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Genetics, Vol. 175, 1163-1174, March 2007, Copyright © 2007
doi:10.1534/genetics.106.067959
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Section of Molecular Cell and Developmental Biology, Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas 78712
4 Corresponding author: Section of Molecular Cell and Developmental Biology, Institute for Cellular and Molecular Biology, University of Texas, Moffett Molecular Biology Bldg., 2500 Speedway, Austin, TX 78712.
E-mail: jaf{at}mail.utexas.edu
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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In Drosophila, Lqf is a core component of the Notch signaling pathway (OVERSTREET et al. 2004; WANG and STRUHL 2004, 2005). Internalization of the transmembrane Notch ligands, Delta and Serrate, is required for signaling (PARKS et al. 2000; LAI et al. 2001; PAVLOPOULOS et al. 2001; LE BORGNE and SCHWEISGUTH 2003a; LI and BAKER 2004; OVERSTREET et al. 2004; WANG and STRUHL 2004, 2005; LAI et al. 2005; LEBORGNE et al. 2005a). Although the role of endocytosis in signaling is a mystery, what is clear is that the essential endocytic event absolutely requires Lqf (OVERSTREET et al. 2004; WANG and STRUHL 2004, 2005).
There are two classes of models to explain why Lqf-dependent endocytosis of Delta or Serrate by the signaling cells is required for Notch activation in the receiving cells: postreceptor-binding models and prereceptor-binding models (reviewed in LE BORGNE and SCHWEISGUTH 2003b; LE BORGNE et al. 2005b; FISCHER et al. 2006). The postreceptor-binding model supposes that internalization of Delta bound to Notch provides a pulling force that exposes the extracellular domain cleavage site on the receptor (PARKS et al. 2000). Cleavage of the Notch extracellular domain is prerequisite for cleavage of the intracellular domain, which is the ultimate step in Notch activation and results in a small portion of the receptor translocating to the nucleus where it regulates transcription (reviewed in FORTINI 2002). The prereceptor-binding model is that Notch ligands are somehow processed within endosomes and then either recycled to the cell surface in active form or secreted in active form as exosomes (LE BORGNE and SCHWEISGUTH 2003a; WANG and STRUHL 2004).
The precise role of Lqf in endocytosis of Notch ligands is unclear. In the eye and the wing, the lqf null mutant phenotype is indistinguishable from the Delta and/or Serrate mutant phenotypes (OVERSTREET et al. 2004; WANG and STRUHL 2004). One explanation for the apparent specificity of Lqf might be that Lqf directs Delta or Serrate into special endosomes for processing and recycling (WANG and STRUHL 2004). Alternatively, Lqf might play a more general role in internalization of plasma membrane proteins that use ubiquitin as an internalization signal, and the Notch ligands are simply the only such proteins whose endocytosis is absolutely necessary for normal development of the fly.
To better understand the role of Lqf in Notch signaling, we performed a mutagenesis screen of the Drosophila genome to identify genes that interact with lqf. By overexpressing lqf+ in the eye with a transgene, we generated an eye-specific lqf mutant. In a screen for dominant enhancers of the eye phenotype, we identified four genes, three of which had not been known to interact genetically with lqf. These results suggest potential roles for the enhancer genes in Lqf function.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Mutant screens:
Meiotic mapping:
Physical mapping of C18:
Twenty-seven stocks with deficiencies in polytene region 21–24 were crossed to C18. The four critical lines are listed below.
Physical mapping of 727:
Twenty-six stocks with deficiencies in polytene region 72C–86D were used. (Bloomington stock numbers are available upon request.)
Male recombination mapping of 727:
2-3/Sco; th st aux727 sr e/TM6B (this study)
Physical mapping of EE1:
The X chromosome duplication kit as well as 11 X chromosome deficiency stocks from Bloomington were used. The critical stocks are listed below.
Complementation tests:
Other strains:
Drosophila genetics:
Fly crosses not explicitly described were performed in a typical manner. All fly crosses were carried out at 25° using standard food unless otherwise indicated.
