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Please cite this article in press as: Parsons et al., Lgl Regulates Notch Signaling via Endocytosis, Independently of the Apical aPKC-Par6-Baz Polarity Complex, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2014.07.075

Lgl Regulates Notch Signalin

Current Biology 24, 1–12, September 22, 2014 ª2014 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2014.07.075

Articleg

via Endocytosis, Independently of theApical aPKC-Par6-Baz Polarity Complex

Linda M. Parsons,1,2,6,8 Marta Portela,1,8

Nicola A. Grzeschik,1,7 and Helena E. Richardson1,3,4,5,*1Cell Cycle and Development Laboratory, Research Division,Peter MacCallum Cancer Centre, 7 St. Andrew’s Place,East Melbourne, Melbourne, VIC 3002, Australia2Department of Genetics, University of Melbourne,1-100 Grattan Street, Parkville, Melbourne, VIC 3010, Australia3Sir Peter MacCallum Department of Oncology, PeterMacCallum Cancer Centre, 7 St. Andrew’s Place,East Melbourne, Melbourne, VIC 3002, Australia4Department of Biochemistry and Molecular Biology,University of Melbourne, 1-100 Grattan Street, Parkville,Melbourne, VIC 3010, Australia5Department of Anatomy and Neuroscience, University ofMelbourne, 1-100 Grattan Street, Parkville, Melbourne,VIC 3010, Australia

Summary

Background: The Drosophila melanogaster junctionalneoplastic tumor suppressor, Lethal-2-giant larvae (Lgl), isa regulator of apicobasal cell polarity and tissue growth.We have previously shown in the developing Drosophila eyeepithelium that, without affecting cell polarity, depletion ofLgl results in ectopic cell proliferation and blockage of devel-opmental cell death due to deregulation of the Hippo signalingpathway.Results:Here, we show that Notch signaling is increased in lgl-depleted eye tissue, independently of Lgl’s function in apico-basal cell polarity. The upregulation of Notch signaling isligand dependent and correlates with accumulation of cleavedNotch. Concomitant with higher cleaved Notch levels in lgl2

tissue, early endosomes (Avalanche [Avl+]), recycling endo-somes (Rab11+), early multivesicular bodies (Hrs+), andacidified vesicles, but not late endosomal markers (Car+ andRab7+), accumulate. Colocalization studies revealed that Lglassociates with early to late endosomes and lysosomes. Upre-gulation of Notch signaling in lgl2 tissue requires dynamin- andRab5-mediated endocytosis and vesicle acidification but isindependent of Hrs/Stam or Rab11 activity. Furthermore, Lglregulates Notch signaling independently of the aPKC-Par6-Baz apical polarity complex.Conclusions: Altogether, our data show that Lgl regu-lates endocytosis to restrict vesicle acidification and pre-vent ectopic ligand-dependent Notch signaling. This Lglfunction is independent of the aPKC-Par6-Baz polaritycomplex and uncovers a novel attenuation mechanismof ligand-activated Notch signaling during Drosophila eyedevelopment.

6Present address: Department of Anatomy and Neuroscience, University of

Melbourne, 1-100 Grattan Street, Parkville, Melbourne, VIC 3010, Australia7Present address: Department of Cell Biology, University Medical Centre

Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, the Netherlands8Co-first author

*Correspondence: [email protected]

Introduction

In epithelial cells, polarization of cell membranes along the api-cobasal axis (apical-basal cell polarity) is crucial for maintain-ing tissue architecture and limiting tissue growth (reviewed by[1, 2]). Endocytosis is also crucial in the regulation of plasmamembrane composition together with apicobasal cell polarityregulation (reviewed by [3, 4]). Although endocytic and cellpolarity pathway components have been defined, how thesepathways interact to regulate cellular architecture and functionis unclear.Apicobasal cell polarity is regulated by a conserved network

of proteins: the Par (Par3 [Bazooka, Baz in Drosophila],atypical protein kinase C [aPKC)], Par6, and Cdc42), Crumbs(Crumbs [Crb], PALS [Stardust, Sdt in Drosophila], andPATJ), and Scribble (Scribble [Scrib], Discs-large [Dlg],and Lgl) modules, which establish and maintain cell polaritythrough mutually antagonistic interactions (reviewed by[5, 6]). In addition to their roles in cell polarity, these proteinsalso regulate tissue growth, and their deregulation leads toneoplastic tumor formation (reviewed by [7, 8]).Links between apicobasal cell polarity and endocytosis have

been revealed from studies in mammalian cells and inverte-brates (reviewed by [3, 9]). In Drosophila, mutants in Par com-plex proteins exhibit trafficking defects of the adherens junc-tion protein, E-cadherin (Ecad) [10, 11], and of apical proteins[12]. Furthermore, mutations in Drosophila endocytic regula-tors exhibit defects in cell polarity and behave as neoplastic tu-mor suppressors similar to dlg, scrib, and lgl (reviewed by [3]).Endocytosis involves trafficking of plasma membrane com-

ponents through different vesicular compartments: early en-dosomes (EEs), multivesicular bodies (MVBs), late endosomes(LEs), and the lysosome, where they are degraded, or, alterna-tively, the recycling endosome (RE), where they return to theplasma membrane (reviewed by [4]). In Drosophila, defects inendocytosis alter signal transduction pathways, and these de-fects in signaling contribute to tumorigenesis (reviewed by [3]).In particular, deregulation of Notch signaling occurs in variousendocytic pathway mutants and contributes to cell prolifera-tion [13–16]. Acidification is another important mechanismin the endocytic pathway. Maturation of EEs to lysosomes ismarked by gradual acidification of their lumen, through theactivity of proton pumps (vacuolar-ATPases [V-ATPases];reviewed by [17]). Deregulation of endocytosis and vesicleacidification affects components of cell signaling networks,in particular, Notch. Consistent with this, mutations in regula-tors or subunits of V-ATPase complexes decrease endosomeacidification, resulting in reduced Notch signaling [18, 19].By contrast, overexpression of the V-ATPase component,Vha44, increases Notch signaling [20]. The links among endo-cytosis, vesicle acidification, and Notch signaling reflectthe importance of Notch intracellular trafficking for correctpathway activation (reviewed by [21, 22]).Notch signaling is an evolutionarily conserved cell-cell

signaling pathway that controls numerous cell-fate specifi-cation events in multicellular organisms (reviewed by [23]).Signaling is triggered when the Notch extracellular domainbinds to Delta (Dl)/Serrate (Ser) ligands, presented by

http://dx.doi.org/10.1016/j.cub.2014.07.075


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Figure 1. lgl Regulates Notch Signaling in the Drosophila Eye Epithelia

Confocal planar cross-sections of larval eye antennal discs. White scale bar

represents 50 mM, unless otherwise stated. Posterior is left, and apical is up;

yellow bar indicates the MF; arrowheads denote mutant tissue; and dots

outline clonal borders in this figure and all other figures. Error bars show

SEM in this figure and all other figures.

(A) The Notch signaling pathway. Ligand binding to the Notch extracellular

domain induces proteolytic cleavage and release of Notch intracellular

domain (Nicd) that enters the nucleus and together with Mam and CSL

(Su(H)) activates target genes, such as (E(spl)-C).

(B–D) Schematic mosaic eye disc (B); wild-type clones GFP+ (C) or lgl2

clones GFP2 (D).

(C and C0) Control mosaic discs stained for b-gal (C, gray; C0, red) showing

endogenous expression of E(spl)lacZ within and posterior to the MF

(merge in C0).

Current Biology Vol 24 No 182


neighboring cells (Figure 1A). After ligand binding, Notch un-dergoes S2 cleavage by disintegrin/metalloprotease (ADAM)releasing the ligand and extracellular domain of the Notch(Necd) into the signal-sending cell. In the signal-receivingcell, further proteolysis of cleaved Notch (Next) by g-secretase(S3 cleavage) generates Notch intracellular domain (Nicd) thattranslocates to the nucleus and activates gene transcription.Notch signaling is also regulated by endocytosis of full-lengthNotch in a ligand-independent manner, where it is recycledto the surface or degraded in the lysosome. Disruption of EEcomponents, such as Dynamin (Shibire), Syntaxin 7 (Avl),and Rab5, block Notch signaling. In contrast, loss of later en-dosomal trafficking components, such as ESCRTI (TSG101)and ESCRTII (Vps25), result in ectopic Notch activation [22].Here, we investigate effects of Lgl depletion on Notch

signaling and endocytosis. We show that in lgl2 tissue,increased Notch target gene expression is concomitant withaccumulation of cleaved Notch, Avl+, Hrs+, Rab11+, and acidicvesicles. Increased Notch signaling in Lgl-depleted tissue isrescued by decreasing vesicle acidification, revealing a novellink among cell polarity, endocytosis, and Notch signaling.Furthermore, we show that Lgl regulates Notch signaling inde-pendently of aPKC-Par6-Baz. Altogether, our results revealthat Lgl plays a role in the regulation of endocytic traffickingand limitingNotchactivationby restricting vesicle acidification.

