1

2

3 binding motifs control Scribble cortical

4 dynamics and polarity function

5

6 Batiste Boëda and Sandrine Etienne Manneville

7

8 Institut Pasteur (CNRS URA 3691-INSERM), France

9 Correspondance to Sandrine Etienne-Manneville. Cell Polarity, Migration and Cancer Unit,

10 Institut Pasteur, 25 rue du Dr Roux, 75724 Paris cedex 15, France; Phone: +33 1 4438 9591;

11 FAX: +33 1 4568 8548.

12 e-mail: [email protected]

13

14 Competing interests statement:

15 The authors declare that no competing interests exist.

16

17

1

18 Abstract

19 The tumor suppressor Scribble (SCRIB) plays an evolutionary conserved role in

20 cell polarity. Despite being central for its function, the molecular basis of SCRIB

21 recruitment and stabilization at the cell cortex is poorly understood. Here we show that

22 SCRIB binds directly to the CH1 domain of , a molecular scaffold that

23 contributes to the cortical and connects it to the plasma membrane.

24 We have identified a short evolutionary conserved peptide motif named SADH motif

25 (SCRIB ABLIMs DMTN Homology) which is necessary and sufficient to mediate protein

26 interaction with  spectrins. The SADH domains contribute to SCRIB dynamics at the

27 cell cortex and SCRIB polarity function. Furthermore, mutations in SCRIB SADH

28 domains associated with spina bifida and cancer impact the stability of SCRIB at the

29 plasma membrane, suggesting that SADH domain alterations may participate in human

30 pathology.

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37

2

38 Introduction

39 The protein SCRIB has been implicated in a staggering array of cellular processes

40 including polarity, migration, proliferation, differentiation, apoptosis, stemcell

41 maintenance, and vesicle trafficking [1]. SCRIB is a membrane associated protein

42 localizing at cell junctions [2, 3]. Alteration of SCRIB localization at the membrane often

43 mimics SCRIB loss-of-function phenotype [4-6] and SCRIB aberrant accumulation in the

44 cytosol strongly correlates with poor survival in human cancers [3, 7-12]. Despite the

45 crucial importance of SCRIB subcellular localization, the molecular basis of SCRIB

46 recruitment and stabilization to the cell cortex is not fully understood.

47 SCRIB has a tripartite domain organization that consists of an N-terminal region

48 composed of leucine-rich repeats (LRR), PDZ domains and a C-terminal region with no

49 identified protein domain. While the LRR region is crucial for membrane targeting [13-

50 15], mutations affecting the PDZ and C-terminal regions have also been reported to

51 affect SCRIB subcellular localization [16, 17]. The LRR and PDZ domains are highly

52 conserved between Drosophila and human (60% of homology). The C-terminal region

53 displays a poor conservation between the two species (13%) (Figure 1—figure

54 supplement 1) and its function remains unclear. This is surprising considering that about

55 40 % (5/12) of all the pathological germline mutations identified in the mouse and

56 human Scrib/SCRIB lies within the C-terminal domain of the protein [15, 17-20].

57 Here we show that the C-terminal part of SCRIB contains three spectrin binding motifs

58 which are crucial for SCRIB cortical dynamics and polarity function.

3

59 Results/Discussion

60 SCRIB C-terminal domain interacts directly with spectrins

61 We have previously reported that over-expression of the C-terminal part of SCRIB

62 impedes the polarized orientation of the centrosome during astrocyte migration [21].

63 The same SCRIB fragment consisting of the last 407 amino acids was used as a bait to

64 screen exhaustively a human fetal brain cDNA library in a two-hybrid screen. Nine

65 independent clones encoding 2-spectrin (SPTBN1 ) and seven independent

66 clones encoding 3-spectrin (SPTBN2 gene) were recovered (Figure 1—figure

67 supplement 2). The overlapping sequences of these clones map to the calponin

68 homology one (CH1) domain of spectrins. GST-SCRIB including the C-terminal

69 fragment (1223-1630aa) or the C1 fragment (1223-1424aa) bound endogenous 2

70 spectrin from cell extracts (Figure 1A and B). In contrast, the C2 fragment (1425-1630)

71 did not interact with spectrin (Figure 1B). These interactions were also confirmed by co-

72 immunoprecipitation in HEK cells (Figure 1C).

73 Insertion of the alternative exon 36 brings an additional 25aa sequence within the

74 SCRIB C2 protein fragment and forms a long SCRIB isoform (Figure 1A). In contrast to

75 the short GFP-C2 fragment (C2 - exon36) domain, the long GFP-C2 isoform (C2 +

76 exon36) strongly interacted with spectrin Flag-CH1 (27-167aa), suggesting that the

77 exon 36 encodes a putative -spectrin binding sequence (Figure 1C). Sequence

78 analysis of the C1 fragment revealed the presence of two 25aa sequences displaying

79 strong similarity with exon 36 sequence. Single deletion of each repeated motif impaired

80 GST-C1 binding to the GFP-tagged CH1 domain of spectrin. The deletion of both 4

81 repeats totally abolished spectrin binding (Figure 1D). GST fusion protein including the

82 25aa peptide corresponding to the exon 36 as well as the two other similar repeat motifs

83 identified in SCRIB C1, but not GST alone, interacted robustly with GFP tagged CH1

84 spectrin domain (Figure 1E).