Mutagenesis and enhancer screening:
Males were treated with EMS (Sigma, St. Louis) according to LEWIS and BACHER (1968). F1 males or females were screened for enhanced eye roughness with a Leica MZ6 dissecting microscope.
Meiotic recombination mapping of C18 and 727 complementation group alleles:
The chromosome 2-linked enhancer C18 was localized near al as follows. Virgin females of the genotype C18/al dp b c px were crossed to al dp b Bl c px/CyO males and 10–20 non-CyO males with parental chromosomes or with each of the 8 single recombinant types were collected. To score each chromosome for the enhancer C18, single males were crossed with w; glrs-lqf virgins and the eyes of the non-Bl progeny were assessed. The key result was that the recombination frequency between al and C18 is very low: no al chromosomes also had C18, and all al+ chromosomes contained C18. The chromosome 3-linked enhancer 727 (aux727) was localized between th and cu as follows. Virgin females of the genotype 727/ru h th st cu sr e ca were crossed to ru h th st cu sr e Pr ca/TM6B males and 10 non-TM6B males with parental chromosomes or with each of the 10 of single recombinant types were collected. To score each chromosome for the enhancer 727, single males were crossed with glrs-lqf; th st cu sr e/TM6B virgin females and the eyes of the non-Pr progeny were assessed. The key results were that no th cu chromosomes contained 727, all th+ cu+ chromosomes contained 727, and th cu+ or th+ cu chromosomes could contain 727 or not.
Physical mapping of C18:
Twenty-seven chromosomes with deficiencies in polytene region 21–24 were tested for complementation of the lethality of C18. Two of them [Df(2L)net-PMF and Df(2L)net-PM47C] failed to complement. Two deficiency chromosomes that do complement [Df(2L)PM45 and Df(2L)al] provided critical additional information. Using the information on FlyBase (GRUMBLING et al. 2006) regarding the breakpoints of these four deficiencies, we localized C18 to a region between the genes Glutamine synthetase 1 and kismet, which contains 12 genes and includes split ends (spen).
Male recombination mapping of 727 alleles:
Enhancer 727 was mapped with respect to 12 different P elements located between polytene positions 78A and 84B on the basis of methods described in B. CHEN et al. (1998). First, a th st 727 sr ca chromosome was generated. Males of the genotype w; CyO, 2-3/Sco; th st 727 sr ca/TM6B were crossed with virgins containing the P element to generate CyO, 2-3/+; th st 727 sr ca/P males. These were crossed with ru h th st cu sr e ca virgins and the progeny were examined for male recombination events. The progeny with chromosomes that had recombined between st and sr could be distinguished easily by their eye colors. The vast majority of the progeny had wild-type (P/ru h th st cu sr e ca) or orange (th st 727 sr ca/ru h th st cu sr e ca) eyes, the latter because they are homozygous st ca. Progeny with rare recombinant chromosomes (th st sr+ ca+/ru h th st cu sr e ca or th+ st+ sr ca/ru h th st cu sr e ca) had bright red (st/st) eyes or brown (ca/ca) eyes, respectively. Recombination between the markers th and sr served as a second check on the origins of the recombinant chromosomes. Recombinant chromosomes were scored for the presence or absence of 727 by crossing with glrs-lqf ; if 727 is present, half of the progeny should have the enhanced rough-eye phenotype and if 727 is absent, none of them should. Two P elements in 82A (P{y+w+SUP}KG08740 and P{y+w+EP}CG14641) were found to flank 727 and these were used to map two other aux alleles (L7 and F37) that were isolated as enhancers. For this analysis, st F37 sr e ca and st L7 sr e ca chromosomes were used in identical experiments. Rare recombinant chromosomes were assayed for both the enhancer functions of F37 and L7 and also for their lethality in trans to 727. Both the enhancer and lethality functions mapped between the two P elements that flanked 727. Males of the genotype w; CyO, 2-3/+; st F37 sr e ca/P were crossed with ru h th st cu sr e Pr ca/TM6B, Bri virgins and rare recombinant chromosomes were identified in flies with bright red (st/ st) or brown (ca/ca) eyes (st sr+ e+ ca+/ru h th st cu sr e Pr ca or st+ sr e ca/ru h th st cu sr e Pr ca, respectively). The sr and e markers served as second checks on the origins of the chromosomes. The recombinant chromosomes were scored for the presence or absence of the lethal function of F37 by crossing with 727/TM6B and determining if any non-Pr and non-TM6B progeny were viable. Male progeny of this cross containing the recombinant chromosomes (st sr+ e+ ca+/TM6B or st+ sr e ca/TM6B) were crossed with glrs-lqf virgin females to assess if they carried the enhancer of function of F37. If so, all of the non-TM6B progeny should have enhanced rough eyes and, if not, none of them should have enhanced rough eyes. Both the enhancer and lethality in trans to 727 functions of L7 and F37 mapped between the two P elements that flank the enhancer function of 727.
