(2003) 22, 9225–9230 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc hScrib is a functional homologue of the Drosophila tumour suppressor Scribble

Lukas E Dow1,2, Anthony M Brumby3, Rosa Muratore1, Michelle L Coombe3, Karin A Sedelies4, Joseph A Trapani4,5, Sarah M Russell5,6, Helena E Richardson3,7 andPatrick O Humbert* ,1,5

1Cell Cycle and Genetics Laboratory, Peter MacCallum Cancer Centre, St Andrew’s Place, East Melbourne, Victoria 3002, Australia; 2Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3005, Australia; 3Cell Cycle and Development Laboratory, Peter MacCallum Cancer Centre, St Andrew’s Place, East Melbourne, Victoria 3002, Australia; 4Cancer Cell Death Laboratory, Peter MacCallum Cancer Centre, St Andrew’s Place, East Melbourne, Victoria 3002, Australia; 5Department of Pathology, University of Melbourne, Parkville, Victoria 3005, Australia; 6Immune Signaling Laboratory, Peter MacCallum Cancer Centre, St Andrew’s Place, East Melbourne, Victoria 3002, Australia; 7Department of Anatomy and Cell Biology, University of Melbourne, Parkville, Victoria 3005, Australia

Scribble (scrib), discs large (dlg) and lethal giant larvae 1996; Bilder and Perrimon, 2000). In addition to the loss (lgl) encode that regulate and have of polarity, epithelial andneural tissues from scrib, lgl or been identified as neoplastic tumour suppressor in dlg mutant larvae overproliferate to form large neoplas- . Here, we have used the tic cell masses (Woods and Bryant, 1991; Bilder et al., Drosophila model system to provide the first functional 2000). In Drosophila models, dlg and lgl evidence that human Scribble (hScrib) can act as a tumour homozygous mutant cells can form invasive tumours suppressor. We show that hScrib displays highly that migrate to distant sites and kill the host polarized localization in mammalian epithelial cells and (Woodhouse et al., 1998). These observations have led colocalizes with mammalian Dlg, similar to D. melano- to the classification of scrib, dlg and lgl as neoplastic gaster Scribble (DmScrib) distribution in Drosophila tumour suppressor genes in Drosophila (Gateff, 1994; . Furthermore, hScrib can rescue the polarity Bilder et al., 2000). and tumorous overgrowth defects of scrib mutant The scrib, lgl and dlg genes are highly conserved Drosophila. hScrib therefore can act as an effective between Drosophila andmammals. Both human Scrib tumour suppressor in vivo, regulating both apical–basal (hScrib) and Dlg (hDlg) are targeted for degradation by polarity and cellular proliferation in a manner similar to viral oncoproteins such as human papillomavirus E6, that of DmScrib in Drosophila. These data demonstrate and recent studies have shown decreased expression of that hScrib is a functional homologue of DmScrib and Dlg homologues in gastric carcinomas anda mouse therefore predict an important role for hScrib in the model of ovarian cancer (Liu et al., 2002; Boussioutas suppression of mammalian tumorigenesis. et al., 2003; Huang et al., 2003). Such correlative data Oncogene (2003) 22, 9225–9230. doi:10.1038/sj.onc.1207154 have ledto the speculation that hDlg, hScrib andhuman Lgl may play a role in human epithelial Keywords: hScrib; Scribble; polarity; ; tumour (Mantovani andBanks, 2001; Humbert et al., 2003). suppressor; Drosophila; discs large Consistent with a functional conservation of this network of proteins, mammalian Dlg homologues can rescue Drosophila Dlg mutants, andhave been impli- catedin the regulation of apical–basal polarity and Loss of cell polarity is one of the hallmarks of cancer, proliferation in mammalian cells (Makino et al., andis correlatedwith more aggressive andinvasive 1997; Thomas et al., 1997; Ishidate et al., 2000; cancers (Bissell andRadisky,2001; Thiery, 2002). In Ludford-Menting et al., 2002). However, little is known Drosophila melanogaster, mutation analysis in three about the functional properties of hScrib. genes, discs large (dlg), scribble (scrib) and lethal giant hScrib is a membrane-associatedprotein that, like its larvae (lgl), has revealeda link between the regulation Drosophila counterpart, contains 16 leucine-rich repeats of cell polarity andcell proliferation control. Scrib, dlg (LRRs) andfour PDZ domains(Figure 1a) (Nakagawa and lgl genetically interact, andhomozygous mutant andHuibregtse, 2000). The recent identification of Scrib larvae (for either scrib, dlg or lgl) show a loss of apical– (scrb1) as the mutatedin the mouse circletail basal cell polarity andepithelial tissues lose their mutant indicates an important role for Scrib in columnar, monolayeredorganization (Woods et al., development (Murdoch et al., 2003). Homozygous circletail mice die perinatally from severe neural tube *Correspondence: PO Humbert, Cell Cycle and Cancer Genetics defects (craniorachischisis) (Murdoch et al., 2003) and Laboratory, Peter MacCallum Cancer Centre, St Andrew’s Place, show defects in tissue patterning in the embryonic East Melbourne, Victoria 3002, Australia; E-mail: [email protected] cochlea (Montcouquiol et al., 2003). These patterning Received3 June 2003; revised24 July 2003; accepted22 August 2003 defects implicate mammalian Scrib in the regulation of Functional conservation of hScrib LE Dow et al 9226 planar cell polarity, a role not establishedfor Drosophila andsuppress tissue overgrowth are conservedbetween melanogaster Scrib (DmScrib). While this hints that the DmScrib andhScrib, suggesting that hScrib is likely to two proteins may have divergent evolutionary function, play an important role in the suppression of human these studies have not examined whether mammalian tumours. Scrib can act to regulate apical–basal polarity and To study the function of hScrib in vivo, we generated proliferation similar to DmScrib. In this paper, we show andcharacterizedan EGFP-hScrib fusion protein. The that the ability to regulate both apical–basal polarity construct was assembledusing the cDNA clone

