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Phosphorylation of the Smo tail is controlled bymembrane localisation and is dispensable forclustering

Adam P. Kupinski1,*,`, Isabel Raabe1,*, Marcus Michel1, Divya Ail1,§, Lutz Brusch2, Thomas Weidemann3 andChristian Bokel1,"

1Center for Regenerative Therapies Dresden (CRTD), Technische Universitat Dresden, Fetscherstrasse 105, 01307 Dresden, Germany2Center for Information Services and High Performance Computing, Technische Universitat Dresden, Helmholtzstrasse 10, 01069 Dresden, Germany3Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany

*These authors contributed equally to this work`Present address: Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK§Present address: Department of Ophthalmology, University of Zuerich, Wagistrasse 14, 8952 Schlieren, Switzerland"Author for correspondence (christian.boekel@crt-dresden.de)

Accepted 25 July 2013Journal of Cell Science 126, 4684–4697� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.128926

SummaryThe Hedgehog (Hh) signalling cascade is highly conserved and involved in development and disease throughout evolution. Nevertheless, incomparison with other pathways, our mechanistic understanding of Hh signal transduction is remarkably incomplete. In the absence of

ligand, the Hh receptor Patched (Ptc) represses the key signal transducer Smoothened (Smo) through an unknown mechanism. Hh bindingto Ptc alleviates this repression, causing Smo redistribution to the plasma membrane, phosphorylation and opening of the Smo cytoplasmictail, and Smo oligomerisation. However, the order and interdependence of these events is as yet poorly understood. We havemathematically modelled and simulated Smo activation for two alternative modes of pathway activation, with Ptc primarily affecting either

Smo localisation or phosphorylation. Visualising Smo activation through a novel, fluorescence-based reporter allowed us to test thesecompeting models. Here, we show that Smo localisation to the plasma membrane is sufficient for phosphorylation of the cytoplasmic tail inthe presence of Ptc. Using fluorescence cross-correlation spectroscopy (FCCS), we also demonstrate that inactivation of Ptc by Hh induces

Smo clustering irrespective of Smo phosphorylation. Our observations therefore support a model of Hh signal transduction whereby Smosubcellular localisation and not phosphorylation is the primary target of Ptc function.

Key words: Activation state reporter, Fluorescence cross-correlation spectroscopy, Hedgehog, Smoothened, Signal transduction

IntroductionHedgehog signalling has been implicated in crucial

developmental and physiological processes in both Drosophila

and vertebrates. Despite these important roles, there are still large

gaps in our mechanistic understanding of Hh signal transduction

(Jiang and Hui, 2008; Ingham et al., 2011). In the absence of

ligand, the Hh receptor Ptc (Ingham et al., 1991; Marigo et al.,

1996; Zheng et al., 2010) inhibits the GPCR-like signal

transducer Smo (Alcedo et al., 1996; van den Heuvel and

Ingham, 1996). Smo inhibition occurs without direct protein

interaction (Taipale et al., 2002) and presumably involves a

small-molecule intermediate, which is probably a lipid (Bijlsma

et al., 2006; Khaliullina et al., 2009). However, it is not known

where in the cell Ptc acts to inactivate Smo. In addition, although

the components of the Hh signalling cascade are largely

conserved across evolution, there are also clear differences in

the way the vertebrate and fly cascades operate. This is illustrated

by the different sensitivities of fly and mammalian Smo to

Cyclopamine and the differences in the relative importance of

Cos2 and Su(Fu) proteins for pathway activity. These differences

might be associated with the absence of primary cilia, which play

a central role in mammalian Hh signalling, from most Drosophila

cells (Ingham et al., 2011; Briscoe and Therond, 2013).

Recently, Drosophila Ptc was postulated to control Smo

localisation and activation indirectly through a wide range of

intermediate protein players acting on Smo. These include lipid-

modifying enzymes (Yavari et al., 2010), cAMP and protein

kinase A (PKA) (Ogden et al., 2008), and the phosphatases PP1,

PP4 and PP2A (Jia et al., 2009; Su et al., 2011). Regardless of the

precise mechanism, Hh binding alleviates the inhibitory activity

of Ptc towards Smo. In its inactive state Smo resides on internal

cell membranes (Denef et al., 2000; Zhu et al., 2003; Nakano

et al., 2004) that are presumably a mixture of early and late

endosomes, and lysosomes (Nakano et al., 2004; Li et al., 2012;

Xia et al., 2012). Electrostatic interactions between four

positively charged arginine clusters collectively termed

Smoothened autoinhibitory domain (SAID) in the membrane

proximal part of the C-terminal cytoplasmic domain and

negatively charged distal patches keep the cytoplasmic tail in a

closed conformation (Zhao et al., 2007).

Upon pathway activation, Smo is phosphorylated by protein

kinase A (PKA) and casein kinase 1 (CK1) at multiple serine

residues within the cytoplasmic tail (Jia et al., 2004; Zhang et al.,

2004; Apionishev et al., 2005; Jia et al., 2010; Su et al., 2011).

Their phosphorylation or phosphomimetic replacement masks the

positive charge of the SAID, releasing the C-terminal tail into an

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open conformation and inducing Smo clustering (Zhao et al.,2007). Smo phosphorylation also activates downstream signal

transduction (Jia et al., 2004; Zhao et al., 2007) by therecruitment of a protein complex centred on Costal2 (Jia et al.,2003; Ogden et al., 2003) (Fan et al., 2012). Assembly of thiscomplex is thought to suppress the proteolytic processing of the

Gli family transcription factor Cubitus interruptus (Ci) into itsrepressor version, thereby stabilising the full-length activatorform (Jiang and Hui, 2008; Ingham et al., 2011). Finally, Smo

phosphorylation is sufficient for the redistribution of Smo to theplasma membrane associated with pathway activation (Denefet al., 2000; Zhu et al., 2003; Nakano et al., 2004). A possible

mechanism is provided by the observations that SAIDphosphorylation prevents the ubiquitylation of adjacent lysineresidues, which promotes Smo internalisation (Li et al., 2012). Inaddition, Hh promotes the recruitment of a deubiquitylating

enzyme to the Smo cytoplasmic tail, thereby suppressingrecruitment of Smo into early endosomes (Xia et al., 2012).

Together, these observations can be condensed into a model of

Hh pathway organisation whereby Ptc primarily controls thephosphorylation state of Smo. Ptc inactivation in response to Hhthen leads to Smo phosphorylation and subsequently to

conformational change, clustering and accumulation at theplasma membrane, where Smo becomes active as a signaltransducer. However, Smo localises to the plasma membrane in a

Hh-independent manner when cellular phosphatidylinositol-4phosphate (PI4P) levels are experimentally increased.Intriguingly, Ptc itself directly or indirectly downregulates PI4Paccumulation (Yavari et al., 2010). This suggests an alternative

order of events, whereby Ptc inactivation by Hh first drives Smomembrane localisation by modulating membrane phospholipids,with Smo phosphorylation and clustering occurring downstream.

