www.sciencemag.org/cgi/content/full/338/6111/1229/DC1
Supplementary Material for A Mutation in EGF Repeat-8 of Notch Discriminates Between Serrate/Jagged and Delta Family Ligands Shinya Yamamoto, Wu-Lin Charng, Nadia A Rana, Shinako Kakuda, Manish Jaiswal, Vafa Bayat, Bo Xiong, Ke Zhang, Hector Sandoval, Gabriela David, Hao Wang, Robert S Haltiwanger, Hugo J Bellen*
*To whom correspondence should be addressed. E-mail:
[email protected] Published 30 November 2012, Science 338, 1229 (2012) DOI: 10.1126/science.1229745 This PDF file includes:
Materials and Methods Figs. S1 to S13 Tables S1 to S3 References (29–53)
Materials and Methods Fly strains and genetics We used the following Drosophila melanogaster strains in this study: y w FRT19A, Df(1)JA27/FM7c Kr-GAL4, UAS-GFP (indicated as l(1)/FM7c Kr>GFP in Fig S1), w sn FRT19A; Ubx-FLP, w sn FRT19A;; Ubx-FLP, w Ubi-GFP Ubx-FLP FRT19A, w Ubi-GFP FRT19A; Ubx-FLP, w Ubi-GFP FRT19A;; Ubx-FLP, hs-FLP Ubi-GFP FRT19A, Ubx-FLP tubGAL80 FRT19A; Actin-GAL4 UAS-CD8::GFP, hs-FLP tub-GAL80 FRT19A; Actin-GAL4 UASCD8::GFP, w N54l9 FRT19A/FM7c, y w N55e11/FM7c, Df(1)64c18, g1 sd1 /Dp(1;2;Y)w+ /C(1)DX, w1118 ; Dp(1;3)DC109; PBac{DC109}VK00033 (29), Ngt-wt-attVK22 (30), msh-lacZ/TM2 Ubx (31), Ser-LacZ (32), UAS-SerRNAi (P{KK108416}VIE-260B) (33), Notch responsive elementGFP (NRE-GreenRabbit) (34), w;; Dpp-GAL4/TM6b Tb, w; UAS-Dl/CyO, w; UAS-Ser/CyO, w; UAS-fng, y w; fng13 mwh jv FRT80B/TM3 Sb, and Bc1/CyO. Experiments over-expressing transgenes in vivo were performed using the GAL4/UAS system (35). Mutant clones negatively marked by GFP were generated using the FLP/FRT system (36). Mutant clones positively marked by GFP, as well as co-overexpression of transgenes or RNAi within the mutant clones were performed with the MARCM technology (37). Flies were kept in standard media and stocks were maintained at room temperature (2123°C). Most crosses were performed at 25°C. Crosses utilizing the GAL4/UAS system were performed at 28°C.
Forward genetic screen on the Drosophila X chromosome and identification of new Notch alleles Mutagenesis was performed by feeding isogenized y w FRT19A males with sucrose solution containing low concentration (7.5-10mM, in contrast to the standard concentration (15-25mM)) of ethyl methanesulfonate (EMS) to minimize introduction of multiple lethal hits per chromosome. After recovery from mutagenesis, these males were mated en masse with Df(1)JA27/FM7c Kr-GAL4 UAS-GFP virgin females. In the F1 generation, y w mut* FRT19A/FM7c Kr-GAL4 UAS-GFP (mut* indicates the EMS-induced mutation) virgins were collected and 33,887 females were crossed with FM7c Kr-GAL4 UAS-GFP males to establish balanced stocks. 5,859 lines carried lethal mutations and the remaining stocks were discarded. We crossed each y w mut* FRT19A/FM7c Kr-GAL4 UAS-GFP virgins with w sn FRT19A; UbxFLP males and screened y w mut* FRT19A/ w sn FRT19A; Ubx-FLP/+ progenies for defects in wings and bristles. 577 lines exhibited wing margin loss (notching) and/or bristle loss (balding) phenotypes and were further subjected to mapping by standard genetic techniques, such as rescue with X chromosome duplications, complementation tests with deficiencies and existing mutant alleles of N. Forty-two lines failed to complement a null allele of Notch (N54l9). Genomic rescue experiments were performed by crossing the mutant lines to w1118 ; Dp(1;3)DC109; PBac{DC109}VK00033 (29) and Ngt-wt-attVK22 (30). Molecular lesions were determined by PCR and Sanger sequencing of genomic DNA isolated from hemizygous or heterozygous flies. 2
Sequence alignment of fly and human Notch paralogs Amino acid sequence of fly Notch and human Notch paralogs were obtained from FlyBase (http://www.flybase.org) and GenBank (http://www.ncbi.nlm.nih.gov/genbank), respectively: Sequence alignments of specific regions of Notch were performed using Clustal X (38) using standard parameters. Immunostaining, Imaging and Quantification Immunostaining of fly tissues and endocytosis assays were performed as previously described (39-41). The following primary antibodies and fluorescently labeled reagents were used at designated concentrations: mouse anti-NotchECD monoclonal (1:100; C458.2H; Developmental Studies Hybridoma Bank (DSHB)) (42), mouse anti-Notchintra monoclonal (1:100; C17.9C6; DSHB) (43), mouse anti-Cut monoclonal (1:100, 2B10, DSHB) (44), mouse anti-Wingless monoclonal (1:100; 4D4; DSHB) (45), mouse anti-DeltaECD monoclonal (1:100, C594.9B; DSHB) (43), rat anti-Serrate polyclonal (1:100, a gift from Marco Milan) (46), guinea pig anti-Senseless polyclonal (1:1000) (47), rat anti-ELAV monoclonal (1:500, 7E8A10, DSHB) (48), anti-Lozenge