Oncogene Functional conservation of hScrib LE Dow et al 9227 KIAA0147 (GenBank Accession No. D63481), Dlg in a polarizedMDCK cell monolayer (Figure 1e). which corresponds to an incomplete hscrib sequence These studies show that EGFP-hScrib possesses proper- (Nakagawa andHuibregtse, 2000). Basedon the ties similar to those of endogenous mammalian Scrib. available hscrib coding sequence (GenBank Accession Importantly, the localization of hScrib at andbasal to No. NM_015356), the missing 50 sequence including the adherens junctions and its colocalization with mamma- initiating ATG codon was PCR amplified from human lian Dlg in epithelial cells suggests a high degree of colon adenocarcinoma (LIM1899) cDNA and inserted conservation in the polarizedlocalization of Scrib into KIAA0147 to create the entire hscrib open reading between Drosophila andmammalian epithelium. frame (ORF). This full-length cDNA was placedin To determine whether hScrib is a functional homo- frame anddownstream of the EGFP ORF in the logue of Drosophila Scrib, we assessedwhether EGFP- pEGFP-C1 expression vector (Clontech). Expression of hScrib couldsubstitute for the Drosophila Scrib protein. the fusion protein at the predicted molecular weight was To achieve this, we generatedthree independentlines of confirmedby Western blot analysis following transfec- transgenic flies carrying the EGFP-hScrib cDNA under tion into 293 cells (data not shown). In Drosophila the control of a GAL4-responsive UAS promoter embryonic epithelium, Scrib localizes basal to the (BrandandPerrimon, 1993). The EGFP-hScrib trans- adherens junctions and plays a role in the establishment gene was then crossedinto flies carrying a strong mutant of this junction complex (Bilder et al., 2000). Polarized allele of scrib (scrib1) (Bilder and Perrimon, 2000). Madin Darby Canine (MDCK) epithelial cells We first investigatedwhether EGFP-hScrib could were analysedby confocal microscopy for the localiza- compensate for the loss of DmScrib in Drosophila eye tion of EGFP-hScrib (expressedby transient transfec- epithelial tissue. Using the ey-FLP/FRT, GAL4/GAL80 tion) andestablishedmarkers of (MARCM) system (Lee andLuo, 1999), we were able to including ZO-1 (tight junctions) (Stevenson et al., generate clonal patches of scrib mutant cells surrounded 1986) and E-cadherin (adherens junctions) (Behrens by otherwise normal epithelial tissue andexpress et al., 1985). As previously reportedwith an incomplete mCD8-GFP (a membrane-targetedGFP construct) or hScrib GFP fusion protein (Nakagawa andHuibregtse, EGFP-hScrib specifically within these cells (Figure 2b). 2000), EGFP-hScrib was localizedpredominantly to the Thus, the patches of mutant tissue in the eye disc could cell membrane of MDCK cells, in contrast to EGFP be identified by the expression of mCD8-GFP or EGFP- alone, which was distributed throughout the cell hScrib. (Figure 1b). EGFP-hScrib partially overlappedwith E- Expression of EGFP-hScrib within wild-type clones cadherin at the basolateral membrane (Figure 1b), of cells showedno aberrant effects on the eye tissue suggesting it may be associatedwith adherens junctions. allowing normal differentiation (Figure 2c, top panel). In Drosophila, Scrib colocalizes andforms a complex Loss of scrib in clonal patches in the eye disc disrupted with Dlg (Bilder et al., 2000; Mathew et al., 2002). In proper differentiation of the photoreceptor cells, as polarizedMDCK cells, EGFP-hScrib overlappedsig- measuredby Elav staining, a marker of differentiated nificantly with Dlg, colocalizing at the basolateral photoreceptor cells (A Brumby andH Richardson, membrane of MDCK cells, distinctly basal to ZO-1 at manuscript in press; Figure 2c, middle). Expression of tight junctions (Figure 1c). To ensure that EGFP-hScrib EGFP-hScrib specifically within scrib mutant tissue was localizedin a manner similar to that of endogenous largely restoredthe differentiation andpatterning of MDCK Scrib, we examinedthe localization of Scrib in the photoreceptors in the eye (Figure 2c, bottom). MDCK cells using a monoclonal antibody we have EGFP-hScrib expression also effectively suppressedthe generated(7C6.D10) anda commercially available tissue organization defects (as assessed by actin staining) polyclonal antibody (Santa Cruz). Both monoclonal associatedwith scrib mutant tissue. In scrib mutant andpolyclonal hScrib antibodiesrecognize a single clones, the columnar, monolayeredepithelial cells protein of the expectedsize by Western blot analysis on became rounded and multilayered, suggesting the cells MDCK whole cell lysates (data not shown). Like the had lost apical–basal polarity (Figure 2d, middle). scrib human fusion protein, endogenous Scrib localized basal mutant cells expressing EGFP-hScrib maintainedtheir to ZO-1 (Figure 1d) and colocalized with endogenous columnar architecture, andthe actin cytoskeleton

