Role of conserved intracellular motifs in Serrate signalling, cis-inhibition and endocytosis
2006; Springer Nature; Volume: 25; Issue: 20 Linguagem: Inglês
10.1038/sj.emboj.7601337
ISSN1460-2075
AutoresMarcus Glittenberg, Chrysoula Pitsouli, Clare Garvey, Christos Delidakis, Sarah J. Bray,
Tópico(s)Developmental Biology and Gene Regulation
ResumoArticle28 September 2006free access Role of conserved intracellular motifs in Serrate signalling, cis-inhibition and endocytosis Marcus Glittenberg Marcus Glittenberg Department of Physiology Development and Neuroscience, University of Cambridge, Cambridge, UK Search for more papers by this author Chrysoula Pitsouli Chrysoula Pitsouli Institute of Molecular Biology and Biotechnology, FORTH, and Department of Biology, University of Crete, Heraklion, Greece Search for more papers by this author Clare Garvey Clare Garvey Department of Physiology Development and Neuroscience, University of Cambridge, Cambridge, UK Search for more papers by this author Christos Delidakis Christos Delidakis Institute of Molecular Biology and Biotechnology, FORTH, and Department of Biology, University of Crete, Heraklion, Greece Search for more papers by this author Sarah Bray Corresponding Author Sarah Bray Department of Physiology Development and Neuroscience, University of Cambridge, Cambridge, UK Search for more papers by this author Marcus Glittenberg Marcus Glittenberg Department of Physiology Development and Neuroscience, University of Cambridge, Cambridge, UK Search for more papers by this author Chrysoula Pitsouli Chrysoula Pitsouli Institute of Molecular Biology and Biotechnology, FORTH, and Department of Biology, University of Crete, Heraklion, Greece Search for more papers by this author Clare Garvey Clare Garvey Department of Physiology Development and Neuroscience, University of Cambridge, Cambridge, UK Search for more papers by this author Christos Delidakis Christos Delidakis Institute of Molecular Biology and Biotechnology, FORTH, and Department of Biology, University of Crete, Heraklion, Greece Search for more papers by this author Sarah Bray Corresponding Author Sarah Bray Department of Physiology Development and Neuroscience, University of Cambridge, Cambridge, UK Search for more papers by this author Author Information Marcus Glittenberg1, Chrysoula Pitsouli2, Clare Garvey1, Christos Delidakis2 and Sarah Bray 1 1Department of Physiology Development and Neuroscience, University of Cambridge, Cambridge, UK 2Institute of Molecular Biology and Biotechnology, FORTH, and Department of Biology, University of Crete, Heraklion, Greece *Corresponding author. Department of Physiology Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK. Tel.: +44 1223 333792; Fax: +44 1223 333786; E-mail: [email protected] The EMBO Journal (2006)25:4697-4706https://doi.org/10.1038/sj.emboj.7601337 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Notch is the receptor in a signalling pathway that operates in a diverse spectrum of developmental processes. Its ligands (e.g. Serrate) are transmembrane proteins whose signalling competence is regulated by the endocytosis-promoting E3 ubiquitin ligases, Mindbomb1 and Neuralized. The ligands also inhibit Notch present in the same cell (cis-inhibition). Here, we identify two conserved motifs in the intracellular domain of Serrate that are required for efficient endocytosis. The first, a dileucine motif, is dispensable for trans-activation and cis-inhibition despite the endocytic defect, demonstrating that signalling can be separated from bulk endocytosis. The second, a novel motif, is necessary for interactions with Mindbomb1/Neuralized and is strictly required for Serrate to trans-activate and internalise efficiently but not for it to inhibit Notch signalling. Cis-inhibition is compromised when an ER retention signal is added to Serrate, or when the levels of Neuralized are increased, and together these data indicate that cis-inhibitory interactions occur at the cell surface. The balance of ubiquitinated/unubiquitinated ligand will thus affect the signalling capacity of the cell at several levels. Introduction Notch is the receptor in a highly conserved cell:cell signalling pathway that is essential in a multitude of developmental processes (Artavanis-Tsakonas et al, 1999; Schweisguth, 2004). Notch activity is also important in maintenance of many tissues, through its activity in stem cells and/or in regulating the production and fates of differentiated cell types. In the majority of processes, Notch receptors are activated by a class of ligands referred to as DSL ligands, which are themselves type I integral membrane proteins (Fleming, 1998). Intriguingly, many of these ligands have the capacity to act as both agonists and antagonists of the receptor (e.g. de Celis and Bray, 1997; Klein et al, 1997; Micchelli et al, 1997; Sakamoto et al, 2002; Li and Baker, 2004). The complex behaviours of Notch ligands have perhaps been best characterised in the Drosophila wing. Drosophila Notch has two ligands, Delta and Serrate. Like all members of this family, their extracellular domain consists largely of EGF repeats and an N-terminal region containing the conserved DSL motif. This motif is necessary for ligands to bind Notch and to confer full function in vivo (Muskavitch, 1994; Henderson et al, 1997; Fleming, 1998; Shimizu et al, 1999), although in one case a ligand lacking the DSL was reported to activate Notch (Kiyota and Kinoshita, 2002). In the developing wing the two ligands preferentially activate Notch in different compartments: Serrate activates Notch in ventral cells and Delta preferentially activates Notch in dorsal cells (Irvine, 1999). This difference in activating capacity occurs because of differential glycosylation of the Notch receptor by Fringe, which is present in dorsal cells and modifies sites in the receptor that alter its affinities for the two ligands (Irvine, 1999; Bruckner et al, 2000; Moloney et al, 2000; Okajima et al, 2003). At late stages in wing development, the domain of Notch activation is also limited by the expression of the ligands themselves, since the pathway becomes ectopically activated in clones of mutant cells that lack both ligands (Micchelli et al, 1997). This demonstrates that Notch can be inhibited by the presence of excess ligand in the same cell (cis-inhibition; de Celis and Bray, 1997; Klein et al, 1997; Micchelli et al, 1997; Sakamoto et al, 2002; Li and Baker, 2004). Ligand signalling activity is regulated post-translationally through the actions of two E3 ubiquitin ligases, Mindbomb1 (Mib1) and Neuralized (Neur) (Lai et al, 2001, 2005; Pavlopoulos et al, 2001; Itoh et al, 2003; Koo et al, 2005; Le Borgne et al, 2005b). In Drosophila the two ligases can functionally substitute for one another in at least some processes, indicating that they perform similar functions even though they share no obvious sequence similarity apart from a canonical RING domain (Lai et al, 2005; Le Borgne et al, 2005b; Pitsouli and Delidakis, 2005). Mib1 and Neur also regulate trafficking of the Notch ligands, but how ubiquitination and/or trafficking renders the ligands active for signalling remains unclear (Overstreet et al, 2004; Lai et al, 2005; Le Borgne et al, 2005b; Pitsouli and Delidakis, 2005; Wang and Struhl, 2005). However, the importance of trafficking is underscored by the ability of an endocytic motif from an unrelated trans-membrane protein to substitute for the Delta intracellular domain (Wang and Struhl, 2004, 2005). It is evident therefore that Notch ligands undergo complex regulation. Here, we have investigated what parts of Serrate are required for regulating its transport and signalling/inhibitory functions. To do this we have examined the effects of expressing altered Serrate ligands in the developing wing. Our results identify a conserved intracellular motif that is essential for Notch activation and for an interaction with Mib1 and Neur. We also show that Serrate mediated cis-inhibition requires the N-terminal/DSL domain, and occurs through Notch–Serrate interactions at the cell surface. Results Effects of ectopic Serrate on Notch activity In the developing wing imaginal disc Serrate is expressed primarily in the dorsal compartment, from where it activates Notch at the d/v boundary to induce expression of genes such as E(spl)mβ, cut and wingless (Figure 1A; e.g. de Celis et al, 1996; Neumann and Cohen, 1996). To investigate which parts of Serrate are required for its functions, we expressed wild-type (SerWT) and