monoclonal (1:10, DSHB) (49), Cy2-, Cy3- and Cy5 or DyLight649conjugated affinity purified donkey secondary antibodies (1:200, Jackson ImmunoResearch Laboratories), Alexa488-conjugated Phalloidin (1:200, Invitrogen) and DAPI (0.5μg/ml, Invitrogen). For immunostaining of Drosophila S2 cells, cells were cultured in Schneider’s media supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma) on a sterilized cover glass. Fixation and subsequent procedures were the same as immunostaining of fly tissue. Images were acquired using LSM510 and LSM710 confocal microscopes (Zeiss) and examined and processed with LSM viewer (Zeiss), ZEN (Zeiss), Photoshop (Adobe) or Image J (NIH) software. To obtain images of the wing, the adult flies were dissected and mounted in DPX medium (Electron Microscopy Sciences). Images of the notum were obtained through dissection of thoraxes in phosphate buffered saline (PBS), followed by incubation of the notum in 10% KOH solution to digest the soft tissues except for the cuticle. Images of the circulating crystal cells were taken by direct imaging of the dorsal abdominal segments of the larvae. Images were obtained using a digital camera (MicroFire; Olympus) mounted on a stereomicroscope (MZ16; Leica) using ImagePro Plus 5.0 acquisition software (Media Cybernetics). The Extended Focus function of the AxioVision software was used to obtain stack images. To quantify the number of circulating crystal cells, we introduced a visible genetic marker (Black cells) to visualize the crystal cells in vivo. We counted the number of black cells, which are melanized crystal cells that can easily be seen. We counted 10 larvae/genotype.
3
To quantify the wing margin defects, we measured the length of the notch at the wing margin and divided this value by the total length of the wing margin to calculate the % of margins lost. Wings with no notching are classified as “Normal”, wings with 0~30% of the wing margin lost are classified as “Mild”, wings with 30~60% are “Moderate” and wings with 60~100% are “Strong”. We counted 50 wings/genotype. Analysis of sugar modification of N by collision-induced dissociation mass spectrometry Expression Constructs: pMTHy plasmids encoding all 36 EGF repeats in the Drosophila Notch extracellular domain (ECD) with a C-terminal triple FLAG tag (N-EGF:FLAG) and Drosophila Fringe with a C-terminal His6 tag (Fng) were gifts from Kenneth Irvine. Construction of these plasmids, transfection of Drosophila S2 cells, and purification of the resulting proteins from medium have been described (18, 23, 24). pMTHy empty vector, used as a control for transfections, was a gift from J. Peter Gergen. We introduced the V361M mutation found in Njigsaw into the NEGF:FLAG plasmid through site-directed mutagenesis (N-EGF:FLAG V361M). Cell culture and protein purification: Stably transfected S2 cells were generated and cultured as described previously (23). Briefly, S2 cells were stably transfected with 4.0 µg of N-EGF:FLAG WT or V361M, and cotransfected with either 4.0 µg pMTHy empty vector or 4.0 µg Fng, using Cellfectin II (Invitrogen) to generate unmodified (N-EGF:FLAG, N-EGF:FLAG V361M) or in vivo Fngelongated N-EGF:FLAG (N-EGF:FLAG+Fng, N-EGF:FLAG V361M+Fng). Cells were cultured in Schneider’s Drosophila media (Invitrogen) supplemented with 10% FBS and 1% penicillin-streptomycin. For purification of N-EGF:FLAG WT or V361M (-/+Fng), stably transfected S2 cells were cultured in 250 ml of Schneider’s Drosophila media, and expression was then induced by addition of 0.7 mM CuSO4. After incubation for 3 days, media was cleared by centrifugation at 4000 rpm for 20 min at 4ºC, and conditioned media was incubated with 250 µl of anti-FLAG beads (Sigma) overnight at 4ºC with rotation. Purified protein was eluted from beads using 3X FLAG peptide (Sigma) as per manufacturer’s instructions. To remove excess 3X FLAG peptide, samples were dialyzed overnight against TBS supplemented with 2 mM CaCl2 and 10% glycerol. 10 µl of eluate was separated by 12% SDS-PAGE alongside BSA standards of varying concentrations and stained with Coomassie blue. Protein yields were estimated by densitometry using ImageJ software (NIH). Analysis of glycopeptides by nano-LC-ESI-MS: Nano-LC-ESI/MS and MS/MS, Constant Neutral Loss (CNL) manual searches, and Extracted Ion Chromatogram (EIC) manual searches were all performed as described (22). Briefly, 1.0 µg N-EGF:FLAG WT or V361M (-/+ Fng) was acetone precipitated, subjected to reduction/alkylation and in-solution protease digest with trypsin for 8 hours, or chymotrypsin for 6 hours. In-solution digests were purified by spin-filtering with a 0.22 μm filter (Agilent). Samples were then subjected to nano-LC-MS/MS using low energy CID on an Agilent 6340 ion trap mass spectrometer with an HPLC Chip-Cube interface. 1.0 μl sample volumes were injected onto a Zorbax 300SB-C18 chip (Agilent), all as described (22). 4