85 We then performed co-localization experiments. In bronchial epithelial cell line (16HBE)

86 SCRIB and both spectrins colocalized to adherens junctions (Figure 1F). In contrast,

87 in HeLa cells which do not express endogenous cadherin [22], GFP-SCRIB was mainly

88 cytosolic. In these cells, exogenously expressed RFP-CH1 domain of 2 spectrin

89 localises into filamentous structures and induces the relocalisation of GFP-SCRIB to

90 these structures (Figure 1G). In these conditions the GFP-SCRIB lacking putative

91 spectrin binding domains remained cytosolic (Figure 1G). Together our results indicate

92 that SCRIB interacts directly with spectrins via three spectrin binding motifs with one

93 of them included in an alternative exon.

94 SCRIB spectrin binding motifs bind to the CH1 domains of the spectrin familly

95 The 2 spectrin N-terminal actin binding region is composed of a tandem calponin

96 homology domain designated CH1-CH2 (Figure 2A). GST coupled C1 SCRIB fragment

97 interacted with GFP tagged spectrin CH1 but neither with CH2 nor with CH1-CH2

98 tandem domains expressed in HEK cells (Figure 2B). In agreement with this

99 observation RFP-CH1 but not RFP-CH1-CH2 2 spectrin constructs strongly

100 colocalises with F-actin and GFP-C1 SCRIB fragment in HeLa cells. Furthermore, C1

101 SCRIB fragment interacted with actin-associated CH1 domain (Figure 2D) suggesting

5

102 that SCRIB as a preferential affinity with actin-associated  spectrin. Structural studies

103 have shown that the CH1-CH2 tandem domains can switch between an open

104 conformation where the CH1 domain binds to F-actin robustly [23] and a closed

105 conformation in which the CH1 and CH2 domains are closely apposed and display a

106 weak F-actin affinity [24, 25].

107 Calponin homology domains are widespread in the (157 domains

108 referenced in SMART database) and are classified in 3 classes CH1, CH2 and CH3.

109 We could not detect any interaction with the 2 spectrin CH2 domain (Figure 2B and F)

110 or the CH3 domain of Vav3 or IQGAP proteins (Figure 2E and F). However, we found

111 that at physiological salt stringency a SCRIB GST-SADH3 bound to the CH1 domains of

112 spectrins , 2,3,5 and - 2 (Figure 2E and F). At high salt stringency (500

113 mM NaCl), only the spectrin , 2 and 3 displayed binding with the GST-SADH3 motif

114 (Figure 2F). These results indicate that SCRIB has a preferential affinity with the CH1

115 domain from the close homologues spectrin , 2 and 3.

116 Identification of the SADH (SCRIB ABLIMs DMTN Homology) motif

117 Phylogenetic analysis revealed that the spectrin binding repeats are conserved in all

118 vertebrate SCRIB sequences tested (primate, rodent, bird, amphibian and fish) but are

119 not be present in Drosophila or C. elegans scribble/LET-413 (Figure 3A—figure

120 supplement 3A). Alignment of all spectrin binding repeats revealed a conserved [+]-X-X-

121 Y-[+]-X--A-A--P sequence (Figure 3A). We examined the consequences of alanine or

122 glycine substitutions in SCRIB repeat 3. Mutations of both positively charged residues

6

123 did not noticeably affect the binding to spectrin CH1 domain. However, mutations of the

124 tyrosine, the alanine doublet or the proline of the repeat 3 severely weakened or

125 completely abolished GST-repeat 3 binding to spectrin CH1 domain (Figure 3B).

126 A search in the human protein database (Swiss-prot) for the simplified Y-[KR]-X-FL-A-

127 A-ILV-P motif sequence identified four other proteins displaying a perfect fit with the

128 SCRIB motif consensus : DMTN (dematin/Band 4.9) and the three member of the

129 ABLIM family of protein (Actin Binding LIM domain containing proteins) (Figure 3C). We

130 found that the DMTN and the ABLIMs motifs retained GFP tagged spectrin CH1 domain

131 from cell extract (Figure 3D). Interestingly DMTN is a constituent of the spectrin-actin

132 junctional complex and was known to bind directly to spectrin [26]. Phylogenetic

133 analyses of the three ABLIMs showed that the spectrin CH1 binding motif was

134 conserved in all vertebrate ABLIMs but could not be detected in invertebrate ABLIMs

135 proteins (Figure 3—figure supplement 3B). We also observed that the noncanonical

136 motifs identified in MyoX, MPP and Afadin did not bind to spectrin CH1 domain (Figure

137 3D). Altogether those results indicate that the consensus sequence Y-[KR]-X-FL-A-A-

138 ILV-P is a spectrin binding motif specific of the vertebrate sub-phylum and present in

139 at least 5 different genes in the human genome. We named this sequence the SADH

140 motif (SCRIB ABLIMs DMTN Homology). No homology between the SADH motif

141 sequences and the previously characterized 10 kDa spectrin-actin-binding domain of

142 the band 4.1 family of proteins could be detected [27].