mutant forms of the ligand in a stripe orthogonal to the d/v boundary (using ptc∷Gal4; Figure 1B) and examined their affect on Notch activity. Two major effects were seen with SerWT as described previously (e.g. Speicher et al, 1994; Klein et al, 1997). Firstly, the ligand induced two stripes of ectopic Notch activation in the ventral compartment, as manifest by expression of Cut adjacent to cells with highest SerWT (trans-activation; Figures 1C and 2A). Ectopic Notch signalling is restricted to ventral cells by the activity of Fringe in dorsal cells, which modifies the receptor making it refractory to activation by Serrate (Irvine, 1999). Secondly, in those cells where SerWT was present at higher levels it inhibited endogenous Notch activation at the d/v boundary (Figures 1C and 2A). This cell autonomous inhibitory effect is referred to as cis-inhibition. Figure 1.Wing disc assay for Serrate functions and details of mutant Serrate proteins tested. Cut (A–C, green) expression in wild type (A, B) and in discs ectopically expressing SerWT (C); ventral (v) and dorsal (d) are indicated. (A) Endogenous Serrate (red); (B) Src-GFP (red) expressed in the ptc∷Gal4 domain (ptc∷GAL4/UAS∷Src-GFP); (C, C′) Ectopic Cut expression (trans-activation, arrows) is induced in cells flanking the domain with highest levels of SerWT (ptc∷GAL4/UAS∷SerWT; red), most evident at the posterior side (right) where there is a sharp boundary between SerWT expressing/non-expressing cells. Endogenous Notch signalling at the d/v boundary is inhibited due to cis-inhibition (arrowhead). Note: At the levels of detection used, endogenous Serrate expression is not visible in (B, C), or in most subsequent images. (D) Diagram depicting domains of Serrate as labelled: N-terminal (NT), green; DSL, yellow; EGF-repeats, grey; cysteine-rich region (Cys) black oval; transmembrane (TM), red. Sites of mutations are indicated. *DSL, RPRDD (residues 251–55) substituted with AAAAA; ΔC20, terminal 20 residues deleted; *LL, LL (1352–3) substituted with AA; Δint, residues 1269–1285 deleted; ΔT, truncation after residue 1290 fused to Serrate C-terminal tripeptide VMV; *KK, both K(1269) and K(1287) substituted with A; KKYL, addition of KKYL tetrapetide to C-terminus. Table summarises results from expression in wing discs: + to +++ indicates increasing strength of effects; no, indicates no effect; * indicates there may also be inhibition of Notch on adjacent cells; ^ indicates possible weak inhibition of Cut at larval stages, this does not affect the adult wing. (E) ClustalW alignment of juxtamembrane region from the intracellular domains of the Serrate/Jagged proteins indicated: black shading, identities; grey shading, similarities; yellow shading indicates region with conservation between invertebrates and vertebrates; blue box, 17amino-acids deleted in Δint; thicker blue lines highlight the NNL (aa1271–3) and NPL (aa1284–6) triplets; red boxes outline lysine residues mutated (K1269 and K1287); green triangle indicates ΔT truncation (T1290). Download figure Download PowerPoint Figure 2.Differential effects of Serrate mutants on Notch activation. (A–D) Effects on Cut (green) when the indicated ligands are expressed using ptc∷Gal4: arrows, ventral stripes of ectopic Cut (trans-activation); arrowheads, differing levels of inhibition. (E–H) Distribution of Serrate proteins (x/z sections), yellow line indicates ptc∷GAL4 domain: SerWT (E) and Ser*DSL (F) are detected in intracellular puncta (arrowheads) and apically at the membrane (arrows); Ser*LL (G) and SerΔint (H) are restricted apically at the membrane (arrows). (I–K) Similar activation characteristics (α-Cut, green) are observed when wild-type (I) and mutant (J, K) Serrate proteins are expressed in clones (red). In (K), SerΔint clones that touch the d/v boundary eliminate activity in both compartments, as occurs with Notch mutant clones (de Celis et al, 1996). Download figure Download PowerPoint To determine whether activation and cis-inhibition both require the conserved DSL domain, we generated a mutant ligand in which five residues were altered to alanines (Ser*DSL; Figure 1D). Ser*DSL had a similar cellular distribution to wild-type ligand, which is present in many intracellular vesicles as well as localizing