CHAPTER SIXTEEN
Genetic Circuitry Modulating Notch Signals Through Endosomal Trafficking Kazuya Hori1, Anindya Sen1, Spyros Artavanis-Tsakonas2 Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA 1 These authors contributed equally to this work 2 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Genetic Screen Using the Exelixis Collection 2.1 The Exelixis collection 2.2 Materials 2.3 Genetic screen for modifiers of Dx–Krz action 3. Notch Localization in Endosomes 3.1 Immunostaining in cultured cells and imaginal discs 3.2 Endocytosis assay in cultured cells and imaginal discs 4. Optical Approaches 4.1 Construction of fluorescently tagged Notch and ligand molecules 4.2 Generation of stable cell lines 4.3 Rescue experiments 5. Ubiquitination Status of Notch 5.1 Materials 5.2 Ubiquitination assay 6. Conclusion Acknowledgments References
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Abstract Genetic modifier screens offer a powerful, indeed a uniquely powerful tool for the analysis and identification of elements capable of modulating specific cellular functions in development. Here, we describe the methodology that allowed us to explore the genetic circuitry that affects a Notch mutant phenotype caused by the abnormal endosomal trafficking of the Notch receptor. Endosomal trafficking events are increasingly appreciated to play a major role in controlling Notch signaling in development.
Methods in Enzymology, Volume 534 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-397926-1.00016-0
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1. INTRODUCTION The Notch pathway is used throughout development to couple the fate choices of a particular cell to those of neighboring cells, ultimately affecting proliferation, apoptosis, and differentiation (Artavanis-Tsakonas, Rand, & Lake, 1999; Bray, 2006; Schweisguth, 2004; Hori et al., 2013). Notch malfunction, which may result either in the up- or downregulation of the signal, has been associated with abnormal development in all metazoans examined and with diseases in humans including cancer (Louvi & Artavanis-Tsakonas, 2012). Notch encodes a single-pass transmembrane receptor and the developmental logic of this signaling pathway relies on the interaction of the receptor expressed on one cell with membrane-bound ligands expressed on its neighboring cell. The canonical signaling model has the Notch receptor being activated through a series of proteolytic events after it interacts with the ligands, Delta or Serrate (Bray, 2006). The crucial cleavage event for signaling depends on g-secretase and results in releasing the intracellular domain of Notch from the membrane, allowing it to translocate to the nucleus, where it participates directly in a core transcriptional complex together with the DNA-binding protein Suppressor of Hairless and the nuclear effector Mastermind, to activate the transcription of target genes (Bray, 2006; Schweisguth, 2004). The role of endocytic trafficking in the regulation of Notch signaling has been increasingly appreciated (Fortini, 2009; Yamamoto, Charng, & Bellen, 2010). Several endocytic factors modulating the degradation of the Notch receptor and consequently the negative attenuation of signaling have been identified (Fortini, 2009; Yamamoto et al., 2010). However, sorting of the receptor through the endocytic compartments has also been shown to result in the activation of the receptor. Such intracellular events have been associated with ligand-dependent (Coumailleau, Fu¨rthauer, Knoblich, & Gonza´lez-Gaita´n, 2009) as well as ligand-independent, that is, noncanonical, activation of the receptor (Childress, Acar, Tao, & Halder, 2006; Hori et al., 2004; Hori, Fuwa, Seki, & Matsuno, 2005; Hori, Sen, Kirchhausen, & Artavanis-Tsakonas, 2011; Thompson et al., 2005; Vaccari & Bilder, 2005; Vaccari, Lu, Kanwar, Fortini, & Bilder, 2008; Vaccari et al., 2009; Wilkin et al., 2004, 2008). Mutations in elements of the endosomal and multivesicular bodies sorting machinery have been shown to be sufficient to trigger ligand independent signaling of the receptor, but the mechanisms underlying these events and the genetic circuitry capable of modulating such intracellular Notch signaling remain opaque.
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We previously showed that Kurtz (Krz), the single nonvisual b-arrestin homolog in Drosophila, together with the ubiquitin ligase Deltex (Dx), affects trafficking of the Notch receptor and regulates Notch signaling by modulating the turnover of the receptor (Mukherjee et al., 2005). To gain further insight into how Krz and Dx regulate the trafficking of the receptor, we carried out genetic screens for modifiers of the Krz and Dx-dependent synergy, which is manifested in vivo as a typical Notch loss of function as evidenced by the notched wing phenotype. Here, we describe the genetic approach that allowed us to search for genetic modifiers of a double mutant krz/dx combination. This specific approach, originally designed to unveil elements of the endocytic machinery, also serves as an experimental paradigm for exploring a genetic circuitry that can affect any phenotype of choice.
2. GENETIC SCREEN USING THE EXELIXIS COLLECTION The advent of genome wide, molecularly characterized, mutant collections [e.g., Bloomington Drosophila Stock Center (BDSC, http://fly stocks.bio.indiana.edu/), Vienna Drosophila RNAi Center (VDRC, http://www.vdrc.at), and Drosophila Genetic Resource Center at Kyoto (DGRC, http://www.dgrc.kit.ac.jp/en/)] over the past few years has revolutionized the way we can elucidate genetic circuitries that affect specific phenotypic parameters. We can now carry out genetic screens with the highest degree of saturation of Drosophila genome, coupled with essentially instantaneous identification of the modifier genes. We have been extensively using the Exelixis collection (http://drosophila.med.harvard.edu/; Artavanis-Tsakonas, 2004) for probing genetic circuitries.
2.1. The Exelixis collection The collection is composed of 15,500 transposon-induced gene disruptions, resulting in mutations in 53% of the Drosophila genome (ArtavanisTsakonas, 2004; Parks et al., 2004; Thibault et al., 2004). Each insertion is derived from one of four vector types, three piggyBac-derived (PB, RB, and WH) and a fourth, a P-element variant (XP) (Parks et al., 2004; Thibault et al., 2004). Currently, the collection is 22% PB, 20% RB, 35% WH, and 23% XP. There are two classes of disruption events, those leading to inactivation of loci or those driving expression of downstream genes when combined with GAL4, due to the presence of Upstream Activation Sequence (UAS) sequences within the insertional transposon
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(WH and XP elements) (Parks et al., 2004; Thibault et al., 2004). In the presence of GAL4, UAS-containing insertions could theoretically inactivate loci even if oriented to drive expression, for example, by generating antisense RNA products. This can be investigated genetically by screening the collection in a background containing a loss-of-function mam or Notch allele without any GAL4 driver. WH insertions also contain splice acceptor sites (splice traps), permitting normal transcription of tagged genes but are designed such that WH, rather than endogenous, splice acceptors are used, allowing for a piece of the piggyBac transposon to be spliced into the final transcript, thereby disrupting translation. In the presence of GAL4, UAS-containing WH and XP insertions may represent hypomorphic, hypermorphic, neomorphic, or antisense alleles. In contrast, PB and RB insertions lack UAS sequences and are likely to represent null or hypomorphic alleles. Therefore, screening in a genetic background containing a GAL4-dependent phenotype allows one to exploit the full potential of the collection and to recover interactors representing both classes of insertional events. In the screen we describe here, we used the Exelixis collection to search for dominant modifiers of the wing-notching phenotype elicited by coexpression of Dx and Krz under C96-GAL4 control (Fig. 16.1B) (Hori et al., 2011). Given that this synergistic phenotype is a result of degradation of the Notch receptor via a ubiquitinrelated mechanism within the endocytic pathway, we expected to identify elements of the endocytic machinery that affect this phenotypic parameter.
2.2. Materials 1. The Exelixis collection and the Exelixis deficiency kits were obtained from Harvard Medical School (https://drosophila.med.harvard.edu). 2. A deficiency kit was obtained from the Bloomington Stock Center (http://flystocks.bio.indiana.edu/). 3. The following mutant alleles were used: N54l9 (Lindsley & Zimm, 1992), dx152 (Fuwa et al., 2006), and krz1 (Roman, He, & Davis, 2000). 4. The UAS lines used were UAS-Flag:Dx (Mukherjee et al., 2005), UASHA:Krz (Mukherjee et al., 2005), and UAS-NFL (Hori et al., 2004). The UAS constructs are driven by C96-Gal4 (Gustafson & Boulianne, 1996). All crosses were carried out at 25 C. 5. The genotypes of fly strains used in the screen were UAS-Flag:Dx/þ; C96-Gal4, UAS-HA:Krz/þ (C96-Dx þ Krz), UAS-Flag:Dx/þ; C96Gal4/þ (C96-Dx), C96-Gal4, UAS-HA:Krz/þ (C96-Krz), dx152; C96-Gal4/þ (dx152, C96), and krz1, C96-Gal4/þ (krz1, C96).