To improve our understanding of Hh signal transduction wetherefore need to identify which of the multiple cell biologicalprocesses downstream of Ptc primarily regulates Smo activation,

and must clarify the connections between the events occurring atthe level of Smo, the key signal transducer of the pathway. Wehave addressed these questions by combining a modellingapproach with the direct visualisation of Smo phosphorylation

status and the biophysical detection of Smo clustering. First, wesimulated Smo activation in response to Hh with the help of asimplified, nondimensionalised equilibrium model, considering

two scenarios corresponding to the alternative roles of Ptc in Smoregulation outlined above. Second, following a previouslyestablished strategy (Michel et al., 2011), we generated a

fluorescence-based Smo activation reporter by inserting theconformation-sensitive core of the Inverse Pericam Ca2+ sensor(Nagai et al., 2001) into the cytoplasmic tail of Smo. Fluorescence

of this reporter strictly reflects the phosphorylation dependentopening of the Smo tail, which can therefore be tracked withsubcellular resolution both live and in fixed samples. We have usedthis reporter to test the alternative models and their underlying

assumptions. Third, we have directly measured the oligomerisationstate of fluorescently tagged Smo on the plasma membrane ofcultured cells by dual-colour fluorescence cross-correlation

spectroscopy (FCCS) (Weidemann et al., 2002; Bacia et al., 2006).

Here, we show that localisation of Smo to the plasmamembrane is by itself sufficient to induce phosphorylation of

the cytoplasmic tail, irrespective of the presence or absence ofPtc. In addition, we demonstrate that inactivation of Ptc by Hhcontrols Smo clustering independent of Smo tail phosphorylation.

These results challenge models that place Smo phosphorylation

at the apex of regulatory events. Our observations insteadstrongly support models of Hh pathway function whereby thesubcellular localisation of Smo is the primary cell biological

target of Ptc activity.

ResultsModelling Smo regulation in response to Hh

Mechanistic dissection of Hh pathway activation is hampered by

the large number of feedback events and regulatory inputs into thepathway. In addition, key steps within the pathway, for example, theinhibition of Smo by Ptc or the activation of stabilised Ci are not

understood at the biochemical level, and have to be treated as blackboxes in attempts to model the pathway. We wondered whether theSmo response to Hh could be modelled at a very abstract level while

still allowing testable predictions about the behaviour of the system.We were particularly interested in any inferences that could be madeabout the role of Ptc from modelling, and therefore devised asimplified, formalised description of Smo behaviour in response to

Hh. First, we treated the total Smo pool in the cells as four discretepopulations that differ in their localisation and phosphorylation state(i.e. localised at the plasma membrane or on intracellular membranes,

and a phosphorylated or nonphosphorylated cytoplasmic tail).Second, we defined these populations as being in pair-wiseequilibrium with each other in exocytosis or endocytosis and

phosphorylation or dephosphorylation (Fig. 1). This highlysimplified treatment allowed us to focus on the position of theseequilibria, subsuming all additional inputs on Smo trafficking

mediated by Cos2 (Liu et al., 2007) or the nonvisual b-arrestinKurtz (Molnar et al., 2011; Li et al., 2012) into the respective rateconstants. By only considering the distribution of the Smo proteinpresent in the cell between the four populations, we sidestepped

for the moment the question of production and degradation rates.This can be justified in first approximation, because proteinphosphorylation and endocytosis occur on shorter time-scales than

protein synthesis or degradation. Third, we assumed that in allinstances, phosphorylation has a positive feedback on Smomembrane localisation (Denef et al., 2000; Jia et al., 2003; Zhao

et al., 2007), presumably through the suppression of Smoubiquitylation and endocytosis (Li et al., 2012; Xia et al., 2012).Within this framework, we then simulated the Smo response toincreasing Hh doses. Importantly, analytic treatment showed that the

overall response is determined only by the ratios defining theequilibria and not the absolute values of the rate constants. Asoutlined above, we considered two distinct cell biological roles of

Ptc: under the phosphorylation model, Ptc activity shifts theequilibria between the different Smo pools towards thenonphosphorylated forms both at the plasma membrane and within

the cell (Fig. 1A). To match the observed intracellular accumulationof Smo in the absence of Hh, we had to additionally assume thattrafficking of nonphosphorylated Smo is constitutively biased

towards endocytosis.

Under the alternative endocytosis model (Fig. 1B), Ptc insteadcontrols the position of the equilibrium between secretion andendocytosis of nonphosphorylated Smo. To recapitulate the

observed ground state we had to additionally demand that thebalance between kinase and phosphatase activities is biasedtowards phosphorylation at the membrane but towards

dephosphorylation within the cell. Importantly, both modelsbreak down when these additional assumptions are omitted fromthe simulation (supplementary material Fig. S1) but capture the

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key features of Smo behaviour when they are incorporated: both

models predict a shift from the nonphosphorylated, intracellular

pool to the phosphorylated, plasma membrane pool when Ptc is

gradually inactivated by increasing concentrations of Hh

(Fig. 1A9–B0). Importantly, a key difference appears between

the two models when Smo distribution is artificially biased

towards the plasma membrane in the absence of Hh,

corresponding to a pharmacological block of endocytosis using

the dynamin inhibitor Dynasore (Macia et al., 2006). Under these

conditions, the phosphorylation model predicts the accumulation

of nonphosphorylated Smo at the plasma membrane: even though

the plasma membrane pool becomes enlarged, Ptc should

continue to bias the equilibrium towards the nonphosphorylated

form of Smo, shifting to the phosphorylated form only when Hh

is present (Fig. 2A–A0).

By contrast, the endocytosis model predicts that Smo retention

at the plasma membrane is by itself sufficient to drive Smo

towards the phosphorylated state: while the presumed activity of

Ptc in promoting endocytosis is counteracted by the drug, the

unchanged local bias towards phosphorylation at the plasma

membrane is predicted to drive Smo phosphorylation even in the

absence of Hh (Fig. 2B–B0). Simultaneously tracking the

localisation and phosphorylation state of Smo in response to

either Hh or endocytosis block would therefore allow

discriminating between these models. This would, in turn,

permit inferences about the cell biological process targeted by

Ptc without requiring prior knowledge of the molecular

mechanism. We therefore decided to generate a fluorescence-

based sensor for Smo activation.

Smo-IP – a fluorescence-based sensor for Smo tail

phosphorylation

We have previously shown that the conformation-sensitive cpYFP

core of the Inverse Pericam (IP) Ca2+ sensor (Nagai et al., 2001)

can be used to detect changes in protein interactions during the

activation of signalling cascades, and have used this to selectively

image active BMP receptors (Michel et al., 2011). We therefore

adapted this strategy for the in vivo visualisation of Smo activation.

To generate the Smo-IP reporter we replaced the bulk of the loop in

the Drosophila Smo cytoplasmic tail between the Smo SAID

domain (Zhao et al., 2007) and the distal, acidic patches (amino

acids 757–915) with the IP cpYFP core. In the inactive state the

closed conformation of the Smo cytoplasmic tail should keep the

cpYFP core in a nonfluorescent conformation, whereas SAID

phosphorylation should allow the IP core to relax into a fluorescent

conformation (Fig. 3A). The Smo-IP construct was able to rescue

amorphic smo alleles and therefore retains full signalling function

(supplementary material Table S1).

Fig. 1. Modelling Smo regulation as a network of equilibria. (A–A0) Phosphorylation model. Trafficking is biased towards internalisation for

nonphosphorylated Smo. Phosphorylation inhibits endocytosis, favouring membrane localisation. (A) In the absence of Hh Ptc shifts the kinase to phosphatase

balance towards Smo dephosphorylation (regulated equilibria indicated by red arrows), leading to the accumulation of the nonphosphorylated, intracellular pool of

Smo (EE Smo, red). (A9) Inactivation of Ptc promotes Smo phosphorylation, leading to accumulation of the phospho-Smo pool at the plasma membrane (PM

Smo-P, green). (A0) Mathematical modelling of Smo response to varying Hh levels for the phosphorylation model. Simulations of the equilibrium distribution

between the four Smo populations correctly predict a shift towards PM Smo-P upon supra-threshold Hh stimulation. Plasma-membrane-associated

nonphosphorylated Smo (PM Smo, black) and intracellular phosphorylated Smo (EE Smo-P, blue) provide minor contributions to the total Smo pool.