to the subapical domain of the cell membrane (Figure 2E and F). However, Ser*DSL failed to elicit Cut expression in adjacent cells (Figure 2B), demonstrating that it could no longer activate the receptor as predicted from the importance of the DSL domain for Notch binding (Muskavitch, 1994; Fleming, 1998; Shimizu et al, 1999). In addition, Ser*DSL had lost the ability to significantly cis-inhibit as Cut was present throughout the ectopic domain of ligand expression (Figure 2B; the levels within the domain are slightly reduced but not dissimilar to variability seen in wild-type discs, see Figure 1A and B) and adult flies were phenotypically wild type. We also tested the effects of a range of other mutations within the DSL and adjacent conserved NT domain of Serrate (data not shown); all had similar consequences of eliminating both aspects of ligand function. A Serrate intracellular motif is essential for activation To investigate key functions within the Serrate intracellular domain, we looked for motifs that might confer important properties. The C-terminal residue of Serrate is valine (VMV). Since C-terminal valine is required for efficient maturation of proTGFα and many V-terminal motifs interact with PDZ domains (e.g. Briley et al, 1997; Sheng and Sala, 2001; Six et al, 2004; Wright et al, 2004), we tested the effects of removing 4 or 20 amino acids from the C-terminus of Serrate (Figure 1D). The resulting proteins (SerΔC4 and SerΔC20) retained activating and cis-inhibitory functions, and showed normal subcellular distribution (data not shown). The C-terminal motif is thus not essential for either activation or cis-inhibition, at least when high levels of Serrate are expressed as in our assay. A second sequence detected in the Serrate intracellular domain (RSSQILL) resembled a di-leucine motif ([D/E/R]x[3−5] L[L/I]), which is involved in intracellular sorting and/or internalisation of proteins (Sandoval et al, 2000; Bonifacino and Traub, 2003). This motif is present in Anopheles gambiae Serrate (RNSQILL) and in Danio rerio Jagged 2 (RTKNGLI) and a related motif was found to regulate endocyotis of Lin12/Notch in Caenorhabditis elegans (SSQHSLL). Changing the two leucines to alanines (Ser*LL; Figure 1D) altered ligand subcellular distribution in a manner that was consistent with the dileucine motif playing a role in endocytosis; Ser*LL was predominantly localised at the cell surface and very little was detected as puncta inside the cell (Figure 2C and G). Surprisingly, this mutation had little effect on ligand function. Ser*LL retained the capability to trans-activate Notch both when expressed using ptc∷Gal4 and when expressed randomly throughout the disc in actin∷Gal4 clones (Figure 2C and J). At all locations, robust cis-inhibition was detected. However, Ser*LL differed from SerWT in that it activated only on the posterior side of the ptc∷Gal4 expression domain. One possible explanation is Ser*LL is less efficient at activating relative to cis-inhibition. The anterior stripe of Cut is induced within the region where ptc∷Gal4 expression gradually tails off towards the anterior. It occurs therefore at the transition between cells with high SerWT and those with lower levels that must be below the threshold for cis-inhibition so that the residual SerWT can transactivate. Therefore, if the balance between cis-inhibition and activation is altered in Ser*LL as suggested above, this threshold for cis-inhibition may not be overcome. Nevertheless, Ser*LL retains widespread ability to activate Notch despite the fact that the *LL mutation severely reduces its endocytosis. Our analysis of the Serrate intracellular domain also identified a short motif conserved between invertebrate and vertebrate Serrate/Jagged proteins ([E/D][E/D]x[2−3]NNx5Nx[3−5]NP[L/I]; Figure 1E). A related motif [E/D]x[2−4]NN[L/I] is present in the intracellular domain of Delta ligands, at a similar position relative to the transmembrane domain. We therefore generated a mutant form of Serrate that deleted this motif (SerΔint; Figure 1D and E). In this case, the mutant ligand completely lost the ability to trans-activate, since no ectopic Cut was detected when SerΔint was expressed in the wing disc (Figure 2D). In contrast to Ser*DSL, however, SerΔint