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Figure 16.1 Interaction of the screening stock with Notch. (A) Wild-type adult wing. (B) Coexpression of Dx and Krz driven by C96-Gal4 shows wing-nicking phenotype. (C) Heterozygous Notch null allele (N54l9/þ) is associated with the typical wing nicking. (D) N54l9/þ enhances Dx- and Krz-mediated wing-nicking phenotype. (E) Expression of full length Notch driven by C96-Gal4 does not affect wing morphology under our experimental conditions. (F) The wing-nicking phenotype associated with coexpression of Dx and Krz is rescued by expressing a transgene encoding wild-type Notch.
2.3. Genetic screen for modifiers of Dx–Krz action 1. Validation of the screening phenotype: In order to explore the feasibility of the screen for modifiers of the synergistic action of Dx and Krz, we needed to ensure that the phenotype was adequate for screening. We first determined that the genotype we generated for the screen (C96Dx þ Krz) is suited for screening given the health of the stock and its sensitivity to Notch pathway modulation, using known Notch pathway mutations. Figure 16.1 shows that the screening phenotype can be either enhanced by reducing the gene dosage of Notch (Fig. 16.1D) as N54l9 is a null allele of Notch or suppressed by expressing a wild-type copy of Notch transgene (Fig. 16.1F).
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2. Validation of the screening strategy: To explore whether modifiers of the Dx–Krz phenotype can be identified in an unbiased fashion, we first used the Bloomington deficiency kit (Bloomington Stock Center at Indiana University) as well as the Exelixis deficiency kit which is composed of smaller deletions (Harvard Medical School). C96-Dx þ Krz virgin females were crossed with males carrying autosomal deficiency. For deficiency on X chromosome, C96-Dx þ Krz males were crossed with virgin females carrying deficiency, and the F1 progeny was scored for phenotypic modifications. Both enhancer and suppressor deficiencies were thus identified, ensuring the feasibility of such a screen. Furthermore, the number of modifiers identified was not inordinately large, something that could render the significance of the results from the designed screens questionable. It is also worth pointing out that the deficiency screens revealed regions harboring known components of the Notch pathway. 3. Primary screen: In the primary screen, C96-Dx þ Krz virgin females were crossed with males carrying autosomal or viable X-linked insertions. C96-Dx þ Krz males were crossed with virgin females carrying lethal insertions on the X chromosome and the F1 progeny was scored for phenotypic modifications (Fig. 16.2). Modifying transposons were
Figure 16.2 Schematic representation of modifier screen. F1 modifier screen to identify genetic modifiers of Dx- and Krz-mediated wing phenotype using the Exelixis collection, composed of 15,500 transposon-induced gene disruptions. Individual Exelixis fly stocks harboring a unique transposon insertion was crossed to UAS-Flag:Dx/CyO; C96-Gal4, UAS-HA:Krz/TM3 and the F1 generation was screened for modifiers.
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categorized as enhancers or suppressors of weak, moderate, or strong intensity based on the observed wing-nicking phenotype. 4. Secondary screens: Mutations identified in the primary screen were crossed to flies carrying C96-Gal4 alone, to identify genes that affected the wing unilaterally and hence would be scored as positives (albeit false-positives) in the primary screen. Moreover, positive secondary tests were performed to examine the interaction with either dx or krz, alone, using wing phenotypes that result from the expression of Dx (C96-Dx), or the expression of Krz (C96-Krz) as well as mutant versions of dx mutant (dx152, C96), or krz (krz1, C96). Modifying transposons were again categorized as enhancers or suppressors of weak, moderate, or strong intensity in these independent screens.
3. NOTCH LOCALIZATION IN ENDOSOMES The genetic screen provided a roster of genes that are involved in different aspects of Notch activation mediated by Dx and Krz. To identify novel molecular players involved in endosomal trafficking of Notch, we probed the Notch localization and signaling output in either Schneider 2 (S2) cells, or Drosophila imaginal discs. S2 cells are derived from a primary culture of late stage (20–24 h old) Drosophila melanogaster embryo, from a macrophage-like lineage (Schneider, 1972). Analysis of Notch localization in endosomes involved colocalization of Notch (antibodies against Notch ECD and ICD) with different endosomal markers—antibodies against Rab5 (Abcam), Hook (Kra¨mer & Phistry, 1996), Hrs (Lloyd et al., 2002), Sara (Coumailleau et al., 2009), Rab7 (Chinchore, Mitra, & Dolph, 2009), LAMP1 (Abcam), Lysotracker (Molecular Probes), and so on. In addition, the signaling output of Notch activity was measured using antibodies against the Notch targets cut and wingless, in wing imaginal discs (Hori et al., 2011).
3.1. Immunostaining in cultured cells and imaginal discs 3.1.1 Materials 1. Drosophila S2 cells are cultured in Schneider’s Drosophila medium (Gibco) with 10% fetal bovine serum (Gibco) and penicillin– streptomycin (Gibco) at 25 C. 2. Wing imaginal discs dissected from third instar Drosophila larvae reared at 25 C. 3. Glass-bottom Petri dishes (MatTek Corporation).
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4. Concanavalin A (ConA, Sigma) treatment: To ConA coat glass-bottom dishes, spread 200 ml of the ConA solution (a 0.5 mg/ml in sterile water) onto the lid-facing side of the glass slide at the bottom of the Petri dish. After 30 min incubation of ConA, rinse the surface of the Petri dish with sterile water. The ConA-coated dishes are stored at room temperature (RT) and can be used months after preparation. 5. Fixative: 10 PBS, pH 7.0 (200 mM KPO4, 140 mM NaCl) 500 mM EGTA pH 6.4, 37% formaldehyde (Sigma). 6. Alternative fixative: 0.5 M PIPES pH 6.9, 500 mM EGTA pH 7.4, 1 M MgSO4, 37% formaldehyde (Sigma). 7. Blocking solution PBT: 10 PBS, pH 7.0 (200 mM KPO4, 140 mM NaCl) 500 mM EGTA pH 7.4, Triton X-100 BSA. 8. Wash buffer PT: 10 PBS, pH 7.0 (200 mM KPO4, 140 mM NaCl) 500 mM EGTA pH 7.4, Triton X-100. 9. Mounting medium: Vectashield (Vector Laboratories). 3.1.2 Methods 1. Drosophila S2 cells were cultured in glass-bottomed Petri dishes treated with ConA. 2. Fixative: Make a stock solution that is 1.33 PBS and 67 mM EGTA (“in situ fix”) or one that is 100 mM PIPES, 2 mM EGTA, and 1 mM MgSO4 (“PEMFA buffer”). These fixatives can be filter sterilized and stored for 1 year. 3. Make PT (1 PBS pH 7.0, 0.1% Triton X-100) and PBT (1 PBS pH 7.0, 0.1% Triton X-100, 2% BSA). Make PT in 1 l volumes, filter sterilize it, and store it at RT. To make PBT, use 50 ml of PT in a 50 ml conical and add 1 g of BSA. PBT spoils very quickly; store it at 4 C for 2–3 days. 4. (a) Drosophila S2 cells were fixed in 4% formaldehyde for 20 min at RT. (b) Drosophila third instar larval wing discs were dissected in cold 1 PBS and fixed in 4% formaldehyde for 40 min. The imaginal discs are processed in 1.5 ml microfuge tubes. 5. The fixative was poured off and the fixed cells or wing imaginal discs washed with four washes of PT, 15 min each. 6. Following this, 1 ml of blocking solution (PBT) was added for 1 h at RT. 7. The blocking solution was removed and primary antibody (diluted in blocking solution) was added directly onto the cells or wing imaginal discs. A total of 200 ml of antibody solution should completely cover
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the cells on a plate. The Petri lid was placed back on dish to prevent evaporation of the antibody solution. The antibody was incubated with the cells overnight at 4 C. The antibody solution was removed and three PT washes were performed. A transfer pipette was used to add 2 ml of PBT to the dish; each wash was 15 min for a total time of 1 h. After the last wash, 200 ml of fluorescent secondary antibody (diluted in blocking solution PBT) was added directly onto the cells or wing imaginal discs. The Petri dish (or 1.5 ml microfuge tubes) was covered with aluminum foil to prevent bleaching of fluorescent secondary antibody. The antibody was incubated with the cells for 2 h at RT. The secondary antibody was removed, followed by four 15-min washes with PT. After the last wash, a few drops of Vectashield were added and the dish was stored in a dark environment at 4 C, ready for light microscopy. Wing imaginal discs were mounted on a glass slide in a drop of Vectashield, covered with a glass cover slip and stored at 4 C.