(B–B0) Endocytosis model. Smo trafficking is intrinsically biased towards secretion for both forms of Smo. (B) Ptc shifts this balance towards endocytosis for

nonphosphorylated Smo, whereas phospho-Smo is resistant to Ptc. Assuming in addition that the kinase to phosphatase equilibrium is biased towards Smo

phosphorylation at the plasma membrane but towards dephosphorylation for the intracellular pool, inactivation of Ptc by Hh (B9) causes accumulation of PM Smo-

P. (B0) Mathematical modelling of Smo behaviour under the endocytosis model also correctly reproduces Smo response to increasing Hh levels.

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Reporter fluorescence intensities under endogenous

expression were too weak for practical use (supplementary

material Fig. S2A). We therefore ubiquitously overexpressed

Smo-IP from a tubulin promoter. Ptc is not expressed in the

posterior compartment of the larval wing imaginal disc, where

Smo is therefore constitutively active (Fig. 3B). In the anterior

compartment, Hh pathway activation is determined by the Hh

protein gradient (Torroja et al., 2005). Consistently, reporter

fluorescence decreased with growing distance from the

anteroposterior (AP) boundary (Fig. 3B,C). The Smo activity

gradients had characteristic decay lengths of 10.861.8 mm

(Fig. 3D and supplementary material Fig. S2B), which is

consistent with previously reported ranges of the Hh gradient

(Wartlick et al., 2011). However, in the wing disc Hh signalling

also controls Smo protein stability. Similar to endogenous Smo,

Smo-IP protein levels were therefore high in the posterior

compartment, gradually decayed in front of the compartment

boundary, and were low in the anterior compartment as a result

of Ptc-mediated degradation (Denef et al., 2000; Li et al., 2012).

Because only activated Smo is protected from degradation,

reporter fluorescence in the anterior compartment necessarily

closely followed protein levels (Fig. 3C). This made it

impossible to unambiguously attribute the observed

fluorescence to Smo phosphorylation and precluded validating

reporter function in the disc by a ratiometric approach. In

addition, although Hh signalling in the wing imaginal discs is

quantitatively well understood (Nahmad and Stathopoulos,

2009), the disc epithelial cells are unsuitable for subcellular

studies because of their pseudostratified arrangement and small

diameter. To circumvent both problems, we instead turned to the

larval salivary glands, whose large epithelial cells have

previously been used for studies of Hh signalling (Zhu et al.,

2003; Yavari et al., 2010).

Smo phosphorylation and localisation in the salivary gland

The larval salivary glands are situated adjacent to the fat body,

which is a major site of Hh production and signalling (Pospisilik

et al., 2010). Correspondingly, wild-type (WT) Smo-IP expressed

in the salivary gland under 71B::Gal4 control was found largely

at the plasma membrane and in its fluorescent state (Fig. 3E),

indicating activation of the Hh pathway. This is at odds with a

previous report which concluded that additional Hh expression is

necessary for ptc::lacZ expression (Zhu et al., 2003). However,

consistent with the presence of an endogenous Hh signal, co-

overexpression of Ptc in the gland cells abolished reporter

fluorescence and caused relocalisation of a large fraction of Smo

from the cell surface onto internal membranes. (Fig. 3F). Both

effects were reverted by overexpression of constitutively active

PKA (Jia et al., 2004) (Fig. 3G), which also enhanced the

phosphorylation and membrane localisation of WT Smo-IP

Fig. 2. Modelling Smo regulation in response to endocytosis block. (A–A0) Phosphorylation model. (A) Ptc drives dephosphorylation of Smo, which

accumulates intracellularly (EE Smo, red). (A9) Inhibition of dynamin-mediated endocytosis by Dynasore. Smo cannot be internalised despite being driven

towards the nonphosphorylated form by Ptc and accumulates at the plasma membrane (PM Smo, black). (A0) Under the phosphorylation model,

mathematical modelling predicts a shift from the PM Smo pool (black) to the plasma-membrane-resident phosphorylated pool (PM Smo-P, green) for supra-

threshold Hh levels. (B–B0) Endocytosis model. (B) Ptc promotes the removal of nonphosphorylated Smo from the plasma membrane. (B9) Dynasore treatment.

Inhibition of dynamin-mediated endocytosis overcomes Ptc function. Smo accumulates at the plasma membrane, where the local bias towards

phosphorylation shifts the equilibrium towards the PM Smo-P pool (green). (B0) Mathematical modelling for the endocytosis model predicts a predominance of

the PM Smo-P pool (green) even in the absence of Hh.

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Fig. 3. Smo-IP, a fluorescent sensor for Smo phosphorylation. (A) In the Smo-IP reporter, the cpYFP core from Inverse pericam (IP) replaces the central loop

of the Smo cytoplasmic tail. In the absence of Hh, Ptc inhibits Smo and forces it onto intracellular membranes. Interactions between the positively charged

SAID and distal, negatively charged patches keep the Smo tail in a closed conformation, inactivating IP fluorescence. In the presence of Hh, the IP core relaxes

into a fluorescent conformation due to phosphorylation of SAID-associated serines. (B) Fluorescence of SmoIP in the wing imaginal disc reflects Hh pathway

activity. Note activation in posterior compartment, decay in front of AP boundary marked by Ptc and SmoIP protein degradation in anterior compartment. Box and

arrow indicate representative area and direction for intensity measurements, respectively. (C) Quantification of normalised intensities for Ptc and GFP

immunostaining and SmoIP fluorescence. Dashed line indicates AP boundary. Note anterior decay of both GFP staining and reporter fluorescence. (D) Averaged,

normalised SmoIP fluorescence can be fitted by a single exponential decay (n58 discs). (E–H) Subcellular localisation of SmoIP reporter expressed in the salivary

gland using 71B::Gal4. (E) In otherwise wild-type glands, both total reporter protein (red) and endogenous Ptc (blue) are enriched near the plasma membrane.

Smo-IP is fluorescent (green). (F) Co-overexpression of Ptc suppresses Smo-IP fluorescence and partially relocalises the protein to the interior of the cells.

(G) Both effects are reverted by constitutively active murine PKA. (H) PKA overexpression also increases fluorescence and membrane localisation of Smo-IP in

the absence of extraneous Ptc. (I) Ratiometric quantification of reporter activity in the salivary gland. The ratio of Smo-IP reporter fluorescence to anti-GFP

immunostaining signal is plotted for both the plasma-membrane-associated (white bars) and intracellular (grey) pools. Note reduction following Ptc

co-overexpression and increase due to activated PKA. (J) Fraction of membrane-associated Smo-IP. Membrane localisation correlates with receptor activation

state. Scale bars: 50 mm (B), 20 mm (E–H). Discs oriented: dorsal, up; anterior, left. Error bars indicate s.d.; *P,0.05; ***P,0.01 (ANOVA).