had enhanced inhibitory effects that most likely reflect a combination of trans and cis-inhibitory interactions (Figure 2D and K). Analysis of SerΔint protein distribution revealed that it was highly concentrated at the membrane but was not present in intracellular puncta (Figure 2H). Thus, this deletion had compromised both trans-activation and endocytosis of Serrate. To further localise residues essential for Serrate function, a series of substitutions were made within the region delineated by Δint (Figure 1E). Since many endocytic motifs require tyrosine residues, we first replaced the single tyrosine residue but found no effect on Serrate function (data not shown). Subsequently, the conserved NNL or NPL triplets were substituted with AAA (Figure 1E). Neither mutation alone eliminated function, although they both resulted in ligands with partially compromised signalling activity (Figure 3A and data not shown). In addition, Ser*NNL accumulated apically and retained robust cis-inhibition (Figure 3A). Combining the two mutations produced a ligand that mimicked the effects of SerΔint. Thus, Ser*NNL/NPL did not trans-activate signalling but it retained inhibition and strongly accumulated apically at the membrane of expressing cells (Figure 3B and C). Figure 3.Role of the Serrate intracellular motif in Notch activation. (A, B, D, E) Cut expression (green) in wing discs expressing mutant Serrate proteins (red) as indicated. Ser*NNL (A) and SerΔT (D) activate to varying extents (arrows) and retain cis-inhibition (arrowheads); Ser*NNL/NPL (B) and Ser*KK (E) no longer trans-activate but still inhibit (arrowhead). (C, F) x/z section showing that Ser*NNL/NPL (C) and Ser*KK (F) are predominantly localised apically at the cell membrane, no prominent intracellular staining is detected (although some Ser*NNL/NPL is visible basally). In (F), Src-GFP (green) expressed in the ptc∷Gal4 domain (ptc∷Gal4/UAS∷Src-GFP) labels cell membranes. Yellow line indicates ptc∷Gal4 domain in all panels. Download figure Download PowerPoint Previous experiments have shown that deletion of the intracellular domain compromises trans-activation by DSL ligands, although they retain inhibitory functions (e.g. Sun and Artavanis-Tsakonas, 1996; Shimizu et al, 2002; Itoh et al, 2003; Wang and Struhl, 2004). We therefore tested whether the ability to trans-activate was retained when Serrate was truncated just C-terminal to the Δint motif (SerΔT; Figure 1D and E) as predicted if this is the essential motif within the intracellular domain. In agreement, SerΔT retained the ability to trans-activate (albeit weakly; Figure 3D) and to cis-inhibit (Figure 3D). These data therefore confirm the importance of the conserved motif for Serrate signalling activity and the dispensability of more C-terminal domains, including the di-leucine motif. SerΔintdefines a region required for an interaction with ubiquitin ligases Mib1 and Neur DSL ligands in both vertebrates and invertebrates are regulated by the ubiquitin ligases Mib1 and Neur (Lai et al, 2001, 2005; Pavlopoulos et al, 2001; Itoh et al, 2003; Koo et al, 2005; Le Borgne et al, 2005b; Pitsouli and Delidakis, 2005). To investigate whether the inactivity of SerΔint could be explained by an impaired ability to interact with Mib1 or Neur, we employed an S2-cell co-expression assay (Figure 4). SerWT was highly enriched at the plasma membrane when expressed in S2 cells (Figure 4A). Co-expression with Drosophila Mib1 or Neur led to a dramatic relocalisation, such that little SerWT was detected on the cell surface (Figure 4D, G, J and K). Both Ser*LL and SerΔint were also enriched at the plasma membrane when expressed alone (Figure 4B and C), so we next compared effects of Mib1 and Neur on their cellular distribution. Like SerWT, Ser*LL was redistributed from the membrane in the presence of Mib1 or Neur (Figure 4E, H, J and K). In contrast, SerΔint remained at the cell surface in the presence of Mib1 or Neur (Figure 4F, I, J and K), indicating that it was no longer recognised by either ligase. Figure 4.The conserved intracellular motif confers an interaction with ubiquitin ligases. (A–K) S2 cells were transfected with the constructs indicated and the subcellular distribution of Serrate monitored. (A–C) All