143

144 7

145 SADH motifs are necessary for SCRIB membrane stability

146 We then investigated the effect of SADH motif mutations (P>A mutant described in

147 Figure 2B) on SCRIB cortical localization. We determined the Cortical Localisation

148 Index (CLI, Figure 4—figure supplement 1), defined as the ratio between the mean GFP

149 fluorescence intensity in the cortical region and the mean fluorescence intensity in the

150 cytoplasm of the cell. A CLI superior to one indicates that GFP is enriched at the cell

151 cortex while a CLI equal to one shows that the GFP is equally distributed between the

152 cytoplasm and the cortex. GFP alone had a CLI around 1 (0.9) whereas a control GFP-

153 CAAX construct which essentially localized at the cell membrane had a CLI of 1.75.

154 Short and long WT SCRIB (-/+ exon36) constructs showed a strong cortical

155 accumulation (CLI=1.8 and 2 respectively). In contrast the P305L SCRIB mutant that

156 carries a mutation in the N-terminal LRR region which impedes SCRIB plasma

157 membrane localization [13] was mainly cytosolic (CLI=1). SCRIB SADH1+2 mutant

158 showed a statistically weaker cortical recruitment (CLI=1.4) than the WT protein (Figure

159 4A) suggesting that SADH mutations may influence SCRIB recruitment and/or

160 stabilization at the membrane. We assessed SCRIB WT and SADH mutant protein

161 dynamics at the membrane by monitoring FRAP (Fluorescence Recovery After

162 Photobleaching) at cell-cell contact in confluent transiently transfected 16HBE cells. A

163 membrane associated GFP-CAAX showed a fast recovery rate (t1/2=10s) and the vast

164 majority of the protein was available for exchange (mobile fraction of 97%) (Figure 4B,

165 C and D). In contrast GFP-WT SCRIB exchanged less rapidly (t1/2=26s) and had a

166 smaller mobile fraction (60%). SCRIB SADH12 mutant displayed a significantly faster

167 exchange (t1/2=18s) than the WT SCRIB protein with 82% of the protein pool available 8

168 for exchange (Figure 4B, C and D). These results indicate that the SADH motifs are

169 important for SCRIB stabilization at the cell cortex.

170 SCRIB is involved in both polarization and orientation of migrating cells in in vitro

171 scratch ‘wound-healing’ assay [2, 21, 28]. Expression of GFP-SCRIB C-terminal

172 constructs and to a lesser extent of GFP SADH3 perturbed centrosome reorientation in

173 migrating astrocytes (Figure 4 E and F) [21]. Mutations of SADH domains strongly

174 reduced the ability to disrupt centrosome reorientation indicating that the SADH

175 domains are involved in SCRIB polarity fonction. 2 spectrin depletion moderately but

176 significantly perturbed scratch-induced centrosome reorientation (Figure 4G). This

177 effect, likely underestimated because of the incomplete knock down or the

178 compensatory role of other spectrins, indicates a role of spectrin in the control of

179 centrosome positioning. Moreover, spectrin 2 depletion impaired SCRIB Cter ability to

180 disrupt centrosome reorientation (Figure 4G). Together, these results suggest that

181 SCRIB interaction with spectrin contributes to SCRIB polarity function.

182 SCRIB is required for the recruitment and the activation of Cdc42 at the cell front edge

183 leading to the centrosome reorientation [21, 28]. GFP SCRIB Cter overexpression but

184 not GFP alone significantly reduced Cherry-Cdc42 membrane recruitment at the leading

185 edge (Figure 4—figure supplement 2) while SCRIB SADH12 mutant Cter construct had

186 significantly weaker effect, suggesting that SCRIB SADH domains are implicated in the

187 SCRIB-mediated recruitment of Cdc42 at the leading edge of migrating cells.

188 In addition to its role in polarity, SCRIB has been previously implicated in tight junction

189 (TJ) assembly in intestinal epithelium [2, 29]. After calcium switch, SCRIB-depleted 9

190 Caco-2 cell monolayers showed short and disconnected areas of ZO-1 labeling at the

191 cell-cell contacts, indicative of a significant delay in TJ reassembly (Figure 4—figure

192 supplement 3A, B and C). This tight junction phenotype could be partially rescued by

193 the expression of a siRNA resistant WT SCRIB but not by SCRIB SADH12 mutant,

194 suggesting that SCRIB interaction with spectrin plays a role in tight junction assembly

195 (Figure 4—figure supplement 3D). Altogether these results strongly suggest that SCRIB

196 SADH motifs control SCRIB dynamics at the cell cortex and are important for polarity

197 and tight junction assembly.