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(Fig. 3H). The apparent reduction in the level of intracellular Ptcis presumably caused by the slightly deeper imaging required as a

result of the strong cell surface distortions induced by PKA. Thus,the Smo-IP reporter is switchable in a Ptc- and phosphorylation-sensitive manner. Importantly, stability of Smo protein in theglands appears to be less tightly linked to activation when

compared with results in the wing disc. This allowed thevalidation of reporter function by a ratiometric approach,comparing reporter fluorescence and protein levels under

different experimental conditions (Fig. 3I,J). The basal ratio ofSmo-IP reporter fluorescence and anti-GFP immunostaining wasreduced both at the plasma membrane and in the interior of the

cell following co-overexpression of Ptc and increased in turn byaddition of activated PKA. Coexpression of PKA also increasedthis ratio for WT Smo-IP (Fig. 3I and supplementary materialTable S2A). This was mirrored by redistribution of the total Smo-

IP pool from the membrane to the cell interior following Ptcoverexpression and back to the plasma membrane after co-overexpression of PKA. The same membrane localisation of

Smo-IP was achieved by expression of PKA in a WT Smo-IPbackground (Fig. 3J and supplementary material Table S2A).The Hh pathway in the glands is thus either endogenously only

partially active or the equilibrium at saturating Hh levels is notnear full Smo phosphorylation. Nevertheless, these experimentsprove that the IP reporter cassette is switchable in the context of

the Smo tail, and that its fluorescence reflects Hh pathwayactivation.

We confirmed this independently with the help ofnonphosphorylatable and phosphomimetic versions of our

reporter. We replaced the serine residues interspersed with thebasic SAID patches (Jia et al., 2004; Zhang et al., 2004) witheither aspartate (SmoSD-IP) or alanine (SmoSA-IP). These

amino acid changes had been shown by FRET to force theSmo tail into an open or closed conformation, respectively, albeitwithout achieving subcellular resolution (Zhao et al., 2007). We

expressed both wild-type and mutant Smo-IP constructsspecifically in the dorsal compartment of the wing disc underap::Gal4 control. The ventral compartment of each disc thusserved as an internal control. Expression of WT Smo-IP

reproduced the graded fluorescence in the anterior compartmentalso seen with ubiquitous expression. Overexpression of thereporter had little effect on the width of the expression domains

of the Hh target genes collier (Fig. 4A) and ptc::lacZ(supplementary material Fig. S3A). By contrast, SmoSD-IP wasfluorescent in the entire dorsal anterior compartment beyond the

range of Hh protein (Fig. 4B). Similar to the equivalentphosphomimetic Smo versions lacking the reporter cassette (Jiaet al., 2004; Zhang et al., 2004; Zhao et al., 2007), SmoSD-IP

acted as a constitutive active protein driving expression of Hhtarget genes (Fig. 4B and supplementary material Fig. S3B).Conversely, the nonphosphorylatable SmoSA-IP reporter wasnonfluorescent in both anterior and posterior compartments, even

though protein levels were increased relative to the wild type(Fig. 4C). As expected (Jia et al., 2004; Zhang et al., 2004;Apionishev et al., 2005), SmoSA-IP strongly suppressed collier

and ptc::lacZ expression (Fig. 4C and supplementary materialFig. S3C).

In the salivary glands, SmoSD-IP was strongly fluorescent

(Fig. 4D and supplementary material Fig. S3D) and enriched atthe plasma membrane (supplementary material Fig. S3E, TableS2A) regardless of Ptc overexpression (Fig. 4E). This confirms

that the phosphomimetic mutations render Smo resistant to Ptc-

mediated clearance from the cell surface (Zhao et al., 2007; Li

et al., 2012; Xia et al., 2012). By contrast, SmoSA-IP expressed

in the glands was only weakly fluorescent, indicating a

nonphosphorylated, closed and inactive conformation. The

Fig. 4. Validation of Smo IP by phosphomimic and nonphosphorylatable

versions. (A–C) Signalling activity and fluorescence of Smo-IP reporter

derivatives in the wing disc. SmoIP expression in the dorsal part of the wing

disc using ap::Gal4 (A) reflects normal Smo activity and expands expression

of Hh and the target Collier only weakly. Reporter fluorescence and Collier

expression are strongly upregulated by the phosphomimetic reporter version

SmoSD-IP (B) and suppressed by the nonphosphorylatable reporter SmoSA-

IP (C). The ventral compartment where ap::Gal4 is inactive serves as an

internal control. (D–G) Subcellular localisation of Smo-IP derivatives in

salivary gland cells. SmoSD-IP is constitutively fluorescent and found at the

cell surface (D). Both properties are resistant to Ptc co-overexpression (E).

(F,G) SmoSA-IP is nonfluorescent and contains a large intracellular pool (F).

Some SmoSA-IP remains at the membrane when Ptc is co-overexpressed (G).

Scale bars: 50 mm (A–C), 20 mm (D-G). Discs oriented: dorsal, up;

anterior, left.

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remaining baseline fluorescence limits the signal to noise ratio of

our reporter (Fig. 4F and supplementary material Fig. S3D).

Even under Ptc overexpression conditions, a considerable

fraction of the reporter was still found outlining the plasma

membrane, both for SmoSA-IP (Fig. 4F,G) and for WT Smo-IP

(Fig. 3F). However, in all cases, the membrane fraction was

reduced relative to WT Smo-IP under signalling conditions

(Fig. 3E,I) or the phosphomimetic SmoSD-IP construct

(Fig. 4D,E and supplementary material Fig. S3E). Together,

these experiments show that, in the glands, the fluorescence of

the conformation-sensitive IP cpYFP core is uncoupled from

reporter protein levels and responds to the charge-dependent

conformation of the Smo cytoplasmic tail. In addition, Smo

phosphorylation itself cannot be required for the transport of Smo

to the plasma membrane. Instead, the observations suggest a

steady state trafficking equilibrium that is shifted towards

internalisation for nonphosphorylated Smo. However, the

observed behaviour of SmoSA-IP and SmoSD-IP is consistent

with either of the two mathematical models (supplementary

material Fig. S4), and is thus insufficient for discriminating

between the alternatives. As argued above, experimentally testing

the two models also requires a means of blocking Smo

endocytosis.

Smo localisation in cultured cells

We first addressed Smo internalisation in cell culture

experiments, where the localisation of Smo to the plasma

membrane can be assayed unambiguously by immunostaining

against the extracellular N-terminal domain performed under

non-permeabilising conditions. S2R+ cells do not express Ci but

contain endogenous Ptc and low amounts of Smo (Cherbas et al.,

2011). In the absence of Hh, a C-terminally tagged Smo-GFP

construct (Smo-GFP) transfected into these cells was therefore

largely excluded from the cell surface (Fig. 5A,J and

supplementary material Table S2B). Smo-GFP translocated to

the plasma membrane following either stimulation by Hh

(Fig. 5B,J) or treatment with Dynasore, a pharmacological

inhibitor of dynamin-mediated endocytosis (Macia et al., 2006)

Fig. 5. Smo-GFP localisation in cultured cells. (A–C) Smo-GFP (green) is present within transfected cells, but cannot be seen at the plasma membrane by

extracellular Smo immunostaining (red) in the absence of Hh (A). Smo-GFP becomes detectable at the membrane following Hh stimulation (B) or Dynasore

treatment (C). (D–F) SmoSD-GFP is found at the membrane in the absence (D) or presence of Hh (E) or Dynasore (F). (G–I) SmoSA-GFP cannot be detected at

the membrane in the absence (G) or presence (H) of Hh, but can be trapped there by Dynasore treatment (I). (J) Quantification of A–I. Error bars indicate s.d.; n.s.

not significant; *P,0.05; ***P,0.01 (ANOVA).

