Genetics: Published Articles Ahead of Print, published on December 18, 2006 as 10.1534/genetics.106.067959

1

Identification of that interact with Drosophila liquid facets

Suk Ho Eun *, Kristi Lea *, 1, Erin Overstreet 2, Samuel Stevens, Ji-Hoon Lee,

Janice A. Fischer *, 3

Section of Molecular Cell and Developmental Biology, Institute for Cellular and Molecular

Biology, The University of Texas at Austin, Austin, Texas 78712 USA

* these authors contributed equally to this work

1 present address: Ambion, Inc., 2130 Woodward, Austin, TX 78744 USA

2 present address: University of California at Los Angeles, Department of Biological

Chemistry, 675 Charles E. Young, 5-784 MRL, Los Angeles, California 90095, USA

3 Corresponding author: Section of Molecular Cell and Developmental Biology, Institute

for Cellular and Molecular Biology, The University of Texas at Austin, Moffett Molecular

Biology Building, 2500 Speedway, Austin, Texas 78712, USA.

E-mail: [email protected]

2 running title: Drosophila lqf overexpression screen

key words: liquid facets/ Epsin/ endocytosis/ Notch signaling/ overexpression screen

corresponding author information:

Janice A. Fischer phone: 512-471-5831 fax: 512-232-3432 e-mail: [email protected]

US postal address: The University of Texas at Austin, 1 University Station, A4800,

Austin, Texas 78712

Fedex address: The University of Texas at Austin, Moffett Molecular Biology Building,

2500 Speedway, Austin, Texas 78712 3

ABSTRACT

We have performed mutagenesis screens of the Drosophila X and the autosomes for dominant enhancers of the rough eye resulting from overexpression of liquid facets. The liquid facets encodes the homolog of vertebrate endocytic Epsin, an endocytic adapter . In Drosophila, Liquid facets is a core component of the

Notch signaling pathway required in the signaling cells for ligand endocytosis and signaling. Why ligand internalization by the signaling cells is essential for signaling is a mystery. The requirement for Liquid facets is a hint at the answer, and the genes identified in this screen provide further clues. Mutant alleles of clathrin heavy chain,

Rala, split ends, and auxilin were identified as enhancers. We describe the mutant alleles and mutant phenotypes of Rala and aux. We discuss the relevance of all of these genetic interactions to the function of Liquid facets in Notch signaling.

4

Drosophila Liquid facets (Lqf) is the homolog of vertebrate endocytic epsin, an adapter for internalization of that span the plasma membrane (CHEN et al. 1998a).

Endocytic Epsin is a modular protein with an ENTH (Epsin N-terminal homology) domain that binds the plasma membrane, and motifs that bind ubiquitin, clathrin, the AP-2 core adapter complex, and EH-domain-containing endocytic factors (reviewed in WENDLAND

2002).

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

2003; LI and BAKER 2004; OVERSTREET et al., 2004; WANG and STRUHL, 2004,

2005; LEBORGNE et al. 2005; LAI et al. 2005). 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: post-receptor binding models and pre-receptor binding models (reviewed in LE

BORGNE et al. 2005; FISCHER et al. 2006). The post-receptor 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 pre-receptor 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 5 active form as exosomes (LE BORGNE and SCHWEISGUTH 2003; 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.

In order 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.

MATERIALS AND METHODS

Drosophila Strains

Mutations and balancers are described in LINDSLEY and ZIMM 1992 and on FLYBASE

(GRUMBLING et al. 2006). BL# means Bloomington Drosophila Stock Center number.

Mutant screens: pr; st (both isogenized prior to the screen; our laboratory stock) st (our laboratory stock) 6 w; P{w+, glrs-lqf} (this study) w; P{w+, glrs-lqf}, Bl/ CyO (this study) w; Sb/ TM6B (our laboratory stock) w; Sco/ CyO (our laboratory stock) chcG0438/ FM7c (BL#12029)

Meiotic mapping: al dp b pr cn c px sp / CyO (BL# 4187) al dp b pr Bl cn c px sp / CyO (BL# 214) ru h th st cu sr e ca (BL#576) ru h th st cu sr e Pr ca / TM6B, Bri (BL# 1711) w; P{w+, glrs-lqf}/ CyO; th st cu sr e /TM6B (this study)

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.

Df(2L)al (BL#3548)

Df(2L)net-PM47C (BL#3636)

Df(2L)PM45 (BL#6133)

Df(2L)net-PMF (BL#3638)

Physical mapping of 727: Twenty-six stocks with deficiencies in polytene region

72C-86D were used. (Bloomington stock numbers available upon request.)

Male recombination mapping of 727:

CyO, Δ2-3/ Sco; th st aux727 sr e/ TM6B (this study) yw; P{w+lacW}l(3)L5541 (78A5/6; BL#10199) yw; P{w+lacW}l(3)L2100 (84B2/3; BL#10219) yw; P{w+lacW}l(3)L1233 (82B1/2; BL#12213) yw; P{y+w+SUP}KG03264 (80A1; BL#12935) 7 y; P{y+w+SUP }KG03229 (80A1; BL#12936) y; P{y+w+SUP}KG00844 (80A2; BL#12963) y; P{y+w+SUP}KG06133a (80B3; BL#14143) y; P{y+w+SUP}KG08740 (82A1; BL#14969) yw; P{y+w+EP}CG14641 (82A1; BL#15525) y; P{y+w+SUP}KG03023 (82A4; BL#14427) yw; P{y+w+EP}CG1103 (82A5; BL#15295) yw; P{w+lacW}j1E6 (82A3/5; BL#10206) yw; P{y+w+EP}EY01545 (82B1; BL#15360)

Physical mapping of EE1: The X chromosome duplication kit as well as eleven

X chromosome deficiency stocks from Bloomington were used. The critical stocks are listed below.