198 SADH motif mutation in human pathology

199 Sixty-one missense mutations in the SCRIB gene coding sequence have been identified

200 so far (COSMIC, [30]). Almost 10% of these mutations (6/61) fall within the SCRIB

201 SADH motifs (Figure 5 A), which account for less than 5% of SCRIB sequence

202 (75/1657aa). One of the mutations (R1322W) identified in a lung cancer patient directly

203 impacts the core SADH2 consensus motif. In contrast to GST-SADH2 WT sequence,

204 GST-SADH2 R1322W mutant did not bind to the spectrin GFP-CH1 domain pointing to

205 the R1322W mutation as a spectrin binding loss of function mutation (Figure 5 B).

206 SCRIB mutations have also been described in congenital diseases [16, 17]. In

207 particular, six SCRIB mutations have been recently described in spina bifida [17] and

208 two of those six fall within the SADH2 motif sequence (Figure 5 C). The A1315T

209 mutation did not affect the ability of a GST-SADH2 to bind to GFP spectrin. In contrast

210 the P1332L mutation noticeably increased GST-SADH2 affinity for GFP spectrin in vitro

211 (Figure 5C and D). These mutations did not impact significantly SCRIB overall

212 recruitment to the cellular cortex (Figure 5—Figure supplement 1). However, FRAP 10

213 experiments showed that GFP R1322W and GFP P1332L SCRIB mutants exchanged

214 more rapidly than the GFP WT SCRIB at the plasma membrane (Figure 5F).

215 Surprisingly, P1332L mutation also increased SCRIB exchange at the plasma

216 membrane, suggesting that, in the context of the SCRIB full length molecule, the

217 P1332L mutation prevents rather than increases spectrin binding. We cannot however

218 exclude the possibility that this mutation also affects other yet-unidentified function of

219 the SADH domain. Altogether these observations indicate that mutations of the SADH

220 motifs may participate in human pathology by impacting the stability of SCRIB at the cell

221 cortex.

222 Dow et al have shown that a transgene encoding the human gene SCRIB was able to

223 partially rescue the Drosophila scribble mutant (23% survival rescue to adult stage),

224 arguing for an indisputable conserved role of scribble/SCRIB during evolution [31].

225 Nevertheless it is clear that some phenotypic differences exist between Drosophila and

226 mouse Scrib mutant models like the severity of apico-basal epithelium polarity

227 phenotype, and that the scribble/SCRIB genes have undergone some intra-phylum

228 specific adaptation [18, 32]. No phylum specific SCRIB interacting partner has been

229 characterized so far that could account for the differences observed between

230 invertebrates and vertebrates SCRIB function. The vertebrate-specific spectrin binding

231 motifs SADH located inside SCRIB divergent C-terminal region appears to be a good

232 candidate to bear such role.

233

11

234 Material and methods

235 Yeast Two-Hybrid Analysis

236 Yeast two-hybrid screening was performed by Hybrigenics Services, S.A.S., Paris,

237 France (http://www.hybrigenics-services.com). The coding sequence for human protein

238 SCRIB (aa 1224-1630) (GenBank accession number gi: 18141296) was PCR-amplified

239 and cloned into pB27 as a C-terminal fusion to LexA (N-LexA-SCRIB-C) and into pB66

240 as a C-terminal fusion to Gal4 DNA-binding domain (N-Gal4-SCRIB-C). The constructs

241 were checked by sequencing and used as a bait to screen a random-primed Human

242 Fetal Brain cDNA library. A confidence score (PBS, for Predicted Biological Score) was

243 attributed to each interaction as previously described [33].

244 Material

245 The following reagents were used in this study: Anti SCRIB C-20 (Goat, Santa Cruz

246 Biotech), Anti-SPTBN1 and E-cadherin (Mouse, BD Transduction Laboratories), Anti-

247 SPTBN2 A301-117A (Rabbit, BETHYL), Anti-GFP HRP ab6663 (Abcam), Anti-FLAG

248 HRP clone M2 (SIGMA), Anti-pericentrin PRB-432C (Rabbit, Covance), Anti-Actin AC-

249 40 (SIGMA). SiRNA sequences: Non-Targeting control siRNA (luciferase)

250 (UAAGGCUAUGAAGAGAUAC), SPTBN1 (UGAUGGCAAAGAGUACCUCTT), SCRIB-

251 ORF1 (GCACUGAGGAGGAGGACAATT),ORF2 (GAACCUCUCUGAGCUGAUCTT),

252 UTR1(GUUCUGGCCUGUGACUAACTT) and UTR2(GGUUUAAGGAGAAUAAAGUTT)

253 were ordered at Eurofins.

254

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255 DNA constructs

256 The SCRIB domains were amplified by PCR from a human SCRIB template provided by

257 Jean Paul Borg and cloned into the NotI-EcoRI sites mammalian expression vectors

258 CB6-N-GFP and pEGFP or the E. Coli expression vector pMW172-GST. SADH motifs

259 were obtained by annealed oligo cloning and cloned likewise in the above-mentioned

260 vectors. The SCRIB internal deletions and point mutagenesis were generated using

261 PCR-based site-directed mutagenesis. The spectrin sequence corresponding to the 27-

262 167aa prey clone encompassing the 2-spectrin CH1 domain was cloned in CB6-N-

263 GFP, CB6-N-RFP or CB6-N-Flag.

264 Sequence analysis

265 For search the Y-[KR]-X-FL-A-A-ILV-P motif was blasted on the

266 Pattern Search program at http://www.expasy.org/ [34] against the Homo sapiens

267 Swiss-prot database. Sequence alignments and phylogeny of calponin homology

268 domains were done on the mobile website [35] and using the seaview program [36].