Serrate variants are predominantly localised at the cell surface when expressed alone. (D–I) Co-expression of Mib1 or Neur causes significant relocalisation of SerWT (D, G) and Ser*LL (E, H), but not of SerΔint (F, I); cells were double labelled to confirm the presence of Mib1 or Neur (not shown). (J, K) Results from analysis of >50 cells for each combination, scored for high (black), low (green) or no (orange) detectable surface Serrate staining in the absence (−, dark shading) or presence (+, light shading) of Mib1 (J) or Neur (K). (L) SerWT and Ser*LL but not SerΔint co-immunoprecipitate with NeurΔRING. Input: Serrate levels (detected as two bands) in extracts from larvae expressing Serrate and Neur constructs as indicated. SerWT and Ser*LL are at reduced levels (asterisks) in the presence of full-length Neur, SerΔint is not affected. None show reduced levels in the presence of NeurΔRING. Extracts probed with antitubulin (α-Tub) as loading control. α-Neur IP: proteins complexed with Neur were immunoprecipitated from larval extracts expressing the constructs shown and probed with α-Neur (upper panel) or α-Ser (lower panel). Lower panel: levels of SerWT, Ser*LL or SerΔint co-immunoprecipitated with Neur (arrowhead) or NeurΔRING (arrows). SerWT and Ser*LL can be co-precipitated with NeurΔRING (black arrows) which does not promote ligand degradation, SerΔint does not co-precipitate even under these conditions (grey arrows). Download figure Download PowerPoint To confirm that SerΔint had lost sequences required for interaction with the E3 ligases, we tested whether SerΔint co-immunoprecipitated with Neur from Drosophila larval extracts. SerWT, Ser*LL or SerΔint were each expressed together with either Neur or NeurΔRING (which retains ligand binding but is expected to lack ubiquitinating activity and is more stable than Neur; Pitsouli and Delidakis, 2005). We observed that total levels of SerWT and Ser*LL were lowered in the presence of Neur, whereas SerΔint was unaffected (Figure 4L, asterisks; the truncated SerWT fragment appears more susceptible to degradation), indicating that the latter is refractory to the activity of Neur. Furthermore, both SerWT and Ser*LL were co-immunoprecipitated with NeurΔRING and weakly with Neur, but SerΔint failed to co-immunoprecipitate with either (Figure 4L). Thus, together these data confirm the importance of the conserved motif in an interaction with both Neur and Mib. Finally, since Mib1 and Neur have E3 ubiquitin ligase activity which modifies lysine residues, we tested the consequences of mutating the two lysine residues most closely associated with the Δint motif (Ser*KK, EEKSNN….NPLK; Figure 1E). Ser*KK was incapable of trans-activation (Figure 3E) indicating that one or both of these lysine residues are likely targets for modification by Mib1/Neur (mutation of the second lysine alone, NPLK, had no effect on Serrate function; data not shown). As with the other mutations in the intracellular domain, Ser*KK accumulated at the membrane (Figure 3F) and strongly inhibited endogenous Notch signalling at the d/v boundary (Figure 3E). Relationship between endocytosis and ligand functions Most mutations that abolish Serrate's ability to signal (SerΔint; Ser*NNL/NPL; Ser*KK) correlate with a severe reduction in internalisation, as shown by the reduced number of intracellular puncta relative to surface ligand. However, in two cases (Ser*LL; Ser*NNL), trafficking is reduced without significantly compromising signalling, and in another (Ser*DSL) trafficking still occurs although signalling is abolished. We therefore investigated further the intracellular puncta and the effects of altering endocytosis and/or intracellular transport on the ability of Serrate to signal. First, we analysed the relationship between Serrate and Notch localisation within cells expressing SerWT (Figure 5A and B, and Supplementary Figure S1A). In wild-type cells, Notch was predominantly localised apically at the cell surface where it overlapped with E-Cadherin (Supplementary Figure S1D). However, little of the Notch remained at the cell membrane in cells producing SerWT (Figure 5A and B). Instead, Notch co-localised with SerWT in intracellular structures (Figure 5B and Supplementary Figure S1A) that