003; Lu et al., 2006).
Fig. 8. Ptc and Sxl are endocytosed with Hh signaling
components.Ci, Cos2, Smo or Sxl IP of embryo (0- to 12-hour)
extractsexpressing the dominant negative Shi (ShiK44A), Rab5
(Rab5SN) orgain-of-function Rab7 (Rab7QL) variant probed for Ptc
and Sxl.Percentage of Sxl (A) and Ptc (B) immunoprecipitated for
eachvariant compared with wild-type (wt) data of Fig. 1. The
average andstandard error are the mean of two or more data sets.
Error barsrepresent ±1 s.e. White bars, wild type; light gray bars,
Shi;white/black dot bars, Rab5; dark gray bars, Rab7.
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Augmenting the Hh signalThe yields of Ptc with the Hh
cytoplasmic components wasgreater in females than males, suggesting
Sxl stabilizes Ptcwithin the complex. Sxl may bind directly to Ptc,
asoverexpression of Ptc can titrate Sxl out of the nucleus.
How does Sxl enhance the Hh signal particularly as itstabilizes
the association of Ptc, a negative component insignaling? We find
that Sxl influences the segregation of theHh components during
endocytosis. Sex-specific differences inthe colocalization of Ci
with both Ptc and Smo are mostlyeliminated by blocking the first
step of endocytosis (Fig. 7B1-2), suggesting that Sxl impacts
signaling events at orimmediately after the plasma membrane.
Additionally, sortingresponses when endocytosis is perturbed using
a Rab5dominant negative or Rab7 gain-of-function variant,
showeddifferences between males and females. Protein sorting
muststill be in progress during these early steps in endocytosis
asperturbing them increases the levels of colocalization of
theproteins relative to the wild type (including Sxl).
When signaling is off, females show lower amounts of full-length
Ci colocalizing with both Ptc and Smo. When signalingis on however,
females show more Ci and Ptc colocalizing nearthe plasma membrane,
and a concomitant increase in thecolocalization of Sxl with Ptc
(Fig. 7A2). As Ptc is the Hhreceptor, this might reflect enhanced
signaling or a delay inendocytosis, or both, relative to males. A
delay at the plasmamembrane would not compromise signaling, but
couldinfluence the interactions and subsequent segregation
ofcomponents within the cell. The Hh complex is capable ofsignaling
at peak levels when endocytosis is blocked by theloss of shi
function (Han et al., 2004; Torroja et al., 2004). Thisindicates
that although the Hh components are trapped at theplasma membrane,
Hh alters the interactions within thecomplex and Smo is no longer
inhibited by Ptc. In keeping withthe idea that Hh modifies
interactions within the complex,alterations that affect Ptc
activity also affect whether itimmunoprecipitates strongly or
weakly with the othercomponents.
In the presence of Hh, females also show higher levels thanmales
of Ci and Smo colocalization within cells, which should
favor Ci activation and signaling. Conversely, the associationof
Ci with Ptc within cells is lower in females. The latter
wouldimplicate a decrease in proteolysis of full-length Ci
becausePtc is proteolyzed when Hh is present. This suggests that
withboth membrane proteins, females favor production of full-length
Ci more than males, sparing more of it from proteolysisas well as
activating more. Consistent with this proposal, theamount of
full-length Ci is higher in females and Hh signalingis enhanced
(Horabin, 2005).
These effects during Hh signaling would appear to mostlyoccur
near the plasma membrane, as the levels ofcolocalization of Sxl
with the Hh components are relativelylow within the cell. Indeed,
IP experiments suggested Sxlbegins to dissociate from the Hh
cytoplasmic complexrelatively early, before the Rab5 stage (Fig.
8A). Theassociation of Sxl with Ptc appeared to be disrupted
evenearlier, because unlike the cytoplasmic complex components,the
Rab7 gain-of-function variant does not reverse itsdissociation from
Ptc (Fig. 8B).