Multiple Reaction Monitoring (MRM) of peptide glycoforms: Semi-quantitative analysis of the different glycoforms of peptides (e.g., unmodified, Ofucose monosaccharide, O-fucose disaccharide forms of the same peptide) was performed using Multiple Reaction Monitoring (MRM) (50). For MRM analysis, 1.0 μg quantities of NEGF:FLAG WT or V361M -/+ Fng were prepared by in-solution digest in parallel, as described above. In MRM, the mass spectrometer is programmed to monitor the level of a specific fragment ion (so called “transition ion”) derived from a selected parent ion (e.g., molecular ion corresponding to a peptide modified with O-fucose disaccharide). We typically use the molecular ion corresponding to the unmodified peptide as transition ion since it is one of the most abundant species in the MS/MS spectra. In addition to quantifying glycopeptides, non-glycosylated peptides were also quantified in minus and plus Fng samples as an injection control. Since a given peptide lacking glycosylation should ionize to the same efficiency independent of the glycosylation state of the sample (i.e., minus or plus Fng), using an internal non-glycosylated peptide as our control allowed us to normalize for run-to-run variations and also served a dual function as an injection control. The pairs of ion used for each MRM run are provided in the figure legends. Ligand-receptor binding assays in Drosophila S2 cells Ligand-receptor binding assays were performed as described previously (17, 24, 41) with subtle modifications. In brief, we introduced the Njigsaw V361M mutation in the N-CD2 expression construct through site-directed mutagenesis. We transfected S2 cells with these constructs alone, or together with constructs that allow expression of Fringe or O-Fut1 using Fugene HD (Promega) with standard protocols. In parallel, we transfected another set of S2 cells with constructs expressing the extracellular domain of Dl or Ser fused to alkaline phosphatase (AP). The next day, transgene expression was induced with 0.7mM CuSO4. Three days after induction, we collected cells expressing N-CD2 and conditioned media containing Dl-AP or SerAP. Conditioned media containing AP-fused ligands was 5 fold concentrated using centrifugal filter units (Millipore). We incubated S2 cells expressing N-CD2 with concentrated conditioned media for 90 minutes. After extensive washing, cell lysis and inactivation of endogenous AP activity, we assayed AP activity that bound to S2 cells using Phospha-light System (Applied Biosystems) and FLUOSTAR Optima (BMG Labtech). Experiments were performed in triplicate and repeated at least twice. Notch exocytosis assay in Drosophila S2 cells Exocytosis of N was measured based on secretion of N-AP (17). We introduced the V361M mutation found in Njigsaw allele in N-AP expressing construct through site-directed mutagenesis. N-AP or Njigsaw -AP were transfected with or without a Fringe expressing construct. On the next day, transgene expression was induced with 0.7mM CuSO4. Three days after induction, conditioned media containing N-AP or Njigsaw-AP was collected and heat-inactivated to inhibit endogenous AP activity. AP activity was assayed using the Phospha-light System (Applied Biosystems) and FLUOSTAR Optima (BMG Labtech). 5
Co-Culture Based Notch Signaling Assay in Mammalian Cells Cell-based Notch signaling assays were performed essentially as described (22, 25, 51). We introduced mutations (V327M, V327A, T314A) into the mouse Notch2 expression plasmid (pTracerCMV-mNotch2-Flag) by site-directed mutagenesis. NIH/3T3 cells (1.2 x 105) were seeded in a 24-well tissue culture plate and transiently transfected using Lipofectamine 2000 (Invitrogen) with 0.2 μg of wild-type or mutant forms of pTracerCMV-Notch2-Flag, 0.15 μg of TP-1 luciferase reporter construct (Ga981-6, a gift form Georg Bornkamm), and 0.15 μg of gWIZ β-galactosidase construct (Gene Therapy Systems) for transfection efficiency normalization. Four hours post-transfection, L cells expressing Jagged1 or Delta-like 1 (a gift from Gerry Weinmaster) were overlaid on the transfected NIH3T3 cells at a density of 1.5 x 105 cells/well. Twenty-four hours post co-culture, cell lysates were prepared and assayed for luciferase and β-galactosidase activities. Luciferase activity was normalized to β-galactosidase activity. All co-cultures were carried out in triplicate and in three independent experiments.