Complementation of AA1 and BB2:
The two X-linked enhancers that were on hemizygous lethal X chromosomes (AA1 and BB2) were tested for complementation by the clathrin heavy chain (chc+) gene in a transgene (P{w+chc+}) and also as a duplication [Dp(1;Y)shi+y+BS] that contains chc+. To test for complementation by the transgene, AA1/FM7c virgin females were crossed to chc3; P{w+chc+}/+ males. Non-FM7c male progeny will be hemizygous for AA1 and will be present only if P{w+chc+} complements the lethality of AA1. The same crosses were performed for AA1 and BB2 and in both cases non-FM7c male progeny that were wild type in appearance were produced. To test Dp(1;Y)shi+y+BS for complementation, chc1/Dp(1;Y)shi+y+BS males were crossed with AA1/FM7c or BB2/FM7c females. Non-FM7c male progeny that appeared wild type were produced, indicating that Dp(1;Y)shi+y+BS complements AA1 and BB2.
Physical mapping of EE1:
All stocks in the X chromosome duplication kit were tested for complementation of the rough eye, bristle, and wing mutant phenotypes of EE1 hemizygous males. In each case, males bearing the duplication were crossed with EE1/FM7c females, and the progeny were scored for the presence of non-FM7c males with normal morphology, indicative of complementation. The males used in the above crosses were simply those present in the duplication stocks, with two exceptions: the stocks of Dp(1;f)y+(BL no. 5459) and Dp(1;f)LJ9 (BL no. 3219) required additional crosses because the males are C(1;Y). For Dp(1;f)y+, males carrying the duplication were generated by crossing virgin females of the genotype C(1)RM,y/Dp(1;f)y+ to yw males to produce virgin female progeny of the genotype C(1)RM,y /Y/Dp(1;f)y+. These females were crossed with yw males to generate males that contain the duplication, which are of the genotype y/Y/Dp(1;f)y+. Analogous crosses were performed to generate males containing Dp(1;f)LJ9. Once a duplication that complemented EE1 was identified [Tp(1;2)w-ec], it was used to test 11 deficiency chromosomes for complementation of EE1 as follows. EE1/FM7c virgin females were crossed with FM6; CyO/Sco males to generate EE1/FM6; CyO virgin females. These were crossed with Tp(1;2)w-ec/+ males to generate males of the genotype EE1; Dp(1:2)w-ec/CyO, which were crossed with females containing various Df chromosomes in trans to an FM balancer. The progeny were examined to determine if there were any EE1/Df; CyO/+ [Dp(1;2)w-ec not present] females present that appear wild type. If so, the Df chromosome complements EE1; if not, the Df chromosome does not complement.
We found that two duplications complement EE1: Tp(1;2)w-ec and Dp(1;3)w+67k. This information, taken together with the observation that two overlapping duplications [Dp(1;2;Y)w+and Dp(1;2)51b] do not complement EE1, defined the region containing EE-1 as 3D6–3E8. In addition, we found that three deficiency chromosomes [Df(1)RR62, Df(1)N264-105, and Df(1)Exel6233] failed to complement EE1. Of these, the breakpoints of only Df(1)Exel6233 are identified molecularly and thus defined the distal endpoint of the region containing EE1. Taken together, the duplication and deficiency data implicate the genes between Ilp7 and CG32781, including Rala, as candidates for EE1.