Results

lgl Regulates Notch Signaling in the Drosophila EyeEpithelium

To determine whether Notch signaling is deregulated in cellscontaining an lgl null allele (lgl27S3, denoted lgl2), we examinedthe Notch reporter, E(spl)m8-lacZ (Figure 1A), in Drosophilathird instar larvaleyeantennaldiscclones.Anti-b-galactosidasestaining inwild-type eye epithelia revealed thatE(spl)m8-lacZ isexpressed throughout the posterior-differentiated region of theeye disc, with increased levels just posterior to the morphoge-netic furrow (MF) (Figures 1C and 1C0). lgl2 clones (GFP2 tissue,Figure 1B) spanning and posterior to the MF showed a 2-fold,cell-autonomous increase in E(spl)m8-lacZ expression, relativeto surrounding wild-type tissue (Figures 1D–1D0 0) compared tocontrol tissue (Figures 1C and 1C0, quantified in Figure 1G).Expression of wild-type lgl (lglWT) restored the elevated E(spl)m8-lacZ to normal in lgl2 clones (Figures 1E–1G). Further-more, expression of a dominant-negative form of mastermind

(D–D0 0) lgl2 mosaic disc (clones outlined in green) stained for b-gal (D0, gray;D andD0 0, red) showing upregulation of E(spl)lacZ in lglmutant tissue (GFP2)

(arrowheads, merge in D0 0).(E, F, H, and J) lgl2 clones GFP+ (E, H, and J); schematic mosaic eye (F);

mutant clones GFP+ and wild-type tissue GFP2 (experiments E–E0 0, H, H0,J, and J0).(E–E0 0) lgl2;UAS-lglWTmosaic discs stained for b-gal (E0, gray; E and E0 0, red)showing normal E(spl)lacZ levels in mutant tissue (GFP+) (arrowheads).

(G) Quantification of b-gal pixel intensity ratio between wild-type versus

mutant clones of the listed genotypes (***p value < 0.0001, differences not

significant [n.s.]).

(H and H0) lgl2 pupal mosaic disc at w40% pupal development stained for

Rst (H, gray; H0, red) showing elevated levels of Rst in mutant tissue

(GFP+) compared to wild-type (arrowheads, merge in H0).(I) lgl2 mosaic adult female eye.

(J and J0) lgl2,UAS-mamDN mosaic disc at w45% pupal development

stained for Rst (J, gray; J0, red) showing similar Rst levels in mutant tissue

(GFP+, arrowheads, merge in J0) compared to wild-type.

(K) lgl2,UAS-mamDN mosaic adult female eye. mamDN expression partially

rescues lgl2 mosaic eye phenotype.

Figure 2. Cleaved Notch Levels Are Elevated in

lgl2 Tissue

(A) Schematic Notch cleavage: full-length Notch

receptor undergoes S2 cleavage, generating

Next. Subsequent S3 cleavage by g-secretase

generates transcriptionally active Nicd. Black

arrowheads show the region of Notch receptor

recognized by the Nintra- and Nextra-specific

antibodies.

(B–B0 0 0) Cross-section of lgl2 mosaic eye disc

stained for Nintra (B, gray; B0, red) and aPKC

(B0 0, blue; B0 0 0, purple) show accumulation of

Notch in lgl2 clones (GFP2, arrowheads, merges

in B0 and B0 0 0).(C–C0 0) Planar section of lgl2 mosaic eye discs

stained forNintra (red) showaccumulationofNotch

in lgl2 clones (GFP2, arrowheads, merge in C0 0).(D–E0 0) lgl2mosaic eye discs stained for Dl (D, red)

or Nextra (E) showing normal Dl or full-length

Notch/Necd levels and localization in lgl2 tissue

(GFP2, arrowheads, merges in D0 0 and E0 0).

Regulation of Notch Signaling and Endocytosis by Lgl3


(mamDN) [24], a component of the Notch transcriptional activa-tion complex, reduced E(spl)m8-lacZ expression to wild-typein lgl2 clones (Figures S1A–S1B0 0 available online; Figure 1G).Thus, Lgl is required to restrictNotchsignaling in thedevelopingeye epithelium.

Upregulation of Notch Signaling Contributes to the lgl2

Phenotype

To determine whether upregulation of Notch signaling contrib-utes to the lgl2 phenotype, we first investigated whether Notchtargets,CyclinA (CycA), a cell-cycle regulator [25, 26], and Irreg-ular chiasm C-Roughest (Rst), a cell survival factor in the pupalretina [27, 28], were altered in lgl2 tissue. Indeed, CycA wasupregulated in lgl2 clones (Figures S1C–S1C0 0). Furthermore,Rst was increased in lgl2 clones atw40% pupal development,which show increased interommaditial cell numbers due todecreased apoptosis [29] compared to wild-type tissue (Fig-ures 1H and 1H0; quantified in Figure S1F). Consistent with Rstbeing a Notch target, reducing Notch signaling by expressingmamDN in lgl2 clones restored Rst levels to normal (Figures

1J, 1J0, S1D, S1D0, andS1F). Importantly,expression of mamDN in lgl2 tissue re-sulted in partial suppression of the lgl2

mosaic adult phenotype; eyes were lessbulgy and rough (Figures 1I and 1K; Fig-ure S1E). However, the adult eye pheno-type was not restored to normal, prob-ably because of continued impairmentin Hippo pathway signaling [30]. Thesedata support the notion that in Lgl-depleted tissue, ectopic Notch signalingcontributes to the lgl2 disorganizedovergrown eye phenotype, possiblythrough upregulated CycA, which drivesproliferation [25, 26], and Rst, whichblocks apoptosis and prevents cell sort-ing when overexpressed [27].

lgl2 Tissue Accumulates CleavedNotch

To examine effects of Lgl depletionon full-length and cleaved Notch, we

generated lgl2 clones in the eye disc and monitored levelsand localization of Notch by immunofluorescence with anti-Notch-intra (Nintra), which detects all forms of Notch(Figure 2A). In wild-type eye tissue, higher Nintra stainingoccurred within the MF and was localized to cell membranesof developing ommatidial clusters (Figure S2A). Cross-sec-tions of lgl2 mosaic eye discs revealed that just posterior toand within the MF, Nintra staining accumulated throughoutapical and basal cell membranes and the cytoplasm (Figures2B and 2B0). Apical localization of aPKC within lgl2 clonesdemonstrated that lgl2 tissue retained cell polarity (Figures2B0 0 and 2B0 0 0). Planar confocal sections of lgl2 clones revealedincreased Nintra staining posterior to and within the MF,compared to wild-type (GFP+) tissue (Figures 2C–2C0 0; FiguresS2D–S2E0 0). Thus, consistent with upregulation of Notch tar-gets, Notch protein accumulates in lgl2 tissue.A possible explanation for increased Notch levels and

signaling in lgl2 clones is via increased levels of the ligand,Dl, in lgl2 tissue. However, Dl staining was not altered in lgl2

clones (Figures 2D–2D0 0) relative to the wild-type clones

Figure 3. Notch Signaling in lgl2 Tissue Is Ligand Dependent and Requires Endocytosis

(A–B0 0 0) lgl2;Dl2,Ser2 mosaic discs stained for b-gal (A and B, gray; merges in blue or purple); lgl2 clones (GFP2, arrows); Dl2,Ser clones (RFP+). Expression

of E(spl)lacZ is abolished in Dl2,Ser clones (B–B0 0 0) and in triple mutant clones (outlined clone shown in A) but is upregulated in lgl2 clones (see Figure 1).

(C and C0) E(spl)lacZ; UAS-shiK44A mosaic disc stained for b-gal (C, gray; C0 red, merge). E(spl)lacZ is not expressed in shiK44A clones (GFP+, arrowheads).

(legend continued on next page)





(Figure S2B). Thus, changes in Notch signaling in lgl2 clonesare not due to increased levels of Dl.