3.2. Endocytosis assay in cultured cells and imaginal discs Either Notch–ligand interaction or ligand-independent activation of Notch in endosomes requires Notch trafficking through different endosomal compartments. To track such trafficking of the Notch receptor, imaginal discs or S2 cells are used to assay antibody uptake. 3.2.1 Materials 1. Drosophila S2 cells are cultured in Schneider’s Drosophila medium (Gibco) with 10% fetal bovine serum (Gibco) and penicillin–streptomycin (Gibco) at 25 C. 2. Wing imaginal discs from third instar Drosophila larvae reared at 25 C. 3. Glass-bottom Petri dishes (MatTek Corporation). 4. 0.5 mg/ml solution of ConA (in sterile water). 5. (1) Fixative: 10 PBS, pH 7.0 (200 mM KPO4, 140 mM NaCl) 500 mM EGTA pH 6.4, 37% formaldehyde (Sigma). (2) Alternative fixative: 0.5 M PIPES pH 6.9, 500 mM EGTA pH 7.4, 1 M MgSO4 37% formaldehyde (Sigma). 6. Blocking solution PBT: 10 PBS, pH 7.0 (200 mM KPO4, 140 mM NaCl) 500 mM EGTA pH 7.4, Triton X-100, BSA. 7. Wash buffer PT: 10 PBS, pH 7.0 (200 mM KPO4, 140 mM NaCl) 500 mM EGTA pH 7.4,Triton X-100.
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8. Mounting medium: Vectashield (Vector Laboratories). 9. Primary antibodies: anti-Notch ECD, an antibody for Notch extracellular domain (2H, Developmental Studies Hybridoma Bank). 3.2.2 Methods 1. (a) Drosophila S2 cells were cultured in glass-bottomed Petri dishes, treated with ConA. (b) Imaginal discs were dissected in standard M3 medium. 2. Pulse: Imaginal discs (or S2 cells) were incubated for either 15 or 40 min (depending on the experimental approach) with anti-Notch ECD (1:500) antibody at 25 C in M3 medium (Gibco). 3. Chase: The anti-Notch ECD antibody was washed off the cells (or wing imaginal discs in 1.5 ml microfuge tubes) with three washes of 10 min each of standard M3 medium at RT. 4. Drosophila S2 cells (or wing imaginal discs in 1.5 ml microfuge tubes) were fixed in 4% formaldehyde for 20 min at RT. 5. The fixative was poured off and fixed cells or wing imaginal discs were washed with four 15 min washes with PT. 6. 1 ml of blocking solution (PBT) was added to the cells (or wing imaginal discs in 1.5 ml microfuge tubes) for 1 h. 7. PBT was removed and 200 ml of fluorescent secondary antibody (diluted in blocking solution PBT) was added directly onto the cells or wing imaginal discs. The Petri dish or 1.5 ml microfuge tubes were covered with aluminum foil to prevent bleaching of fluorescent secondary antibody. The antibody was incubated with the cells for 2 h at RT. 8. The secondary antibody was removed, followed by four 15-min washes with PT. 9. After the last wash, a few drops of Vectashield were added and the dish was stored in a dark environment at 4 C, ready for light microscopy. For wing imaginal disc, the discs were mounted on a glass slide in a drop of Vectashield, covered with a glass cover slip and stored at 4 C.
4. OPTICAL APPROACHES To follow the trafficking of the Notch and Delta in live cells, we constructed and utilized fluorescently tagged Notch and Delta probes. Crucial to this is not only the construction of the probes but also a demonstration that they are functional and display the proper subcellular localization. We thus expressed each transgene in cultured cells to evaluate the subcellular localization of the corresponding tagged protein compared to the wild type
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and probed functionality by examining their ability to rescue loss-offunction phenotypes.
4.1. Construction of fluorescently tagged Notch and ligand molecules 1. We have tagged the Notch receptor and the ligands in multiple sites to ensure that we can generate functional molecules. A summary of the successful constructs is depicted in Fig. 16.3A.
Figure 16.3 Construction and validation of the Notch and ligands transgenes. (A) Schematic representation of EGFP-tag (green) in Notch and tdTomato-tag (red) in Delta and Serrate. (B) nd3/Y; C96-GAL4/þ males show characteristic notching at the wing tip. nd3 is a hypomorphic allele of Notch. (C) The nd3 phenotype was rescued by UAS-Notch-EGFP transgene expressed in the wing margin under the control of the C96-GAL4 driver.
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2. All constructs were expressed in cultured cells via transgenes under a metallothionein promoter (Invitrogen) or in vivo via transgenes under a UAS promoter.
4.2. Generation of stable cell lines 4.2.1 Materials 1. Drosophila S2Rþ cells are cultured in Schneider’s Drosophila medium (Gibco) with 10% fetal bovine serum (Gibco) and penicillin– streptomycin (Gibco) at 25 C. 2. DNA constructs: pMK33-Notch þ EGFP, pMK33-Dl þ tdTomato, pMK33-Ser þ tdTomato. The pMK33-derived vector contains a Hygromycin resistance gene that can be used for selection. As a negative control for selection, any vector that does not contain a Hygromycin resistant gene can be used. 3. Transfection reagent: TransIT-2020 (Mirus). We have evaluated several transfection reagents and found that the TransIT-2020 shows the highest efficiency of transfection in our assay. 4. Hygromycin (Invitrogen). 4.2.2 Methods 1. S2Rþ cells were seeded in 6-well plate at 0.5 106 cells/ml. 2. DNA constructs were transfected using TransIT-2020 (Mirus) following manufacturer’s recommended protocol. 3. After 48 h of transfection, cells were collected by gently pipetting, transferred to 15 ml falcon tube, and centrifuged at 500 rpm for 5 min. 4. The cells were resuspended in 6 ml medium with Hygromycin (100 mg/ml) and transferred into a T25 flask. 5. Every 2–3 days, cells were changed into fresh medium containing Hygromycin (100 mg/ml). 6. For 2–3 weeks, continuous cell death was visible. Controls (e.g., transfected with any empty vector) completely die out after 3–4 weeks. 7. After about a month, the stable line was established and they were expanded as necessary. 8. Expression of transgene was induced by 0.35 mM CuSO4 for 16–24 h. The subcellular localization of the receptors and ligands was confirmed to be on the membrane as well as in some intracellular endosomes. 9. The cells were passaged at 1:5 dilutions every 5 days into fresh medium containing Hygromycin (100 mg/ml).
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4.3. Rescue experiments 1. To confirm the functionality of the fluorescently tagged Notch and ligands, we performed rescue experiments. For this purpose, we established transgenic flies carrying pUAST-Notch þ EGFP, pUASTDelta þ tdTomato, and pUAST-Serrate þ tdTomato. 2. The UAS-Notch þ EGFP construct was driven by C96-Gal4 in the mutant background of Notch. Figure 16.3B and C shows an example showing rescue of the mutant phenotype with fluorescence-tagged Notch construct. 3. We have also demonstrated the biological activity of Delta þ tdTomato and Serrate þ tdTomato transgenes, by eliciting gain-of-function phenotypes in the wing.