269 Statistical analysis was performed using GraphPad Prism 5.0. SCRIB somatic

270 mutations in cancers were obtained at www.sanger.ac.uk [30].

271 Protein Expression, Purification, and Resin Production

272 All proteins were expressed in E. coli BL21(DE3) Rosetta strain. Bacterial cell pellets

273 were lysed 1hr at RT in 150 mM NaCl, 50 mM Tris pH8 and 25% sucrose supplemented

274 with 5000 units of lyzozyme (SIGMA). Cleared supernatants were mixed for 90 min with

13

275 glutathione-Sepharose 4B beads (GE healthcare). The resulting resins were washed

276 three times with PBS containing 200 mM NaCl and 0.1% Triton (buffer A).

277 Interaction Assays and Immunoprecipitation

278 HEK 293 cells were transiently transfected using the phosphate calcium method. Cell

279 lysates were prepared by scraping cells in lysis buffer 50mM Tris pH7,5, triton 2%,

280 NP40 1%, 200 mM NaCl with Complete protease inhibitor tablet (Roche, Indianapolis,

281 IN) and centrifuged for 10 min at 13,000 rpm 4°C to pellet cell debris. Soluble detergent

282 extracts were either incubated with GST-SCRIB resins or Anti FLAG coupled protein G-

283 Sepharose (GE healthcare) for 2 hrs at 4°C prior to washing three times with buffer A

284 and processed for western blot analysis. For the binding

285 experiment (Figure 2B) the high stringency binding was done in lysis buffer containing

286 500mM NaCl.

287 Cell Culture, Nucleofection and Immunofluorescence

288 16HBE cells were maintained in DMEM/F12 medium (Invitrogen), Caco-2 and HEK cells

289 in DMEM supplemented with 10% FBS (Invitrogen) and penicillin (100 U/ml)-

290 streptomycin (100 μg/ml; Invitrogen) at 37°C in 5% CO2. For DNA and siRNA

291 transfection 5x106 16HBE or Caco-2 cells were nucleofected with DNA (5µg) using

292 Lonza kitT (program A-23 and B-024 respectively) and nucleofector device, according to

293 manufacturer’s protocol. Primary rat astrocytes were prepared as described previously

294 [37]. For immunofluorescence, cells were fixed in 4% paraformaldehyde, permeabilized

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295 with 0.1% Triton X-100 in PBS, and blocked in PBS 10% Serum for 1hr before

296 incubation with .

297 Scratch assay and Calcium Switch

298 For scratch-induced assays, primary astrocytes were seeded on poly-L-ornithine-coated

299 coverslips and were grown in serum to confluence. The medium was changed 16 hr

300 before scratching. Individual wounds (approximately 300 mm wide) were made with a

301 microinjection needle and front row migrating astrocytes were immediately micro-

302 injected with the indicated GFP tagged constructs. Centrosome reorientation was

303 determined as described previously [38]. Briefly, 8h after the wound, centrosomes

304 located in front of the nucleus of GFP positive front row cells, within the quadrant facing

305 the wound were scored as correctly oriented. In these assays, a score of 25%

306 (astrocytes) represents the absolute minimum corresponding to random centrosome

307 positioning. Calcium Switch was performed as described previously [29]. Briefly, Caco-2

308 cells were incubated for 1 hour in the low-calcium medium and supplemented with 2

309 mmol/L EGTA before being returned to normal cell culture media (calcium repletion) for

310 indicated times at 37°C. Fixed cells were imaged on a microscope (DM6000 B; Leica)

311 using an HCX Plan Apochromat 40×/1.25 NA oil confocal scanning or HCX Plan

312 Apochromat 63×/1.40 NA oil confocal scanning objective (Leica). Microscopes were

313 equipped with a camera (DFC350FX; Leica), and images were collected with LAS

314 software (Leica).