Tp(1;2)w-ec (BL#1319)

Dp(1;2;Y)w+ (BL#54)

Dp(1;3)w+67k (BL#902)

Dp(1;2)51b (BL#937)

Df(1)Exel6233 (BL#7707)

Complementation tests: w chc1 / FM7c/ Dp(1;Y)shi+y+BS (BL#4166) w; P{w+chc+}/+ (on chr. 3; BAZINET et al. 1993; obtained from B. Zhang) w; P{w+, Act5C-Gal4} (on chr. 2; BL#4414) w; P{w+, UAS-Rala+} (on chr. 3; SAWAMOTO et al. 1999; obtained from H. Okano) w; P{w+, UAS-aux+} on chr. 2 (CHANG et al. 2004; obtained from I. Mellman) spen14O1 / CyO (BL#5808) spen16H1 / CyO (BL#5809) 8 yw; P{w+, lacW}spenk06805 (BL#10646)

P{ry+, PZ}spen03350/CyO; ry506 (BL#11295)

FM6; CyO/Sco (BL#438)

Other strains: w; P{w+, UAS-RalaG20V} (on chromosome 3; SAWAMOTO et al. 1999; obtained from H.

Okano) w; P{w+, UAS-RalaS25N} (on ; SAWAMOTO et al. 1999; obtained from H.

Okano)

TM6B, GFP (BL# 4887) w1118 (our laboratory stock)

Drosophila Genetics

Fly crosses not explicitly described were performed in a typical manner. All fly crosses were carried out at 25oC using standard food unless otherwise indicated.

Mutagenesis and enhancer screening: Males were treated with EMS (Sigma) 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 to 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 9 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 twelve genes and includes split ends.

Male recombination mapping of 727 alleles: Enhancer 727 was mapped with respect to 12 different P elements located between polytene position 78A and 84B based on the methods described in CHEN et al. 1998. First, a th st 727 sr ca chromosome was generated. Males of the genotype w; CyO, Δ2-3/ Sco; th st 727 sr ca/

TM6B were crossed with virgins containing the P element to generate CyO, Δ2-3/ +; th st 727 sr ca/ P males. These were crossed with ru h th st cu sr e ca virgins and the progeny 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 10 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, Δ2-3/ +; 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 in order 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 11 should. 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#5459) and Dp(1;f)LJ9 (BL#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 12 containing Dp(1;f)LJ9. Once a duplication that complemented EE1 was identified

(Tp(1;2)w-ec), it was used to test eleven 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 through 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, Roche, or Invitrogen. Oligonucleotides were synthesized by Integrated DNA Technologies.

Construction of the glrs-lqf transgene and transformants: An ~3.4 kb Asc I fragment containing a Flag-tagged lqf cDNA (CADAVID et al. 2000) was obtained from 13 pBSKII-Flag-lqf (CADAVID et al. 2000) and ligated into the Asc I 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 in order 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 Nhe I and Sac II. The aux+ DNA fragment was ligated into the vector pFOW (S. H. E., B. CHO, and J. A. F., manuscript in preparation) restricted with Nhe I and Sac II. The Sac II site in the resulting plasmid was changed to

Nhe I by ligating annealed oligonucleotides of the sequence 5’-TGCTAGCAGC-3’ into the Sac II site. An ~ 21 kb Nhe I fragment containing the aux+ gene was obtained from the resulting plasmid and ligated into the Xba I site of pCasper4 (THUMMEL and

PIRROTTA 1992). P element transformation was performed by Genetic Services, Inc.

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 non-fluorescing larvae were collected and homogenized in SB (10mM TrisCl pH8.2, 1 mM EDTA, 25 mM NaCl). Template in SB (2-

4 ul) was mixed with 2 ul primer (200 ng) and 45 ul of Platinum PCR SuperMix

(Invitrogen). PCR conditions were: 1 cycle 1 min. @95oC ; 30 cycles of 1 min. at 95oC/ 1 min. at 50oC/ 1 min. 40 sec. at 72oC; 1 cycle of 10 min. at 72oC. PCR products were purified by agarose gel electrophoresis and the QIAquick Gel Extraction Kit (Qiagen).

Four PCR primer pairs were used to amplify the aux gene in 4 overlapping parts: 5’-

AGCAAACTGATTCCGCTCCAC-3’ and 5’-GCATTGTTTGTTCTGAAGCAGTCC-3’; 5’- 14

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

90oC; 30 cycles 1 min. at 95oC/ 1 min. at 55oC/ 1 min. at 72oC; 1 cycle 10 min. at 72oC.

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.

15

RESULTS

Overexpression of lqf+ in the eye by a glrs-lqf transgene: A glrs-lqf transgene that expresses lqf+ specifically in the eye disc was constructed (Figure 1O). The glrs expression vector (HUANG and FISCHER-VIZE 1996), which is nearly identical to the more widely-used GMR vector (HAY et al. 1994), activates in all cells of the third instar larval eye disc 3-4 rows posterior to the morphogenetic furrow. By mobilizing a primary transformant line, many independent glrs-lqf lines were generated in order to obtain one with a strong rough eye phenotype (Figure 1, A and D).

Overexpression of a wild-type lqf gene results in a mutant phenotype most likely because Lqf is performing its normal function too efficiently, and/or because Lqf is titrating from the cell proteins with which it forms complexes. The idea behind the modifier screen is that lowering by half (in heterozygous mutants) the dose of proteins that Lqf requires to function, or that Lqf binds, will enhance the rough eye phenotype. As flies with two copies of the transgene (2Xglrs-lqf) have a much rougher eye than flies with one copy (1Xglrs-lqf) (Figure 1, D and H), the rough eye is lqf+ dosage sensitive, and therefore likely also responsive to changes in the dosage of other genes that interact with lqf+. In order to test this idea, we used mutations in the clathrin heavy chain (chc) gene, as it encodes a protein that we know Lqf binds (DRAKE et al. 2000). We found that two different chc mutations (chc1 and chc4) are strong dominant enhancers of

1Xglrs-lqf (data not shown; see below). This result suggests that a screen for dominant enhancers of the 1Xglrs-lqf rough eye should identify genes encoding proteins that interact with Lqf.