5. UBIQUITINATION STATUS OF NOTCH Membrane trafficking and ubiquitination are generally intimately linked processes and a gene that affects Notch by modulating ubiquitylation is encoded by shrub. shrub encodes a core component of the ESCRT-III complex and was identified as a modifier of the dx–krz synergistic wing phenotype (Hori et al., 2011). To understand the role of Shrub in Notch signaling, we examined the subcellular localization of Notch by modulating the expression level of Shrub. We found that expression of Shrub leads to the accumulation of Notch in endosomes, which are positive for FK1 (Biomol), an antibody that recognizes poly-ubiquitinated proteins (Hori et al., 2011). This result suggested that Shrub regulates the trafficking of Notch though a ubiquitinylation process. Furthermore, we showed that Dx can also influence the ubiquitination status of Notch, which is a phenotype paralleled by an upregulation of Notch signaling (Hori et al., 2011). Utilizing antibody staining in vivo, we understood that Dx and Shrub regulate the trafficking of Notch and its activity. However, we still need to directly assess the relative roles of Dx and Shrub in the ubiquitinylation of Notch. For the purpose, we relied on S2Rþ cultured cells, which do not express Notch endogenously.
5.1. Materials 1. DNA constructs: pMT-NotchFL, pMT-Dx, and pMT-Flag-UbiquitinWT (UbWT), pMT-Flag-UbMono. Flag-UbMONO is a Flag-tagged mutated version of ubiquitin that can only participate in monoubiquitination events.
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2. Lysis buffer: 50 mM Tris pH 7.5, 125 mM NaCl, 1.5 mM MgCl2, 5% glycerol, 0.2% NP-40, 1 mM DTT, 25 mM NaF, 1 mM Na3VO4, and complete protease inhibitor (Roche). 3. Proteasome inhibitor: MG132 (Calbiochem).
5.2. Ubiquitination assay 1. S2R þ cells were seeded at 1 107 cells/ml into 100 mm dishes. 2. pMT-NotchFL, pMT-Dx, and pMT-Flag-UbWT (or pMT-FlagUbMono) are transfected by using the TransIT-2020 reagent (Mirus). Equal amounts of DNA constructs are transfected, and the total amount of DNA is kept constant by adding empty vector (15 mg of DNA constructs are transfected in total). One day after transfection, plasmid expression was induced with 0.35 mM CuSO4 overnight. 3. Cells were treated for 4 h at 25 C with a 50 mM concentration of the proteasome inhibitor MG132 (Calbiochem). 4. The cell culture dishes were placed on ice. The medium was drained, and cells were collected by pipetting in cold PBS. 5. The cells were washed using cold PBS twice and then gently transferred into precooled 1.5 ml microfuge tubes. 6. The cells were lysed in ice-cold lysis buffer. The lysates were incubated on ice for 20 min. 7. The lysates were cleared by centrifugation at 2000 rpm for 10 min at 4 C. 8. The supernatant was collected and placed in a fresh tube kept on ice. 9. On ice, 1000 mg cell lysate was added to the anti-Notch ICD (9C6, Developmental Studies Hybridoma Bank) (1:1000). 10. The sample was incubated with the antibody at 4 C under rotary agitation overnight. 11. The protein G-agarose beads were prepared (Roche) (20 ml of the slurry) by washing twice in 1 ml cold lysis buffer. 12. The beads were added to each sample and incubated at 4 C under rotary agitation for 2–3 h. 13. The precipitates were washed in 1 ml cold lysis buffer five times. 14. Protein complexes were eluted with 30 ml of 2 LDS sample buffer (Invitrogen) and heated at 70 C for 10 min. 15. The samples were run on a 3–7% Tris–Acetate gel (Invitrogen) and transferred onto PVDF membrane (Invitrogen). 16. The ubiquitinated Notch is detected by western blot using rabbit antiFLAG (Sigma, 1:1000).
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6. CONCLUSION Recent genetic and molecular studies have led to the increased appreciation of how important and indeed diverse, trafficking events related to the receptor and the ligands are for the developmental control of Notch signals. Here we described paradigmatic methodology to obtain an informative picture regarding the complexity and the nature of the genetic circuitry that affects Notch trafficking controlled by Dx and Krz. Such genetic screens while focused on a specific phenotype affecting one particular tissue, the value of the screen is more general. This particular screen recovered more than 250 modifiers of the wing phenotype we screened with only less than 20 modifiers associated with membrane trafficking as judged by Gene Ontology analysis (Flybase, http://flybase.org/) with the rest falling into diverse functional categories including, for example, gene regulations, metabolism, cytoskeleton, as well as several genes of unknown function (unpublished data). Linking molecularly each category or each gene with Notch is a nontrivial task (see e.g., Hori et al., 2011), notwithstanding the value of genetic, that is, functional links unveiled by genetic analyses.
ACKNOWLEDGMENTS We would like to thank Robert A. Obar and K. G. Guruharsha for critically reading the manuscript. This work was supported by NIH Grants NS26084 and CA98402 (S. A. -T.), a JSPS Postdoctoral Fellowship for Research Abroad (K. H.), and a Postdoctoral Fellowship from the FSMA (A. S.).
REFERENCES Artavanis-Tsakonas, S. (2004). Accessing the Exelixis collection. Nature Genetics, 36, 207. Artavanis-Tsakonas, S., Rand, M. D., & Lake, R. J. (1999). Notch signaling: Cell fate control and signal integration in development. Science, 284, 770–776. Bray, S. J. (2006). Notch signalling: A simple pathway becomes complex. Nature Reviews Molecular Cell Biology, 7, 678–689. Childress, J. L., Acar, M., Tao, C., & Halder, G. (2006). Lethal giant discs, a novel C2-domain protein, restricts notch activation during endocytosis. Current Biology, 16, 2228–2233. Chinchore, Y., Mitra, A., & Dolph, P. J. (2009). Accumulation of rhodopsin in late endosomes triggers photoreceptor cell degeneration. PLoS Genetics, 5, e1000377. Coumailleau, F., Fu¨rthauer, M., Knoblich, J. A., & Gonza´lez-Gaita´n, M. (2009). Directional Delta and Notch trafficking in Sara endosomes during asymmetric cell division. Nature, 458, 1051–1055. Fortini, M. E. (2009). Notch signaling: The core pathway and its posttranslational regulation. Developmental Cell, 16, 633–647.