Model of Hh signaling and endocytosisObservations by others
taken with the data described here,suggest a model for Hh signaling
(Fig. 9). In the absence ofHh Ptc, Smo and the Hh cytoplasmic
components areendocytosed. All the components, except Ptc, are
degradedwhile Ci is proteolyzed to its repressor form. Most of the
Ptcrecycles (cyloheximide slowly decreases Ptc levels in theabsence
of Hh so low levels of Ptc are likely to also turn over)back to the
plasma membrane (Denef et al., 2000; Incardonaet al., 2002; Zhu et
al., 2003), permitting Ptc to repeat the cycleand regulate Smo
levels in a ‘catalytic’ manner (Taipale et al.,2002).
In the presence of Hh the initial events are similar. Ptc isnow
sorted for degradation while Smo is activated, and full-length Ci
is generated. Membrane and microtubule associationof Cos2 and Fu
decreases (Stegman et al., 2004) favoring theirearly release from
Smo and vesicles. Activated Smo recyclesto the plasma membrane
where it can activate more Ci.Activated Smo destabilizes Cos2 and
Fu (Lum et al., 2003;Ruel et al., 2003), suggesting that once Ci is
activated, Cos2
Fig. 9. Model of endocytosis and Hh signaling. Inthe absence of
Hh Ptc, Smo and the cytoplasmiccomponents are endocytosed. All the
componentsincluding some of the Ptc, are degraded; Ci isproteolyzed
to its repressor form (Ci75). Most of thePtc recycles to the plasma
membrane (broken arrow)permitting Ptc to repeat the cycle and
regulate Smolevels in a ‘catalytic’ manner. In the presence of
Hhthe initial events are similar. Ptc bound to Hh is nowsorted for
degradation while Smo splits apart, isactivated and full-length Ci
(Ci155) is generated.Membrane and microtubule association of Cos2
andFu decreases upon Hh signaling (Stegman et al.,2004) favoring
their early release from Smo andvesicles. Activated Smo recycles to
the plasmamembrane where it activates more Ci; Cos2 and Fuare
destabilized (Lum et al., 2003; Ruel et al., 2003),suggesting that
once Ci is activated, Cos2 and Fu aredegraded. Shi, Rab5 and Rab7
denote where thesecomponents function within the endocytic
cycle.
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and Fu are degraded. The fate of Ci (and associated
Hhcytoplasmic components) thus depends on whether Ptc or Smoreturns
to the cell surface by progression through the endocyticcycle.
Ptc has been described as cycling to and from the
membraneindependently of Hh (Strutt et al., 2001). Cells that are
notexposed to Hh maintain Ptc at the cell surface and
inunidentified internal stores, while Smo colocalizes with
lateendosomes and lysosomal markers. Vertebrate cells
transfectedwith the Hh membrane proteins show that in the presence
ofShh (the vertebrate Hh homolog), Ptc and Smo segregate in thelate
endosomal compartment. Smo recycles back to themembrane while Ptc
is fated for lysosomal degradation(Incardona et al., 2002).
Endocytosis has thus been proposedto segregate Ptc from Smo,
relieving Smo of its repressionwhile inducing the degradation of
the Hh ligand (reviewed byPiddini and Vincent, 2003; Torroja et
al., 2005).
Hh signaling elevates Smo levels and induces itsphosphorylation
at the plasma membrane, while lowering thelevels of Ptc (Denef et
al., 2000; Zhu et al., 2003; Jia et al.,2004; Zhang et al., 2004).
The oncogenic Smo proteins,which signal constitutively, do not
cycle with Ptc and remainat the plasma membrane (Incardona et al.,
2002; Zhu et al.,2003). These observations are accommodated by the
abovemodel.