Molecular biology and histology:
Molecular biology manipulations were performed using standard techniques or instructions from the manufacturers of enzymes and kits. Enzymes used for cloning were obtained from New England BioLabs (Beverly, MA), Roche, or Invitrogen (San Diego). Oligonucleotides were synthesized by Integrated DNA Technologies.
Construction of the glrs-lqf transgene and transformants:
An 
3.4-kb AscI fragment containing a Flag-tagged lqf cDNA (CADAVID et al. 2000) was obtained from pBSKII-Flag-lqf (CADAVID et al. 2000) and ligated into the AscI site of pglrs (HUANG and FISCHER-VIZE 1996). P element transformation of w1118 was performed using standard methods. A primary glrs-lqf transformant line obtained was mobilized to generate the line used for the mutant screen.
Construction of the genomic aux+ transgene and transformants:
A 21,081-bp DNA fragment containing the aux+ gene was obtained by restricting BACR15O02 (GRUMBLING et al. 2006) with NheI and SacII. The aux+ DNA fragment was ligated into the vector pFOW (S. H. EUN and J. A. FISCHER, unpublished results) restricted with NheI and SacII. The SacII site in the resulting plasmid was changed to NheI by ligating annealed oligonucleotides of the sequence 5'-TGCTAGCAGC-3' into the SacII site. An 21-kb NheI fragment containing the aux+ gene was obtained from the resulting plasmid and ligated into the XbaI site of pCasper4 (THUMMEL and PIRROTTA 1992). P element transformation was performed by Genetic Services (Sudbury, MA).
DNA analysis of aux alleles and RalaEE1:
Templates for DNA sequence determination of aux alleles were prepared by polymerase chain reaction (PCR) of genomic DNA from homozygous larvae or from a single adult fly (L7). As most aux homozygotes die before the Tb marker on TM6B is evident in the larvae, stocks were balanced using TM6B GFP and 5–10 small nonfluorescing larvae were collected and homogenized in SB (10 mM Tris–HCl, pH 8.2, 1 mM EDTA, 25 mM NaCl). Template in SB (2–4 µl) was mixed with 2 µl primer (200 ng) and 45 µl of Platinum PCR SuperMix (Invitrogen). PCR conditions were 1 cycle of 1 min at 95°; 30 cycles of 1 min at 95°/1 min at 50°/1 min 40 sec at 72°; 1 cycle of 10 min at 72°. PCR products were purified by agarose gel electrophoresis and the QIAquick Gel extraction kit (QIAGEN, Chatsworth, CA). Four PCR primer pairs were used to amplify the aux gene in four overlapping parts: 5'-AGCAAACTGATTCCGCTCCAC-3' and 5'-GCATTGTTTGTTCTGAAGCAGTCC-3'; 5'-TTGTCGCCTTTGTGGGTTCCAG-3' and 5'-TAAACTCGCAGGACCCAAGCACTG-3'; 5'-AAGTGGATGTCTCTTGCCGACG-3' and 5'-TGTGCCCGAACTTTTGGTG-3'; 5'-agcacgctaagtggaaagtctccc-3' and 5'-acagggataccaatgagtcacagag-3'. The same primers were used for automated fluorimetric DNA sequencing, and also an additional primer was used for the longest template: 5'-TTTCACGCCCGCAAAGGAATGG-3'.
The Rala allele in EE1 mutants was amplified by PCR from one adult male fly. The template was prepared as described above. PCR conditions were 1 cycle 1 min at 90°; 30 cycles 1 min at 95°/1 min at 55°/1 min at 72°; 1 cycle 10 min at 72°. Four primer pairs were used for both PCR and DNA sequencing: 5'-CTGTGAGCCGACTCCATAAGTTG-3' and 5'-CCTGAGAGGAAAGCAAAACGC-3'; 5'-GCTACTTCGTTGCCATAACTCCC-3' and 5'-TCCAGTGATGTTCTCGTTCGTAAG-3'; 5'-ATGTTGGTTCGGTCCTTG-3' and 5'-CTGAAATGCTGCTGTGAAA-3'; 5'-TGACGGTTCTCTGGTGAATAAAGG-3' and 5'-CGTCTGTGTGCTTTTCGCTTG-3'.