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Fuwa, T. J., Hori, K., Sasamura, T., Higgs, J., Baron, M., & Matsuno, K. (2006). The first deltex null mutant indicates tissue-specific deltex-dependent Notch signaling in Drosophila. Molecular Genetics & Genomics, 275, 251–263. Gustafson, K., & Boulianne, G. L. (1996). Distinct expression patterns detected within individual tissues by the GAL4 enhancer trap technique. Genome, 39, 174–182. Hori, K., Fostier, M., Ito, M., Fuwa, T. J., Go, M. J., Okano, H., et al. (2004). Drosophila deltex mediates suppressor of Hairless-independent and late-endosomal activation of Notch signaling. Development, 131, 5527–5537. Hori, K., Fuwa, T. J., Seki, T., & Matsuno, K. (2005). Genetic regions that interact with lossand gain-of-function phenotypes of deltex implicate novel genes in Drosophila Notch signaling. Molecular Genetics & Genomics, 272, 627–638. Hori, K., Sen, A., Kirchhausen, T., & Artavanis-Tsakonas, S. (2011). Synergy between the ESCRT-III complex and Deltex defines a ligand-independent Notch signal. Journal of Cell Biology, 195, 1005–1015. Hori, K., Sen, A., & Artavanis-Tsakonas, S. (2013). Notch signaling at a glance. Journal of Cell Science, 126, 2135–2140. Kra¨mer, H., & Phistry, M. (1996). Mutations in the Drosophila hook gene inhibit endocytosis of the boss transmembrane ligand into multivesicular bodies. Journal of Cell Biology, 133, 1205–1215. Lindsley, D. L., & Zimm, G. G. (1992). The genome of Drosophila melanogaster. (pp. 492–497) San Diego, CA: Academic Press. Lloyd, T. E., Atkinson, R., Wu, M. N., Zhou, Y., Pennetta, G., & Bellen, H. J. (2002). Hrs regulates endosome membrane invagination and tyrosine kinase receptor signaling in Drosophila. Cell, 108, 261–269. Louvi, A., & Artavanis-Tsakonas, S. (2012). Notch and disease: A growing field. Seminars in Cell & Developmental Biology, 23, 473–480. Mukherjee, A., Veraksa, A., Bauer, A., Rosse, C., Camonis, J., & Artavanis-Tsakonas, S. (2005). Regulation of Notch signalling by non-visual beta-arrestin. Nature Cell Biology, 7, 1191–1201. Parks, A. L., Cook, K. R., Belvin, M., Dompe, N. A., Fawcett, R., Huppert, K., et al. (2004). Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome. Nature Genetics, 36, 288–292. Roman, G., He, J., & Davis, R. L. (2000). kurtz, a novel nonvisual arrestin, is an essential neural gene in Drosophila. Genetics, 155, 1281–1295. Schneider, I. (1972). Cell lines derived from late embryonic stages of Drosophila melanogaster. Journal of Embryology and Experimental Morphology, 27, 353–365. Schweisguth, F. (2004). Regulation of notch signaling activity. Current Biology, 14, R129–R138. Thibault, S. T., Singer, M. A., Miyazaki, W. Y., Milash, B., Dompe, N. A., Singh, C. M., et al. (2004). A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac. Nature Genetics, 36, 283–287. Thompson, B. J., Mathieu, J., Sung, H. H., Loeser, E., Rørth, P., & Cohen, S. M. (2005). Tumor suppressor properties of the ESCRT-II complex component Vps25 in Drosophila. Developmental Cell, 9, 711–720. Vaccari, T., & Bilder, D. (2005). The Drosophila tumor suppressor vps25 prevents nonautonomous overproliferation by regulating notch trafficking. Developmental Cell, 9, 687–698. Vaccari, T., Lu, H., Kanwar, R., Fortini, M. E., & Bilder, D. (2008). Endosomal entry regulates Notch receptor activation in Drosophila melanogaster. Journal of Cell Biology, 180, 755–762. Vaccari, T., Rusten, T. E., Menut, L., Nezis, I. P., Brech, A., Stenmark, H., et al. (2009). Comparative analysis of ESCRT-I, ESCRT-II and ESCRT-III function in
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Drosophila by efficient isolation of ESCRT mutants. Journal of Cell Science, 122, 2413–2423. Wilkin, M. B., Carbery, A. M., Fostier, M., Aslam, H., Mazaleyrat, S. L., Higgs, J., et al. (2004). Regulation of notch endosomal sorting and signaling by Drosophila Nedd4 family proteins. Current Biology, 14, 2237–2244. Wilkin, M., Tongngok, P., Gensch, N., Clemence, S., Motoki, M., Yamada, K., et al. (2008). Drosophila HOPS and AP-3 complex genes are required for a Deltex-regulated activation of notch in the endosomal trafficking pathway. Developmental Cell, 15, 762–772. Yamamoto, S., Charng, W. L., & Bellen, H. J. (2010). Endocytosis and intracellular trafficking of Notch and its ligands. Current Topics in Developmental Biology, 92, 165–200.
Genetic Circuitry Modulating Notch Signals Through Endosomal Trafficking Kazuya Hori1, Anindya Sen1, Spyros Artavanis-Tsakonas2 Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA 1 These authors contributed equally to this work 2 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Genetic Screen Using the Exelixis Collection 2.1 The Exelixis collection 2.2 Materials 2.3 Genetic screen for modifiers of Dx–Krz action 3. Notch Localization in Endosomes 3.1 Immunostaining in cultured cells and imaginal discs 3.2 Endocytosis assay in cultured cells and imaginal discs 4. Optical Approaches 4.1 Construction of fluorescently tagged Notch and ligand molecules 4.2 Generation of stable cell lines 4.3 Rescue experiments 5. Ubiquitination Status of Notch 5.1 Materials 5.2 Ubiquitination assay 6. Conclusion Acknowledgments References
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Abstract Genetic modifier screens offer a powerful, indeed a uniquely powerful tool for the analysis and identification of elements capable of modulating specific cellular functions in development. Here, we describe the methodology that allowed us to explore the genetic circuitry that affects a Notch mutant phenotype caused by the abnormal endosomal trafficking of the Notch receptor. Endosomal trafficking events are increasingly appreciated to play a major role in controlling Notch signaling in development.
Methods in Enzymology, Volume 534 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-397926-1.00016-0
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1. INTRODUCTION The Notch pathway is used throughout development to couple the fate choices of a particular cell to those of neighboring cells, ultimately affecting proliferation, apoptosis, and differentiation (Artavanis-Tsakonas, Rand, & Lake, 1999; Bray, 2006; Schweisguth, 2004; Hori et al., 2013). Notch malfunction, which may result either in the up- or downregulation of the signal, has been associated with abnormal development in all metazoans examined and with diseases in humans including cancer (Louvi & Artavanis-Tsakonas, 2012). Notch encodes a single-pass transmembrane receptor and the developmental logic of this signaling pathway relies on the interaction of the receptor expressed on one cell with membrane-bound ligands expressed on its neighboring cell. The canonical signaling model has the Notch receptor being activated through a series of proteolytic events after it interacts with the ligands, Delta or Serrate (Bray, 2006). The crucial cleavage event for signaling depends on g-secretase and results in releasing the intracellular domain of Notch from the membrane, allowing it to translocate to the nucleus, where it participates directly in a core transcriptional complex together with the DNA-binding protein Suppressor of Hairless and the nuclear effector Mastermind, to activate the transcription of target genes (Bray, 2006; Schweisguth, 2004). The role of endocytic trafficking in the regulation of Notch signaling has been increasingly appreciated (Fortini, 2009; Yamamoto, Charng, & Bellen, 2010). Several endocytic factors modulating the degradation of the Notch receptor and consequently the negative attenuation of signaling have been identified (Fortini, 2009; Yamamoto et al., 2010). However, sorting of the receptor through the endocytic compartments has also been shown to result in the activation of the receptor. Such intracellular events have been associated with ligand-dependent (Coumailleau, Fu¨rthauer, Knoblich, & Gonza´lez-Gaita´n, 2009) as well as ligand-independent, that is, noncanonical, activation of the receptor (Childress, Acar, Tao, & Halder, 2006; Hori et al., 2004; Hori, Fuwa, Seki, & Matsuno, 2005; Hori, Sen, Kirchhausen, & Artavanis-Tsakonas, 2011; Thompson et al., 2005; Vaccari & Bilder, 2005; Vaccari, Lu, Kanwar, Fortini, & Bilder, 2008; Vaccari et al., 2009; Wilkin et al., 2004, 2008). Mutations in elements of the endosomal and multivesicular bodies sorting machinery have been shown to be sufficient to trigger ligand independent signaling of the receptor, but the mechanisms underlying these events and the genetic circuitry capable of modulating such intracellular Notch signaling remain opaque.
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We previously showed that Kurtz (Krz), the single nonvisual b-arrestin homolog in Drosophila, together with the ubiquitin ligase Deltex (Dx), affects trafficking of the Notch receptor and regulates Notch signaling by modulating the turnover of the receptor (Mukherjee et al., 2005). To gain further insight into how Krz and Dx regulate the trafficking of the receptor, we carried out genetic screens for modifiers of the Krz and Dx-dependent synergy, which is manifested in vivo as a typical Notch loss of function as evidenced by the notched wing phenotype. Here, we describe the genetic approach that allowed us to search for genetic modifiers of a double mutant krz/dx combination. This specific approach, originally designed to unveil elements of the endocytic machinery, also serves as an experimental paradigm for exploring a genetic circuitry that can affect any phenotype of choice.
2. GENETIC SCREEN USING THE EXELIXIS COLLECTION The advent of genome wide, molecularly characterized, mutant collections [e.g., Bloomington Drosophila Stock Center (BDSC, http://fly stocks.bio.indiana.edu/), Vienna Drosophila RNAi Center (VDRC, http://www.vdrc.at), and Drosophila Genetic Resource Center at Kyoto (DGRC, http://www.dgrc.kit.ac.jp/en/)] over the past few years has revolutionized the way we can elucidate genetic circuitries that affect specific phenotypic parameters. We can now carry out genetic screens with the highest degree of saturation of Drosophila genome, coupled with essentially instantaneous identification of the modifier genes. We have been extensively using the Exelixis collection (http://drosophila.med.harvard.edu/; Artavanis-Tsakonas, 2004) for probing genetic circuitries.