The homology of Ptc to bacterial transporter proteins takenwith
the findings that small molecules such as cyclopamine caninhibit
mammalian Smo, and Ptc acts catalytically in itsinhibition of Smo,
led to the suggestion that a small moleculetransported into the
cell by Ptc serves to inhibit Smo (Taipaleet al., 2002). This
obviated the need for a close interaction.[Note that Casali and
Struhl (Casali and Struhl, 2004) suggestthat in vivo, the degree of
the ‘catalytic effect’ of Ptc on Smomay be smaller than proposed.]
Our model does not precludePtc from inhibiting Smo through a small
molecule. Indeed,their proposed proximity would enhance the
efficacy, elevatingthe local concentration. Additionally, within
small endocyticvesicles, Ptc would more rapidly affect Smo as it
would requirefewer molecules to alter the concentration.
The association of Ptc with Smo need not be direct.
Cellsincapable of giving a Hh response show very highcolocalization
of the two human proteins, but the two proteinsdo not
co-immunoprecipitate (Incardona et al., 2002). A directinteraction
between the two membrane components may notexist or it may be
unstable. Alternatively, the data of Incardonaet al. (Incardona et
al., 2002) may suggest a requirement for(some of) the cytoplasmic
components to bridge the two Hhmembrane proteins. Future analysis
will determine which is thecase.
Materials and MethodsFly stocks and clone generationFlies were
raised at 25°C; OreR was the wild-type control.
Immunoprecipitationsand embryo analysis used the following strains:
UAS-ptc1130X, UAS-ptcC, UAS-ptc+ (Johnson et al., 2000),
UAS-ptcD584N (Johnson et al., 2002), UAS-ptc�Loop2(Page, 2002),
UAS-shiDN (Moline et al., 1999), UAS-rab5S43N, UAS-rab7Q67L
(Entchev et al., 2000), UAS-hh (Azpiazu et al., 1996), UAS-ptc13
(Strutt et al.,2001). Transgenes were expressed using the UAS-Gal4
system (Brand andPerrimon, 1993). da-Gal4 (Wodarz et al., 1995), or
the salivary gland driver sgs3-gal4 were used for expression.
Description of genes can be found in
FlyBase(http://www.flybase.org/).
To generate clones SmoD16, DCOH2, FRT40/CyO were mated to y, w,
hs70-FLPflies and first instars heat shocked for 30 minutes.
Salivary glands from third instarswere used for immunostaining.
Immunoprecipitation, western blots and immunofluorescenceExcept
for the wild type which used 50 �l, immunoprecipitations used 75 �l
of 0-12 hour embryo extracts. Western blots, immunoprecipitations
and whole mountstains were done as described (Vied and Horabin,
2001) varying only the lysis andwash buffers: lysis buffer 100 mM
Tris-HCl pH 8.0, 300 mM NaCl, 1% NP40, 2mM EGTA plus protease
inhibitors; wash buffer 50 mM Tris-HCl pH 8.0, 300 mMNaCl, 0.5%
NP40, 1 mM EGTA. Antibody dilutions were as described (Horabin
etal., 2003) except for anti-Sxl (1:600; western blot), anti-Ptc
(1:400; from P. Ingham,Johns Hopkins University, Baltimore, MD),
anti-Smo (immunoprecipitation 1:3,from P. Beachy, University of
Sheffield, Sheffield, UK; western blot 1:100, from J.Hooper,
University of Colorado Health Science Center, Boulder, CO),
anti-Cos2(immunoprecipitation 1:3, from P. Beachy; western blot
1:250, from D. Robbins,Dartmouth Medical School, Hanover, NH) and
Fu rabbit polyclonal serum (1:100).The anti-Fu serum was made as
described (Robbins et al., 1997). Quantification
ofimmunoprecipitates used IP lab from Scanalytics, taking the
average of two or moreexperiments, except for the Ptc variants
where amounts are for samples shown.Whole mount stains used anti-Ci
at 1:2.5, anti-Ptc 1:300, anti-Smo 1:100, anti-Sxl1:250 and anti-Wg
at 1:10.