Because increased Notch signaling in various mutants isdue to defects in the recycling and degradation of full-lengthNotch from the plasma membrane (reviewed by [21, 22]), wetested whether full-length Notch was accumulating in lgl2

mosaic eye discs by staining with anti-Notch-extra (Nextra),which only detects full-length Notch and Necd (Figure 2A). Incontrol eye discs, Nextra staining predominantly accumulatedin the MF (Figure S2C). In lgl2 clones, the localization and levelof Nextra staining were not altered within and posterior to theMF (Figures 2E–2E0 0; Figures S2F–S2F0 0), suggesting that accu-mulation observed with Nintra staining represents cleavedintracellular forms of Notch.

To extend this result, we performed a live trafficking assayusing the Nextra antibody in cultured eye discs (FiguresS2G–S2H0 0). Comparison of Notch staining between wild-type and adjacent lgl2 clones revealed that there was no dif-ference in the level and localization of Notch upon initialsurface labeling (Figures S2G–S2G0 0). After incubating eyediscs for 1 hr in the absence of anti-Nextra antibody in themedia, surface-labeled Notch was depleted in the most api-cal membrane region but was localized subapically (FiguresS2H–S2H0 0). Importantly, there was no difference in the accu-mulation and distribution of Nextra-labeled Notch in wild-type and lgl2 clones 1 hr after initial labeling (Figures S2H–S2H0 0), and at 6 hr after labeling, Nextra-labeled Notch waseliminated from both wild-type and lgl2 clones (data notshown). These data confirm that internalization and traf-ficking of full-length Notch or Necd were unaffected in lgl2

tissue. Thus, processing and endocytosis of full-lengthNotch or Necd into the signal receiving or sending cell,respectively, were not perturbed in lgl2 tissue. Together,with Nintra staining results, these data indicate that lgl2 tis-sue specifically accumulates cleaved forms of Notch (Nextand/or Nicd).

Notch Signaling Is Ligand Dependent in lgl2 TissueAccumulation of cleavedNotch in lgl2 tissue strongly suggeststhat ectopic activation of Notch signaling observed in theabsence of lgl is ligand dependent. To confirm this, we gener-ated lgl2 clones lacking Notch ligands, Dl (Dlrev10, denotedDl2)and Ser (SerRX106, denoted Ser2), and examined E(spl)m8-lacZexpression. In order to identify lgl;Dl,Ser triple mutant tissue,we generated lgl2 clones lacking GFP and labeled Dl2,Ser2

clones with red fluorescent protein (RFP); thereby, triplemutant clones were red (RFP+GFP2). Consistent with previousresults, single lgl2 clones showed increased E(spl)m8-lacZ(Figures 3A and 3B, clones lacking GFP and RFP, blue inmerge). In contrast, lgl2 tissue that overlapped Dl,Ser doublemutant clones lacked E(spl)m8-lacZ expression in their center,but, at the periphery of these clones, E(spl)m8-lacZ expressionwas observed, due to rescue by Dl/Ser in adjacent wild-typetissue (Figures 3A–3B0 0 0, RFP only). As expected,Dl,Ser doublemutant clones lacked E(spl)m8-lacZ expression (Figures 3A–3B0 0 0, RFP and GFP tissue). Thus, Notch signaling in lgl2 tissueis ligand dependent.

(D and D0) lgl2,E(spl)lacZ; UAS-shiK44A mosaic disc stained for b-gal (D, gray; D

arrowheads).

(E and E0) E(spl)lacZ; UAS-Rab5RNAimosaic disc stained for b-gal (E, gray; E0, red(F and F0) lgl2,E(spl)lacZ; UAS-Rab5RNAimosaic disc stained for b-gal (F, gray; F

arrowheads).

(G) Quantification of b-gal pixel intensity ratio between wild-type versus mutan

Notch Signaling Requires Endocytosis in lgl2 TissueTo determine whether endocytosis is required for activation ofNotch target genes in lgl2 tissue, we used a transgene ex-pressing a GTP-binding defective (dominant-negative) formof dynamin (UAS-shiK44A) [31]. shiK44A prevents fission of ves-icles and formation of clathrin-coated pits, arresting endocy-tosis at the plasma membrane and transgolgi trafficking. Wealso disrupted EE formation by lowering endogenous Rab5levels via UAS-Rab5RNAi expression. Expression of shiK44A orRab5RNAi alone in eye discs caused some disruption to tissuemorphology, as judged by aPKC staining (data not shown);however, as expected, Notch signaling, monitored by E(spl)m8-lacZ expression, was blocked (Figures 3C, 3C0, 3E,and 3E0, quantified in Figure 3G). Importantly, expression ofshiK44A or Rab5RNAi in lgl2 tissue abolished E(spl)m8-lacZexpression (Figures 3D, 3D0, 3F, 3F0, and 3G). Thus, blockingtrafficking of Notch through EE pathways prevents upregula-tion of Notch signaling in lgl2 cells.

Lgl Associates with Nintra and Endosomal CompartmentsWe then investigated whether Lgl might function at endosomalcompartments by examining whether Lgl colocalized with Nin-tra and endocytic markers. As expected, Lgl antibody stainingrevealed no detectable signal in lgl null clones (Figures 4A–4B).In wild-type tissue, Lgl was detected in large puncta in baso-lateral junctions and in smaller puncta throughout the cyto-plasm and membranes of photoreceptor cells (Figure 4C).Importantly, costaining of Lgl and Nintra revealed partial over-lap of Nintra and Lgl+ smaller puncta (Figures 4C–4C0 0). Cos-taining of Lgl with markers of EE (Avl), early MVB (Hrs), RE(Rab11), LE (deep-orange, Dor) [32], or LE-lysosome (Lamp1)[33], showed that Lgl puncta overlapped with a subset of allendocytic compartments (Figures 4D–4H0 0). Although enlargedcytoplasmic and membrane-associated Lgl+ puncta were de-tected in close proximity to Avl, Hrs, Rab11, or Lamp1 vesicles,colocalization of large Lgl+ puncta was most obvious with theLE marker, Dor. These data show that Lgl is concentrated notonly at basal-lateral membranes but also in a spectrum ofendocytic compartments.

lgl2 Tissue Accumulates Avl, Hrs, and Rab11 Endosomes

We then examined whether endocytic compartments werealtered in lgl2 clones. To investigate the EE compartmentin lgl2 tissue, we generated lgl2 clones in the eye discand examined the localization of the EE marker, Avl [34]. Inwild-type tissue, punctate staining of Avl was observedthroughout the cytoplasm of eye disc cells, with highestlevels accumulating at the most-apical region of cell mem-branes (Figure S3A). In planar sections, lgl2 clones withinand just posterior to the MF accumulated Avl+ vesicles (Fig-ures 5A and 5A0). These changes were also observed incross-section, where increased Avl staining was presentapically and basolaterally throughout the membranes andcytoplasm of lgl2 cells (Figure 5B). aPKC staining confirmedthat changes in Avl staining occurred in lgl2 clones withoutcell polarity loss (Figure 5B0). Thus, lgl2 tissue shows accu-mulation of EE vesicles.

0, red, merge). shiK44A inhibits expression of E(spl)lacZ in lgl2 tissue (GFP+,

, merge). E(spl)lacZ is not expressed inRab5RNAi clones (GFP+, arrowheads).0, red, merge).Rab5RNAi inhibits expression of E(spl)lacZ in lgl2 tissue (GFP+,

t clones of the listed genotypes (***p value < 0.0001).

Figure 4. Lgl Colocalizes with EE, MVB, RE, LE, and Lysosomal

Compartments

(A and B) Lgl antibody staining (red) of lgl2 mosaic eye discs. lgl2 tissue

(GFP2) shows no staining, revealing the specificity of the Lgl antibody.

(C–H) Wild-type (wt [w1118] or GMR>GFP-lamp1) eye discs stained with Lgl

and Nintra (C), Lgl and Avl (D), Lgl and Rab11 (E), Lgl and Hrs (F), Lgl and Dor

(G), and Lgl and Lamp1-GFP (H). Scale bars of (C)–(H) represent 2 mm.

Lgl (gray or green in merges); Nintra or endocytic markers (gray or red in

merges). Magenta circles denote close juxtaposition or colocalization of

Lgl and Nintra or endocytic vesicle markers (see inset).