Mutations found in aux or Rala alleles were confirmed by repeating the PCR and sequencing reactions.
Analysis of eye and wing morphology:
Scanning electron micrography and plastic sectioning of adult eyes were as described in HUANG et al. (1995). Eye disc immunohistochemistry was as described in OVERSTREET et al. (2004). Wings were mounted as described in CADAVID et al. (2000). Light photomicrographs of eyes was with an Olympus SZX12 microscope and a Kodak DC120 digital camera. Wings and eye sections were photographed with a Zeiss Axioplan and Axiocam HRc. Immunostained eye discs were photographed with a Leica TCSSP2 confocal microscope. Adobe Photoshop 7.0 was used for processing images.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Screens for dominant enhancers of the glrs-lqf eye phenotype:
Separate screens were performed for dominant enhancers of the 1Xglrs-lqf rough eye on the autosomes or the X chromosome. (The 1Xglrs-lqf rough eye is too weak to screen reliably for suppressors.) Approximately 30,000 F1 males were screened for autosomal enhancers (Figure 2); 16 chromosome 2-linked mutants and 74 chromosome 3-linked mutants were recovered. An example of an enhanced eye phenotype is shown in Figure 1, K–N.
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We found that 13 of the 16 enhancers on chromosome 2 and 31 of the 74 enhancers on chromosome 3 were on homozygous lethal chromosomes and these enhancer mutants were used in the complementation matrices. Also included in the chromosome 3 complementation analysis was one homozygous viable enhancer chromosome (L7) where the homozygotes, in an otherwise wild-type background, initially appeared to have slightly rough eyes. The result was that one complementation group containing 12 mutants was identified among the chromosome 2 enhancers, and one complementation group containing 12 enhancers was identified among the chromosome 3 enhancers (Figure 3). As there are 12 members of each lethal complementation group, it is likely that we would have isolated at least two alleles of most essential autosomal genes that enhance glrs-lqf. Thus, we reason that the lethal chromosomes that complement every other enhancer chromosome in the matrices most likely contain at least two mutations: an enhancer mutation in a nonessential gene and an unrelated EMS-induced homozygous lethal mutation.
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Identification of the chromosome 3 complementation group as auxilin:
By meiotic recombination with a multiply marked third chromosome, enhancer 727, a representative of the chromosome 3 complementation group, was localized between thread (th) and curled (cu) (MATERIALS AND METHODS). Readily available deficiency chromosomes in polytene chromosome region 72C–86D (MATERIALS AND METHODS) all complement the lethality of enhancer 727, but there were several gaps in the coverage of the region. Male recombination mapping of 727 with four P elements at polytene positions 78A, 80A, 82B, and 84B (MATERIALS AND METHODS) localized 727 within 80A–82B, a region including the centromere not uncovered by any of the deficiencies that we used. Another round of male recombination mapping with nine P elements within 80A–82B (MATERIALS AND METHODS) localized 727 to a region within 82A that includes eight genes (CG12581–CG181430) (GRUMBLING et al. 2006). Among the eight genes, auxilin (aux) was chosen as a candidate because in vitro and in other organisms, Auxilin is involved in clathrin-mediated endocytosis (PRASAD et al. 1993; HONING et al. 1994; UNGEWICKELL et al. 1995; HOLSTEIN et al. 1996; GALL et al. 2000; PISHVAEE et al. 2000; UMEDA et al. 2000; GREENER et al. 2001; LEMMON et al. 2001). As there were no existing aux mutant alleles to use for complementation tests, we tested 727/D136 heterozygotes (a lethal combination) for complementation with Act5C-gal4>UAS-aux transgenes and with a genomic aux+ transgene (MATERIALS AND METHODS). Either the aux+ cDNA or the genomic DNA was sufficient for complete rescue of all 727/D136 phenotypes (data not shown). We also determined the DNA sequences of the aux genes present on each enhancer-containing third chromosome within the 727 complementation group. Nonsense or missense mutations were found in the aux open reading frame in 11 of the 12 enhancer chromosomes (Figure 5). We conclude that the 727 complementation group is aux.