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(Fig. 5C,J). As expected (Jia et al., 2004), SmoSD-GFP was

constitutively found at the plasma membrane (Fig. 5D–F,J),

whereas the nonphosphorylatable SmoSA-GFP fusion protein

remained at the detection limit at the plasma membrane of cells

stimulated by Hh (Fig. 5G,H,J). However, significant amounts

of SmoSA-GFP became trapped at the surface when endocytosis

was inhibited by Dynasore (Fig. 5I,J).

This confirms earlier observations showing that both inhibition

of dynamin-dependent endocytosis (Xia et al., 2012) and

phosphomimetic mutations in the SAID-associated serines (Jia

et al., 2004; Zhao et al., 2007) can enrich Smo at the plasma

membrane. The observation that SmoSA can also be trapped at

the membrane shows, first, that some exchange between the

intracellular and plasma membrane bound pools must also occur

for nonphosphorylated Smo, and second, that the forced

membrane retention of WT Smo by Dynasore cannot be

dependent on Smo phosphorylation.

Membrane localisation and phosphorylation of Smo

Finally, to test experimentally the two models of Smo activation,

we combined the Smo reporter with inhibition of endocytosis. We

treated S2R+ cells transfected with the Smo-IP reporter either with

Hh to inactivate Ptc or with Dynasore to block dynamin-dependent

endocytosis. As with the Smo-GFP fusion proteins (Fig. 5A–C),

both treatments induced a significant increase in the levels of Smo

protein detectable at the plasma membrane (Fig. 6A,B). As

expected, stimulation with Hh caused increased phosphorylation

of the Smo tail, which could be detected by Smo-IP reporter

fluorescence. Importantly, treatment with Dynasore in the absence

of ligand was equally sufficient to induce Smo phosphorylation

(Fig. 6C,D).

To verify that this Hh-independent Smo activation is not

merely an artefact of the pharmacological experiments in

cultured cells, we turned to transgenic flies. Inactivation of Hh

signalling in the salivary glands by Ptc overexpression

suppressed Smo-IP fluorescence (Fig. 6E). In this background,

endocytosis was inhibited by co-overexpression of a dominant-

negative version of the Drosophila dynamin Shibire

(UAS::shibireK44A) (Moline et al., 1999). Successful inhibition

of endocytosis was reflected by Ptc accumulation at the

membrane. Consistent with the cell culture results, this also

induced reporter fluorescence, indicating Smo activation

(Fig. 6F). Blocking Smo internalisation is therefore sufficient

to induce Smo phosphorylation despite the presence of large

amounts of Ptc both in vivo and in cultured cells. This challenges

the traditional view that Smo membrane localisation is strictly a

consequence of phosphorylation. We therefore also investigated

the relationship between Smo phosphorylation and Smo

clustering.

Smo clustering measured by FCCS

Phosphorylation in response to Hh has been shown by FRET

microscopy to promote Smo clustering at the plasma membrane

(Zhao et al., 2007). However, FRET-based techniques do not

provide data on cluster size and do not possess great sensitivity.

We instead used two-colour fluorescence cross-correlation

Fig. 6. Membrane retention drives

Smo phosphorylation. (A,B) In cells

transfected with Smo-IP, the intensity of

extracellular Smo staining (red) is low in

the absence of Hh. Treatment with Hh or

blocking of endocytosis with Dynasore

both increase the levels of Smo-IP

detectable at the cell surface by

extracellular immunostaining

(B) Quantification of immunostaining in

A. (C) Hh stimulation and inhibition of

endocytosis equally activate Smo-IP

reporter fluorescence. (D) Quantification

of reporter signal in C. (E,F) Inhibition of

endocytosis drives Smo phosphorylation

in transgenic flies. Co-overexpression of

Ptc with Smo-IP under 71B::Gal4 control

inactivates reporter fluorescence in

salivary glands (E). Additional co-

overexpression of the dominant-negative

dynamin ShibireK44A leads to the

accumulation of Ptc at the cell surface

and the activation of Smo-IP reporter

fluorescence (F). Scale bars: 20 mm.

Error bars indicate s.d., n.s. not

significant; ***P,0.01 (ANOVA

followed by Tukey’s HSD).

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spectroscopy (FCCS), which allowed us to quantitatively analyse

Smo diffusion and oligomerisation under different experimental

conditions (Bacia et al., 2006). This method is based on the

statistical analysis of intensity fluctuations that arise when two

spectrally distinct fluorophores diffuse through a microscopic

detection volume (supplementary material Fig. S5A–B9).

Whereas autocorrelation analysis of the individual fluorescence

signals yields average dwell times and numbers of the observed

particles, cross-correlation between the colour channels indicates

co-diffusion of differently labelled molecules (Fig. 7A;

supplementary material Fig. S5C). A useful readout is the ratio

between auto- and cross-correlation amplitude, which is linked to

various binding schemes and stoichiometries and can be used to

measure the degree of binding (Weidemann et al., 2002;

Weidemann et al., 2011).

To probe homotypic Smo interactions we generated C-

terminally-tagged Smo-mRFP, SmoSD-mRFP and SmoSA-

mRFP constructs analogous to the corresponding GFP fusion

proteins and performed FCCS measurements in cells co-

expressing green and red fluorescent Smo versions.

Fluorescence was recorded at the membrane in the periphery of

adherent S2R+ cells to maximise the contribution of plasma-

membrane-resident proteins to the total signal (supplementary

material Figs S5D, S6A–E). Co-transfection of independently

membrane-anchored non-interacting GFP and RFP constructs

(supplementary material Fig. S6A) fixed the baseline cross-

correlation level, whereas a membrane-anchored GFP-mRFP

fusion protein was used to define the maximum cross-correlation

achievable under the experimental conditions (Fig. 7B and

supplementary material Table S2C).

The phosphomimetics SmoSD-GFP and SmoSD-RFP exhibited

strong cross-correlation, as expected for oligomerisation of

constitutively active Smo at the plasma membrane (Zhao et al.,

2007). Cross-correlation between the WT Smo constructs was also

increased relative to the negative control, indicating a low level of

Smo clustering in the absence of pathway activation. However,

stimulation with Hh further increased WT Smo crosscorrelation,

comparable to but weaker than the levels seen for the SmoSD

constructs. This difference suggests that in contrast to the

constitutively open phosphomimetic construct, a fraction of the

total WT Smo pool exists as monomers or dimers even under Hh-

induced signalling conditions, presumably in a nonphosphorylated

state. Thus, the kinase and phosphatase equilibrium at the surface

cannot be fully shifted towards phosphorylation and clustering.

Steady-state levels of SmoSA at the surface were low in

comparison to the corresponding WT and SmoSD constructs

(supplementary material Fig. S6B–E), reflecting the preferential

partitioning of nonphosphorylatable Smo to intracellular

membranes (Fig. 4F). In the absence of ligand, SmoSA

constructs showed clustering at the level of non-stimulated WT

Smo. Importantly, treatment with Hh also induced significant

oligomerisation of SmoSA that is comparable with the WT

response (Fig. 7B and supplementary material Table S2C). As

expected, blocking endocytosis with Dynasore, which was

sufficient to trap Smo on the plasma membrane and induce

phosphorylation (Fig. 5C), also increased Smo clustering (Fig. 7B

and supplementary material Table S2C).