6
Fig. S1. A forward genetic screen to isolate new recessive lethal Notch alleles. Isogenized y w FRT19A males were mutagenized using 7.5-10mM EMS (ethyl methanesulfonate), and mated en masse with females with l(1)/FM7c Kr>GFP genotype. In the F1 generation, we selected virgin females that carry the mutated y w FRT19A chromosome (y w mut* FRT19A) over the FM7c Kr>GFP balancer and set up 33,887 single crosses with FM7c Kr>GFP males to establish stocks that carry unique mutations. Stocks that produced y- w- and B+ males, indicating that the mutation was viable, were discarded. 5,859 lines were maintained as recessive lethal stocks. We collected virgin females from these individual recessive lethal stocks and crossed them to w sn FRT19A; Ubx-FLP males to generate progenies with homozygous mutant clones in the wing and thorax while avoiding the lethality associated with the mutation using the FLP/FRT system (36). 577 lines exhibited wing margin loss (notching) and/or bristle loss (balding) phenotypes. 7
Fig S2. The roles of Notch signaling during mechanosensory bristle development in Drosophila (A) An image of mechanosensory bristles on the notum. The shaft cell extends a long hair that is surrounded by the socket cell. Smaller hair-like structures that are seen between the bristles are trichomes, an actin-rich apical cuticle that is secreted by individual epithelial cells. The sheath cells and neurons cannot be seen in this image since they are located beneath the cuticle under the shaft and hair cells. (B) A schematic diagram of a mechanosensory bristle. The shaft cell extends a long hair apically, which is supported by the socket cell as shown in (A). The neuron projects its dendrites to the base of the shaft and extends its axon to the central nervous system. The sheath cell functions as a glia-like cell by encircling the neuron. (C) Development of the bristles through lateral inhibition and lineage decisions through N signaling. Bristles develop from group of epithelia cells that begin to express proneural genes, referred to as the proneural cluster. Through Dl mediated lateral inhibition, one cell is selected as a sensory organ precursor (SOP) cell. The SOP undergoes series of asymmetric cell division and subsequent cell fate specification through Dl- and Ser-mediated lineage decisions. The cell that receives the highest amount of Notch activity becomes the socket cell, and the cell that receives the lowest amount of Notch activity becomes the neuron. Ser is not involved in lateral inhibition of SOP, and Dl and Ser act redundantly during lineage decisions (14). 8
Fig. S3 Cell fate determination in the bristle lineage occurs normally in Njigsaw mutant tissue (A) Four cells (socket, shaft, sheath and neuron) of the sensory organ are labeled by Cut (red). Neurons are also positive for Elav (blue). Njigsaw mutant cells are labeled by absence of GFP. (A’) A higher magnification image of a sensory organ cluster. Scale bars indicate 20 μm.
9
Fig. S4 10
Fig. S4 Notch signaling defects and wing margin loss in Njigsaw clones that are present in the ventral compartment of the wing. (A-C) Immunofluorescence staining of third instar larval wing imaginal disc with Njigsaw MARCM clones (marked by the presence of GFP). N activation (monitored by Cut expression, red) is lost when Njigsaw clones span both the dorsal and ventral compartment (A) or are limited to the ventral compartment (C). In contrast, N signaling is not affected when Njigsaw clones are restricted to the dorsal compartment (B). Single channel representations of Cut expression are shown in white in (A’-C’). (D-E) Differential interference contrast (DIC) and confocal microscopy images of pharate adult wings with Njigsaw MARCM clones (marked by GFP). When Njigsaw clones are located in the ventral domain at the dorsal-ventral boundary, the wing margin is lost (E-E”). In contrast, the wing margin is not affected when the clone is located in the dorsal domain (D-D”). DIC images are shown in (D) and (E), optical confocal sections are shown in (D’) and (E’), and Z section along the marked dotted line in (D’) and (E’) are shown in (D”) and (E”). Nuclei of wing cells are marked by DNA staining with DAPI (blue).
11
Fig. S5 Trans-activation of Notch by Serrate, but not by Delta, is defective in Njigsaw mutant cells. (A-F) Overexpression of Delta or Serrate along the anterior-posterior (A-P) boundary of the wing imaginal disc of wild-type and Njigsaw mutant clones by Decapentaplegic(Dpp)-GAL4. Overexpression of Serrate leads to ectopic Notch activation in the ventral (V) domain, while overexpression of Delta leads to ectopic Notch activation in the dorsal (D) domain. Schematic diagrams of the Notch activation pattern in wild-type discs are shown in (A, D) with Dpp-GAL4 expression domains indicated in blue. Immunofluorescence staining of wing discs (B, C, E, F). N mutant clones are labeled by the absence of GFP (green) in (C, F). Single channel representations of Wingless (Wg) expression in the boxed area are shown in white in (C’, F’).
12
Fig. S6 Notch-Serrate signaling is defective in lymph and salivary glands in Njigsaw mutant larvae. (A-E) Crystal cell specification is defective in Njigsaw mutant lymph glands. (A-B) Immunofluorescence stainings of 3rd instar larval lymph glands, the hematopoietic organs. Lozenge (green) is expressed in crystal cells via N-Ser signaling in wild-type. Njigsaw mutant larvae fail to upregulate Lozenge expression. Cell nuclei are marked with DAPI (blue). Single channel representation of Lozenge expression is shown in white in (A’) and (B’). (C-E) Visualization and quantification of circulating crystal cells using a genetic marker, Black cell. Crystal cells are shown in black and many are lost in Njigsaw mutant larvae, quantified in (E). (C’) and (D’) are higher magnification images of the boxed area in (C) and (D), respectively. (F-G) Notch activation in imaginal ring cell is severely impaired in Njigsaw mutant larva. Differential interference contrast (DIC) images are shown in (F) and (G), and confocal fluorescence images are shown in (F’) and (G’). Error bars indicate S.E.M.
13
Fig. S7 Upregulation of Serrate in Njigsaw mutant clones is post-transcriptional and nonautonomous Notch activation can be suppressed by reducing Serrate levels in Njigsaw mutant clones. (A) Immunofluorescence staining of a third instar larval wing imaginal disc with Njigsaw MARCM clones (marked by GFP) expressing Ser-lacZ (blue). Ser-lacZ levels are not altered in Njigsaw mutant clones, while non-autonomous activation of N signaling (monitored by Cut expression shown in red, arrows point to ectopically expressed Cut) are seen in neighboring wild-type cells. Single channel representation of Ser-lacZ expression is shown in white in (A’). (B) Immunofluorescence staining of a 3rd instar larval wing imaginal disc with Njigsaw MARCM clones (marked by GFP) expressing RNAi against Ser specifically in mutant clones. Note the decrease in Ser (blue) expression in GFP positive cells (B’). In these animals, non-autonomous Notch expression is suppressed and we do not observe ectopic Cut expression in wild-type cells neighboring Njigsaw clones (B”). Wild-type cells that would express ectopic Cut in the absence of Ser RNAi are indicated with arrowheads in (B & B”). Single channel representation of Ser and Cut expression are shown in white in (B’) and (B”), respectively.