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 16 autosomes or the X chromosome. Approximately 30,000 F1 males were screened for autosomal enhancers (Figure 2); sixteen chromosome 2-linked mutants and seventy four chromosome 3-linked mutants were recovered. An example of an enhanced eye phenotype is shown in Figure 1, K - N.

The first goal in analyzing the autosomal enhancers was to sort them into complementation groups by performing pairwise crosses of all of the mutants on each chromosome. When knocked out, most Drosophila genes will not result in an obvious mutant phenotype or lethality, and are considered redundant genes (ADAMS et al.

2000). Thus, we expected that most of the enhancers we identified were not likely to result in a homozygous mutant phenotype, and therefore, even though they are probably multiple mutant alleles of several genes, would not sort into complementation groups in the cross matrix. Although these redundant genes may be interesting factors in Lqf function, as the mutants have phenotypes only in a glrs-lqf background, the genes are difficult to identify and their functions are difficult to study. For this reason, we decided to pursue only the genes corresponding to the complementation groups that we could identify in our matrix. Thus, we tested each enhancer-containing chromosome for homozygous phenotypes (morphology or lethality) in an otherwise wild-type background.

Only those enhancers on chromosomes that result in a mutant phenotype when homozygous were entered into the matrix. If the homozygous mutant phenotype is due to the enhancer rather than to an unrelated EMS-induced mutation, then multiple alleles are likely to be identified in the complementation matrix.

We found that thirteen of the sixteen enhancers on chromosome 2 and thirty-one of the seventy-four 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 17 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 twelve mutants was identified among the chromosome 2 enhancers, and one complementation group containing twelve enhancers was identified among the chromosome 3 enhancers (Figure 4).

Approximately 15,000 F1 females were screened for X-linked enhancers (Figure

3) and eight mutants were identified. Of these eight mutant chromosomes, two were homozygous lethal (AA1 and BB2), one was hemizygous viable with morphological phenotypes and male sterility (EE1), and five were homozygous viable with no obvious mutant phenotypes. As it is impossible to perform complementation crosses with the X- linked lethals (adult males are unobtainable), we determined whether or not AA1, BB2, and EE1 are allelic in a different manner (see below).

Identification of the chromosome 2 complementation group as split ends:

By meiotic recombination analysis using a multiply marked second chromosome, the enhancer C18, as a representative of the complementation group on chromosome 2, was localized close to aristaless (al) at polytene position 21C3 (Materials and Methods).

Physical mapping with twenty-seven deficiency chromosomes covering polytene region

21-24 defined proximal and distal boundaries for the C18 region (Materials and

Methods). Available genetic data regarding loci included in and excluded from the regions of the deficiencies localized C18 to an area that includes twelve genes (Materials and Methods). Among them, split ends (spen) was identified as a candidate for C18 because it is the largest gene in the region, amorphic spen alleles are homozygous lethal, spen is known to play a role in Notch signaling (KUANG et al. 2000), and spen was the only gene in the region for which mutant alleles were available. The C18 18 chromosome failed to complement three homozygous lethal spen alleles (spen14O1, spen16H1, and spen03350) or Df(2L)net-PM47C, which deletes spen (Materials and

Methods). Also, the other eleven members of the C18 complementation group were tested for complementation by spen14O1 and Df(2L)net-PM47C. In trans to spen14O1, the three alleles C18, N28, and I55 are completely lethal, while the other eight alleles produce viable escapers (0.5 – 10% of progeny) with no noticeable morphological phenotypes. (Similarly, the alleles N18, L13, and L19 in heterozygous combinations with each other produce normal-appearing escapers.) By contrast, every allele in the C18 complementation group is lethal in trans to Df(2L)net-PM47C. We conclude that the C18 complementation group is spen, and that at least eight of the alleles are hypomorphic.

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 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 it was the largest, and also because in vitro and in other organisms, Auxilin is involved in clathrin-mediated endocytosis (PRASAD et al. 19

1993; UNGEWICKELL et al. 1995; HOLSTEIN et al. 1996; HONING et al. 1994; UMEDA et al. 2000; GALL et al. 2000; LEMMON et al. 2001; GREENER et al. 2001; PISHVAEE et al. 2000). 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 also 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 eleven of the twelve enhancer chromosomes (Figure 5). We conclude that the 727 complementation group is aux.

Identification of the genes corresponding to the X-linked complementation groups: As we already knew that X-linked chc mutations are lethal when amorphic

(BAZINET et al. 1993) and are dominant enhancers of glrs-lqf rough eyes (see above), we tested whether AA1, BB2, or EE1 were chc mutants. We asked if a P element containing chc+ genomic DNA that rescues amorphic chc mutants, or if Dp(1;Y)shi+y+BS, a Y chromosome containing a duplication of X that includes chc+, would complement any of the three enhancers (Materials and Methods). We found that AA1 and BB2 were complemented completely in both cases, while EE1 was not. We conclude that AA1 and

BB2 are chc mutants.

Enhancer EE1 was mapped physically by testing twenty-seven stocks that constitute the X chromosome duplication kit from Bloomington and subsequently, eleven deficiency chromosomes, for complementation of EE1 (Materials and Methods). Taken 20 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 referred to also as DRal in the literature, we use here 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 through 156 are required for nucleotide binding (SAWAMOTO et al.