Figure 1 hScrib localizes along the basolateral membrane of polarizedMDCK cells with endogenousmammalian Dlg. ( a) Schematic representation of the EGFP-hScrib fusion protein used in this study is shown with reference to DmScrib. Like DmScrib, hScrib is predicted to contain 16 LRRs and four PDZ domains. Within these defined domains, hScrib shares a high level of sequence with DmScrib; between 50 and75% amino-acididentity,comparedto 37% overall. ( b) and (c) MDCK cells culturedon polyester membranes were transiently transfected with EGFP or EGFP-hscrib and allowed to form a confluent monolayer over 3 days. Cells were fixed in 3.7% paraformaldehyde and probed with antibodies to ZO-1 (rat monoclonal, Chemicon), E-cadherin (mouse monoclonal, Transduction Laboratories) or mammalian Dlg (PSD-95 family mouse monoclonal, Upstate Biotech). The cells were analysedby confocal microscopy andimages processedwith Image J andAdobePhotoshop 7.0 software. (b) Localization of EGFP-hScrib is predominantly at the plasma membrane of MDCK cells in contrast to EGFP alone, which is distributed throughout the cell. EGFP-hScrib is found at the basolateral membrane partially overlapping with E-cadherin (red) at points of cell–cell contact (adherens junctions). (c) EGFP-hScrib (green) localization is basal to anddistinctfrom ZO-1 (blue) at tight junctions. Dlg homologues (red) also localize at the basolateral membrane of polarized epithelial cells, overlapping with EGFP-hScrib. (d) and (e) Confluent MDCK cells were fixedandprobedwith antibodiesto hScrib (mouse monoclonal, 7C6.D10, raisedagainst PDZ2 of hScrib) andZO-1 or hScrib (goat polyclonal, Santa Cruz) andDlg. ( d) Endogenous Scrib (green) localizes to the basolateral membrane of MDCK cells basal to ZO-1 (red). (e) Endogenous Scrib (green) colocalizes with Dlg (red) at the basolateral membrane of these cells. Scale bars: 20 mm(xy), 5 mm(xz)