14
Fig. S8 fringe modifies the Njigsaw phenotype in adult wings. (A-D) Njigsaw mutant clonal phenotype is enhanced by a reduction of Fringe activity and suppressed by Fringe overexpression in mutant clones. Adult wing phenotypes are shown in (AC), and quantification is shown in (D). Scale bars indicate 100 μm in (A-C).
15
Fig. S9 16
Fig. S9 The Njigsaw phenotype is modified by fringe in the wing disc. (A-C) Third instar imaginal discs with clones of Njigsaw (marked by the presence of GFP) occasionally contain cells that still activate N signaling (arrow, (A’)). However, when one copy of fringe is removed, most cells in the mutant clones do not activate N (arrowhead, (B’)). When we overexpress Fringe specifically in Njigsaw mutant cells, the N signal is activated only in those cells located in the ventral domain (asterisk, (C, C’)). (D-G) N activation (monitored by Cut, white) at the wing margin of hemizygous wild-type (D) or mutant (E-G) 3rd instar larval imaginal discs. Njigsaw hemizygous wing disc exhibit a severe loss or reduction of Notch activation at the wing margin (F). One copy of fringe enhances the defect (G), leading to a further loss of Cut expression. One copy of fringe in a NWT background has no effect (E).
17
Fig. S10 18
Fig. S10 Sugar modifications of EGFr-8 in Njigsaw are minimally affected. (A) Mass spectrum of O-fucose monosaccharide form of the peptide, 328 CQDDVDECAQRDHPVCQNGATCTNTHGSY356, from EGFr-8. Top panel: MS of parent ion, m/z = 886.6, corresponding to 4+ form of the glycopeptide. Bottom panel: MS2 of 886.6, showing a major product at m/z = 850.2, corresponding to the 4+ form of the unmodified peptide. (B) Mass spectrum of O-fucose disaccharide form of the peptide, 328 CQDDVDECAQRDHPVCQNGATCTNTHGSY356, from EGFr-8. Top panel: MS of parent ion, m/z = 937.4, corresponding to 4+ form of the glycopeptide. Bottom panel: MS2 of 937.4, showing major product ions corresponding to the peptide with O-fucose monosaccharide (4+form, m/z = 887.3; 3+-form, m/z = 1181.7) and the unmodified peptide (4+-form, m/z = 850.3; 3+-form, m/z = 1133.1). Symbols: Black rectangle, peptide. Red triangle, fucose. Blue square, GlcNAc. The ions used for MRM in Figs. 3A and 3B are as follows: O-fucose disaccharide glycoform (blue line): 937.5 Æ 1133.2; O-fucose monosaccharide glycoform (red line): 886.8 Æ 850.4; unmodified peptide (black line): 850.4 Æ1534.7. (C-D) MRM analyses of NWT (C) or Njigsaw (D) samples using the ions described above in quintuplicate. Top panels show relative levels of unmodified (black line), O-fucose monosaccharide (red line), or O-fucose disaccharide (blue line) glycoforms of peptides from EGFr-8. Bottom panels show MRM of a control, unglycosylated peptide for normalization purposes. Estimates of the amount of the disaccharide and monosaccharide glycoforms were determined by calculating the area under the respective curves (top panels) and dividing by the area under the curve for control peptide (bottom panel). The average of these results is plotted in Fig. 3C.
19
Fig. S11 20
Fig. S11 Njigsaw mutation does not cause significant changes in glycosylation at several other EGFrs. MRM traces of ions show relative levels of unmodified (black line), O-fucose monosaccharide (red line), or O-fucose disaccharide (blue line) glycoforms of peptides from EGFr-3, EGFr-5, EGFr-7, and EGFr-23 generated in the presence or absence of Fng. Mass spectra identifying the relevant ions can be found in (23). The level of glycosylation shown here is similar to what was observed on WT protein previously (23). The lack of unmodified peptide in all samples is a strong indication that each of these EGFr is properly folded. MRM of a control, unglycosylated peptide is shown as a loading control to demonstrate similar amounts of material were analyzed in the +/- Fng samples. Parent and transition ions for each of these peptides used in the MRM analysis are as follows: EGFr-3: O-fucose disaccharide glycoform (blue line): 1330.3 Æ 1214.0; O-fucose monosaccharide glycoform (red line): 1262.5 Æ 1214.0; unmodified peptide (black line): 1214.0 Æ 764.1. EGFr-5: O-fucose disaccharide glycoform (blue line): 984.1 Æ 867.6; Ofucose monosaccharide glycoform (red line): 916.4 Æ 867.6; unmodified peptide (black line): 867.6 Æ 787.6. EGFr-7: O-fucose disaccharide glycoform (blue line): 1316.4 Æ 1199.9; Ofucose monosaccharide glycoform (red line): 1248.4 Æ 1199.9; unmodified peptide (black line): 1199.9 Æ1313.7. EGRr-23: O-fucose disaccharide glycoform (blue line): 1245.6 Æ 1128.8; Ofucose monosaccharide glycoform (red line): 1178.0 Æ 1128.8; unmodified peptide (black line): 1128.8 Æ 579.6.