315

15

316 Image acquisition and FRAP

317 16HBE cells grown on MatTek (P35G-1.5-14-C) Petri dishes were analyzed 72h after

318 nucleofection. Live cell imaging was performed on a spinning disk confocal microscope

319 Zeiss Axiovert 200 with UltraView ERS (Perkin-Elmer), at 37°C with a Plan-Apochromat

320 X63/1.4 objective. To calclulate the CLI we used the following equation : CLI =(i/(i +

321 I))/(•a/(a + A)) where (i) represents the pixel intensity contained in a 0.625µm thick

322 cortical band encompassing the cell edge, (I) the cytoplasmic pixel intensity, (a) the

323 cortical region area and (A) the cytoplasmic area (Figure 3—supplement figure1). The

324 CLI was calculated for each cell on 3 different Z plan and then averaged. FRAP

325 experiment was performed using the FRAP module of confocal Volocity software

326 (Perkin-Elmer). A 9 µm2 square region of interest to be bleached was defined for the

327 FRAP and maximum laser power at 488 nm for one iteration was used to bleach

328 signals. After bleaching, images were taken within the same focal plane at regular

329 intervals (between 3 and 10 s) to monitor fluorescence recovery. After background

330 subtraction the recovery of the GFP signal was measured using ImageJ and fitted using

2kt 331 the Prism software and the equation Y(t)=(Ymax-Ymin)(1-e )-Ymin [39], where Y(t) is the

332 intensity of fluorescence at time t, Ymax and Ymin are respectively the maximum and

333 minimum intensities of fluorescence post-bleaching and k is the rate constant of

334 recovery. Mobile fraction was determined as Mf=(Ymax-Y0)/(1-Y0). All data are presented

335 as the mean ± s.e.m. One-way ANOVA analysis of the variance was followed by the

336 Tukey's multiple comparison post-hoc test. A P value of <0.05 was considered as

337 statistically significant.

16

338 Acknowledgments

339 We are grateful to JP Borg, Norbert Frey and A El Amraoui for plasmids and thank

340 Jean-Yves Tinevez from the Plate-Forme d’Imagerie Dynamique/Imagopole of Institut

341 Pasteur for technical support. This work was supported by the Institut National du

342 Cancer, l’Association pour la Recherche contre le Cancer, and La Ligue contre le

343 Cancer. B. Boëda is supported by Institut National de la Santé et de la Recherche

344 Médicale.

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401 22. Lock, J.G. and J.L. Stow, Rab11 in recycling endosomes regulates the sorting and basolateral 402 transport of E-cadherin. Mol Biol Cell, 2005. 16(4): p. 1744-55. 403 23. Way, M., B. Pope, and A.G. Weeds, Evidence for functional homology in the F-actin binding 404 domains of and alpha-actinin: implications for the requirements of severing and capping. 405 J Cell Biol, 1992. 119(4): p. 835-42. 406 24. Sjoblom, B., J. Ylanne, and K. Djinovic-Carugo, Novel structural insights into F-actin-binding and 407 novel functions of calponin homology domains. Curr Opin Struct Biol, 2008. 18(6): p. 702-8. 408 25. Galkin, V.E., et al., Opening of tandem calponin homology domains regulates their affinity for F- 409 actin. Nat Struct Mol Biol, 2010. 17(5): p. 614-6. 410 26. Koshino, I., N. Mohandas, and Y. Takakuwa, Identification of a novel role for dematin in 411 regulating red cell membrane function by modulating spectrin-actin interaction. J Biol Chem, 412 2012. 287(42): p. 35244-50. 413 27. Gimm, J.A., et al., Functional characterization of spectrin-actin-binding domains in 4.1 family of 414 proteins. Biochemistry, 2002. 41(23): p. 7275-82. 415 28. Dow, L.E., et al., The tumour-suppressor scribble dictates cell polarity during directed epithelial 416 migration: regulation of Rho GTPase recruitment to the leading edge. Oncogene, 2007. 26(38): 417 p. 5692. 418 29. Ivanov, A.I., et al., Tumor suppressor scribble regulates assembly of tight junctions in the 419 intestinal epithelium. Am J Pathol, 2010. 176(1): p. 134-45. 420 30. Forbes, S.A., et al., COSMIC: exploring the world's knowledge of somatic mutations in human 421 cancer. Nucleic Acids Res, 2014. 422 31. Dow, L.E., et al., hScrib is a functional homologue of the Drosophila tumour suppressor Scribble. 423 Oncogene, 2003. 22(58): p. 9225-30. 424 32. Bilder, D., M. Li, and N. Perrimon, Cooperative regulation of cell polarity and growth by 425 Drosophila tumor suppressors. Science, 2000. 289(5476): p. 113-6. 426 33. Formstecher, E., et al., Protein interaction mapping: a Drosophila case study. Genome Res, 2005. 427 15(3): p. 376-84. 428 34. Sigrist, C.J., et al., PROSITE, a protein domain database for functional characterization and 429 annotation. Nucleic Acids Res, 2010. 38(Database issue): p. D161-6. 430 35. Neron, B., et al., Mobyle: a new full web bioinformatics framework. Bioinformatics, 2009. 25(22): 431 p. 3005-11. 432 36. Gouy, M., S. Guindon, and O. Gascuel, SeaView version 4: A multiplatform graphical user 433 interface for sequence alignment and phylogenetic tree building. Mol Biol Evol, 2010. 27(2): p. 434 221-4. 435 37. Etienne-Manneville, S., In vitro assay of primary astrocyte migration as a tool to study Rho 436 GTPase function in cell polarization. Methods Enzymol, 2006. 406: p. 565-78. 437 38. Etienne-Manneville, S. and A. Hall, Integrin-mediated activation of Cdc42 controls cell polarity in 438 migrating astrocytes through PKCzeta. Cell, 2001. 106(4): p. 489-98. 439 39. Weisswange, I., et al., The rate of N-WASP exchange limits the extent of ARP2/3-complex- 440 dependent actin-based motility. , 2009. 458(7234): p. 87-91.