Oncogene Functional conservation of hScrib LE Dow et al 9228

Figure 2 Suppression of the scrib mutant phenotype by expression of EGFP-hScrib in mosaic eye discs. (a) Genotypes of the F1 progeny analysed. (b) Schematic representation of the generation of scrib mosaic eye tissue using the MARCM system. Expression of FLP from the eyeless promoter induces mitotic recombination at FRT82B sites on homologous . When a scrib-null cell is produced, the GAL80 repressor gene is lost and UAS-responsive transgenes (i.e. mCD8-GFP or EGFP-hScrib) become irreversibly activated, marking the scrib mutant clonal tissue. In control flies (‘EGFP-hScrib’) where the scrib locus is wildtype, expression of EGFP-hScrib marks wild-type tissue. (c) The thirdinstar eye imaginal discswere harvested,immunostainedfor the expression of Elav (mouse monoclonal (9F8A9), Developmental Studies Hybridoma Bank, University of Iowa), F-actin (phalloidin-TRITC, Sigma) or Dlg (mouse monoclonal (4F3), Developmental Studies Hybridoma Bank, University of Iowa) and examined by confocal microscopy. Representative clonal tissue is highlighted by dotted outline. Elav staining (red) marks differentiated photoreceptor cells in the developing eye disc. Expression of EGFP-hScrib in wild-type clones does not interfere with normal differentiation (top panel). In the scrib mutant tissue, differentiation is disrupted and in some cases no Elav expression is detected in the mutant clones (middle). In scrib mutant clones expressing EGFP-hScrib, proper differentiation is restored and is comparable with that seen in wild-type clonal regions (bottom). (d) Transverse sections of mosaic eye imaginal discs show the columnar epithelial monolayer of the eye disc. In scrib mutant clones (GFP), the actin cytoskeleton (red) is disorganized and the cells become rounded and multilayered (middle). Actin organization andtissue structure in scrib mutant tissue expressing EGFP-hScrib was similar to wild-type clonal tissue (compare bottom and top). (e) In scrib mutant clones (GFP), the cells become multilayered and the strong apical expression of Dlg (red) is lost (middle). In rescued clonal patches, the scrib mutant cells maintain their columnar monolayeredorganization andthe polarizedexpression of Dlg (bottom)

appearednormal (Figure 2d,bottom). To investigate in (Figure 2e, top). In multilayered scrib mutant clonal more detail, the extent of the functional rescue on tissue the strong subapical staining of Dlg was lost polarity, Dlg localization was examinedon transverse (Figure 2e, middle). EGFP-hScrib expression in the scrib sections of third instar eye imaginal discs. In wild-type mutant tissue was sufficient to maintain the columnar eye epithelium, Dlg was highly polarizedandconcen- monolayeredorganization of the epithelium andthe tratedat the subapical region of the cell membrane polarizedexpression of endogenous Dlg (Figure 2e,