Image capture and analysisImages were obtained on a Leica TCS_NT
or Zeiss LSM 510 Laser Scanningconfocal microscope. For
colocalization analysis, salivary gland cell images werecaptured
with a 63� objective lens and a zoom of 1-2X. Plasma membrane
sectionscapture the region near the membrane; within cell sections
included cross sectionsof the nucleus. Colocalization percentages
used IP Lab colocalization software(Scanalytics). For each image,
the threshold value to eliminate background first useda visual
guide. The threshold exclusion value was then raised or lowered
todetermine whether the percentage of signal pixels was
significantly altered. The cutoff was set as the value that did not
significantly alter the percentage of signal pixelsincluded when it
was increased (indicating inclusion of significant signal), but
didchange significantly if it was lowered by two or more increments
(indicatinginclusion of noise as signal). For a given antibody most
images had relatively similarthreshold values. The overlap
percentage for two probes was generated by thesoftware. Two or more
different images were averaged for each genotype andsection
type.
ChromatographyFractionations were done as described (Gay et al.,
1988).
We are grateful to D. Page, G. Struhl, I. Guerrero, J. Jiang,
M.Gonzales-Gaitan, K. Ho and M. Scott, for fly stocks. Thanks also
toR. Holmgren, J. Hooper, D. Robbins, P. Ingham for antibodies,
R.Huijbregts and I. Chesnokov for help with the
fractionationexperiment, and G. Marques and J. Engler for input on
the manuscript.The anti-Smo and Cos2 antibodies developed by P.
Beachy, anti-Wgdeveloped by S. M. Cohen, were from the
Developmental StudiesHybridoma Bank maintained by the University of
Iowa, Departmentof Biological Sciences, Iowa City, IA 52242. We
also thank AlbertTousson from the UAB imaging facility. This work
was supported bya grant from NIH to J.I.H. S.L.W. was supported by
a fellowship fromthe Comprehensive Minority Faculty Student
Development Programat UAB.
ReferencesAlcedo, J., Zou, Y. and Noll, M. (2000).
Posttranscriptional regulation of smoothened is
part of a self-correcting mechanism in the Hedgehog signaling
system. Mol. Cell 6,457-465.
Aza-Blanc, P., Ramirez-Weber, F. A., Laget, M. P., Schwartz, C.
and Kornberg, T.B. (1997). Proteolysis that is inhibited by
hedgehog targets Cubitus interruptus proteinto the nucleus and
converts it to a repressor. Cell 89, 1043-1053.
Azpiazu, N., Lawrence, P. A., Vincent, J. P. and Frasch, M.
(1996). Segmentation andspecification of the Drosophila mesoderm.
Genes Dev. 10, 3183-3194.
Boggs, R. T., Gregor, P., Idriss, S., Belote, J. M. and McKeown,
M. (1987). Regulationof sexual differentiation in D. melanogaster
via alternative splicing of RNA from thetransformer gene. Cell 50,
739-747.
Brand, A. H. and Perrimon, N. (1993). Targeted gene expression
as a means of alteringcell fates and generating dominant
phenotypes. Development 118, 401-415.
Casali, A. and Struhl, G. (2004). Reading the Hedgehog morphogen
gradient bymeasuring the ratio of bound to unbound Patched protein.
Nature 431, 76-80.
Chen, C. H., von Kessler, D. P., Park, W., Wang, B., Ma, Y. and
Beachy, P. A. (1999).Nuclear trafficking of Cubitus interruptus in
the transcriptional regulation of Hedgehogtarget gene expression.
Cell 98, 305-316.
Cline, T. (1984). Autoregulatory functioning of a Drosophila
gene product that establishesand maintains the sexually determined
state. Genetics 107, 231-277.
Denef, N., Neubuser, D., Perez, L. and Cohen, S. M. (2000).
Hedgehog induces oppositechanges in turnover and subcellular
localization of patched and smoothened. Cell 102,521-531.