1999). We conclude that EE1 is a mutant allele of Rala.

aux mutants represent an allelic series: The two missense mutants, auxK47 and auxL7, are clearly weaker than any of the nonsense mutants or auxF37; at 25oC, auxL7 homozygotes and auxL7/auxK47 heterozygotes are viable, while all of the nonsense mutants are homozygous lethal and lethal in heterozygous combinations with each other. By observing the eye phenotypes of missense/nonsense mutant escapers, we find that auxK47 is a stronger mutant than auxL7 (Figure 7), and we also ordered the nonsense mutants into an allelic series (data not shown). From strongest to weakest, the allelic series is: [D128, 727], [K5, L24], [K48, D136, C2, J26, F37], N7, K47, L7. Although the 21 variability of wing phenotypes makes it difficult to be certain, the positions of K47 and L7 in the allelic series may be reversed in the wing.

auxL7 is cold sensitive. As described above, at 25oC, heterozygotes between auxL7 and any nonsense mutant are semi-viable and escapers have mutant eyes and wings. By contrast, at 18oC these same heterozygous combinations are completely lethal.

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).

spen and aux mutations are not dominant enhancers of hypomorphic lqf phenotypes: Because the enhancer screen was performed in a background where lqf is overexpressed in the eye, we were curious to know if the enhancer mutants we isolated could also have been identified in a screen for enhancers of hypomorphic lqf mutations.

We determined previously that the mutant phenotypes of lqfFDD9 homozygotes (lqfFDD9 is hypomorphic), are strongly enhanced by chc loss-of-function mutants (CADAVID et al.

2000). A weak chc allele dominantly enhances the eye and wing malformations in lqfFDD9, while the dominantly enhanced phenotype caused by strong chc alleles is death. By contrast, none of the spen or aux alleles we tested (spenC18, spenJ47, spenL13, spen14O1, spen16H1 and aux727, auxD136, auxC2, auxN7, auxD128, auxL7, auxF37) enhance lqfFDD9 noticeably (data not shown). We conclude that neither spen nor aux mutants could have been isolated in a screen for enhancers of lqfFDD9 homozygous phenotypes. 22

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 semi-lethal at 25oC 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).

DISCUSSION

In a mutagenesis screen of the entire Drosophila genome, we identified mutations in four essential genes as enhancers of the mutant eye phenotype resulting from lqf+ overexpression: chc, Rala, spen, and aux. While we expected to identify chc alleles in the screen, the other three loci were unexpected. We discuss below the implications of these genetic interactions for the role of Lqf in Notch signaling.

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- 23

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 a 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 interact not only with glrs-lqf, but also interact with lqf loss-of- function mutations. Also, the phenotypes of aux hypomorphs described here and in 24

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. E., B. CHO, J. A.

F., manuscript in preparation). 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 in order 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 possiblity 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. 1995;

WIELLETTE et al. 1999; REBAY et al. 1999; STAEHLING-HAMPTON et al. 1999;

KUANG et al. 2000; CHEN and REBAY 2000; THERRIEN et al. 2000; LANE et al. 2000;

PROKOPENKO 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. 1995) .

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. 25

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).

This suggests that the interaction between Lqf and Spen is close and thus that Lqf might function in the nucleus with Spen.

ACKNOWLEDGEMENTS

We thank Soojin Kim for isolating aux727 in a pilot screen, and Shelley Acosta for performing some eye sectioning and mounting some wings. For fly stocks we thank the

Bloomington Drosophila Stock Center, Hideyuki Okano, Ira Mellman, and Bing Zhang.

We are grateful to John Mendenhall of the ICMB Microscopy Facility for scanning electron microscopy, the ICMB DNA Facility for DNA sequencing, and Paul Macdonald for use of his confocal microscope. We thank members of our laboratory for discussions.

This work was supported by a grant from the National Institutes of Health (HD30680) to

J. A. F. Support for E. O., S. H. E., and J.-H. L. came also from The University of Texas at Austin.

LITERATURE CITED

ADAMS, M. D., et al., 2000 The genome sequence of Drosophila melanogaster.

Science 287:2185-2195.

AGUILAR, R. C., LONGHI S. A., SHAW, J. D., YEH, L.-Y., KIM, S., SCHON, A.,

FREIRE, E., HSU, A., MCCORMICK, W. K., WATSON, H. A. and B. WENDLAND, 2000

Epsin N-terminal homoogy domains perform an essential function regulating Cdc42 26 through binding Cdc42 GTPase-activating proteins. Proc. Natl. Acad. Sci. USA

103:4116-4121.

AHLE, S. and E. UNGEWICKELL, 1990 Auxilin, a newly identified clathrin-associated protein in coated vesicles from bovine brain. J. Cell Biol. 111:19-29.

BAKER, N. E., 2002 Notch and the patterning of ommatidial founder cells in the developing Drosophila eye, pp. 35-58 in Drosophila Eye Development, Springer-

Verlag, Berlin.

BAZINET, C., A. L. KATZEN, M. MORGAN, A. P. MAHOWALD and S. K. LEMMON,

1993 The Drosophila clathrin heavy chain gene: clathrin function is essential in a multicellular organism. Genetics 134: 1119-1134.

BENMERAH, A., M. SCOTT, V. POUPON and S. MARULLO, 2003 Nuclear functions for plasma membrane-associated proteins? Traffic 4: 503-511.

BENMERAH, A., 2004 Endocytosis:signaling from endocytic membranes to the nucleus.

Curr Biol. 14: R314-316.

CADAVID, A. L. M., A. GINZEL and J. A. FISCHER, 2000 The function of the

Drosophila Fat facets deubiquitinating enzyme in limiting photoreceptor cell number is intimately associated with endocytosis. Development 127: 1727-1736.

27

CHANG, H. C., M. HUL and I. MELLMAN, 2004 The J-domain protein Rme-8 interacts with Hsc70 to control clathrin-dependent endocytosis in Drosophila. J. Cell Biol. 164:

1055-1064.

CHEN, B., T. CHU, E. HARMS, J. P. GERGEN and S. STRICKLAND, 1998 Mapping of

Drosophila mutations using site-specific male recombination. Genetics 149: 157-163.