Finally, the maximum expected cross-correlation signal for

homotypic dimerisation events is limited to roughly one third of

the corresponding value of a dual-coloured fusion protein

(Weidemann et al., 2002). The observed cross-correlation levels

therefore indicate higher-order stoichiometries than dimers, but

precise quantification of cluster sizes is difficult. Cross-

correlation fractions suggest an average cluster size of around

Fig. 7. FCCS analysis of Smo clustering.

(A) Representative examples of

autocorrelation (red, green) and

crosscorrelation (blue) curves for WT Smo

(left) and SmoSD (right). Note increased

crosscorrelation amplitude indicating stronger

clustering for SmoSD (arrow).

(B) Quantification of crosscorrelation fractions

(CCg). Expression of a membrane bound GFP-

mRFP fusion protein serves as positive control

(blue dashed line), whereas co-transfection of

separate membrane-bound GFP and RFP

constructs establishes the measurement

baseline (red dashed line). In the absence of

Hh, SmoSD shows increased crosscorrelation

relative to WT Smo. In response to Hh,

crosscorrelation increases for both WT Smo

and the nonphosphorylatable version SmoSA.

Blocking endocytosis with Dynasore also

increases Smo oligomerisation in the absence

of Hh. Boxplot shows 1st and 3rd quartile

(box), median (line) and mean (square).

Whiskers represent 1.56 interquartile distance

and circles individual measurements.

***P,0.01, n.s. not significant (ANOVA

followed by Tukey’s HSD).

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two Smo molecules in the absence of Hh, increasing to about

five following Hh exposure (supplementary material Fig. S7A).However, these values are known to underestimate the true sizeof the oligomers, as the model function assumes ideal conditions

that are typically not achieved under live-cell conditions(Weidemann et al., 2002). Smo cluster sizes in the range oftens of molecules are also consistent with the observed changesin diffusion times (Ramadurai et al., 2009), albeit with large

uncertainty margins for both approaches (supplementarymaterial Fig. S7B and Table S2C). In summary, as expectedfrom the literature (Zhao et al., 2007), clustering of Smo is

induced by the open, phosphorylated conformation. However,phosphorylation is per se not required for Smo oligomerisation,because Hh also induces clustering of SmoSA at the plasma

membrane.

DiscussionPrevious observations have shown that phosphorylation of the

cytoplasmic tail exerts positive feedback on Smo membraneaccumulation (Jia et al., 2004; Zhao et al., 2007), presumably byregulating the Smo ubiquitylation state and subsequentendocytosis (Li et al., 2012; Xia et al., 2012). These studies

have also provided evidence that interfering with Smoubiquitylation or endocytosis leads to increased Smo membranelevels, Ci stabilisation and Hh target gene expression (Li et al.,

2012; Xia et al., 2012). This was generally interpreted asaffecting Smo protein stability by preventing internalisation andproteolytic degradation, thereby increasing the concentration of

membrane resident, active Smo. These observations can thus becondensed into a simplified ‘phosphorylation’ model of Hh signaltransduction, whereby Hh signalling first leads to Smo

phosphorylation and translocation to the membrane. Smophosphorylation then prevents clearance of the active Smo poolby counteracting ubiquitylation and endocytosis.

However, other studies have shown that modulation of the

lipid environment can govern the subcellular localisation of Smo(Khaliullina et al., 2009; Yavari et al., 2010). Specifically,artificially increased PI4P levels can force Smo localisation to the

membrane and activation of the Hh pathway in the absence ofHh. Intriguingly, inactivation of Ptc is sufficient to increaselevels of this phospholipid (Yavari et al., 2010). Theseobservations support an ‘endocytosis’ model of Hh pathway

activation, whereby inactivation of Ptc primarily affects Smoredistribution to the plasma membrane, presumably by regulatingthe local lipid content of either the plasma membrane or Smo-

containing endosomes.

We have developed a genetically encoded, fluorescence-basedreporter for Smo phosphorylation. By simultaneously trackingSmo localisation and activation at the subcellular level we could

demonstrate that enforced membrane localisation is sufficient todrive Smo phosphorylation, irrespective of the presence of Ptc.With the help of mathematical simulations, we show that this

response is compatible with the endocytosis model but not thephosphorylation model. Importantly, the endocytosis modeldemands that the balance between the kinases and phosphatases

acting on Smo differs between plasma membrane and internalmembranes. Intriguingly, spatially differentiated regulation ofenzyme activity at the subcellular level is a hallmark of the PKA

system, at least as far as the membrane-associated AKAP-anchored type II isoforms are concerned (Wong and Scott, 2004).We therefore propose that Ptc controls activation of the Hh

pathway by regulating access of the Smo substrate to theenzymes (kinases or phosphatases), thereby regulating its

phosphorylation state, rather than by controlling the activity ofthe enzymes per se. To test this hypothesis, additional tools willhave to be developed to directly measure PKA activity withsubcellular resolution and independent of Smo. However, the

assumption of asymmetric kinase to phosphatase equilibriarequired by our model helps to reconcile the two apparentlycontradicting mechanisms of Hh pathway activation. Although it

is formally possible that Ptc regulates localisation andphosphorylation independently by separate downstreammechanisms, we would instead like to propose that Hh-induced

translocation of Smo to the membrane by means of lipidmodification (Khaliullina et al., 2009; Yavari et al., 2010) causesphosphorylation (Jia et al., 2004; Zhang et al., 2004; Apionishevet al., 2005) and opening (Zhao et al., 2007) of the Smo tail. This

would, in turn, prevent Smo ubiquitylation and endocytosis (Liet al., 2012; Xia et al., 2012), leading to downstream signaltransduction.

Nevertheless, our observations suggest that both active andinactive Smo are continuously shuttling between intracellularcompartments and the plasma membrane. First, even the

inactive SmoSA variant can readily be trapped at the cellsurface by blocking endocytosis, although its detection at themembrane under normal conditions requires single-moleculesensitivity. Second, even though activation of Smo appears to

occur at the membrane, the ratio of reporter activity to GFPlevel co-varies in the intracellular and plasma-membrane-associated fractions. Phosphorylation of the cytoplasmic tail is

known to be sufficient for Smo clustering (Zhao et al., 2007).However, our FCCS experiments have shown Hh can alsoinduce Smo clustering independent of phosphorylation. Thus,

some unidentified activity of Ptc appears to directly inhibit Smoclustering. Future experiments must test whether this role isdistinct from the regulation of Smo localisation, or whether

these activities represent two aspects of the same molecularfunction. Finally, the higher quantitative resolution of FCCScompared with FRET approaches showed that SmoSD (whereall monitored molecules are expected to be in the open

conformation) shows significantly higher crosscorrelation thanWT Smo exposed to Hh. The latter pool must therefore containa significant fraction of monomeric Smo molecules, suggesting

that the position of the equilibrium between kinase andphosphatase activities yields a considerable fraction ofnonphosphorylated Smo even in the presence of Hh.

Importantly, this does not affect the conclusions of ouranalytically robust modelling approach.

Smo is related to the GPCR family of signalling receptors, andit is therefore interesting to note that recently the formation of

higher-order complexes has been recognised as a major mode ofGPCR regulation. Consistently, the observed degree of cross-correlation of phosphorylated Smo cannot be explained by simple

homodimerisation, but must involve the formation of higher-orderoligomers (Worch et al., 2010; Weidemann et al., 2011).Intriguingly, while we were finishing this manuscript a study has

shown, by an unrelated biochemical approach, that Hh can induceSmo oligomerisation within lipid rafts, and that this is required forsignal transduction (Shi et al., 2013). It will therefore be interesting

to test whether Smo clustering is related to downstream signallingthrough the G-protein cascade (Ayers and Therond, 2010). Webelieve that our approach of combining the direct visualisation of

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the activation state of individual pathway components through

fluorescent sensors with microscopy-based biophysical techniques

shows great promise for uncovering the cell biological machinery

underlying intercellular communication, both for the Hh pathway

and other signalling cascades.