Oncogene Functional conservation of hScrib LE Dow et al 9229 bottom). In addition, we observed that similar to DmScrib, hScrib localizedwith endogenous Dlg at the subapical region of the epithelial tissue. Together, these observations indicate that hScrib can effectively sub- stitute for DmScrib to regulate tissue organization and apical–basal polarity in Drosophila epithelium. We next considered whether ubiquitous expression of hScrib couldrescue all of the phenotypes of Drosophila scrib mutants. To allow ubiquitous expression of EGFP- hScrib in scrib-null animals, the EGFP-hScrib transgenic lines carrying the scrib1 allele were crossedto an actin- GAL4 activator line carrying a secondmutant scrib allele (scribj7B3revB). The scribj7B3revB allele was generated from the scribj7B3 hypomorphic allele following imprecise excision of the P-element. scrib1/scribj7B3revB animals are larval lethal indicating that this allelic combination results in a Scrib-null phenotype. We examinedF1 progeny of three genotypes: flies that expressedEGFP- hScrib andwere heterozygous for scrib (essentially wild type), scrib mutants and scrib mutants expressing EGFP-hScrib (Figure 3a). At the thirdinstar stage of larval development, scrib mutants fail to pupate and continue to grow to form giant larvae (Bilder et al., 2000). Ubiquitous expression of EGFP-hScrib effec- tively suppressedthe larval overgrowth phenotype of scrib mutants (Figure 3b); no giant larvae were observed that were both transgenic for EGFP-hScrib andmutant for scrib. The thirdinstar imaginal wing discstaken from scrib mutants showedloss of monolayered structure andgrowth in three dimensions (Figure 3c, middle). In contrast, expression of EGFP-hScrib in scrib mutant Drosophila suppressedthe tumorous overgrowth of the epithelial tissues andallowedthe formation of monolayeredepithelial structure closely resembling that of wild-type discs (Figure 3c, compare left and right panels). The neoplastic overgrowth of scrib neural tissue Figure 3 EGFP-hScrib suppresses the giant larvae phenotype and previously reported(Bilder et al., 2000), was also neoplastic overgrowth of scrib mutant wing discs. EGFP-hScrib transgenic flies carrying a strong mutant allele of scrib (scrib1) were suppressedby the ubiquitous expression of EGFP- crossedto an actin-GAL4 activator line carrying a secondmutant hScrib (data not shown). scrib allele (scribj7B3revB).(a) Genotypes of F1 progeny analysed. (b) To measure the efficiency of rescue by EGFP-hScrib The thirdinstar homozygous scrib mutant larvae fail to pupate and we assessedthe development of scrib heterozygous and continue to grow to form giant larvae. scrib mutant larvae that express EGFP-hScrib are comparable in size to control transgenic scrib null progeny beyondthe thirdinstar larval stage. larvae. (c) Wing imaginal discs taken at the third instar stage and The relative Mendelian ratio (observed-to-expected) of stainedfor F-actin (phalloidin)to highlight tissue structure, were EGFP-hScrib/scrib mutant progeny surviving to pupa- analysedby confocal microscopy. Control discsshow a regular tion was equivalent to wild-type survival (99 and 95% monolayeredepithelial structure (left panel), while discstaken from scrib homozygous scrib mutants are disorganized and grow in three respectively), while less than 9% of mutants dimensions forming large amorphous cell masses (middle panel). reachedthis stage of development (Table 1). Expression Expression of EGFP-hScrib in scrib mutants restoredthe normal of EGFP-hScrib in scrib mutants allowedthe majority folded epithelial structure and suppressed the three-dimensional (59%) of these animals to form adult structures (pharate neoplastic overgrowth of wing imaginal discs (right panel) adults) and 23% of the EGFP-hScrib/scrib mutant animals eclosed as mature adult flies. These data confirm that EGFP-hScrib can substitute for DmScrib function throughout development. While we show that the expression of EGFP-hScrib is sufficient to rescue the indicated that both the male and female rescued animals scrib phenotype to adulthood, we note that the rescue is were sterile. These observations are consistent with not as effective as that observedwith expression of those made in Drosophila rescue experiments with DmScrib (our unpublishedobservation). We observed rodent Dlg1 and Dlg3 (Thomas et al., 1997). Taken some developmental defects in the eyes and wings on a together, the above data demonstrate that hScrib can proportion of rescuedanimals as well as ectopic substitute for DmScrib to regulate tissue organization scutellar bristles, suggesting the rescue effect is not fully andstructure andsuppress tissue overgrowth in penetrant (data not shown). In addition, our data Drosophila.

Oncogene Functional conservation of hScrib LE Dow et al 9230 Table 1 Suppression of the scrib mutant lethality by expression of EGFP-hScrib Genotypes Animals surviving Animals surviving Animals surviving Expected to pupation (%)a to pharate adult (%)a to adulthood (%)a numberb

scribi7B3revB;actinÀGal4 UAS-EGFP-hscrib; þ 95 95 95 171

i7B3revB scrib ;actinÀGal4 c scrib1 8.5 0083

scribi7B3revB;actinÀGal4 UAS-EGFP-hscrib; scrib1 99 59 23 93

aObservedcomparedto expectednumber of progeny. bExpectednumber of specifiedgenotype calculatedfrom the total number of F1 progeny scored. cRemaining progeny die as giant larvae