We then examined the localization of Rab11 (RE) and Hrs (anESCRT-0 component involved in maturation of the EE to theearly MVB; reviewed by [4, 35]). In wild-type tissue, Rab11and Hrs were detected within the cytoplasm and cell mem-branes of all eye disc cells (Figures S3B and S3C). In lgl2

clones, Rab11 and Hrs accumulated within the cytoplasmand membranes of cells within or posterior to the MF (planarsections, Figures 5C, 5C0, 5E, and 5E0, respectively; cross-sec-tions, Figures 5D, D0, 5F, and 5F0, where aPKC staining showedthat polarity was maintained). Costaining with Hrs andNintra antibodies revealed Hrs and Notch partially colocalizedin lgl2 tissue (Figures S3D–S3DV). The altered level and local-ization of markers of the EE, early MVB, and RE in lgl2 clonesreveals that lgl depletion affects multiple endocytic compart-ments. However, Rab7, a marker of maturing LE [36] andCarnation (Car), required for LE to lysosome fusion [32, 37],was unchanged in lgl2 tissue (Figures S3E–S3E0 0 and S3F–S3F0 0, respectively). Furthermore, the localization and levelsof Ecad, which are regulated by aPKC-dependent endocytosis[10, 11], were unaffected in lgl2 mutant tissue (Figures S3G–S3H0). Thus, EEs, early MVBs, and REs specifically accumulatein lgl2 tissue.

Lgl Regulates Notch Signaling Independently of theHrs/Stam Complex

Because Lgl colocalizes with a subset of Hrs+ vesicles and Hrsaccumulates in lgl2 tissue (Figures 4D, 4F, and 4F0 0; Figures 5E–5F0), we hypothesized that upregulated Hrs might causeincreased Notch signaling in lgl2 tissue. To determine whetherthis was the case, we used a null allele, hrsD28 (denoted hrs2),combined with knockdown of the Hrs binding protein, Stam[38], to reveal the importance of the Hrs/Stam (ESCRT-0) com-plex for ectopic Notch signaling in lgl2 clones (Figures S4A–S4I0; quantified in Figure 5G). Consistent with reports that Hrsand Stam form a stable complex [39], UAS-StamRNAi-depletedclones stained with Hrs had low Hrs levels (Figure S4D).In lgl2,hrs2; UAS-StamRNAi clones (Figures S4G and S4G0,

quantified in Figure 5G), elevated levels of E(spl)m8-lacZ,similar to lgl2 clones, were observed (Figures S4A–S4F0 and5G). Importantly, Notch still accumulated in lgl2,hrs2; UAS-StamRNAi clones (Figures S4H–S4J, S4K, and S4K0). Thus,upregulation of Hrs is not responsible for ectopic Notchsignaling or accumulation of Notch in lgl2 tissue.

Lgl Regulates Notch Signaling Independently of Rab11We then investigated whether the accumulation of Rab11 wasresponsible for increased Notch signaling in lgl2 tissue. Deple-tion of Rab11 alone or in lgl2 clones (using UAS-Rab11RNAi,which strongly decreased Rab11 protein; data not shown)did not reduce E(spl)m8-lacZ expression (Figures S4L–S4M0;quantified in Figure 5G) or Nintra accumulation (Figures S4N–S4O0). Thus, increased Notch activity and levels in lgl2 tissueare not due to accumulation of Rab11.

The Acidic Compartment Is Altered in lgl2 TissueBecause acidification of vesicles is important for g-secretaseactivity and Notch cleavage [20, 40], we examined whetherlgl depletion affected vesicle acidity by using a pH-sensitive,vital dye lysotracker, which detects lysosomes and otheracidic endosomes. In wild-type cells, acidic organelleswere evident by lysotracker uptake in cell membranes andthe cytoplasm (Figure S5A). Importantly, in apical planar sec-tions in lgl2 clones, overlapping and posterior to the MF,strong incorporation of lysotracker was observed (Figures

Figure 5. lgl2 Tissue Accumulates Avl, Hrs,

Rab11, and Lysotracker, but Increased Hrs/

Stam or Rab11 Are Not Responsible for

Increased Notch Signaling

Planar sections (A, C, E, and H) and cross-sec-

tions stained with endocytic markers (B, D, F,

and I). Merges show aPKC staining (B0, D0, andF0, blue).(A–B0) lgl2mosaic discs stained for Avl (red) show

increased Avl in lgl2 tissue (GFP2, arrowheads,

merges in A0 and B0).(C–D0) lgl2 mosaic discs stained for Rab11 (red)

show increased Rab11 in lgl– clones (GFP2,

arrowheads, merges in C0 and D0).(E–F0) lgl2mosaic discs stained for Hrs (red) show

increased Hrs in lgl2 clones (GFP2, arrowheads,

merges in E0 and F0).(G) Quantification of b-gal pixel intensity ratio

between wild-type versus mutant tissue of the

listed genotypes from experiments in Figures

S4A–S4G0 and S4L–S4M0 (***p value < 0.001).

(H–I0) lgl2 mosaic discs stained for lysotracker

(red) show increased lysotracker incorporation

in lgl2 (GFP2, arrowheads, merges in H0 and I0).



5H and 5H0). In cross-sections, apical accumulation of lyso-tracker was also observed in lgl2 tissue (Figures 5I and 5I0),and, also, intense lysotracker staining was detected basally,

consistent with the presence of basallyextruded apoptotic cells (which incor-porate lysotracker) present at theclonal borders [29]. Thus, lgl2 tissueaccumulates acidic intracellularcompartments.

Lgl Upregulation of Notch SignalingRequires Acidification of Endosomes

Because we observed accumulationof acidic vesicles in lgl2 tissue (Fig-ures 5H–5I0), this raised the possibilitythat changes in acidic compartmentsin lgl2 tissue mediate upregulationof Notch signaling. Therefore, weblocked acidification of vesicles in lgl2

tissue, using chloroquine, which ac-cumulates within endosomes and lyso-somes, raising the pH of these intra-cellular compartments.

Reducing vesicle acidity, by exposinglarvae to chloroquine during feeding,was demonstrated by isolating thirdinstar larval eye antennal discs anddetermining lysotracker incorporation.Wild-type or lgl2 mosaic eye discsfrom larvae that were fed control foodincorporated lysotracker (Figures S5A,S5B, and S5B0). In contrast, wild-typeor lgl2 mosaic discs dissected fromlarvae exposed to chloroquine showedreduced lysotracker incorporation (Fig-ures S5C, S5D, and S5D0).

Consistent with our previous results,lgl2 clones in eye discs, from larvaeraised on control food, showedincreased levels of E(spl)m8-lacZ

(Figures 6A–6A0 0, quantified in Figure 6C). Strikingly, exposureof lgl2 tissue to chloroquine reduced E(spl)m8-lacZ to wild-type levels (Figures 6B–6B0 0, quantified in Figure 6C). However,

Figure 6. Increased Notch Signaling in lgl2

Tissue Requires Vesicle Acidification

(A–A0 0) Control lgl2 mosaic discs stained for b-gal

(red) show increased E(spl)lacZ in lgl2 clones

(GFP2, arrowheads, merge in A0 0).(B–B0 0) lgl2 mosaic discs from larvae exposed to

chloroquine stained for b-gal (red) show normal

expression levels of E(spl)lacZ reporter in lgl2

tissue (GFP2, arrowheads, merge in B0 0).(C) Quantification of b-gal pixel intensity ratio

between wild-type and lgl2 clones of the listed

samples (***p value < 0.0001).

(D and E) Adult eyes from wild-type (D) and lgl2

(E) mosaic larvae reared in control food.