CHEN F. and I. REBAY, 2000 split ends, a new component of the Drosophila EGF receptor pathway, regulates development of midline glial cells. Curr. Biol. 10: 943-946.

CHEN, H., S. FRE, V. I. SLEPNEV, M. R. CAPUA, K. TAKEI, M. H. BUTLER, P. P. DI

FIORE and P. DE CAMILLI, 1998a Epsin is an EH-domain-binding protein implicated in clathrin-mediated endocytosis. Nature 394: 793-797.

DICKSON, B. J., A. VAN DER STRATEN, M. DOMINGUEZ and E. HAFEN, 1996.

Mutations modulating Raf signaling in Drosophila eye development. Genetics 142: 163-

171.

DRAKE, M. T., M. A. DOWNS and L. M. TRAUB, 2000 Epsin binds to clathrin by associating directly with the clathrin-terminal domain: evidence for cooperative binding through two discrete sites. J. Biol. Chem. 275: 6479-6489.

FEIG, L. A., 2003 Ral-GTPases: approaching their 15 minutes of fame. Trends Cell Biol.

13: 419-425.

28

FISCHER, J. A., S. K. LEAVELL and Q. LI, 1997 Mutagenesis screens for interacting genes reveal three roles for fat facets during Drosophila eye development. Dev. Gen.

21: 167-174.

FISCHER, J. A., S. H. EUN and B. DOOLAN, 2006 Endocytosis, endosome trafficking and the regulation of Drosophila development. Ann. Rev. Cell Dev. Biol. 22:181-206.

FORTINI, M. E., 2002 Gamma-secretase-mediated proteolysis in cell-surface-receptor signaling. Nat. Rev. Mol. Cell Biol. 3: 673-684.

GALL, W. E., M. A. HIGGINBOTHAM, C.-Y. CHEN, M. F. INGRAM, D. M. CYR and T. R.

GRAHAM, 2000 The auxilin-like phosphoprotein Swa2p is required for clathrin function in yeast. Curr. Biol. 10: 1349-1358.

GREENER, T., X. ZHAO, H. NOJIMA, E. EISENBERG and L. E. GREENE, 2000 Role of cyclin G-associated in uncoating clathrin-coated vesicles from non-neuronal cells. J. Biol. Chem. 275: 1365-1370.

GREENER, T., B. GRANT, Y. ZHANG, X. WU, L. E. GREENE, D. HIRSH and E.

EISENBERG, 2001 Caenorhabditis elegans auxilin: a J-domain protein essential for clathrin-mediated endocytosis in vivo. Nat. Cell Biol. 3: 215-219.

GRUMBLING, G., V. STRELETS and THE FLYBASE CONSORTIUM, 2006 FlyBase: anatomical data, images and queries. Nucl. Acids Res. 34: D484-D488; doi:10.1093/nar/gkj068. http://flybase.org/ 29

HAGEDORN, E. J., J. L. BAYRAKTAR, V. R. KANDACHAR, T. BAI, D. M. ENGLERT and H. C. CHANG, 2006 Drosophila melanogaster auxilin regulates the internalization of

Delta to control activity of the Notch signaling pathway. J. Cell Biol. 173: 443-452.

HAY, B. A., T. WOLFF and G. M. RUBIN, 1994 Expression of baculovirus P35 prevents cell death in Drosophila. Development 120:2121-2129.

HOLSTEIN, S. E., H. UNGEWICKELL and E. UNGEWICKELL, 1996 Mechanism of clathrin basket dissociation: separate functions of protein domains of the DnaJ homolog auxilin. J. Cell Biol. 135: 925-937.

HONING, S., G. KREIMER, H. ROBENEK and B. M. JOCKUSCH, 1994 Receptor- mediated endocytosis is sensitive to antibodies against the uncoating ATPase (Hsc70).

J. Cell Sci. 107: 1185-1196.

HUANG, Y., R. T. BAKER and J. A. FISCHER-VIZE, 1995 Control of cell fate by a deubiquitinating enzyme encoded by the fat facets gene. Science 270: 1828-1831.

HUANG, Y. and J. A. FISCHER-VIZE, 1996 Undifferentiated cells in the developing

Drosophila eye influence facet assembly and require the Fat facets deubiquitinating enzyme. Development 122: 3207-3216.

HYMAN, J., H. CHEN , P. P. DI FIORE, P. DE CAMILLI and A. T. BRUNGER, 2000

Epsin 1 undergoes nucleocytosolic shuttling and its eps15 interactor NH(2)-terminal 30 homology (ENTH) domain, structurally similar to Armadillo and HEAT repeats, interacts with the transcription factor promyelocytic leukemia Zn(2)+ finger protein (PLZF). J. Cell

Biol. 149: 537-546.

JULLIEN-FLORES, V., Y. MAHE, G. MIREY, C. LEPRINCE, B. MEUNIER-BISCEUIL, A.

SORKIN and J. H. CAMONIS, 2003 RLIP76, an effector of the GTPase Ral, interacts with the AP2 complex: involvement of the Ral pathway in receptor endocytosis. J. Cell

Sci. 113: 2837-2844.

KANAOKA, Y., S. H. KIMURA, I. OKAZAKI, M. IKEDA and H. NOJIMA, 1997 GAK: a cyclin G associated kinase contains a tensin/auxilin-like domain. FEBS Lett. 402: 73-80.

KIMURA S. H., H. TSURUGA, N. YABUTA, Y. ENDO and H. NOJIMA, 1997 Structure, expression, and chromosomal localization of human GAK. Genomics 44: 179-187.

KUANG, B., S. C. WU, Y. SHIN, L. LUO and P. KOLODZIEJ, 2000 split ends encodes large nuclear proteins that regulate neuronal cell fate and axon extension in the

Drosophila embryo. Development 127: 1517-1529.