2.1. The Exelixis collection The collection is composed of 15,500 transposon-induced gene disruptions, resulting in mutations in 53% of the Drosophila genome (ArtavanisTsakonas, 2004; Parks et al., 2004; Thibault et al., 2004). Each insertion is derived from one of four vector types, three piggyBac-derived (PB, RB, and WH) and a fourth, a P-element variant (XP) (Parks et al., 2004; Thibault et al., 2004). Currently, the collection is 22% PB, 20% RB, 35% WH, and 23% XP. There are two classes of disruption events, those leading to inactivation of loci or those driving expression of downstream genes when combined with GAL4, due to the presence of Upstream Activation Sequence (UAS) sequences within the insertional transposon
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(WH and XP elements) (Parks et al., 2004; Thibault et al., 2004). In the presence of GAL4, UAS-containing insertions could theoretically inactivate loci even if oriented to drive expression, for example, by generating antisense RNA products. This can be investigated genetically by screening the collection in a background containing a loss-of-function mam or Notch allele without any GAL4 driver. WH insertions also contain splice acceptor sites (splice traps), permitting normal transcription of tagged genes but are designed such that WH, rather than endogenous, splice acceptors are used, allowing for a piece of the piggyBac transposon to be spliced into the final transcript, thereby disrupting translation. In the presence of GAL4, UAS-containing WH and XP insertions may represent hypomorphic, hypermorphic, neomorphic, or antisense alleles. In contrast, PB and RB insertions lack UAS sequences and are likely to represent null or hypomorphic alleles. Therefore, screening in a genetic background containing a GAL4-dependent phenotype allows one to exploit the full potential of the collection and to recover interactors representing both classes of insertional events. In the screen we describe here, we used the Exelixis collection to search for dominant modifiers of the wing-notching phenotype elicited by coexpression of Dx and Krz under C96-GAL4 control (Fig. 16.1B) (Hori et al., 2011). Given that this synergistic phenotype is a result of degradation of the Notch receptor via a ubiquitinrelated mechanism within the endocytic pathway, we expected to identify elements of the endocytic machinery that affect this phenotypic parameter.
2.2. Materials 1. The Exelixis collection and the Exelixis deficiency kits were obtained from Harvard Medical School (https://drosophila.med.harvard.edu). 2. A deficiency kit was obtained from the Bloomington Stock Center (http://flystocks.bio.indiana.edu/). 3. The following mutant alleles were used: N54l9 (Lindsley & Zimm, 1992), dx152 (Fuwa et al., 2006), and krz1 (Roman, He, & Davis, 2000). 4. The UAS lines used were UAS-Flag:Dx (Mukherjee et al., 2005), UASHA:Krz (Mukherjee et al., 2005), and UAS-NFL (Hori et al., 2004). The UAS constructs are driven by C96-Gal4 (Gustafson & Boulianne, 1996). All crosses were carried out at 25 C. 5. The genotypes of fly strains used in the screen were UAS-Flag:Dx/þ; C96-Gal4, UAS-HA:Krz/þ (C96-Dx þ Krz), UAS-Flag:Dx/þ; C96Gal4/þ (C96-Dx), C96-Gal4, UAS-HA:Krz/þ (C96-Krz), dx152; C96-Gal4/þ (dx152, C96), and krz1, C96-Gal4/þ (krz1, C96).
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Figure 16.1 Interaction of the screening stock with Notch. (A) Wild-type adult wing. (B) Coexpression of Dx and Krz driven by C96-Gal4 shows wing-nicking phenotype. (C) Heterozygous Notch null allele (N54l9/þ) is associated with the typical wing nicking. (D) N54l9/þ enhances Dx- and Krz-mediated wing-nicking phenotype. (E) Expression of full length Notch driven by C96-Gal4 does not affect wing morphology under our experimental conditions. (F) The wing-nicking phenotype associated with coexpression of Dx and Krz is rescued by expressing a transgene encoding wild-type Notch.
2.3. Genetic screen for modifiers of Dx–Krz action 1. Validation of the screening phenotype: In order to explore the feasibility of the screen for modifiers of the synergistic action of Dx and Krz, we needed to ensure that the phenotype was adequate for screening. We first determined that the genotype we generated for the screen (C96Dx þ Krz) is suited for screening given the health of the stock and its sensitivity to Notch pathway modulation, using known Notch pathway mutations. Figure 16.1 shows that the screening phenotype can be either enhanced by reducing the gene dosage of Notch (Fig. 16.1D) as N54l9 is a null allele of Notch or suppressed by expressing a wild-type copy of Notch transgene (Fig. 16.1F).
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2. Validation of the screening strategy: To explore whether modifiers of the Dx–Krz phenotype can be identified in an unbiased fashion, we first used the Bloomington deficiency kit (Bloomington Stock Center at Indiana University) as well as the Exelixis deficiency kit which is composed of smaller deletions (Harvard Medical School). C96-Dx þ Krz virgin females were crossed with males carrying autosomal deficiency. For deficiency on X chromosome, C96-Dx þ Krz males were crossed with virgin females carrying deficiency, and the F1 progeny was scored for phenotypic modifications. Both enhancer and suppressor deficiencies were thus identified, ensuring the feasibility of such a screen. Furthermore, the number of modifiers identified was not inordinately large, something that could render the significance of the results from the designed screens questionable. It is also worth pointing out that the deficiency screens revealed regions harboring known components of the Notch pathway. 3. Primary screen: In the primary screen, C96-Dx þ Krz virgin females were crossed with males carrying autosomal or viable X-linked insertions. C96-Dx þ Krz males were crossed with virgin females carrying lethal insertions on the X chromosome and the F1 progeny was scored for phenotypic modifications (Fig. 16.2). Modifying transposons were
Figure 16.2 Schematic representation of modifier screen. F1 modifier screen to identify genetic modifiers of Dx- and Krz-mediated wing phenotype using the Exelixis collection, composed of 15,500 transposon-induced gene disruptions. Individual Exelixis fly stocks harboring a unique transposon insertion was crossed to UAS-Flag:Dx/CyO; C96-Gal4, UAS-HA:Krz/TM3 and the F1 generation was screened for modifiers.
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categorized as enhancers or suppressors of weak, moderate, or strong intensity based on the observed wing-nicking phenotype. 4. Secondary screens: Mutations identified in the primary screen were crossed to flies carrying C96-Gal4 alone, to identify genes that affected the wing unilaterally and hence would be scored as positives (albeit false-positives) in the primary screen. Moreover, positive secondary tests were performed to examine the interaction with either dx or krz, alone, using wing phenotypes that result from the expression of Dx (C96-Dx), or the expression of Krz (C96-Krz) as well as mutant versions of dx mutant (dx152, C96), or krz (krz1, C96). Modifying transposons were again categorized as enhancers or suppressors of weak, moderate, or strong intensity in these independent screens.
3. NOTCH LOCALIZATION IN ENDOSOMES The genetic screen provided a roster of genes that are involved in different aspects of Notch activation mediated by Dx and Krz. To identify novel molecular players involved in endosomal trafficking of Notch, we probed the Notch localization and signaling output in either Schneider 2 (S2) cells, or Drosophila imaginal discs. S2 cells are derived from a primary culture of late stage (20–24 h old) Drosophila melanogaster embryo, from a macrophage-like lineage (Schneider, 1972). Analysis of Notch localization in endosomes involved colocalization of Notch (antibodies against Notch ECD and ICD) with different endosomal markers—antibodies against Rab5 (Abcam), Hook (Kra¨mer & Phistry, 1996), Hrs (Lloyd et al., 2002), Sara (Coumailleau et al., 2009), Rab7 (Chinchore, Mitra, & Dolph, 2009), LAMP1 (Abcam), Lysotracker (Molecular Probes), and so on. In addition, the signaling output of Notch activity was measured using antibodies against the Notch targets cut and wingless, in wing imaginal discs (Hori et al., 2011).