(F) Adult eye from an lgl2 mosaic larva reared in

chloroquine food.



lgl2 tissue exposed to chloroquine still accumulated Nintra,Avl, Rab11, and Hrs (Figures S5E–S5H, compared to Figures2C–2C0 0 and 5A–5F0, respectively). Thus, vesicle acidificationis not responsible for accumulation of endocytic compart-ments in lgl2 tissue. Strikingly, lgl2 adult mosaic eyes fromchloroquine-treated larvae were restored to near wild-type(Figures 6D–6F). Thus, reducing vesicle acidity in lgl2 tissueprevented upregulation of Notch signaling and rescued thelgl2 adult mosaic eye phenotype.

lgl Regulates Notch Signaling Independently of the aPKC-Baz-Par6 Complex

Becausemost functions of Lgl are attributed to its regulation ofthe aPKC-Baz-Par6 complex, we investigated whether thisrelationship was maintained with respect to regulation ofNotch targets in the eye disc. Accordingly, we reduced aPKCactivity via a kinase-dead (dominant-negative) aPKC trans-gene (aPKCCAAX-DN) [41] in lgl2 clones and examined E(spl)m8-lacZ expression. Surprisingly, aPKCCAAX-DN expressiondid not suppress upregulation of E(spl)m8-lacZ in lgl2 clones(Figures 7A–7A0 0 0 and 1D–1D0 0, quantified in Figure 7D).Furthermore, depleting Baz (UAS-bazRNAi) failed to suppress

ectopic Notch activation in lgl2 clones(Figures 7B, 7B0, and 7D). Expressionof aPKCCAAX-DN or bazRNAi in lgl2 clonespartially rescued the adult eye defectsof lgl2 mosaics, demonstrating thatthese transgenes are functional (FiguresS6A–S6E). Because we have shown thatexpression of lglWT rescued increasedE(spl)m8-lacZ in lgl2 clones (Figures1E–E00 and 1G) and suppressed the lgl2

mosaic adult eye phenotype (Fig-ure S6F), the failure of aPKCCAAX-DN

and BazRNAi to suppress E(spl)m8-lacZexpression was not due to delayedexpression of transgenes or perduranceof b-galactosidase activity. Consistentwith our findings that reduced aPKCactivity in lgl2 clones had no effect onE(spl)m8-lacZ levels, clones overex-pressing an activated form of aPKC(aPKCCAAX-WT) [41] did not upregulateE(spl)m8-lacZ (Figures 7C, 7C0, and7D). Thus, in contrast to Lgl’s regulationof the Hippo pathway [42], thesedata show that depletion of lgl

upregulates E(spl)m8-lacZ independently of the aPKC-Baz-Par6 complex.

Discussion

In this study, we demonstrate a novel function for the cellpolarity regulator lgl in regulation of endocytosis and Notchsignaling. We show in the developing Drosophila eye epithe-lium that (1) Notch targets, E(spl)m8, CycA, and Rst, are upre-gulated in lgl2 tissue; (2) Notch upregulation contributes to thelgl2mosaic adult eye phenotype; (3) Notch upregulation in lgl2

clones is ligand dependent and requires endocytosis; (4) Lglcolocalizes with intracellular Notch and endocytic markers;(5) lgl2 tissue accumulates Avl+ EEs, Hrs+ MVBs, Rab11+

REs, endocytic compartments, and acidified vesicles, butnot LE markers, Rab7, and Car; (6) Notch upregulation in lgl2

clones is independent of the ESCRT-0 complex (Hrs/Stam)or Rab11, but it requires Rab5 function and acidification of en-dosomes; and (7) Notch upregulation in lgl2 clones is indepen-dent of aPKC-Baz-Par6. Altogether, our data reveal a novelrole for Lgl in attenuating ligand-activated Notch signalingvia restricting the acidification of endocytic compartments,

Figure 7. lgl Regulates Notch Signaling Independently of the aPKC-Par6-Baz Complex

(A andA0 0 0) lgl2;UAS-aPKCCAAX-DNmosaicdisc stained forb-gal (red) shows increasedE(spl)lacZ in lgl2;aPKCCAAX-DN clones (GFP+, arrowheads,merge inA0 0 0).(B and B0) lgl2;UAS-bazRNAi mosaic disc stained for b-gal (red) shows increased E(spl)lacZ in lgl-,bazRNAi clones (GFP+, arrowheads, merge B’).

(C andC0)UAS-aPKCCAAX-WTmosaic disc stained for b-gal (red) shows normalE(spl)lacZ expression in aPKCCAAX-WT clones (GFP+, arrowheads,merge inC0).(D) Quantification of b-gal pixel intensity ratio between wild-type and mutant clones of the listed genotypes (***p value < 0.0001).

(E) Model: Lgl regulates endocytosis and the Notch signaling pathway. Our results show that Lgl regulates Notch signaling by limiting vesicle acidification

and attenuating signaling from ligand-activated Notch, probably at the EE stage (blocking arrows). Lgl also promotes endosomalmaturation, probably at the

early MVB to MVB stage (arrow). Increasing yellow intensity in vesicles indicates increasing acidification.



independently of its role in regulating the aPKC-Baz-Par6 cellpolarity complex (Figure 7E).

Lgl-Mediated Activation of Notch SignalingOur data have revealed that increased vesicle acidificationin lgl2 tissue is responsible for elevated Notch signaling.Because S3 cleavage (by g-secretase) of Next to formNicd de-pends on acidification of endosomes [18, 20, 40], this suggeststhat increased vesicle acidification in lgl2 tissue leads to

aberrant g-secretase activity and cleavage of Next to Nicd, re-sulting in upregulation of Notch signaling (Figure 7E).The precise endocytic compartment in which the Notch re-

ceptor undergoes g-secretase-mediated S3 proteolytic pro-cessing is controversial (reviewed by [21, 22]). In Drosophilaepithelial tissues, V-ATPase function is implicated in Notchactivation in the EEs or the MVBs [40]; however, whether thisis ligand dependent or independent is unclear. In contrast,ligand-independent generation of Nicd in the LE and/or



lysosome compartment has been described [43]. Becauseincreased Notch signaling in lgl2 tissue depends on Rab5 EEactivity, but Rab7 and Car LE compartments were not per-turbed in lgl2 tissue, we favor a model in which Lgl restrictsvesicle acidification and activation of Notch signaling in theEE and/or early MVB compartments.

We speculate that Lgl regulates Notch signaling by twopossible mechanisms: (1) via direct regulation of vesicle acid-ification or (2) via regulation of endosomal maturation, whichindirectly affects vesicle acidification. In the first model, Lglmight inhibit V-ATPase activity by regulating levels and/or sub-unit composition or the association and/or dissociation of theV-ATPase complex. In the second model, Lgl might regulateendosomal maturation, and alterations in this process subse-quently lead to accumulation of acidic vesicles and ectopicNotch activation. Our data showing that lgl2 tissue accumu-lates EEs (Avl+), REs (Rab11+), and early MVBs (Hrs+), but notLEs (Rab7+ or Car+), suggest that Lgl regulates a specificstep in endosome maturation after early MVB formation.Further studies are required to determine whether Lgl controlsvesicle acidification by affecting the V-ATPase or endosomematuration to regulate Notch signaling.

The Relationship among Cell Polarity, Endocytosis, andNotch Signaling

Previous work has revealed that alterations in componentsof endocytic compartments, such as Rab5 overexpression ormutation of tsg101/ept or vps25, disrupt epithelial cell polarityand upregulate Notch signaling (reviewed by [3]). However,in these cases, it is unclear whether perturbation of endo-cytosis alters Notch signaling via cell polarity disruption orwhether changes in endocytic compartments directly impacton Notch. We show that without cell polarity loss, lgl2 tissuedisplays altered endocytic compartments and upregulatesNotch signaling, indicating that changes in endocytosisalone are sufficient to upregulateNotch pathway activity.More-over, we show that reducing aPKC-Baz-Par6 complex activitydoes not rescue Notch pathway upregulation in lgl2 tissue,revealing that Lgl’s roles in regulating cell polarity and endocy-tosis are separable. Interestingly, the Crb polarity protein alsohas separable roles in the regulation of cell polarity and Notchsignaling and endocytosis [44], via differentmechanisms to Lgl.

Lgl, Notch, and Tumorigenesis

Our discovery that Lgl depletion increases Notch activationwithout cell polarity loss has implications for tumorigenesis.In the developing eye epithelium, increased Notch signalingresults in upregulation of the cell cycle regulator, CycA, andthe cell survival regulator, Rst [25, 26, 28], which in lgl2 tissue,is expected to contribute to increased cell proliferation andsurvival, concomitant with impaired Hippo signaling [30].Because elevated Notch signaling is associated with varioushuman cancers (reviewed by [45, 46]), our finding that Lglregulates Notch signaling warrants investigation of whetherelevated Notch signaling in human cancer is associated withLgl depletion. Notably, Lgl1 knockout in the mouse braininduces hyperproliferation and decreased differentiation,associated with increased Notch signaling [47], and mutationof zebrafish Lgl disrupts retinal neurogenesis, dependent onincreased Notch signaling [48]. Moreover, our finding that Lglplays a novel role in regulating endosomal acidificationand the striking suppressive effect of chloroquine on the adultlgl2mosaic phenotype reveal the importance of acidification intumor growth, perhaps by also modulating Hippo signaling.

Our data, together with evidence that many cancers showhigher acidity due to increased V-ATPase activity, which con-tributes to tumorigenesis (reviewed by [49]), posit the questionof whether Lgl dysfunction might contribute to acidificationdefects in human cancer.