were of endocytic origin, as they co-labelled with dextran in uptake assays (Supplementary Figure S1B) and were reduced in cells where endocytosis was compromised by a mutation in dynamin (shits; Supplementary Figure S1C). The amount of Notch in vesicles was increased 1.5-fold by the presence of SerWT. The distribution of SerWT and Notch was also altered by the expression of the small GTPases Rab5 and Rab7, which regulate endocytic and intracellular trafficking (Entchev et al, 2000; Supplementary Figure S1E–H). Co-expression of Rab5 with SerWT resulted in the accumulation of Notch and SerWT in large apical puncta (Supplementary Figure S1E and F), whereas co-expression with Rab7 led to their accumulation in Rab7 positive basal puncta (Supplemetnary Figure S1G and H). Together, these data indicate that a large proportion of Notch and Serrate travels through the Rab5/Rab7 endocytic pathway. Interestingly, however, neither increased Rab7 nor increased Rab5 had a significant effect on SerWT mediated signalling or cis-inhibition (data not shown). Likewise, the bulk of intracellular trafficking occurs independent of Notch signalling, since Ser*DSL also colocalised with Notch in intracellular puncta even though it cannot make a productive interaction (Figure 5C and D). Thus, trafficking through these compartments does not appear to be limiting for Serrate mediated activation. However, in agreement with previous studies where endocytosis was linked to ligand activity (Parks et al, 2000; Wang and Struhl, 2004; Le Borgne et al, 2005a), co-expression of a dominant-negative Rab5 (Rab5SN) was able to severely reduce signalling by SerWT when larvae were maintained under conditions to maximise its dominant-negative effects (24 h at 29°C, data not shown). Figure 5.Differential effects of Serrate mutants on Notch distribution. (A–K) Notch protein (A–H, anti-NotchICD white/green; I–K, anti-NotchECD) distribution in discs expressing SerWT (A, B, I), Ser*DSL (C, D), Ser*LL (E, F, J) and SerΔint (G, H, K). Yellow line marks ptc∷Gal4 domain, yellow arrows indicate d/v boundary. (A, C, E, G, I–K) Apical x/y sections: Notch is depleted from the membrane in the domain of ectopic SerWT but is enriched at the membrane in cells expressing Ser*LL or SerΔint. In (I–K), discs were not permeablised so that only surface protein is detected. (B, D, F, H) x/z sections, arrowheads indicate Notch puncta which colocalise with SerWT (B, B′) and Ser*DSL (D, D′). Download figure Download PowerPoint Cis-inhibition by Serrate at the cell surface When SerWT is expressed in the wing disc, it inhibits endogenous Notch signalling within the domain of highest ligand expression (Figures 1C and 2A). Ligand mediated cis-inhibition also plays a role in modulating Notch activation in wild-type wing discs, as illustrated by the ectopic Notch signalling detected within clones that are mutant for both ligands (Figure 6I; Micchelli et al, 1997). The change in Notch distribution elicited by SerWT (Figure 5A) initially suggested a model where cis-inhibitory interactions resulted in the downregulation of Notch by promoting its endocytosis and degradation. However, several data suggest that depletion of Notch is not essential for cis-inhibition, and support an inhibitory interaction between Serrate and Notch taking place at the cell surface. Figure 6.Cis-inhibition is compromised by an ER retention signal, enhanced by NeurΔRING and independent of Fringe. (A–C) Expression of Cut (green) in discs expressing SerWT (red, A) and SerKKYL (red B, C). Addition of KKYL motif results in reduced/no activation (arrows) and reduced cis-inhibition (arrowheads). (D, E, E′) SerKKYL has different intracellular distribution from SerWT. In x/z sections, SerWT (D) is detected at the membrane (arrow) and in vesicles (arrowhead); SerKKYL (E, E′) accumulates apically and basally within the cells (white lines). (F, G) SerWT (red, F) induces E(spl)mβ∷CD2 (green F, white F′) in flanking ventral cells (arrows) and inhibits expression throughout the ptc∷GAL4/UAS∷Ser stripe (yellow line). Wild-type E(spl)mβ∷CD2 (G, white) is shown for comparison. To aid comparison, yellow asterisks indicate area of high expression around L3 sensilla, yellow li
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