Jour
nal o
f Cel
l Sci
ence
-
837Large Hh signaling complex
Dominguez, M., Brunner, M., Hafen, E. and Basler, K. (1996).
Sending and receivingthe hedgehog signal: control by the Drosophila
Gli protein Cubitus interruptus. Science272, 1621-1625.
Entchev, E. V., Schwabedissen, A. and González-Gaitán, M.
(2000). Gradientformation of the TGF-beta homolog Dpp. Cell 103,
981-991.
Gay, N. J., Poole, S. and Kornberg, T. (1988). Association of
the Drosophilamelanogaster engrailed protein with specific soluble
nuclear protein complexes. EMBOJ. 7, 4291-4297.
Han, C., Belenkaya, T. Y., Wang, B. and Lin, X. (2004).
Drosophila glypicans controlthe cell-to-cell movement of Hedgehog
by a dynamin-independent process.Development 131, 601-611.
Hooper, J. E. (2003). Smoothened translates Hedgehog levels into
distinct responses.Development 130, 3951-3963.
Horabin, J. I. (2005). Splitting the Hedgehog signal: sex and
patterning in Drosophila.Development 132, 4801-4810.
Horabin, J. I., Walthall, S., Vied, C. and Moses, M. (2003). A
positive role for Patchedin Hedgehog signaling revealed by the
intracellular trafficking of Sex-lethal, theDrosophila sex
determination master switch. Development 130, 6101-6109.
Incardona, J. P., Gruenberg, J. and Roelink, H. (2002). Sonic
hedgehog induces thesegregation of patched and smoothened in
endosomes. Curr. Biol. 12, 983-989.
Jia, J., Tong, C. and Jiang, J. (2003). Smoothened transduces
Hedgehog signal byphysically interacting with Costal2/Fused complex
through its C-terminal tail. GenesDev. 17, 2709-2720.
Jia, J., Tong, C., Wang, B., Luo, L. and Jiang, J. (2004).
Hedgehog signaling activityof Smoothened requires phosphorylation
by protein kinase A and casein kinase I.Nature 432, 1045-1050.
Jiang, J. and Struhl, G. (1998). Regulation of the Hedgehog and
Wingless signallingpathways by the F-box/WD40-repeat protein Slimb.
Nature 391, 493-496.
Johnson, R. L., Milenkovic, L. and Scott, M. P. (2000). In vivo
functions of the patchedprotein: requirement of the C terminus for
target gene inactivation but not Hedgehogsequestration. Mol. Cell
6, 467-478.
Johnson, R. L., Zhou, L. and Bailey, E. C. (2002). Distinct
consequences of sterolsensor mutations in Drosophila and mouse
patched homologs. Dev. Biol. 242, 224-235.
Lebrand, C., Corti, M., Goodson, H., Cosson, P., Cavalli, V.,
Mayran, N., Faure, J.and Gruenberg, J. (2002). Late endosome
motility depends on lipids via the smallGTPase Rab7. EMBO J. 21,
1289-1300.
Lessing, D. and Nusse, R. (1998). Expression of wingless in the
Drosophila embryo: aconserved cis-acting element lacking conserved
Ci-binding sites is required forpatched-mediated repression.
Development 125, 1469-1476.
Lu, X., Liu, S. and Kornberg, T. B. (2006). The C-terminal tail
of the Hedgehogreceptor Patched regulates both localization and
turnover. Genes Dev. 20, 2539-2551.
Lucchesi, J. C., Kelly, W. G. and Panning, B. (2005). Chromatin
remodeling in dosagecompensation. Annu. Rev. Genet. 39,
615-651.
Lum, L., Zhang, C., Oh, S., Mann, R. K., von Kessler, D. P.,
Taipale, J., Weis-Garcia,F., Gong, R., Wang, B. and Beachy, P. A.