Experimental Procedures

Mutants and Transgenes

Fly stocks were as follows: lgl27S3 [29]; E(spl)lacZm8-2.61 (on 2R [50]); UAS-

GFP-lamp1 (H. Kramer); UAS-aPKCCAAX-WT and UAS-aPKCCAAX-DN [41];

UAS-bazRNAi (5055R-1, National Institute of Genetics); UAS-lglWT (J. Kno-

blich); FRT82B Dlrev10SerRX106 (N. Baker); hrsD28FRT40 (H. Bellen); UAS-

stamRNAi (v22497); UAS-Rab5RNAi (v34096) and UAS-Rab11RNAi (v22198)

(Vienna Drosophila Resource Center); w1118; GMR-GAL4; UAS-shiK44A;

UAS-mamDN (Bloomington Stock Center). Eye disc clones were generated

using the following: ey-FLP, UAS-GFP; tub-GAL80,FRT40A; tub-GAL4/

TM6B and ey-FLP,UAS-mCD8-GFP;;tub-GAL4,FRT82B,tub-GAL80/TM6B

[51]; ey-FLP/FM7; UAS-myrRFP,tub-GAL4,FRT82B,tub-GAL80/TM6B [52]

or ey-FLP; FRT40,Ubi-GFP (A. Bergmann).

Antibodies for Immunofluorescence

Third instar larval eye antennal discs were dissected in PBS, fixed in 4%

paraformaldehyde for 30 min, washed in PBS plus 0.1% or 0.3% Triton

X-100 (PBT), and blocked in PBT plus 1% BSA.

Antibodies used were as follows: mouse b-galactosidase (Sigma, 1:500),

rabbit Lgl (J. Knoblich, 1:500), mouse Nintra (Developmental Studies

Hybridoma Bank [DSHB], 1:50), mouse Nextra (DSHB, 1:50), mouse Dl

(DSHB, 1:10), rabbit E-Cad (DSHB, 1:50), rabbit aPKC (Santa Cruz, PKCz,

1:500), chicken Avl (D. Bilder, 1:1,000), guinea pig Hrs (H. Bellen, 1:500),

mouse Rab11 (BD Biosciences, 1:100), mouse Rab7 (A. Nakamura,

1:2,000), rabbit Car and guinea pig Dor (H. Kramer, 1:100), mouse anti-IrreC-

Rst (mAb 24A5.1) (K. Fischbach, 1:50), and lysotracker red DND-99

(Invitrogen).

Secondary antibodies used were as follows: anti-mouse Alexa 488, 568,

and 633, anti-rabbit Alexa 488, 568, and 633, anti-chicken Alexa 568, and

anti-guinea pig Alexa 568 and 633. DNA was stained with 2-(4-amidino-

phenyl)-1H-indole-6-carboxamidine (DAPI, 1mM).

Supplemental Information

Supplemental Information includes Supplemental Experimental Procedures

and six figures and can be found with this article online at http://dx.doi.org/

10.1016/j.cub.2014.07.075.

Author Contributions

L.M.P. designed and performed the experiments, analyzed the data, and

wrote and edited the paper. M.P. designed and performed the experiments,

analyzed the data, compiled the figures, and wrote and edited the paper.

N.A.G. designed and performed the experiments, analyzed the data, and

edited the paper. H.E.R. conceived the study, analyzed the data, and wrote

and edited the paper.

Acknowledgments

We thank Sarah Russell and Kirsten Allan for critiquing the manuscript, and

we thank Sonsoles Campuzano, Thomas Vaccari, Sally Dunwoodie, Paul

Gleeson, Patrick Humbert, and Annette Shewan for discussions. We are

grateful to all those who contributed fly stocks or antibodies to this study,

to Bloomington, VDRC, and National Institute of Genetics Stock Centers,

toOzDros, and to Flybase. H.E.R. is supported by a Senior Research Fellow-

ship from the National Health and Medical Research Council (NHMRC)

Australia. This work was supported by grants from the NHMRC (#628401

to H.E.R. and N.A.G.), the Cancer Council Australia (#APP1041817 to

H.E.R.), and the Contributing to Australian Scholarship and Science Foun-

dation (#SM/13/4847 to L.M.P.).

Received: January 28, 2014

Revised: July 1, 2014

Accepted: July 28, 2014

Published: September 11, 2014





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Current Biology, Volume 24

Supplemental Information

Lgl Regulates Notch Signaling

via Endocytosis, Independently of the

Apical aPKC-Par6-Baz Polarity Complex

Linda M. Parsons, Marta Portela, Nicola A. Grzeschik, and Helena E. Richardson

Figure S1: MamDN rescues increased Notch signalling and the Notch target CycA is upregulated in lgl- tissue – related to Figure 1

Confocal planar sections of larval eye-antennal discs. White scale bar represents 50μM unless otherwise stated. Posterior is to the left and apical is up in this and all other figures. Yellow bar indicates morphogenetic furrow (MF). Arrowheads denote mutant tissue in this and all other figures. Dots outline clonal borders in this and all other figures.

(A) UAS-mamDN mosaic disc stained for βGal (red in A and A’’ merge, gray in A’) showing inhibition of E(spl)lacZ levels in (GFP+) mutant tissue compared to wild-type (arrowheads). Genotype: eyFLP, UAS-GFP; FRT40, E(spl)m8-lacZ; UAS-mamDN/tubgal4.

(B) lgl-, UAS-mamDN mosaic disc stained for βGal (red in B and B’’ merge, gray in B) showing similar E(spl)lacZ levels in (GFP+) lgl mutant tissue to wild-type (arrowheads). Genotype: eyFLP, UAS-GFP; lgl27S3, FRT40, E(spl)m8-lacZ; UAS-mamDN/tubgal4.

(C) lgl- mosaic disc (clones outlined in green) stained for CycA (red in C and C’’ merge, gray in C) showing upregulation of CycA in lgl (GFP-) mutant tissue (arrowheads). Genotype: eyFLP; lgl27S3, FRT40, E(spl)m8-lacZ / Ubi-GFP, FRT40.

(D) UAS-mamDN mosaic disc at ~45% pupal development stained for Rst (gray in D, red in D’, merge) showing similar Rst levels in (GFP+) mutant tissue to wild-type (arrowheads), suggesting that endogenous Rst expression is not as sensitive to Mam inhibition as E(spl)m8-lacZ (see Figure S1A). Genotype: eyFLP, UAS-GFP; FRT40, E(spl)m8-lacZ; UAS-mamDN/tubgal4.

(E) UAS-mamDN mosaic adult female eye image showing slight disorganization. Genotype: eyFLP, UAS-GFP; FRT40, E(spl)m8-lacZ; UAS-mamDN/tubgal4.

(F) Quantification of Rst staining pixel intensity ratio between wild-type clones versus mutant clones from the experiments shown in Figure 1H-J and Figure S1D (*** P-value<0.0001, differences not significant (n.s.)).

Figure S2: Notch signaling pathway components localization in wild-type or lgl-

mosaic eye tissue and localization of other proteins – related to Figure 2

Genotype in panels (A-C): w1118.

(A) wild-type eye disc stained with Nintra (red).

(B) wild-type eye disc stained with Delta (red).

(C) wild-type eye disc stained with Nextra (red).

Genotype in panels (D-H): eyFLP; lgl27S3, FRT40, E(spl)m8-lacZ / Ubi-GFP, FRT40. Mutant tissue is GFP negative.

(D) lgl-, E(spl)lacZ mosaic disc stained with Nintra (red). Nintra staining accumulated in lgl mutant clones (GFP-, arrowheads). The merge is shown in D”.

(E) Magnification of lgl-, E(spl)lacZ mosaic disc stained with Nintra (red). The merge is shown in E”.

(F) lgl- mosaic discs stained for Nextra (red) showing normal Nextra levels and localization in lgl- clones (GFP-, arrowheads). The merge is shown in F''.

(G, H) Nextra antibody uptake assay: lgl- mosaic disc stained with the Nextra antibody (red) reveals no differences between lgl- (GFP-) and wild-type clones at 0hr (G) and 1hr (H) after uptake (arrowheads, G’’ and H’’ merges).

Figure S3: Endocytic marker localization in wild-type discs, Notch and Hrs colocalization, and localization of Rab7, Car and ECad in lgl mutant mosaic tissue – related to Figure 5

Confocal planar sections of lgl mutant mosaic larval eye-antennal disc. Mutant tissue is GFP negative.

Genotype in panels (A-C): w1118.

(A, B and C) wild-type eye discs stained with Avl, Rab11 or Hrs (gray), respectively.