LANE, M. E., M. ELEND, D. HEIDMANN, A. HERR, S. MARZODKO, A. HERZIG and C.

F. LEHNER, 2000 A screen for modifiers of cyclin E function in Drosophila melanogaster identifies Cdk2 mutations, revealing the insignificance of putative phosphorylation sites in Cdk2. Genetics 155: 233-244.

31

LAI, E. C., G. A. DEBLANDRE, C. KINTNER and G. M. RUBIN, 2001 Drosophila

Neuralized is a ubiquitin ligase that promotes the internalization and degradation Delta.

Dev. Cell 1: 783-794.

LAI, E. C., F. ROEGIERS, X. QIN, Y. N. JAN and G. M. RUBIN, 2005 The ubiquitin ligase Drosophila Mind bomb promotes Notch signaling by regulating the localization and activity of Serrate and Delta. Development 132: 2319-2332.

LE BORGNE, R. and F. SCHWEISGUTH, 2003a Unequal segregation of neuralized biases Notch activation during asymmetric cell division. Dev. Cell 5: 139-148.

LE BORGNE, R. and F. SCHWEISGUTH, 2003b Notch signaling: endocytosis makes

Delta signal better. Curr. Biol. 13: R273-275.

LE BORGNE, R., S. REMAUD, S. HAMEL and F. SCHWEISGUTH, 2005a Two distinct

E3 ubiquitin ligases have complementary functions in the regulation of Delta and Serrate signaling in Drosophila. PLOS Biol. 4: e96.

LE BORGNE, R., A. BARDIN and F. SCHWEISGUTH, 2005b The roles of receptor and ligand endocytosis in regulating Notch signaling. Development 132: 1751-1762.

LEE, D. W., X. ZHAO, F. ZHANG, E. EISENBERG and L. E. GREENE, 2005 Depletion of GAK/auxilin 2 inhibits receptor-mediated endocytosis and recruitment of both clathrin and clathrin adaptors. J. Cell Sci. 118: 4311-4321.

32

LEMMON, S. K., 2001 Clathrin uncoating: Auxilin comes to life. Curr. Biol. 11: R49-52.

LEWIS, E. B. and F. BACHER,1968 Method of feeding ethane methylsulfonate (EMS) to

Drosophila males. Dros. Inf. Serv. 43: 193.

LI, Y. and N. E. BAKER, 2004 The roles of cis-inactivation by Notch ligands and of

Neuralized during eye and bristle patterning in Drosophila. BMC Dev. Biol. 4: 5.

LIN, H. V., D. B. DOROQUEZ, S. CHO, F. CHEN, I. REBAY and K. M. CADIGAN, 2003

Splits ends is a tissue/promoter specific regulator of Wingless signaling. Development

130: 3125-3135.

LINDSLEY, D. L. and G. G. ZIMM, 1992 The Genome of Drosophila melanogaster.

Academic Press, San Diego.

MIREY, G., M. BALAKIREVA, S. L'HOSTE, C. ROSS, S. VOEGELING and J.

CAMONIS, 2003 A Ral guanine exchange factor-Ral pathway is conserved in

Drosophila melanogaster and sheds new light on the connectivity of the Ral, Ras, and

Rap pathways. Mol. Cell Biol. 23: 1112-1124.

NEWMYER, S. L., A. CHRISTENSEN and S. SEVER, 2003 Auxilin-dynamin interactions link the uncoating ATPase chaperone machinery with vesicle formation. Dev. Cell 4:

929-940.

33

OVERSTREET, E., X. CHEN, B. WENDLAND and J. A. FISCHER, 2003 Either part of a

Drosophila epsin (Liquid facets) protein, divided after the ENTH domain, functions in the internalization of Delta in the developing eye. Curr. Biol. 13: 854-860.

OVERSTREET, E., E. FITCH and J. A. FISCHER, 2004 Fat facets and Liquid facets promote Delta endocytosis and Delta signaling in the signaling cells. Development 131:

5355-5366.

PARKS, A. L., K. M. KLUEG, J. R. STOUT and M. A. MUSKAVITCH, 2000 Ligand endocytosis drives receptor dissociation and activation in the Notch pathway.

Development 127: 1373-1385.

PAVLOPOULOS, E., C. PITSOULI, K. M. KLUEG, M. A. MUSKAVITCH, N. K.

MOSCHONAS and C. DELIDAKIS, 2001 neuralized encodes a peripheral membrane protein involved in Delta signaling and endocytosis. Dev. Cell 1: 807-816.

PISHVAEE, B., G. COSTAGUTA, B. G. YEUNG, S. RYAZANTSEV, T. GREENER, L. E.

GREENE, E. EISENBERG, J. M. MCCAFFERY and G. S. PAYNE, 2000 A yeast DNA J protein required for uncoating of clathrin-coated vesicles in vivo. Nat. Cell Biol. 2: 958-

963.

PRASAD, K., W. BAROUCH, L. GREENE and E. EISENBERG, 1993 A protein cofactor is required for uncoating of clathrin baskets by uncoating ATPase. J. Biol. Chem. 268:

23758-23761.

34

PROKOPENKO, S. N., Y. HE, Y. LU and H. J. BELLEN, 2000 Mutations affecting the development of the peripheral nervous system in Drosophila: a molecular screen for novel proteins. Genetics 156: 1691-1715.

REBAY, I., F. CHEN, F. HSIAO, P. A. KOLODZIEJ, B. H. KUANG, T. LAVERTY, C.

SUH, M. VOAS, A. WILLIAMS and G. M. RUBIN, 2000 A genetic screen for novel components of the Ras/Mitogen-activated protein kinase signaling pathway that interact with the yan gene of Drosophila identifies Split ends, a new RNA recognition motif- containing protein. Genetics 154: 695-712.