21
Fig S12 Expression, cell surface localization and endocytosis of Njigsaw mutant protein are normal. (A-B) Immunofluorescence staining of 3rd instar larval imaginal discs containing Njigsaw homozygous mutant clones. Both total staining (A) and extracellular staining (B) against N (red) do not exhibit any difference between WT and mutant cells. No intracellular accumulation of N protein is observed (A). (C) Endocytosis of N occurs normally in 3rd instar larval imaginal discs. Imaginal discs containing Njigsaw mutant clones were incubated with anti-N antibody at room temperature (red) and fixed and stained after 15 minutes. Absence of GFP (green) marks the Njigsaw homozygous mutant cells. A single focal plane is shown in each image. Single channel representations of N (A’, B’, C’) are shown in white. Scale bars correspond to 10 μm. 22
Fig S13
23
Fig S13 Notch exocytosis is unaffected by the Njigsaw mutation. (A) A schematic diagram of N-CD2 fusion protein. The extracellular domain of Notch containing the 36 EGFr was fused to human CD2 protein. (B-C) Immunostaining of Notch (red) in cells transfected with NWT -CD2 (B, B’) or Njigsaw-CD2 (C, C’) shows that the majority of the fusion protein is at the plasma membrane regardless of the presence or absence of the V361M mutation. F-Actin, enriched in the cell cortex as well as in intracellular puncta and vesicles, was visualized with phalloidin conjugated with a fluorescent dye (green). Single channel representation of Notch (B’, C’) is shown in white. Scale bars correspond to 10 μm. (D) A schematic diagram of N-AP fusion protein. The extracellular domain of Notch containing the 36 EGFr was fused to human placental alkaline phosphatase (AP) and expressed in S2 cells. Measurement of AP activity was performed to determine whether the Njigsaw mutation affects the exocytosis of the mutant N protein. (E) Measurement of AP activity in the conditioned media revealed no difference in the secretion of NWT-AP and Njigsaw-AP. Furthermore, co-transfection of Fringe did not affect the exocytosis of NWT-AP and Njigsaw-AP.
24
Table S1. Comparison of EGFr of Drosophila and human Notch paralogs Fly Notch, human NOTCH1 and human NOTCH2 carry 36 EGFrs which are homologous, while human NOTCH3 and human NOTCH4 carry a fewer number of EGFrs, 34 and 29, respectively. The EGFr-2 of human NOTCH3 is thought to be a fusion between EGFr-2 and -3, while an EGFr that corresponds to EGFr-21 of fly Notch is absent (52). Homology of EGFrs for human NOTCH4 EGFrs-14 to -21 and EGFr-25 cannot be assigned due to divergence in sequence from the prototype Notch molecules that contain 36 EGFrs (53). EGFr-25 may be homologous to either EGFr-32 or -33.
25
Table S2 26
Table S2. Twenty-one new missense mutations in Notch isolated from the forward genetic screen on the X chromosome and their classification. (A) Molecular definition of novel missense alleles and conservation in the human NOTCH paralogs. 21 missense mutations were subdivided into 8 classes based on the type of mutation and their associated phenotypes. Allele names were given based on the domain and the amino acid changes, except in the case of Njigsaw. Homologous residues in 4 human NOTCH paralogs are listed along with the Drosophila mutation. The red coloring indicates that the fly and corresponding human amino acids are identical, the yellow indicates that they are similar and the blue indicates that they are different. Grey coloring with a “?” indicates that the corresponding residues cannot be identified due to the fewer number of EGFrs in NOTCH4 compared to fly Notch. (B) Definition and characteristic features of the 8 classes of new Notch alleles. Types of the molecular lesions, phenotypes and molecular mechanisms of the defect underlying the mutations are listed when applicable. N.D. stands for “not determined”.
27
Table S3 28
Table S3. Phenotypic characterization of newly isolated Notch missense alleles. Lethal phase analysis, documentation of dominant phenotypes in heterozygous flies and recessive phenotypes seen via clonal analysis of homozygous mutant cells are listed. In addition, results from immunofluorescence staining experiments to determine the level and cell surface localization of the mutant Notch proteins in homozygous mutant clones are also documented. N.D. stands for “not determined”.