Genotype in panels (D-H): eyFLP; lgl27S3, FRT40, E(spl)m8-lacZ / Ubi-GFP, FRT40.

(D) Nintra staining (gray) showing increased Nintra staining in lglmutant tissue (arrowheads).

(D’) Hrs staining (gray) showing increased Hrs staining in lglmutant tissue (arrowheads).

(D’’) Nintra (red) and Hrs (cyan) co-staining showing partial colocalization of Nintra and Hrs (arrowheads).

(D’’’) Nintra (red) and GFP merge showing the mosaic tissue and the accumulation of Nintra in lglmutant tissue (GFP-, arrowheads). Inset shows DAPI in blue.

(Div) Hrs (blue) and GFP merge showing the mosaic tissue and the accumulation of Hrs in lglmutant tissue (GFP-, arrowheads). Inset shows DAPI in blue.

(Dv) Merge showing Nintra (red), Hrs (blue) and GFP.

(E) lgl- mosaic discs stained for Rab7 (red) showing normal levels and localization of Rab7 in lgl mutant tissue (GFP-, arrowheads). The merge is shown in E’’.

(F) lgl- mosaic discs stained for Car (red in F and F’’ merge, gray in F’) showing normal levels and localization of Car in lgl mutant tissue (GFP-, arrowheads). The merge is shown in F’’

(G-H) lgl- mosaic discs stained for ECad (red in G, and in the merges G’’ and H’, gray in G’, H) showing normal levels and localization of ECad in lgl mutant tissue (GFP-, arrowheads) in a planar section (F) and in a cross-section (G). The merges are shown in G’’ and H’.

Figure S4: Increased Notch signaling in lgl- tissue is independent of the ESCRT-0 complex proteins (Hrs/Stam) and Rab11 – related to Figure 5

(A-B) hrsD28 or UAS-stam-RNAi mosaic disc stained for βGal (red) show normal E(spl)lacZ expression (arrowheads, merge A’-B’). hrsD28 mutant tissue is GFP-negative, UAS-stam-RNAi mutant tissue is GFP-positive. Genotypes: eyFLP; hrsD28, E(spl)m8-lacZ, FRT40/ Ubi-GFP, FRT40. eyFLP, UAS-GFP; FRT40, E(spl)m8-lacZ; UAS-stamRNAi/tubgal4.

(C-D) hrsD28 or UAS-stam-RNAi mosaic disc stained for Hrs, show that mutant clones (GFP- for hrsD28 or GFP+ for UAS-stam-RNAi) lack Hrs protein (gray). Insets shows merge, Hrs staining purple. Genotypes: eyFLP; hrsD28, E(spl)m8-lacZ, FRT40/ Ubi-GFP, FRT40 or eyFLP, UAS-GFP; FRT40, E(spl)m8-lacZ; UAS-stamRNAi/tubgal4.

(E-F) lgl-, hrsD28 or lgl-; UAS-stam-RNAi mosaic disc stained for βGal (red) show upregulation of E(spl)lacZ reporter in mutant clones (GFP- for lgl-, hrsD28 or GFP+ for lgl-; UAS-stam-RNAi, arrowheads, merges E’,F’). Genotypes: eyFLP; lgl27S3,hrsD28, FRT40, E(spl)m8-lacZ / Ubi-GFP, FRT40 or eyFLP, UAS-GFP; lgl27S3, FRT40, E(spl)m8-lacZ; UAS-stamRNAi/tubgal4.

(G) lgl-, hrs-; UAS-stam-RNAi mosaic disc stained for βGal (red) showing upregulation of E(spl)lacZ reporter in mutant clones (GFP+, arrowheads, merge G’). Genotype: eyFLP, UAS-GFP; lgl27S3, hrsD28, FRT40, E(spl)m8-lacZ; UAS-stamRNAi/tubgal4.

(H) hrsD28 mosaic disc stained for Nintra (red) showing normal Notch localization (GFP-, merge H’). Genotype: eyFLP; hrsD28, FRT40, E(spl)m8-lacZ / Ubi-GFP, FRT40.

(I-J) lgl-, hrsD28 or lgl-; UAS-stam-RNAi mosaic disc stained for Nintra (red) shows accumulation of Notch in mutant clones (GFP- for lgl-, hrsD28 or GFP+ for lgl-; UAS-stam-RNAi, merges I’ and J’). Genotypes: eyFLP; lgl27S3, hrsD28, FRT40, E(spl)m8-lacZ / Ubi-GFP, FRT40 or eyFLP, UAS-GFP; lgl27S3, FRT40, E(spl)m8-lacZ; UAS-stamRNAi/tubgal4.

(K) lgl-, hrs-; UAS-stam-RNAi mosaic disc stained for Nintra (red) showing accumulation of Notch in mutant clones (GFP- for lgl-, hrsD28 or GFP+ for lgl-; UAS-stam-RNAi, merge K’). Genotype: eyFLP, UAS-GFP; lgl27S3, hrsD28, FRT40, E(spl)m8-lacZ; UAS-stamRNAi/tubgal4.

(L) E(spl)lacZ; Rab11RNAi mosaic disc stained for βGal (red) showing normal expression of E(spl)lacZ reporter in Rab11RNAi clones (GFP+, arrowheads). The merge is shown in L’. Genotype: eyFLP, UAS-GFP; FRT40, E(spl)m8-lacZ; UAS-Rab11RNAi/tubgal4.

(M) lgl-, E(spl)lacZ; Rab11RNAi mosaic disc mosaic disc stained for βGal (red) showing upregulation of E(spl)lacZ in lgl; Rab11RNAi clones (GFP+, arrowheads). The merge is shown in M’. Genotype: eyFLP, UAS-GFP; lgl27S3, FRT40, E(spl)m8-lacZ; UAS-Rab11RNAi/tubgal4.

(N) E(spl)lacZ; Rab11RNAi mosaic disc stained for Nintra (red) showing normal Notch localization in Rab11RNAi clones (GFP+, arrowheads). The merge is shown in N’. Please note that there is a hole in the tissue (assessed by DAPI staining) where the antibodies accumulate showing a false increased staining. Genotype: eyFLP, UAS-GFP; FRT40, E(spl)m8-lacZ; UAS-Rab11RNAi/tubgal4.

(O) lgl-, E(spl)lacZ; Rab11RNAi mosaic disc mosaic disc stained for Nintra (red) showing accumulation of Notch in lgl; Rab11RNAi clones (GFP+ , arrowheads). The merge is shown in O’. Genotype: eyFLP, UAS-GFP; lgl27S3, FRT40, E(spl)m8-lacZ; UAS-Rab11RNAi/tubgal4.

Figure S5: Chloroquine treatment prevents Lysotracker incorporation but does not affect the accumulation of endocytic markers and Nintra - related to Figure 6

Genotype in (A, C) w1118, and (B, D-H), eyFLP; lgl27S3, FRT40, E(spl)m8-lacZ / Ubi-GFP, FRT40. lgl mutant tissue is GFP negative

(A) wild-type disc incubated with Lysotracker (gray) from larvae reared in fly food with PBS (control).

(B) lgl- mosaic disc incubated with Lysotracker (LT, gray) from larvae reared in fly food with PBS (control), show accumulation of Lysotracker in lgl mutant clones (GFP-). The Merge is shown in B’.

(C) wild-type disc incubated with Lysotracker (gray) from larvae reared in fly food with 1mg/ml of chloroquine showing no Lysotracker incorporation in the cells throughout the whole tissue.

(D) lgl- mosaic discs incubated with Lysotracker (gray) from larvae reared in fly food with 1mg/ml of chloroquine, showing no Lysotracker incorporation in the wild-type tissue and in lgl (GFP-) mutant tissue as well. The Merge is shown in D’.

(E-H) lgl- mosaic discs incubated with Nintra (E), Avl (F), Rab11(G) and Hrs (H) (red) from larvae reared in fly food with 1mg/ml of chloroquine, still showing increased levels of Nintra (E), Avl (F), Rab11(G) and Hrs (H) staining in lgl mutant clones (GFP-, arrowheads). The Merges are shown in E’, F’, G’ and H’.

Figure S6: Suppression of the lgl mutant mosaic adult phenotype by blocking aPKC or Baz – related to Figure 7

Adult eye images (side views and top views), showing the effect of expression of Lgl wild-type (lglWT), aPKC dominant negative (aPKCCAAX-DN) and knockdown of Baz (baz-RNAi) using the MARCM system on the lgl mutant mosaic phenotype. Expression of aPKCCAAX-DN shows strong rescue of the lgl mutant mosaic phenotype (B-B’), but defects in ommatidial alignment are still observed (C-C’), whereas baz-RNAi shows only a partial rescue (D-D’). Expression of lglWT fully rescues the lgl mutant mosaic phenotype (F-F’). Expression of baz-RNAi alone does not affect the adult eye phenotype (D-D’) relative to the control (A-A’). aPKCCAAX-DN also does not alter the adult eye phenotype (not shown).