441

442

443

444 19

445 Figure Titles and Legends

446 Legend Figure 1

447 Dissection of the SCRIB -spectrin interaction

448 (A) Schematic representation of SCRIB and different C-terminal constructs used in this

449 study. (B) GST-C, GST-C1 and GST-C2 pull-down on astrocytes cell extract. Samples

450 were analyzed by ponceau staining and immunoblotting using the indicated .

451 (C) Immunoprecipitation was performed with anti Flag antibody using HEK293 cell

452 lysates co-expressing Flag CH1 spectrin domain with indicated GFP-SCRIB constructs.

453 Samples were analyzed by immunoblotting using the indicated antibodies. (D)

454 Schematic representation of the C1 SCRIB constructs with internal deletions of putative

455 spectrin binding motif sequences. GST-C1, GST-, GST-GST- pull-down

456 assay on HEK293 cell lysates expressing GFP-CH1 spectrin domain. Samples were

457 analyzed by immunoblotting using anti GFP. (E) GST and GST-SCRIB repeat 1, 2 and

458 3 pull down assay on HEK293 cell lysates expressing GFP-CH1 spectrin domain. .

459 Samples were analyzed by ponceau staining and immunoblotting using the indicated

460 antibody. (F) Immunofluorescence images of 16HBE monolayers fixed and stained for

461 SCRIB, SPTBN1 (spectrin  and SPTBN2 (spectrin . (G) HeLa cells were singly

462 transfected with indicated GFP-SCRIB constructs (upper panel) or in combination with

463 spectrin RFP-CH1 domain (bottom panels). Images are representative of at least three

464 independent experiments. Bars, 10 µm.

465

20

466

467 Legend Figure 2

468 SCRIB spectrin binding motifs bind to CH1 domains of the  spectrin family

469 (A) Schematic representation of full length human 2 spectrin protein (left) and different

470 2 spectrin deletion constructs used in the study (right). (B) GST pull down using a

471 GST- SCRIB C1 resin from HEK293 cell lysates expressing indicated GFP-tagged 2

472 spectrin CH domains. Samples were analyzed by ponceau staining and immunoblotting

473 using anti GFP. (C) Fluorescence images showing HeLa cells expressing GFP-SCRIB

474 C1 construct together with RFP-NCH1CH2 or SID 2 spectrin domains and stained with

475 phalloidin. (D) GST-SCCRIB C1 pull down from HEK293 cell lysates expressing

476 indicated GFP tagged 2 spectrin CH domains. Samples were analyzed by ponceau

477 staining and immunoblotting using anti GFP. (E) Phylogenetic analysis of the Calponin

478 Homology (CH) domains type1, 2 and 3 used in this study. (F) GST pull down from

479 HEK293 cell lysates expressing the indicated CH domains using a GST-SCRIB SADH3

480 resin. Pull down was done in low or high stringency conditions (LSB: Low Salt Buffer,

481 HSB: High Salt Buffer). Ponceau staining indicates the relative amount of GST tagged

482 proteins bound to the resin. Samples were analyzed by ponceau staining and

483 immunoblotting using anti GFP. Bars, 10 µm.

484

485

486

21

487

488 Legend Figure 3

489 The SADH motif (SCRIB, ABLIM, DEMATIN Homology motif)

490 (A) Alignment of vertebrate SCRIB spectrin binding repeats 1 (pink), 2 (yellow) and 3

491 (green). Conserved residues are shown in blue. [+] indicates positively charged amino

492 acids and  represents hydrophobic residues. (B) Alanine or glycine substitutions (in

493 red) performed on SCRIB repeat 3 conserved residues. WT and mutants GST-repeat 3

494 pull down assay on HEK293 cell lysates expressing spectrin GFP-CH1 domain.

495 Samples were analyzed by ponceau staining and immunoblotting using the indicated

496 antibody. (C) Schematic representation and sequence of DMTN and ABLIM1, 2 and 3

497 proteins displaying strong homology with the SCRIB putative spectrin binding motif Y-

498 [KR]-X-FL-A-A-ILV-P (boxed in pink). Position of the motif in the protein is indicated

499 in brackets in aa. MyoX, MPP7 and Afadin sequences with noncanonical motifs

500 consensus are shown on the bottom. VHP (Villin Hedapiece domain). (D) Pull down

501 assay with GST resin fused to 25 aa motif from indicated proteins performed in HEK293

502 cell lysates expressing spectrin GFP-CH1 domain. Samples were analyzed by ponceau

503 staining and immunoblotting using the indicated antibody.