Materials and MethodsDrosophila stocks

UAS::ptc (Martın et al., 2001),UAS::ptc1130x(Johnson et al., 2000),UAS::shibireK44A

(Molineet al., 1999),ap::GAL4(Callejaet al., 1996),71B::GAL4 (BrandandPerrimon,1993), dpp::LacZBS3.0 (Blackman et al., 1991),ptc::LacZ (Chen and Struhl, 1996), smo2

and smo3 (Nusslein-Volhard et al., 1984) have all been described.

Transgenes and plasmid constructs

The SmoIP reporter was generated by replacing aa 757–915 of the Smo cytoplasmic tailwith the cpYFP core of Inverse Pericam (Nagai et al., 2001) via fusion PCR. TheSmoSA-IP and SmoSD-IP mutants were subsequently created by replacing the serines atSmo aa positions 667, 670, 673, 687, 690, 693, 740, 743 and 746 by fusion PCR withalanines and aspartates, respectively. Transgenic flies were generated by BestGene(Chino Hills, CA) or at the MPI-CBG, Dresden. To generate pUAS::SmoIP the reporterwas inserted into pWRpA (gift from N. Brown, Gurdon Institute, Cambridge).pCaSpeR-tub::Smo-IP was generated by inserting smo-IP into pCaSpeR-tub. To createpUAST-SmoGFP and pUAST-SmoRFP, the eGFP and mRFP ORFs were fused to theC-terminus of Smo by fusion PCR and inserted into the pUAST vector. The SmoSA-RFP/GFP and SmoSD-RFP/GFP versions were derived analogous to the correspondingSmo-IP constructs. pUAST::memGFP, pUAST::memRFP and pUAST::memGFP-RFPwere generated by fusing the respective ORFs to a Lyn palmytoylation/myrystoylationsite (plasmid was a gift from G. Weidinger, Ulm) by fusion PCR.

Immunocytochemistry and microscopy

Established procedures for immunostaining of imaginal discs and testes (Michelet al., 2011; Michel et al., 2012) were followed also for salivary glands andcells. Primary antisera were then applied overnight at 4 C at the followingdilutions: anti-GFP (Clontech 632460; 1:500), anti-b-galactosidase (PromegaZ3781; 1:1000), anti-Col (1:100) (Dubois et al., 2007), anti-Sal (1:1000),(DSHB) anti-Smo (1:100) (Lum et al., 2003), anti-Ptc (1:100) (Capdevila et al.,1994), anti-En (1:100) (Patel et al., 1989) and anti-Ci (1:10) (Motzny andHolmgren, 1995). Secondary antisera (Santa Cruz) were used at 1:500 dilution.Confocal images were collected using Leica SP5/II or Zeiss LSM780microscopes and processed with ImageJ/Fiji. For quantification, images werecollected under nonsaturating conditions for each experimental setting.Membrane and intracellular areas were manually outlined in the anti-GFPchannel on central sections through salivary gland cells intersecting the nucleus.After subtracting the nuclear signals in each channel as background, the averageintensities (in a.u.) of the anti-GFP and reporter fluorescence channels wereseparately measured and their ratio averaged for 8–10 cells. To estimate themembrane-bound fraction, the average intensities and areas of the intracellularand membrane ROIs were multiplied, the membrane value divided by two to

account for the contribution from neighbouring cells that cannot be resolved bylight microscopy, and the ratios determined for the individual cells averaged.Assuming a roughly cuboidal geometry for the gland cells, this is a conservativeestimate of the membrane-associated fraction, because the bottom and topsurfaces are not taken into account. Observed differences were tested forsignificance by ANOVA followed by Tukey’s HSD post-hoc test.

Insect cell culture

Drosophila S2R+ cells (a kind gift from Elisabeth Knust, MPI-CBG, Dresden)were cultured at 25 C, without CO2 in Schneider’s Drosophila medium with L-Glutamine (Invitrogen or PAN BIOTECH) supplemented with 10% fetal bovineserum (FBS, Invitrogen) and transfected using the calcium phosphate method(Graham and van der Eb, 1973; Chen and Okayama, 1987).

Blocking endocytosis by Dynasore and ligand stimulation

Transiently transfected S2R+ cells were incubated with 25 mM Dynasore (Sigma-Aldrich) for up to 1 hour before imaging or fixation. Hh conditioned medium wasgenerated by incubating S2R+ cells transfected with UAS::Hh and Actin5C::Gal4plasmids for 6 days. Cells were stimulated by addition of conditioned medium12 hours before fixation or imaging.

Sample preparation for FCCS

Cells were seeded into concanavalin-A-coated eight-well LabTek chambers (no.1.5, 0.16–0.19 mm, Thermo Scientific) and allowed to adhere to the substrate for1 hour. Before measurements, the medium was replaced with air buffer (20 mMHEPES, pH 7.4, 150 mM NaCl, 15 mM glucose, 20 mM trehalose, 0.15 mg/mlBSA, 5.4 mM KCl, 0.85 mM MgSO4, 0.75 mM CaCl2).

FCCS data acquisition

FCCS was performed at room temperature using a Zeiss LSM780 microsocope witha ConfoCor3 module and a Zeiss C-Apochromat 406, N.A. 1.2 objective (CarlZeiss). Fluorescence was recorded using avalanche photodiodes. Instrument settingswere optimised before each session for maximum particle brightness using 25 nMsolutions of Alexa Fluor 488 (Life Technologies) and CF568 (Biotium) dyes.

FCCS data analysis

Fluorescence signals were recorded in two colour channels for GFP (g) and mRFP(r) and correlated following the definition for auto- (j) and cross-correlation (x):

Gj(t)~SdFj(t)dFj(tzt)T

SFjT2

G|(t)~SdFg(t)dFr(tzt)T

SFgTSFrT:

ð1Þ

Runs showing drift of the count rates due to photobleaching or membranemovements were discarded. To derive parameters a model function for twomolecular species diffusing in a two-dimensional plane and a factor accounting forblinking behaviour at short time-scales was fitted to the data using the Zeiss ZEN

Table 1. Parameter set

Symbol Description Endocytosis model Phosphorylation model

Variables Ptc Active Ptc at plasma membrane 1 (initial) 1 (initial)PMSmo Nonphosphorylated Smo at plasma membrane 0.25 (initial) 0.25 (initial)

PMSmoP Phosphorylated Smo at plasma membrane 0.25 (initial) 0.25 (initial)EESmo Nonphosphorylated Smo at internal membranes 0.25 (initial) 0.25 (initial)

EESmoP Phosphorylated Smo at internal membranes 0.25 (initial) 0.25 (initial)Parameters Hh Extracellular Hh concentration Controlled parameter Controlled parameter

kact Ptc activation rate 1 1kinact Hh binding and Ptc inactivation rate 1 1

kPMkin Kinase activity at plasma membrane 1 NAkPMphos Phosphatase activity at plasma membrane 0.01 NAkEEkin Kinase activity at internal membranes 0.01 NA

kEEphos Phosphatase activity at internal membranes 1 NAkkin1 General kinase activity NA 0.1kkin2 General kinase inhibition scale NA 0.1kphos General phosphatase activity NA 0.01ken0 Constitutive PMSmo endocytosis rate 0.2 1ken1 Ptc-dependent PMSmo endocytosis rate 1 NAken2 Reduced PMSmoP endocytosis rate 0.01 0.01kex General exocytosis rate 0.1 0.1

NA, not applicable.