(A-A’) Control. Genotype: eyFLP; FRT40/ Ubi-GFP, FRT40.

(B-B’) lgl-. Genotype: eyFLP; lgl27S3, FRT40/ Ubi-GFP, FRT40.

(C-C’) lgl-; aPKCCAAX-DN. Genotype: eyFLP, UAS-GFP; lgl2733, FRT40, E(spl)m8-lacZ; UAS-aPKC CAAX-DN/tubgal4.

(D-D’) bazRNAi. Genotype: eyFLP, UAS-GFP; FRT40, E(spl)m8-lacZ; UAS-bazRNAi/tubgal4.

(E-E’) lgl27S3; bazRNAi. Genotype: eyFLP, UAS-GFP; lgl2733, FRT40, E(spl)m8-lacZ; UAS-bazRNAi/tubgal4.

(F-F’) lgl27S3; lglWT. Genotype: eyFLP, UAS-GFP; lgl2733, FRT40, E(spl)m8-lacZ; UAS-lglWT/tubgal4.

Supplemental Experimental Procedures

Genotypes of images in Main Figures

Figure 1: lgl regulates Notch signaling in the Drosophila eye epithelia

1C. eyFLP; FRT40, E(spl)m8-lacZ /Ubi-GFP, FRT40

1D. ; lgl27S3, FRT40, E(spl)m8-lacZ/Ubi-GFP, FRT40. eyFLP

1E. eyFLP, UAS-GFP; lgl27S3, FRT40, E(spl)m8-lacZ; UAS-lglWT/tubgal4.

1H eyFLP, UAS-GFP; lgl27S3, FRT40, E(spl)m8-lacZ; +/tubgal4. .

1I. UAS-GFP; lgl27S3, FRT40, E(spl)m8-lacZ; +/tubgal4.

1J. eyFLP, UAS-GFP; lgl27S3, FRT40, E(spl)m8-lacZ; UAS-mamDN/tubgal4.

1

K. eyFLP, UAS-GFP; lgl27S3, FRT40, E(spl)m8-lacZ; UAS-mamDN/tubgal4.

Figure 2: Cleaved Notch levels are elevated in lgl- tissue

G

enotype in all panels eyFLP; lgl27S3, FRT40, E(spl)m8-lacZ / Ubi-GFP, FRT40.

Figure 3: Notch signaling in lgl- tissue is ligand-dependent and requires endocytosis

(A, B) eyFLP/+; lgl27S3, FRT40, E(spl)m8-lacZ /Ubi-GFP, FRT40; FRT82B, Dlrev10, SerRX105/ UAS-RFP, tubgal4, FRT82B.

(C) eyFLP, UAS-GFP; FRT40, E(spl)m8-lacZ; UAS-shiK44A/tubgal4.

(D) eyFLP, UAS-GFP; lgl27S3, FRT40, E(spl)m8-lacZ; UAS-shiK44A /tubgal4.

(E, F) eyFLP, UAS-GFP; FRT40, E(spl)m8-lacZ; UAS-Rab5-RNAi/tubgal4.

Figure 4: Lgl co-localizes with EE, MVB, RE, LE and Lysosomal compartments

(A,B) eyFLP; lgl27S3, FRT40, E(spl)m8-lacZ / Ubi-GFP, FRT40

(C-G) w1118

(H) GMR-GAL4 UAS-GFP-lamp1

Figure 5: lgl- tissue accumulates Avl, Hrs, Rab11 and lysotracker but increased Hrs/Stam or Rab11 are not responsible for the increased Notch signaling

Genotype in all panels eyFLP; lgl27S3, FRT40, E(spl)m8-lacZ / Ubi-GFP, FRT40.

Figure 6: Increased Notch signaling in lgl- tissue requires vesicle acidification

A,B) eyFLP; lgl27S3, FRT40, E(spl)m8-lacZ / Ubi-GFP, FRT40.

Figure 7: lgl regulates Notch signaling independently of the aPKC-Par6-Baz complex

A) eyFLP, UAS-GFP; lgl27S3, FRT40, E(spl)m8-lacZ; UAS-aPKCDN/tubgal4.

B) eyFLP, UAS-GFP; lgl27S3, FRT40, E(spl)m8-lacZ; UAS-bazRNAi/tubgal4.

C) eyFLP, UAS-GFP; FRT40, E(spl)m8-lacZ, UAS-aPKCCAAXWT; +/tubgal4.

Clonal Analysis

Negatively marked mutant clones were generated using the ey-FLP/FRT system [S1].

Mosaic analysis with a repressible cell marker (MARCM (GFP+)) clones were

generated as previously described [S2]. Flies were raised on standard cornmeal agar

food at 25ºC unless stated otherwise.

Notch Uptake Assay

Third-instar larval eye-antennal discs were dissected on ice in S2 cell medium

(Schneider Gibco, Life Technologies), incubated with anti-Nextra (DSHB, 1:50) for

2h at 4C (pulse). An aliquot was fixed in 4% paraformaldehyde for 30’ (0h time

point). Warm medium was added to the remaining sample and incubated 1h at RT (1h

chase). An aliquot was fixed (1h time point). The sample was incubated a further 4h

at RT (6h chase). The last aliquot was fixed (5h time point). After fixation samples

were subjected to standard immunofluorescence protocol (see above).

Lysotracker Assay

Third-instar larval eye-antennal discs were dissected in PBS, incubated with

lysotracker red DND-99 (Invitrogen) for 5min. Fixed in 4% paraformaldehyde for

30min, washed in PBS and mounted in 80% glycerol.

Chloroquine Experiment

1mg/ml of chloroquine (Sigma) diluted in PBS or just PBS (control) was added to

standard cornmeal agar food. Crosses were set up in normal vials and transferred 24h

later to chloroquine or PBS vials. F1 third-instar larvae were collected, dissected and

processed as described above.

Imaging

Fluorescent labeled samples were mounted in 80% glycerol and analyzed by Confocal

microscopy (Bio-Rad MRC1000, Olympus FV1000 or LEICA TCS SP5). Images

were processed using Confocal AssistantR, Fluorview software and Leica LAS AF

Lite. Images were assembled using Adobe Photoshop CS5.1. Adult eyes were imaged

with a Scitec Infinity1 camera.

Statistical Analysis of Signal Intensity

Relative E(spl)m8-lacZ staining within eye discs was determined from images taken

at the same confocal settings. Average pixel intensity was measured using

measurement log tool from Photoshop 5.1 in an area between 400 and 2500 pixels

depending on clone size, Clones were chosen just posterior to the MF of each eye

disc. Average pixel intensity was measured in mutant clones and the wt tissue

adjacent tissue (N=~15 for each sample) and expressed as a ratio of mutant clone

pixel intensity /wt tissue pixel intensity. Data was analysed and plotted using

Microsoft Excel 2003, using T-Test statistical analysis. Error bars represent Standard

Error of the Mean, significance was p≤0.0001.

Statistical Analysis of Rst Signal Intensity

Relative Rst staining within pupal eye discs was determined from images taken at the

same confocal settings. Average pixel intensity along a line drawn between the

borders IOCs, or IOCs and primary pigment cells was measured using measurement

tool from Fiji. Average pixel intensity was measured from a minimum of twenty

independent cell borders from 5 mutant clones and the wild-type adjacent tissue

(N=~100 cell borders for each sample) and expressed as a ratio of mutant clone pixel

intensity/wt tissue pixel intensity. Data was analysed and plotted using Graphpad

Prism, unpaired t test with Welch’s correction. Error bars represent Standard Error of

the Mean, significance was p≤0.05.

Supplemental References

S1. Xu, T., and Rubin, G.M. (1993). Analysis of genetic mosaics in developingand adult Drosophila tissues. Development 117, 1223‐1237.

S2. Grzeschik, N.A., Parsons, L.M., Allott, M.L., Harvey, K.F., and Richardson, H.E. (2010). Lgl, aPKC, and Crumbs regulate the Salvador/Warts/Hippo pathway through two distinct mechanisms. Curr Biol 20, 573‐581.

lgl regulates notch signaling via endocytosis ... · outline clonal borders in this ﬁgure and all...

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