3.1. Immunostaining in cultured cells and imaginal discs 3.1.1 Materials 1. Drosophila S2 cells are cultured in Schneider’s Drosophila medium (Gibco) with 10% fetal bovine serum (Gibco) and penicillin– streptomycin (Gibco) at 25 C. 2. Wing imaginal discs dissected from third instar Drosophila larvae reared at 25 C. 3. Glass-bottom Petri dishes (MatTek Corporation).
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4. Concanavalin A (ConA, Sigma) treatment: To ConA coat glass-bottom dishes, spread 200 ml of the ConA solution (a 0.5 mg/ml in sterile water) onto the lid-facing side of the glass slide at the bottom of the Petri dish. After 30 min incubation of ConA, rinse the surface of the Petri dish with sterile water. The ConA-coated dishes are stored at room temperature (RT) and can be used months after preparation. 5. Fixative: 10 PBS, pH 7.0 (200 mM KPO4, 140 mM NaCl) 500 mM EGTA pH 6.4, 37% formaldehyde (Sigma). 6. Alternative fixative: 0.5 M PIPES pH 6.9, 500 mM EGTA pH 7.4, 1 M MgSO4, 37% formaldehyde (Sigma). 7. Blocking solution PBT: 10 PBS, pH 7.0 (200 mM KPO4, 140 mM NaCl) 500 mM EGTA pH 7.4, Triton X-100 BSA. 8. Wash buffer PT: 10 PBS, pH 7.0 (200 mM KPO4, 140 mM NaCl) 500 mM EGTA pH 7.4, Triton X-100. 9. Mounting medium: Vectashield (Vector Laboratories). 3.1.2 Methods 1. Drosophila S2 cells were cultured in glass-bottomed Petri dishes treated with ConA. 2. Fixative: Make a stock solution that is 1.33 PBS and 67 mM EGTA (“in situ fix”) or one that is 100 mM PIPES, 2 mM EGTA, and 1 mM MgSO4 (“PEMFA buffer”). These fixatives can be filter sterilized and stored for 1 year. 3. Make PT (1 PBS pH 7.0, 0.1% Triton X-100) and PBT (1 PBS pH 7.0, 0.1% Triton X-100, 2% BSA). Make PT in 1 l volumes, filter sterilize it, and store it at RT. To make PBT, use 50 ml of PT in a 50 ml conical and add 1 g of BSA. PBT spoils very quickly; store it at 4 C for 2–3 days. 4. (a) Drosophila S2 cells were fixed in 4% formaldehyde for 20 min at RT. (b) Drosophila third instar larval wing discs were dissected in cold 1 PBS and fixed in 4% formaldehyde for 40 min. The imaginal discs are processed in 1.5 ml microfuge tubes. 5. The fixative was poured off and the fixed cells or wing imaginal discs washed with four washes of PT, 15 min each. 6. Following this, 1 ml of blocking solution (PBT) was added for 1 h at RT. 7. The blocking solution was removed and primary antibody (diluted in blocking solution) was added directly onto the cells or wing imaginal discs. A total of 200 ml of antibody solution should completely cover
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the cells on a plate. The Petri lid was placed back on dish to prevent evaporation of the antibody solution. The antibody was incubated with the cells overnight at 4 C. The antibody solution was removed and three PT washes were performed. A transfer pipette was used to add 2 ml of PBT to the dish; each wash was 15 min for a total time of 1 h. After the last wash, 200 ml of fluorescent secondary antibody (diluted in blocking solution PBT) was added directly onto the cells or wing imaginal discs. The Petri dish (or 1.5 ml microfuge tubes) was covered with aluminum foil to prevent bleaching of fluorescent secondary antibody. The antibody was incubated with the cells for 2 h at RT. The secondary antibody was removed, followed by four 15-min washes with PT. After the last wash, a few drops of Vectashield were added and the dish was stored in a dark environment at 4 C, ready for light microscopy. Wing imaginal discs were mounted on a glass slide in a drop of Vectashield, covered with a glass cover slip and stored at 4 C.
3.2. Endocytosis assay in cultured cells and imaginal discs Either Notch–ligand interaction or ligand-independent activation of Notch in endosomes requires Notch trafficking through different endosomal compartments. To track such trafficking of the Notch receptor, imaginal discs or S2 cells are used to assay antibody uptake. 3.2.1 Materials 1. Drosophila S2 cells are cultured in Schneider’s Drosophila medium (Gibco) with 10% fetal bovine serum (Gibco) and penicillin–streptomycin (Gibco) at 25 C. 2. Wing imaginal discs from third instar Drosophila larvae reared at 25 C. 3. Glass-bottom Petri dishes (MatTek Corporation). 4. 0.5 mg/ml solution of ConA (in sterile water). 5. (1) Fixative: 10 PBS, pH 7.0 (200 mM KPO4, 140 mM NaCl) 500 mM EGTA pH 6.4, 37% formaldehyde (Sigma). (2) Alternative fixative: 0.5 M PIPES pH 6.9, 500 mM EGTA pH 7.4, 1 M MgSO4 37% formaldehyde (Sigma). 6. Blocking solution PBT: 10 PBS, pH 7.0 (200 mM KPO4, 140 mM NaCl) 500 mM EGTA pH 7.4, Triton X-100, BSA. 7. Wash buffer PT: 10 PBS, pH 7.0 (200 mM KPO4, 140 mM NaCl) 500 mM EGTA pH 7.4,Triton X-100.
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8. Mounting medium: Vectashield (Vector Laboratories). 9. Primary antibodies: anti-Notch ECD, an antibody for Notch extracellular domain (2H, Developmental Studies Hybridoma Bank). 3.2.2 Methods 1. (a) Drosophila S2 cells were cultured in glass-bottomed Petri dishes, treated with ConA. (b) Imaginal discs were dissected in standard M3 medium. 2. Pulse: Imaginal discs (or S2 cells) were incubated for either 15 or 40 min (depending on the experimental approach) with anti-Notch ECD (1:500) antibody at 25 C in M3 medium (Gibco). 3. Chase: The anti-Notch ECD antibody was washed off the cells (or wing imaginal discs in 1.5 ml microfuge tubes) with three washes of 10 min each of standard M3 medium at RT. 4. Drosophila S2 cells (or wing imaginal discs in 1.5 ml microfuge tubes) were fixed in 4% formaldehyde for 20 min at RT. 5. The fixative was poured off and fixed cells or wing imaginal discs were washed with four 15 min washes with PT. 6. 1 ml of blocking solution (PBT) was added to the cells (or wing imaginal discs in 1.5 ml microfuge tubes) for 1 h. 7. PBT was removed and 200 ml of fluorescent secondary antibody (diluted in blocking solution PBT) was added directly onto the cells or wing imaginal discs. The Petri dish or 1.5 ml microfuge tubes were covered with aluminum foil to prevent bleaching of fluorescent secondary antibody. The antibody was incubated with the cells for 2 h at RT. 8. The secondary antibody was removed, followed by four 15-min washes with PT. 9. After the last wash, a few drops of Vectashield were added and the dish was stored in a dark environment at 4 C, ready for light microscopy. For wing imaginal disc, the discs were mounted on a glass slide in a drop of Vectashield, covered with a glass cover slip and stored at 4 C.
4. OPTICAL APPROACHES To follow the trafficking of the Notch and Delta in live cells, we constructed and utilized fluorescently tagged Notch and Delta probes. Crucial to this is not only the construction of the probes but also a demonstration that they are functional and display the proper subcellular localization. We thus expressed each transgene in cultured cells to evaluate the subcellular localization of the corresponding tagged protein compared to the wild type
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and probed functionality by examining their ability to rescue loss-offunction phenotypes.
4.1. Construction of fluorescently tagged Notch and ligand molecules 1. We have tagged the Notch receptor and the ligands in multiple sites to ensure that we can generate functional molecules. A summary of the successful constructs is depicted in Fig. 16.3A.