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software package (Weidemann et al., 2011):

G(t)~1

cVeff

GT (t)G3D(t)

GT (t)~1{fnf zfnf e{t=tnf

1{fnf

� �

G2D(t)~X2

i

fi 1zt

tdiff ,i

� �{1

ð2Þ

Here, Veff denotes the effective focal detection volume, c the fluorophoreconcentration, tdiff,i and fi the dwell times and molar fractions of the two diffusionspecies, and tT and fT the lifetime and fraction of molecules in the dark state. The GFPautocorrelation and the cross-correlation curves were fitted with a two-componentdiffusion model (i52), whereas in the mRFP channel, one diffusion component (i51)was sufficient. Since blinking contributions of mRFP showed significant scatterbetween individual measurements (Hendrix et al., 2008), we fixed our experimentalaverage value to 300 mseconds for evaluation of the particle numbers N.

The amplitudes G(0) were corrected for non-correlating background intensity B inthe plasma membrane as determined in control cells under the same excitation power.

GDj (0)~

1

cVeff ,j

Fj

Fj{Bj

� �{2

G|(0)~d

cVeff ,|

Fg

Fg{Bg

� �{1Fr

Fr{Br

� �{1ð3Þ

Here, the total mean count rate is composed of emitted fluorescence and thebackground intensity F~FcorzB. The background corrected mean fluorescence andamplitudes Gcor

g (0) were further corrected for spectral crosstalk (Bacia et al., 2012)

GGg(t)~Gcorg (t) ð4Þ

GGr(0)~F2

r:Gcor

r (0)zb2F2g:Gcor

g (0){2bFrFg:Gcor

| (0)

Fr{bFg

� �2ð5Þ

GG|(0)~FgFr

:Gcor| (0){bFg

:Gcorg (0)

FgFr{bF2g

ð6Þ

Under our experimental conditions, bleed-through from the green into the redchannel was 9% (b50.09), whereas crosstalk from the red into the green channel wasnegligible. For homogeneous fluorescent particles, the inverse of the correctedamplitudes reflect number of fluorescent particles in the detection volume

GGg(0)~1

NgzNgr

GGr(0)~1

NrzNgr

ð7Þ

GGCC(0)~Ngr

NgzNgr

� �: NrzNgr

� � : ð8Þ

where Ng is the number of exclusively green, Nr the number of exclusively red and

Ngr the number of double labelled particles. However, when particles interact, the

molecular brightness distribution broadens and Eqns (7,8) are not valid. A usefulreadout to evaluate binding scenarios is to normalise the cross-correlation by thesimultaneously measured autocorrelation amplitude (Worch et al., 2010; Weidemannet al.l 2002). Because the green, GFP-tagged particles were more abundant (Ng.Nr)we used this channel for normalisation. Assuming a binomial distribution of red andgreen labels within the complex, this ratio can be linked to the degree ofoligomerisation

CCg:GG|(0)

GGg(0)~

n{1

n{1z1

pg

ð9Þ

For example, evenly expressed GFP and mRFP constructs (pg51/2) and asimple saturated dimerisation (n52) will produce CCg51/3. Uneven expressionslightly increases this ratio but cannot exceed 1/2. Thus, measured CCg.1/2 forhomotypic interactions indicate higher order stoichiometries (n.2). Differencesbetween crosscorrelation levels were tested for significance by ANOVA followedby Tukey’s HSD post-hoc test.

Mathematical modelling of Smo activation

We considered the following two non-dimensionalised models.

Endocytosis model

dPtc

dt~kact{kinact

:Hh:Ptc

dPMSmo

dt~{kPMkin

:PMSmozkPMphos:PMSmoP

{ ken0zken1:Ptcð Þ:PMSmozkex

:EESmo

dPMSmoP

dt~zkPMkin

:PMSmo{kPMphos:PMSmoP

{ken2:PMSmoPzkex

:EESmoP

dPEESmo

dt~{kEEkin

:EESmozkEEphos:EESmoP

{ ken0zken1:Ptcð Þ:PMSmo{kex

:EESmo

dEESmoP

dt~zkEEkin

:EESmo{kEEphos:EESmoP

zken2:PMSmoP{kex

:EESmoP

with the analytical equilibrium solution

Ptc Hhð Þ~ kact

kinact:Hh

PMSmoP&Smototal

1zkPMphos

kPMkin

zken2z ken0zken1

:Ptc Hhð Þð Þ:kPMphos

�kPMkin

kex

that solely depends on the parameter ratios

kact

kinact

,kPMphos

kPMkin

, andkenX

kex

in simple combination and not on their absolute values. Hence, the presentedmodel behaviour is general.

Phosphorylation model

dPtc

dt~kact{kinact

:Hh:Ptc

dPMSmo

dt~{

kkin1

kkin2zPtc:PMSmozkphos

:PMSmoP

{ken0:PMSmozkex

:EESmo

dPMSmoP

dt~

kkin1

kkin2zPtc:PMSmo{kphos

:PMSmoP

{ken2:PMSmoPzkex

:EESmoP

dEESmo

dt~

kkin1

kkin2zPtc:EESmozkphos

:EESmoP

{ken0:PMSmoP{kex

:EESmo

dEESmoP

dt~

kkin1

kkin2zPtc:EESmo{kphos

:EESmoP

zken2:PMSmoP{kex

:EESmoP

Parameter setTo visualise the general model behaviour we have chosen the following particularparameter values listed in Table 1. We confirmed independence of qualitative

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model behaviour from the particular choice of parameter by varying eachparameter tenfold (data not shown).

To simulate experimental manipulations, particular parameter values were set tozero. Thus, for SmoSA-IP: kPMkin~kEEkin~kkin1~0, for SmoSD-IP:kPMphos

~kEEphos~kphos~0 and for Dynasore treatment: ken0~ken1~ken2~0. Simulationswere performed with the help of two independent modelling and simulation tools,Copasi (Hoops et al., 2006) and PottersWheel (Maiwald and Timmer, 2008). Resultscoincided and the diagrams show the steady state solutions as a function of Hhstimulation level. Our new endocytosis regulation model is available as SBML filefor further analysis and use independent of specific simulation software.

AcknowledgementsWe thank Nicholas H. Brown, Michele Crozatier, ChristianDahmann, Suzanne Eaton, Elisabeth Knust, Gilbert Weidinger, theBloomington stock centre and the DSHB for providing flies andreagents, Raquel Perez-Palencia for expert technical assistance andmembers of the Junior European Drosophila Investigators (JEDI)initiative for discussions.

Author contributionsA.K. and M.M. performed the Drosophila experiments; I.R.performed the cell culture and FCS experiments and analysed theFCS data together with T.W.; D.A. generated the proof of principlereporter construct; L.B. performed the mathematical modelling; C.B.initiated the project and wrote the manuscript.

FundingThe project was supported by the Center for Regenerative TherapiesDresden (CRTD); the Federal Ministry of Education and Research(BMBF) Consortium Mesenchymal Stem Cells; and the DeutscheForschungsgemeinschaft [grant number BO 3270/2-1 to C.B.].

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.128926/-/DC1

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