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Enhancer EE1 was mapped physically by testing 27 stocks that constitute the X chromosome duplication kit from Bloomington and, subsequently, 11 deficiency chromosomes for complementation of EE1 (MATERIALS AND METHODS). Taken together, the duplication and deficiency data implicated only a few genes, including Rala (MATERIALS AND METHODS).
We considered Rala as a candidate for EE1 because some of the phenotypes reported for overexpression of a dominant-negative Rala transgene (SAWAMOTO et al. 1999) are similar to some of the EE1 phenotypes (reduced rough eyes and missing bristles and hairs; Figure 6) and because Rala encodes a small Ras-like GTPase that regulates vesicle trafficking (reviewed in FEIG 2003). (In vertebrates, there are two Ral proteins, Rala and Ralb; in Drosophila, there is only one and although it has been also referred to as DRal in the literature, here we use the gene name on FlyBase, Rala.) Expression of a wild-type Rala cDNA (Act5C-gal4; UAS-Rala) complements the morphological phenotypes of EE1. We determined the DNA sequence of the Rala allele on the EE1 X chromosome and found a missense mutation: Ser154 (TCG) is mutated to Leu154 (TTG). Ser154 is conserved in human Ral proteins and also in human Kras, and amino acids 152–156 are required for nucleotide binding (SAWAMOTO et al. 1999). We conclude that EE1 is a mutant allele of Rala.
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aux hypomorphs resemble lqf hypomorphs:
The eye and wing phenotypes of aux hypomorphic escapers resemble those of lqf hypomorphs (CADAVID et al. 2000). The eyes of aux hypomorphs are rough externally and each facet has more than the normal complement of eight R-cells (Figure 7); the wings have extra vein material and are notched (Figure 8).
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Genetic interactions between lqf and aux loss-of-function mutants:
Although strong aux mutant alleles are not dominant enhancers of lqfFDD9, amorphic lqf mutants (lqfARI) are strong dominant enhancers of hypomorphic aux mutations. Flies hypomorphic for aux in these experiments were heterozygous for combinations of auxK47 or auxL7, the two weakest mutant alleles, and each stronger aux allele. As described above, these combinations of aux alleles are semilethal at 25° and produce escapers with mutant eyes and wings. In contrast, the heterozygous combination of an auxK47 lqfARI or an auxL7 lqfARI chromosome with any aux allele is lethal with no escapers, except for auxN7, auxL7, and auxK47, which did give escapers. In these escapers, the rough-eye phenotype is enhanced only slightly, and the wing phenotype is enhanced severely (Figure 9 and data not shown).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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A potential function for Rala in Notch signaling:
The genetic interaction between Rala and lqf suggests the possibility that Rala GTPase might regulate Lqf-dependent Delta signaling through one of three mechanisms. First, Rala might regulate Lqf-dependent endocytosis. In vertebrate and Drosophila cultured cells, Ral, acting through the effector RalBP1 (also called RLIP76), regulates endocytosis through the formation of a complex containing Epsin and the EH-domain protein POB-1 (JULLIEN-FLORES et al. 2000; ROSSE et al. 2003). Second, Rala could regulate endosome recycling. In vertebrate cells, also through the RalBP1 effector, Ral stimulates recycling through the related EH-protein REPS1 (NAKASHIMA et al. 1999). In Drosophila, there is a unique POB-1/REPS homolog encoded by an uncharacterized locus called CG6192 (MIREY et al. 2003), which could participate in the regulation of Lqf-dependent Delta endocytosis or in subsequent recycling of Delta-containing endosomes. Third, Rala might regulate actin organization that is critical for Delta internalization and signaling. In yeast, an Epsin complex containing RalBP1 and the GTPase Cdc42 organizes the actin cytoskeleton and cell polarity (AGUILAR et al. 2006). The function of Epsin is to stabilize Cdc42•GTP and thereby link sites of endocytosis and cell polarity. In Drosophila, experiments using dominant-negative and constitutively active Rala transgenes led to the conclusion that Rala regulates actin cyoskeleton organization through the JNK pathway (SAWAMOTO et al. 1999). However, it is not known if Drosophila Rala works through the RalBP1 homolog or Cdc42. The genetic interaction between Rala and lqf may provide a critical clue to understanding why Lqf-dependent Delta internalization is required for Delta signaling.