Figure 16.3 Construction and validation of the Notch and ligands transgenes. (A) Schematic representation of EGFP-tag (green) in Notch and tdTomato-tag (red) in Delta and Serrate. (B) nd3/Y; C96-GAL4/þ males show characteristic notching at the wing tip. nd3 is a hypomorphic allele of Notch. (C) The nd3 phenotype was rescued by UAS-Notch-EGFP transgene expressed in the wing margin under the control of the C96-GAL4 driver.
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2. All constructs were expressed in cultured cells via transgenes under a metallothionein promoter (Invitrogen) or in vivo via transgenes under a UAS promoter.
4.2. Generation of stable cell lines 4.2.1 Materials 1. Drosophila S2Rþ cells are cultured in Schneider’s Drosophila medium (Gibco) with 10% fetal bovine serum (Gibco) and penicillin– streptomycin (Gibco) at 25 C. 2. DNA constructs: pMK33-Notch þ EGFP, pMK33-Dl þ tdTomato, pMK33-Ser þ tdTomato. The pMK33-derived vector contains a Hygromycin resistance gene that can be used for selection. As a negative control for selection, any vector that does not contain a Hygromycin resistant gene can be used. 3. Transfection reagent: TransIT-2020 (Mirus). We have evaluated several transfection reagents and found that the TransIT-2020 shows the highest efficiency of transfection in our assay. 4. Hygromycin (Invitrogen). 4.2.2 Methods 1. S2Rþ cells were seeded in 6-well plate at 0.5 106 cells/ml. 2. DNA constructs were transfected using TransIT-2020 (Mirus) following manufacturer’s recommended protocol. 3. After 48 h of transfection, cells were collected by gently pipetting, transferred to 15 ml falcon tube, and centrifuged at 500 rpm for 5 min. 4. The cells were resuspended in 6 ml medium with Hygromycin (100 mg/ml) and transferred into a T25 flask. 5. Every 2–3 days, cells were changed into fresh medium containing Hygromycin (100 mg/ml). 6. For 2–3 weeks, continuous cell death was visible. Controls (e.g., transfected with any empty vector) completely die out after 3–4 weeks. 7. After about a month, the stable line was established and they were expanded as necessary. 8. Expression of transgene was induced by 0.35 mM CuSO4 for 16–24 h. The subcellular localization of the receptors and ligands was confirmed to be on the membrane as well as in some intracellular endosomes. 9. The cells were passaged at 1:5 dilutions every 5 days into fresh medium containing Hygromycin (100 mg/ml).
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4.3. Rescue experiments 1. To confirm the functionality of the fluorescently tagged Notch and ligands, we performed rescue experiments. For this purpose, we established transgenic flies carrying pUAST-Notch þ EGFP, pUASTDelta þ tdTomato, and pUAST-Serrate þ tdTomato. 2. The UAS-Notch þ EGFP construct was driven by C96-Gal4 in the mutant background of Notch. Figure 16.3B and C shows an example showing rescue of the mutant phenotype with fluorescence-tagged Notch construct. 3. We have also demonstrated the biological activity of Delta þ tdTomato and Serrate þ tdTomato transgenes, by eliciting gain-of-function phenotypes in the wing.
5. UBIQUITINATION STATUS OF NOTCH Membrane trafficking and ubiquitination are generally intimately linked processes and a gene that affects Notch by modulating ubiquitylation is encoded by shrub. shrub encodes a core component of the ESCRT-III complex and was identified as a modifier of the dx–krz synergistic wing phenotype (Hori et al., 2011). To understand the role of Shrub in Notch signaling, we examined the subcellular localization of Notch by modulating the expression level of Shrub. We found that expression of Shrub leads to the accumulation of Notch in endosomes, which are positive for FK1 (Biomol), an antibody that recognizes poly-ubiquitinated proteins (Hori et al., 2011). This result suggested that Shrub regulates the trafficking of Notch though a ubiquitinylation process. Furthermore, we showed that Dx can also influence the ubiquitination status of Notch, which is a phenotype paralleled by an upregulation of Notch signaling (Hori et al., 2011). Utilizing antibody staining in vivo, we understood that Dx and Shrub regulate the trafficking of Notch and its activity. However, we still need to directly assess the relative roles of Dx and Shrub in the ubiquitinylation of Notch. For the purpose, we relied on S2Rþ cultured cells, which do not express Notch endogenously.
5.1. Materials 1. DNA constructs: pMT-NotchFL, pMT-Dx, and pMT-Flag-UbiquitinWT (UbWT), pMT-Flag-UbMono. Flag-UbMONO is a Flag-tagged mutated version of ubiquitin that can only participate in monoubiquitination events.
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2. Lysis buffer: 50 mM Tris pH 7.5, 125 mM NaCl, 1.5 mM MgCl2, 5% glycerol, 0.2% NP-40, 1 mM DTT, 25 mM NaF, 1 mM Na3VO4, and complete protease inhibitor (Roche). 3. Proteasome inhibitor: MG132 (Calbiochem).
5.2. Ubiquitination assay 1. S2R þ cells were seeded at 1 107 cells/ml into 100 mm dishes. 2. pMT-NotchFL, pMT-Dx, and pMT-Flag-UbWT (or pMT-FlagUbMono) are transfected by using the TransIT-2020 reagent (Mirus). Equal amounts of DNA constructs are transfected, and the total amount of DNA is kept constant by adding empty vector (15 mg of DNA constructs are transfected in total). One day after transfection, plasmid expression was induced with 0.35 mM CuSO4 overnight. 3. Cells were treated for 4 h at 25 C with a 50 mM concentration of the proteasome inhibitor MG132 (Calbiochem). 4. The cell culture dishes were placed on ice. The medium was drained, and cells were collected by pipetting in cold PBS. 5. The cells were washed using cold PBS twice and then gently transferred into precooled 1.5 ml microfuge tubes. 6. The cells were lysed in ice-cold lysis buffer. The lysates were incubated on ice for 20 min. 7. The lysates were cleared by centrifugation at 2000 rpm for 10 min at 4 C. 8. The supernatant was collected and placed in a fresh tube kept on ice. 9. On ice, 1000 mg cell lysate was added to the anti-Notch ICD (9C6, Developmental Studies Hybridoma Bank) (1:1000). 10. The sample was incubated with the antibody at 4 C under rotary agitation overnight. 11. The protein G-agarose beads were prepared (Roche) (20 ml of the slurry) by washing twice in 1 ml cold lysis buffer. 12. The beads were added to each sample and incubated at 4 C under rotary agitation for 2–3 h. 13. The precipitates were washed in 1 ml cold lysis buffer five times. 14. Protein complexes were eluted with 30 ml of 2 LDS sample buffer (Invitrogen) and heated at 70 C for 10 min. 15. The samples were run on a 3–7% Tris–Acetate gel (Invitrogen) and transferred onto PVDF membrane (Invitrogen). 16. The ubiquitinated Notch is detected by western blot using rabbit antiFLAG (Sigma, 1:1000).
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6. CONCLUSION Recent genetic and molecular studies have led to the increased appreciation of how important and indeed diverse, trafficking events related to the receptor and the ligands are for the developmental control of Notch signals. Here we described paradigmatic methodology to obtain an informative picture regarding the complexity and the nature of the genetic circuitry that affects Notch trafficking controlled by Dx and Krz. Such genetic screens while focused on a specific phenotype affecting one particular tissue, the value of the screen is more general. This particular screen recovered more than 250 modifiers of the wing phenotype we screened with only less than 20 modifiers associated with membrane trafficking as judged by Gene Ontology analysis (Flybase, http://flybase.org/) with the rest falling into diverse functional categories including, for example, gene regulations, metabolism, cytoskeleton, as well as several genes of unknown function (unpublished data). Linking molecularly each category or each gene with Notch is a nontrivial task (see e.g., Hori et al., 2011), notwithstanding the value of genetic, that is, functional links unveiled by genetic analyses.
ACKNOWLEDGMENTS We would like to thank Robert A. Obar and K. G. Guruharsha for critically reading the manuscript. This work was supported by NIH Grants NS26084 and CA98402 (S. A. -T.), a JSPS Postdoctoral Fellowship for Research Abroad (K. H.), and a Postdoctoral Fellowship from the FSMA (A. S.).
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