(2003). Hedgehog signal transduction viaSmoothened association with
a cytoplasmic complex scaffolded by the atypical kinesin,Costal-2.
Mol. Cell 12, 1261-1274.
Martin, V., Carrillo, G., Torroja, C. and Guerrero, I. (2001).
The sterol-sensingdomain of Patched protein seems to control
Smoothened activity through Patchedvesicular trafficking. Curr.
Biol. 11, 601-607.
McKeown, M., Belote, J. M. and Baker, B. S. (1987). A molecular
analysis oftransformer, a gene in Drosophila melanogaster that
controls sexual differentiation.Cell 48, 489-499.
Methot, N. and Basler, K. (1999). Hedgehog controls limb
development by regulatingthe activities of distinct transcriptional
activator and repressor forms of Cubitusinterruptus. Cell 96,
819-831.
Methot, N. and Basler, K. (2000). Suppressor of fused opposes
hedgehog signaltransduction by impeding nuclear accumulation of the
activator form of Cubitusinterruptus. Development 127,
4001-4010.
Moline, M. M., Southern, C. and Bejsovec, A. (1999).
Directionality of Winglessprotein transport influences epidermal
patterning in the Drosophila embryo.Development 126, 4375-4384.
Murone, M., Rosenthal, A. and de Sauvage, F. J. (1999). Sonic
hedgehog signaling bythe patched-smoothened receptor complex. Curr.
Biol. 9, 76-84.
Nybakken, K. E., Turck, C. W., Robbins, D. J. and Bishop, J. M.
(2002). Hedgehog-stimulated phosphorylation of the kinesin-related
protein Costal2 is mediated by theserine/threonine kinase fused. J.
Biol. Chem. 277, 24638-24647.
Ogden, S. K., Ascano, M., Jr, Stegman, M. A., Suber, L. M.,
Hooper, J. E. andRobbins, D. J. (2003). Identification of a
functional interaction between the
transmembrane protein Smoothened and the kinesin-related protein
Costal2. Curr. Biol.13, 1998-2003.
Ohlmeyer, J. T. and Kalderon, D. (1998). Hedgehog stimulates
maturation of Cubitusinterruptus into a labile transcriptional
activator. Nature 396, 749-753.
Page, D. T. (2002). Inductive patterning of the embryonic brain
in Drosophila.Development 129, 2121-2128.
Piddini, E. and Vincent, J. P. (2003). Modulation of
developmental signals byendocytosis: different means and many ends.
Curr. Opin. Cell Biol. 15, 474-481.
Rink, J., Ghigo, E., Kalaidzidis, Y. and Zerial, M. (2005). Rab
conversion as amechanism of progression from early to late
endosomes. Cell 122, 735-749.
Robbins, D. J., Nybakken, K. E., Kobayashi, R., Sisson, J. C.,
Bishop, J. M. andTherond, P. P. (1997). Hedgehog elicits signal
transduction by means of a largecomplex containing the kinesin
related protein costal2. Cell 90, 225-234.
Ruel, L., Rodriguez, R., Gallet, A., Lavenant-Staccini, L. and
Therond, P. P. (2003).Stability and association of Smoothened,
Costal2 and Fused with Cubitus interruptusare regulated by
Hedgehog. Nat. Cell Biol. 5, 907-913.
Sanchez, L. and Nöthiger, R. (1983). Sex determination and
dosage compensation inDrosophila melanogaster: production of male
clones in XX females. EMBO J. 2, 211-214.
Sisson, J. C., Ho, K. S., Suyama, K. and Scott, M. P. (1997).
Costal2, a novel kinesin-related protein in the Hedgehog signaling
pathway. Cell 90, 235-245.
Stegman, M. A., Vallance, J. E., Elangovan, G., Sosinski, J.,
Cheng, Y. and Robbins,D. J. (2000). Identification of a tetrameric
hedgehog signaling complex. J. Biol. Chem.275, 21809-21812.