29
References and Notes 1. S. Artavanis-Tsakonas, M. A. Muskavitch, Notch: The past, the present, and the future. Curr. Top. Dev. Biol. 92, 1 (2010). doi:10.1016/S0070-2153(10)92001-2 Medline 2. R. Kopan, M. X. Ilagan, The canonical Notch signaling pathway: Unfolding the activation mechanism. Cell 137, 216 (2009). doi:10.1016/j.cell.2009.03.045 Medline 3. I. Rebay et al., Specific EGF repeats of Notch mediate interactions with Delta and Serrate: Implications for Notch as a multifunctional receptor. Cell 67, 687 (1991). doi:10.1016/0092-8674(91)90064-6 Medline 4. Z. Pei, N. E. Baker, Competition between Delta and the Abruptex domain of Notch. BMC Dev. Biol. 8, 4 (2008). doi:10.1186/1471-213X-8-4 Medline 5. A. Joutel et al., Notch3 mutations in CADASIL, a hereditary adult-onset condition causing stroke and dementia. Nature 383, 707 (1996). doi:10.1038/383707a0 Medline 6. R. McDaniell et al., NOTCH2 mutations cause Alagille syndrome, a heterogeneous disorder of the notch signaling pathway. Am. J. Hum. Genet. 79, 169 (2006). doi:10.1086/505332 Medline 7. S. A. Mohamed et al., Novel missense mutations (p.T596M and p.P1797H) in NOTCH1 in patients with bicuspid aortic valve. Biochem. Biophys. Res. Commun. 345, 1460 (2006). doi:10.1016/j.bbrc.2006.05.046 Medline 8. S. H. McKellar et al., Novel NOTCH1 mutations in patients with bicuspid aortic valve disease and thoracic aortic aneurysms. J. Thorac. Cardiovasc. Surg. 134, 290 (2007). doi:10.1016/j.jtcvs.2007.02.041 Medline 9. K. L. McBride et al., NOTCH1 mutations in individuals with left ventricular outflow tract malformations reduce ligand-induced signaling. Hum. Mol. Genet. 17, 2886 (2008). doi:10.1093/hmg/ddn187 Medline 10. N. Agrawal et al., Exome sequencing of head and neck squamous cell carcinoma reveals inactivating mutations in NOTCH1. Science 333, 1154 (2011). doi:10.1126/science.1206923 Medline 11. N. J. Wang et al., Loss-of-function mutations in Notch receptors in cutaneous and lung squamous cell carcinoma. Proc. Natl. Acad. Sci. U.S.A. 108, 17761 (2011). doi:10.1073/pnas.1114669108 Medline 12. N. Stransky et al., The mutational landscape of head and neck squamous cell carcinoma. Science 333, 1157 (2011). doi:10.1126/science.1208130 Medline 13. J. F. de Celis, R. Barrio, A. del Arco, A. García-Bellido, Genetic and molecular characterization of a Notch mutation in its Delta- and Serrate-binding domain in Drosophila. Proc. Natl. Acad. Sci. U.S.A. 90, 4037 (1993). doi:10.1073/pnas.90.9.4037 Medline 14. C. Zeng, S. Younger-Shepherd, L. Y. Jan, Y. N. Jan, Delta and Serrate are redundant Notch ligands required for asymmetric cell divisions within the Drosophila sensory organ lineage. Genes Dev. 12, 1086 (1998). doi:10.1101/gad.12.8.1086 Medline 0
15. J. Kim, K. D. Irvine, S. B. Carroll, Cell recognition, signal induction, and symmetrical gene activation at the dorsal-ventral boundary of the developing Drosophila wing. Cell 82, 795 (1995). doi:10.1016/0092-8674(95)90476-X Medline 16. T. Klein, A. M. Arias, Interactions among Delta, Serrate and Fringe modulate Notch activity during Drosophila wing development. Development 125, 2951 (1998). Medline 17. K. Brückner, L. Perez, H. Clausen, S. Cohen, Glycosyltransferase activity of Fringe modulates Notch-Delta interactions. Nature 406, 411 (2000). doi:10.1038/35019075 Medline 18. D. J. Moloney et al., Fringe is a glycosyltransferase that modifies Notch. Nature 406, 369 (2000). doi:10.1038/35019000 Medline 19. T. Lebestky, S. H. Jung, U. Banerjee, A Serrate-expressing signaling center controls Drosophila hematopoiesis. Genes Dev. 17, 348 (2003). doi:10.1101/gad.1052803 Medline 20. A. S. Haberman, D. D. Isaac, D. J. Andrew, Specification of cell fates within the salivary gland primordium. Dev. Biol. 258, 443 (2003). doi:10.1016/S0012-1606(03)00140-4 Medline 21. I. Becam, U. M. Fiuza, A. M. Arias, M. Milán, A role of receptor Notch in ligand cisinhibition in Drosophila. Curr. Biol. 20, 554 (2010). doi:10.1016/j.cub.2010.01.058 Medline 22. N. A. Rana et al., O-glucose trisaccharide is present at high but variable stoichiometry at multiple sites on mouse Notch1. J. Biol. Chem. 286, 31623 (2011). doi:10.1074/jbc.M111.268243 Medline 23. A. Xu et al., In vitro reconstitution of the modulation of Drosophila Notch-ligand binding by Fringe. J. Biol. Chem. 282, 35153 (2007). doi:10.1074/jbc.M707040200 Medline 24. T. Okajima, A. Xu, K. D. Irvine, Modulation of notch-ligand binding by protein Ofucosyltransferase 1 and fringe. J. Biol. Chem. 278, 42340 (2003). doi:10.1074/jbc.M308687200 Medline 25. C. Hicks et al., Fringe differentially modulates Jagged1 and Delta1 signalling through Notch1 and Notch2. Nat. Cell Biol. 2, 515 (2000). doi:10.1038/35019553 Medline 26. D. L. Lindsley, G. G. Zimm, The Genome of Drosophila melanogaster (Academic Press, New York, 1992) 27. P. McQuilton, S. E. St Pierre, J. Thurmond; FlyBase Consortium, FlyBase 101—The basics of navigating FlyBase. Nucleic Acids Res. 40, D706 (2012). doi:10.1093/nar/gkr1030 Medline 28. J. F. de Celis, A. Garcia-Bellido, S. J. Bray, Activation and function of Notch at the dorsalventral boundary of the wing imaginal disc. Development 122, 359 (1996). Medline 29. K. J. Venken et al., Versatile P[acman] BAC libraries for transgenesis studies in Drosophila melanogaster. Nat. Methods 6, 431 (2009). doi:10.1038/nmeth.1331 Medline