ROSSE, C., S. L'HOSTE, N. OFFNER, A. PICARD and J. CAMONIS , 2003 RLIP, an effector of the Ral GTPases, is a platform for Cdk1 to phosphorylate epsin during the switch off of endocytosis in mitosis. J. Biol. Chem. 278: 30597-30604.

SAWAMOTO, K., P. WINGE, S. KOYAMA, Y. HIROTA, C. YAMADA, S. MIYAO,, S.

YOSHIKAWA, M. JIN, A. KIKUCHI and H. OKANO, 1999 The Drosophila Ral GTPase

regulates developmental cell shape changes through the Jun NH2-terminal kinase pathway. J. Cell Biol. 146: 361-372.

SCHRODER, S., S. A. MORRIS, R. KNORR, U. PLESSMANN, K. WEBER, G. V.

NGUYEN and E. UNGEWICKELL, 1995 Primary structure of the neuronal clathrin- associated protein auxilin and its expression in bacteria. Eur. J. Biochem. 228: 297-304.

STAEHLING-HAMPTON, K., P. J. CIAMPA, A. BROOK and N. DYSON, 1999 A genetic screen for modifiers of E2F in Drosophila melanogaster. Genetics 153: 275-287. 35

THERRIEN, M., D. K. MORRISON, A. M. WONG and G. M. RUBIN, 2000 A genetic screen for modifiers of a kinase suppressor of Ras-dependent rough eye phenotype in

Drosophila. Genetics 156: 1231-1242.

THUMMEL, C. S. and V. PIRROTTA, 1992 Dros. Inf. Serv. 71: 150.

UMEDA, A., A. MEYERHOLZ and E. UNGEWICKELL, 2000 Identification of the universal cofactor (auxilin 2) in clathrin coat dissociation. Eur. J. Cell Biol. 79: 339-342.

UNGEWICKELL, E., H. UNGEWICKELL, S. E. HOLSTEIN, R. LINDNER, K. PRASAD,

W. BAROUCH, B. MARTIN, L. E. GREENE and E. EISENBERG, 1995 Role of auxilin in uncoating clathrin-coated vesicles. Nature 378: 632-635.

VECCHI, M., S. POLO, V. POUPON, J. W. VAN DE LOO, A. BENMERAH and P. P. DI

FIORE, 2001 Nucleocytoplasmic shuttling of endocytic proteins. J. Cell Biol. 153: 1511-

1517.

WANG, W. and G. STRUHL, 2004 Drosophila Epsin mediates a select endocytic pathway the DSL ligands must enter to activate Notch. Development 132: 2883-2894.

WANG, W. and G. STRUHL, 2005 Distinct roles for Mind bomb, Neuralized and Epsin in mediating DSL endocytosis and signaling in Drosophila. Development 131: 5367-

5380.

36

WENDLAND, B., K. E. STEECE and S. D. EMR, 1999 Yeast epsins contain an essential N-terminal ENTH domain, bind clathrin and are required for endocytosis.

EMBO J. 18: 4383-4393.

WENDLAND, B., 2002 Epsins: adaptors in endocytosis? Nat. Rev. Mol. Cell Biol. 3:

971-977.

WIELLETTE E. L., K. W. HARDING, K. A. MACE, M. R. RONSHAUGEN, F. Y. WANG and W. MCGINNIS, 1999 spen encodes an RNP motif protein that interacts with Hox pathways to repress the development of head-like sclerites in the Drosophila trunk.

Development 126: 5373-5385.

37

FIGURE LEGENDS

FIGURE 1.- The glrs-lqf eye phenotype. Scanning electon micrographs (A, D, H, K) and tangential sections (B, C, E-G, I, J, L-N) of adult eyes are shown. The genotypes are:

(A-C) wild-type (wt); (D-G) 1Xglrs-lqf; (H-J) 2Xglrs-lqf; (K-N) 2Xglrs-lqf; 727/+. The boxes in B, E, I, and L correspond to the enlargements in C, F-G, J, and M-N, respectively. The numbers in C refer to R-cells. In F and G, asterisks indicate ectopic R-cells and arrows indicate degenerated R7s. The plus signs in J indicate R-cells. The 2Xglrs-lqf eyes are homozygous for the 1Xglrs-lqf P element. The rough eye phenotype of 2Xglrs-lqf is unlikely to be affected by a mutation caused by the P element insertion because flies with one copy of this P element and an additional copy at a different location show similarly rough eyes. In eyes with 1Xglrs-lqf, some facets have more than the normal number of 6 outer R-cells (Figure 1, B, C and E – G). Adult eyes with 2Xglrs-lqf have more severely disrupted eye morphology; organized facets are absent, although some

R-cells do form (Figure 1I, J). Also, degeneration of R7 is observed in glrs-lqf transformants (Figure 1B, C, and E-G). (O) The P element containing glrs-lqf used to transform flies. P are the P element ends, white is the white gene marker, the lqf cDNA is FLAG-tagged, pA is the polyadenylation site, and the arrows indicate the direction of transcription.

FIGURE 2.- Mutagenesis screen for autosomal enhancers of the glrs-lqf eye phenotype.

A cross scheme is shown for the screen of the autosomes for dominant enhancers of the glrs-lqf rough eye. See Materials and Methods for information about fly strains.

38

FIGURE 3.- Mutagenesis screen for X-linked enhancers of the glrs-lqf eye phenotype. A cross scheme is shown for the screen of the X chromosome for dominant enhancers of the glrs-lqf rough eye. See Materials and Methods for information about fly strains.

FIGURE 4.- Complementation groups of glrs-lqf enhancers. The glrs-lqf enhancers sort into three lethal complementation groups corresponding to the genes chc, spen, and aux. In addition, a single viable mutant allele of Rala was identified. Lines represent chromosomes, and the filled boxes centromeres. Numbers beneath the lines are polytene bands. Boxed numbers and letters are allele names.