Stegman, M. A., Goetz, J. A., Ascano, M., Jr, Ogden, S. K.,
Nybakken, K. E. andRobbins, D. J. (2004). The Kinesin-related
protein Costal2 associates with membranesin a Hedgehog-sensitive,
Smoothened-independent manner. J. Biol. Chem. 279, 7064-7071.
Stenmark, H., Parton, R. G., Steele-Mortimer, O., Luetcke, A.,
Gruenberg, J. andZerial, M. (1994). Inhibition of rab5 GTPase
activity stimulates membrane fusion inendocytosis. EMBO J. 13,
1287-1296.
Stone, D. M., Hynes, M., Armanini, M., Swanson, T. A., Gu, Q.,
Johnson, R. L.,Scott, M. P., Pennica, D., Goddard, A., Phillips, H.
et al. (1996). The tumour-suppressor gene patched encodes a
candidate receptor for Sonic hedgehog. Nature384, 129-134.
Strutt, H., Thomas, C., Nakano, Y., Stark, D., Neave, B.,
Taylor, A. M. and Ingham,P. W. (2001). Mutations in the
sterol-sensing domain of Patched suggest a role forvesicular
trafficking in Smoothened regulation. [erratum appears in Curr.
Biol. (2001).11, 1153] Curr. Biol. 11, 608-613.
Taipale, J., Cooper, M. K., Maiti, T. and Beachy, P. A. (2002).
Patched acts catalyticallyto suppress the activity of Smoothened.
Nature 418, 892-897.
Therond, P. P., Knight, J. D., Kornberg, T. B. and Bishop, J. M.
(1996).Phosphorylation of the fused protein kinase in response to
signaling from hedgehog.Proc. Natl. Acad. Sci. USA 93,
4224-4228.
Torroja, C., Gorfinkiel, N. and Guerrero, I. (2004). Patched
controls the Hedgehoggradient by endocytosis in a dynamin-dependent
manner, but this internalization doesnot play a major role in
signal transduction. Development 131, 2395-2408.
Torroja, C., Gorfinkiel, N. and Guerrero, I. (2005). Mechanisms
of Hedgehog gradientformation and interpretation. J. Neurobiol. 64,
334-356.
van der Bliek, A. M. and Meyerowitz, E. M. (1991). Dynamin-like
protein encoded bythe Drosophila shibire gene associated with
vesicular traffic. Nature 351, 411-414.
Vied, C. and Horabin, J. I. (2001). The sex determination master
switch, Sex-lethal,responds to Hedgehog signaling in the Drosophila
germline. Development 128, 2649-2660.
Vitelli, R., Santillo, M., Lattero, D., Chiariello, M., Bifulco,
M., Bruni, C. B. andBucci, C. (1997). Role of the small GTPase Rab7
in the late endocytic pathway. J.Biol. Chem. 272, 4391-4397.
Wang, Q. T. and Holmgren, R. A. (1999). The subcellular
localization and activity ofDrosophila cubitus interruptus are
regulated at multiple levels. Development 126, 5097-5106.
Wodarz, A., Hinz, U., Engelbert, M. and Knust, E. (1995).
Expression of crumbsconfers apical character on plasma membrane
domains of ectodermal epithelia ofDrosophila. Cell 82, 67-76.
Yao, S., Lum, L. and Beachy, P. A. (2006). The ihog cell-surface
proteins bind Hedgehogand mediate pathway activation. Cell 125,
343-357.
Zhang, C., Williams, E. H., Guo, Y., Lum, L. and Beachy, P. A.
(2004). Extensivephosphorylation of Smoothened in Hedgehog pathway
activation. Proc. Natl. Acad.Sci. USA 101, 17900-17907.
Zhu, A. J., Zheng, L., Suyama, K. and Scott, M. P. (2003).
Altered localization ofDrosophila Smoothened protein activates
Hedgehog signal transduction. Genes Dev.17, 1240-1252.
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