A growing body of data, both direct and correlative, of DmScrib that allow it to function as a tumour now implicate Scrib andassociatedproteins, Dlg and suppressor, emphasizing the needto investigate whether Lgl, as important mammalian tumour suppressors a loss of Scrib function is associatedwith epithelial (reviewedin Humbert et al, 2003). Here, we demonstrate cancers andmalignant progression. that hScrib is a functional homologue of the Drosophila tumour suppressor Scribble. hScrib protein is highly polarizedin mammalian epithelial cells andoverlaps Acknowledgements We thank G Poortinga for discussions and for comments on with Dlg homologues, similar to the distribution of the manuscript. We thank the Kazuza DNA Research Institute DmScrib in Drosophila epithelium. We demonstrate for the KIAA0147 cDNA clone, D Bilder for the scrib1 fly using mosaic systems in the Drosophila eye disc that stock andJ Treisman for the ey-FLP; GAL4,FRT82B,GAL80 hScrib can functionally rescue the scrib mutant defects stock. LED was supportedby a Cancer Council Victoria in differentiation, tissue architecture and apical–basal Postgraduate Cancer Research Scholarship. JAT is a Principal polarity in Drosophila epithelium. Finally, we show that Research Fellow of the National Health & Medical Research hScrib can effectively suppress the neoplastic over- Council of Australia, SMR andHER are Wellcome Senior growth of Drosophila scrib mutant tissue. These studies Research Fellows in Medical Science and POH is a Special therefore indicate that hScrib possesses all the properties Fellow of the Leukemia andLymphoma Society of America.

References

Behrens J, Birchmeier W, Goodman SL and Imhof BA. (1985). Makino K, Kuwahara H, Masuko N, Nishiyama Y, Morisaki J. Cell Biol., 101, 1307–1315. T, Sasaki J, Nakao M, Kuwano A, Nakata M, Ushio Y and Bilder D, Li M and Perrimon N. (2000). Science, 289, 113–116. Saya H. (1997). Oncogene, 14, 2425–2433. Bilder D and Perrimon N. (2000). Nature, 403, 676–680. Mantovani F andBanks L. (2001). Oncogene, 20, 7874–7887. Bissell MJ andRadiskyD. (2001). Nat. Rev. Cancer, 1, 46–54. Mathew D, Gramates LS, PackardM, Thomas U, BilderD, Boussioutas A, Li H, Liu J, Waring P, Lade S, Holloway AJ, Perrimon N, Gorczyca M andBudnikV. (2002). Curr. Biol., Taupin D, Gorringe K, Haviv I, DesmondPV andBowtell 12, 531–539. DD. (2003). Cancer Res., 63, 2569–2577. Montcouquiol M, Rachel RA, LanfordPJ, CopelandNG, BrandAH andPerrimon N. (1993). Development, 118, Jenkins NA andKelley MW. (2003). Nature, 423, 173–177. 401–415. Murdoch JN, Henderson DJ, Doudney K, Gaston-Massuet C, Gateff E. (1994). Int. J. Dev. Biol., 38, 565–590. Phillips HM, Paternotte C, Arkell R, Stanier P andCopp Huang JH, Rajkovic A, Szafranski P, Ochsner S, Richards J AJ. (2003). Hum. Mol. Genet., 12, 87–98. andGoodeS. (2003). Patterns, 3, 3–11. Nakagawa S andHuibregtse JM. (2000). Mol. Cell. Biol., 20, Humbert P, Russell S andRichardsonH. (2003). Bioessays, 8244–8253. 25, 542–553. Stevenson BR, Siliciano JD, Mooseker MS andGoodenough Ishidate T, Matsumine A, Toyoshima K and Akiyama T. DA. (1986). J. Cell Biol., 103, 755–766. (2000). Oncogene, 19, 365–372. Thiery JP. (2002). Nat. Rev. Cancer, 2, 442–454. Lee T andLuo L. (1999). Neuron, 22, 451–461. Thomas U, Phannavong B, Muller B, Garner CC and Liu LX, Liu ZH, Jiang HC, Qu X, Zhang WH, Wu LF, Zhu Gundelfinger ED. (1997). Mech. Dev., 62, 161–174. AL, Wang XQ andWu M. (2002). World J. Gastroenterol., Woodhouse E, Hersperger E and Shearn A. (1998). Dev. Genes 8, 580–585. Evol., 207, 542–550. Ludford-Menting MJ, Thomas SJ, Crimeen B, Harris LJ, Woods DF and Bryant PJ. (1991). Cell, 66, 451–464. LovelandBE, Bills M, Ellis S andRussell SM. (2002). Woods DF, Hough C, Peel D, Callaini G and Bryant PJ. J. Biol. Chem., 277, 4477–4484. (1996). J. Cell Biol., 134, 1469–1482.

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