FIGURE 5.- Molecular analysis of aux mutant alleles. The Drosophila Auxilin protein is diagrammed, numbers are amino acids 1 – 1165. Arrows indicate approximate positions of the codon changes in each mutant allele. The table shows each nucleotide change and the corresponding codon changes. The functional domains of Drosophila Auxilin were inferred by amino acid sequence similarity to vertebrate Auxilin 2, also known as

GAK (cyclin G-associated kinase) (AHLE and UNGEWICKELL 1990; UNGEWICKELL et al. 1995; KANAOKA et al. 1995; KIMURA et al. 1997; GREENER et al. 2000; UMEDA et al. 2000). Vertebrates also have Auxilin 1, which is neural-specific and lacks the kinase domain (PRASAD et al. 1993; UNGEWICKELL et al. 1995). Drosophila have the single Auxilin 2-like protein shown. Auxilin binds clathrin (HOLSTEIN et al. 1996;

GREENER et al. 2000) and through its DnaJ domain binds Hsc70 (UNGEWICKELL et al. 1995; HOLSTEIN et al. 1996). The amino acid (C671) mutated in allele K47 is not

1111 conserved in vertebrate GAK, but P , which is mutated in allele L7, is conserved in

GAK and in Auxilin 1 from C. elegans and yeast (PISHAVEE et al., 2000; GALL et al.

2000; GREENER et al. 2001). 39

FIGURE 6.- RalaEE1 morphological phenotypes. External body parts of wild-type (wt) and

RalaEE1 hemizygous males are shown. (A, B) RalaEE1 males have rough eyes, and bristles on the head and humerus are either missing or severely truncated. (C, D)

RalaEE1 male are missing notal bristles and hairs. (E, F) The wings of RalaEE1 males are short and curved.

FIGURE 7.- Eye phenotypes of aux hypomorphs. Scanning electron micrographs (A, C,

E) and tangential sections (B, D, F) of the aux mutant genotypes indicated are shown.

(A, B) auxL7 eyes are nearly wild-type. (C-F) Escapers with heterozygous combinations of weak and strong aux alleles have rough external eyes with eye patterning defects typical of Notch pathway mutants: disorganized ommatidia, most with too many R-cells, some with too few, and some fused ommatidia. auxK47 (E, F) has stronger effects in the eye than does auxL7 (C, D).

FIGURE 8.- Wing phenotypes of aux hypomorphs. Wings of wild-type (wt) flies (A), auxL7 homozygotes (B), or escapers (C-E) with the genotypes indicated are shown. (D,E)

For all escaper genotypes, the wing phenotypes show widely variable expressivity.

FIGURE 9.- Genetic interactions between aux and lqf loss-of-function mutations. (A)

Wings heterozygous for auxL7/auxK47 have a wild-type phenotype but are sensitive to the dose of lqf+ activity. (B) Halving the normal lqf+ gene dosage from two gene copies to one copy in a auxL7/auxK47 background results in wings with a strong Notch pathway-like mutant phenotype. FIGURE 1 FIGURE 2

Autosomal Screen EMS

P pr ; st X v v w ; glrs-lqf, Bl CyO

v v screen for F1 w ; pr ; st X w ; glrs-lqf, Bl enhanced glrs-lqf* , Bl *+ rough eyes CyO

do enhanced F2 w ; pr or CyO ; st or + v v rough eyes * * X w ; glrs-lqf, Bl breed through? glrs-lqf, Bl + CyO

on which autosome is F3 If on chr. 2, no CyO are enhanced. enhancer? * If * on chr. 3, some CyO are enhanced. balance v pr *CyO

w ; glrs-lqf, Bl ; st v v X w ; Sb CyO *+ TM6B

v w ; glrs-lqf, Bl ; st F4 + TM6B* * = (potential) enhancer mutation FIGURE 3

EMS X chromosome Screen

P st X v v w ; glrs-lqf

screen for F1 + ; + ; st X w ; glrs-lqf enhanced *w glrs-lqf* *+ rough eyes CyO

do enhanced F2 + ; + glrs-lqf ; st rough eyes or X w ; Sb *w glrs-lqf* * CyO *+ breed through? TM6B

eliminate F3 + ; glrs-lqf ; st X w ; Sb [ + ] chromosome 2 * * * * w + TM6B TM6B

eliminate + ; glrs-lqf ; Sb [ st ] chromosome F4 * * X w ; CyO * w + TM6B Sco

F5 + ; glrs-lqf ; Sb or TM6B X w ; glrs-lqf eliminate *w * CyO + [ glrs-lqf ] *chromosome + ; glrs-lqf ; Sb or TM6B F6 X FM7c *w CyO +

+ ; glrs-lqf balance F7 X FM7c FM7c* CyO FIGURE 4

Rala chc EE1 AA1 BB2 X 3E5/6 13F5/7

spen C18 I55 L13 D108 J28 L19 E26 J43 N18 E29 J47 N28 2 21B3/4 aux 727 F37 K48 C2 J26 L7 D128 K5 L24 D136 K47 N7 3 82A FIGURE 5 auxilin mutant alleles

weak alleles (missense mutants) K47 L7

1 1165 protein kinase tensin-like clathrin binding DnaJ

D128 727 K5 L24 K48 N7 C2 J26 strong alleles D136 (nonsense mutants)

allele aa change codon change

D128 Q172 → stop CAG → TAG 727 W254 → stop TGG → TAG K5 Q404 → stop CAG → TAG L24 Q486 → stop CAG → TAG K48 Q594 → stop CAG → TAG N7 K734 → stop AAG → TAG C2 Q1018 → stop CAG → TAG D136 Q1018 → stop CAG → TAG J26 Q1037 → stop CAA → TAA eF37 ? ?

K47 C671Y TGT → TAT L7 P1111S CCT → TCT FIGURE 6 FIGURE 7 FIGURE 8 FIGURE 9