1
30. J. Leonardi, R. Fernandez-Valdivia, Y. D. Li, A. A. Simcox, H. Jafar-Nejad, Multiple Oglucosylation sites on Notch function as a buffer against temperature-dependent loss of signaling. Development 138, 3569 (2011). doi:10.1242/dev.068361 Medline 31. M. Milán, U. Weihe, S. Tiong, W. Bender, S. M. Cohen, msh specifies dorsal cell fate in the Drosophila wing. Development 128, 3263 (2001). Medline 32. S. J. Yan, Y. Gu, W. X. Li, R. J. Fleming, Multiple signaling pathways and a selector protein sequentially regulate Drosophila wing development. Development 131, 285 (2004). doi:10.1242/dev.00934 Medline 33. G. Dietzl et al., A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448, 151 (2007). doi:10.1038/nature05954 Medline 34. B. E. Housden, K. Millen, S. J. Bray, G3 (Bethesda) 2, 79 (2012). 35. A. H. Brand, N. Perrimon, Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401 (1993). Medline 36. T. Xu, G. M. Rubin, Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117, 1223 (1993). Medline 37. T. Lee, C. Winter, S. S. Marticke, A. Lee, L. Luo, Essential roles of Drosophila RhoA in the regulation of neuroblast proliferation and dendritic but not axonal morphogenesis. Neuron 25, 307 (2000). doi:10.1016/S0896-6273(00)80896-X Medline 38. J. D. Thompson, T. J. Gibson, F. Plewniak, F. Jeanmougin, D. G. Higgins, The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876 (1997). doi:10.1093/nar/25.24.4876 Medline 39. N. Giagtzoglou et al., dEHBP1 controls exocytosis and recycling of Delta during asymmetric divisions. J. Cell Biol. 196, 65 (2012). doi:10.1083/jcb.201106088 Medline 40. T. Vaccari, H. Lu, R. Kanwar, M. E. Fortini, D. Bilder, Endosomal entry regulates Notch receptor activation in Drosophila melanogaster. J. Cell Biol. 180, 755 (2008). doi:10.1083/jcb.200708127 Medline 41. M. Acar et al., Rumi is a CAP10 domain glycosyltransferase that modifies Notch and is required for Notch signaling. Cell 132, 247 (2008). doi:10.1016/j.cell.2007.12.016 Medline 42. R. J. Diederich, K. Matsuno, H. Hing, S. Artavanis-Tsakonas, Cytosolic interaction between deltex and Notch ankyrin repeats implicates deltex in the Notch signaling pathway. Development 120, 473 (1994). Medline 43. R. G. Fehon et al., Molecular interactions between the protein products of the neurogenic loci Notch and Delta, two EGF-homologous genes in Drosophila. Cell 61, 523 (1990). doi:10.1016/0092-8674(90)90534-L Medline 44. K. Blochlinger, R. Bodmer, L. Y. Jan, Y. N. Jan, Patterns of expression of cut, a protein required for external sensory organ development in wild-type and cut mutant Drosophila embryos. Genes Dev. 4, 1322 (1990). doi:10.1101/gad.4.8.1322 Medline
2
45. W. J. Brook, S. M. Cohen, Antagonistic interactions between wingless and decapentaplegic responsible for dorsal-ventral pattern in the Drosophila leg. Science 273, 1373 (1996). doi:10.1126/science.273.5280.1373 Medline 46. M. Milán, S. M. Cohen, Temporal regulation of apterous activity during development of the Drosophila wing. Development 127, 3069 (2000). Medline 47. R. Nolo, L. A. Abbott, H. J. Bellen, Senseless, a Zn finger transcription factor, is necessary and sufficient for sensory organ development in Drosophila. Cell 102, 349 (2000). doi:10.1016/S0092-8674(00)00040-4 Medline 48. S. Robinow, K. White, Characterization and spatial distribution of the ELAV protein during Drosophila melanogaster development. J. Neurobiol. 22, 443 (1991). doi:10.1002/neu.480220503 Medline 49. T. Lebestky, T. Chang, V. Hartenstein, U. Banerjee, Specification of Drosophila hematopoietic lineage by conserved transcription factors. Science 288, 146 (2000). doi:10.1126/science.288.5463.146 Medline 50. R. D. Unwin, C. A. Evans, A. D. Whetton, Relative quantification in proteomics: New approaches for biochemistry. Trends Biochem. Sci. 31, 473 (2006). doi:10.1016/j.tibs.2006.06.003 Medline 51. R. Rampal, J. F. Arboleda-Velasquez, A. Nita-Lazar, K. S. Kosik, R. S. Haltiwanger, Highly conserved O-fucose sites have distinct effects on Notch1 function. J. Biol. Chem. 280, 32133 (2005). doi:10.1074/jbc.M506104200 Medline 52. M. Lardelli, J. Dahlstrand, U. Lendahl, The novel Notch homologue mouse Notch 3 lacks specific epidermal growth factor-repeats and is expressed in proliferating neuroepithelium. Mech. Dev. 46, 123 (1994). doi:10.1016/0925-4773(94)90081-7 Medline 53. H. Uyttendaele et al., Notch4/int-3, a mammary proto-oncogene, is an endothelial cellspecific mammalian Notch gene. Development 122, 2251 (1996). Medline
3