A role for Auxilin in Notch signaling:
The identification of aux mutants as genetic interactors with lqf provides another clue as to why Delta signaling requires Lqf-dependent Delta endocytosis. Auxilin functions in uncoating clathrin-coated vesicles (LEMMON et al. 2001) and also may play an earlier role in endocytosis in the internalization step at the plasma membrane (NEWMYER et al. 2003; LEE et al. 2005). In favor of the idea that aux+ and lqf+ functions are related, we have shown that aux loss-of-function mutations not only interact with glrs-lqf, but also interact with lqf loss-of-function mutations. Also, the phenotypes of aux hypomorphs described here and in HAGEDORN et al. (2006) are similar to those of lqf hypomorphs and are typical of Notch pathway mutants. Like lqf+, aux+ is required in the signaling cells (S. H. EUN and J. A. FISCHER, unpublished results). More experiments are required to determine what potential functions of Drosophila Auxilin (internalization or uncoating) are essential for Delta signaling. Moreover, it remains to be determined if, like Lqf (Epsin), Auxilin plays a direct role in Delta signaling or if the vesicle-uncoating function of Auxilin is required indirectly, for example, to maintain a pool of clathrin available for ligand internalization.
The genetic interaction between spen and lqf suggests a nuclear role for Lqf:
The identification of spen alleles in this screen suggests the exciting possibility that Lqf may have an additional function in the nucleus. Spen is a conserved nuclear protein that regulates many different signaling pathways, including Notch (DICKSON et al. 1996; STAEHLING-HAMPTON et al. 1999; WIELLETTE et al. 1999; CHEN and REBAY 2000; KUANG et al. 2000; LANE et al. 2000; PROKOPENKO et al. 2000; REBAY et al. 2000; THERRIEN et al. 2000; LIN et al. 2003). Spen is thought to function in the Notch signal-receiving cells by modulating the levels of Suppressor of Hairless, a Notch effector protein (KUANG et al. 2000). As is typical of Notch pathway mutants, some ommatidia in spen mutant eyes have extra R-cells (DICKSON et al. 1996).
Vertebrate Epsin (and other plasma-membrane-associated endocytic proteins) shuttles between the cytoplasm and the nucleus, and Epsin has been shown to bind the vertebrate transcription factor PLZF (promyelocytic leukemia Zn(2)+ finger) protein in a yeast two-hybrid screen (HYMAN et al. 2000; VECCHI et al. 2001; BENMERAH et al. 2003; BENMERAH 2004). However, it is unknown whether or not the Epsin/PLZF interaction is physiologically relevant.
It is striking that Spen, a nuclear protein, is one of only four essential proteins in the fly whose levels become limiting for proper eye development when Lqf is overexpressed. No other known Notch pathway genes were identified, not even those known to interact closely with lqf, for example, neuralized (OVERSTREET et al. 2004). [As neuralized is on chromosome 3 and Neuralized protein is 60% the size of Aux protein (700 and 1100 amino acids, respectively), the fact that we obtained 12 independent alleles of aux suggests strongly that we are likely to have identified at least two alleles of neuralized, were it possible.] This suggests that the interaction between Lqf and Spen is close and thus that Lqf might function in the nucleus with Spen.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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FOOTNOTES |
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2 Present address: Ambion, 2130 Woodward, Austin, TX 78744.
3 Present address: Department of Biological Chemistry, University of California, 675 Charles E. Young, 5-784 MRL, Los Angeles, CA 90095.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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ADAMS, M. D., S. E. Celniker, R. A. Holt, C. A. Evans, J. D. Gocayne et al., 2000 The genome sequence of Drosophila melanogaster. Science 287: 2185–2195.
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