504

505

506

22

507

508 Legend Figure 4

509 SADH motif influence SCRIB membrane stability and is required for SCRIB polarity

510 function

511 (A) 16HBE cells were transiently nucleofected with the indicated GFP constructs and

512 analyzed by live confocal microscopy to calculate their cortical localization index. (n=50

513 for each conditions). (B) FRAP experiment on 16HBE cells nucleofected with the

514 indicated GFP constructs. High magnification images of adherens junction before and at

515 the indicated time points after photobleaching. (C) Quantitative analysis of FRAP from

516 experiments similar to those shown in B (n=30 for each conditions). (D) The mobile

517 fraction and t1/2 of recovery for each protein were calculated from the recovery curves in

518 C. (E) Centrosome reorientation in migrating astrocytes expressing the indicated DNA

519 constructs (up). Results shown are means SEM of 3–4 independent experiments, with a

520 total of at least of 150 cells. (F) Representative images of astrocytes expressing the

521 indicated constructs and stained with anti-pericentrin (centrosome, red) and Hoechst

522 (nucleus, blue). (G) Primary astrocytes were nucleofected with control (Ctrl) or 2

523 spectrin (si SPTBN1) siRNA and incubated for 72 hr. Protein levels were analyzed by

524 western blots using anti-SPTBN1 antibody and anti-actin. (H) Centrosome reorientation

525 assay in migrating astrocytes nucleofected with control or 2 spectrin (SPTBN1) siRNA

526 and microinjected with GFP SCRIB Cter construct. Results shown are means +/- SEM

527 of 3 independent experiments, with a total of at least 100 cells. ***P<0.001; **P<0.01;

528 *P<0.05. Bars, 10µm. 23

529

530 Legend Figure 5

531 SADH motifs mutations in human pathology

532 (A) Table of identified somatic SCRIB SADH motifs mutations in human cancers. Data

533 sourced from COSMIC (http://cancer.sanger.ac.uk/cosmic). (B) GST-SCRIB SADH2

534 WT and SADH2 R1322W pull down assay on HEK293 cell lysates expressing spectrin

535 GFP-CH1 domain. Ponceau staining indicates the relative amount of GST tagged

536 proteins bound to the resin. Samples were analyzed by immunoblotting using anti GFP.

537 (C) Table of identified germinal SCRIB SADH motifs mutations in spina bifida. (D) GST-

538 SCRIB SADH2 WT, A1315T and P1332L pull down assay on HEK293 cell lysates

539 expressing spectrin GFP-CH1 domain. Ponceau staining indicates the relative amount

540 of GST tagged proteins bound to the resin. Samples were analyzed by immunoblotting

541 using anti GFP. (E) GST-SCRIB SADH2 WT and P1332L pull down assays similar to D

542 performed in indicated salt stringency. (F) Quantitative analysis of FRAP experiments in

543 16HBE adherens junction expressing the indicated GFP constructs (n=30 for each

544 conditions). The mobile fraction and t1/2 of recovery for R1322W and P1332L proteins

545 were calculated from the recovery curves in Supplementary Figure 5.

546

547

548

549

550

24

551

552 Supplementary figure Titles and Legends

553 Legend Figure 1—figure supplement 1

554 SCRIB Cterminal sequence alignment.

555 Sequence alignment of SCRIB C-terminal domain of indicated different species. Boxes

556 indicate the emplacement of the SADH motifs. Note that no sequences fitting the SADH

557 consensus can be detected in Drosophila Scribble C-terminal domain sequence

558 Legend Figure 1—figure supplement 2

559 Schematic representation of SCRIB, 2-spectrin and 3 spectrin proteins.

560 (A) The SCRIB C-terminal sequence encompassing the 1223-1630 region was used as

561 a bait in the two hybrid screen. (B) Minimal 2-spectrin and 3 spectrin sequences

562 recovered in the two hybrid screen are indicated (SID) as well as the exact start and

563 stop of each individual selected clones.

564 Legend Figure 3—figure supplement 1

565 SADH domains in vertebrates

566 (A) Schematic representation of SCRIB gene with spectrin binding motifs (up). Table

567 displaying the distribution of SADH domains in SCRIB vertebrate protein sequences.

568 SADH motifs could not be identified in drosophila and C. elegans Scribble/LET-413

569 ortholog genes. (B) Schematic representation of SADH motifs in DMTN and in

570 ABLIM123 genes (up) and conservation of SADH motif sequence in vertebrates. 25

571

572 Legend Figure 4—figure supplement 1

573 Cortical localization of SCRIB SADH mutant

574 (A) Representative images of 16HBE cells nucleofected with the indicated GFP

575 constructs acquired live and used for the CLI analysis. (B) Schematic representation of

576 the process used for CLI calculation.

577 Legend Figure 4—figure supplement 2

578 SADH domains are implicated in SCRIB –mediated control of Cdc42 localization

579 (A) Cherry-Cdc42 recruitment at the leading edge of migrating astrocytes cells

580 expressing the indicated constructs. Results as shown are means +/- SEM of 3

581 independent experiments, with a total of at least 200 cells. Representative images are

582 shown on the right. White dotted lines show scratch positions. The scale bar represents

583 10 m.

584 Legend Figure 4—figure supplement 3

585 SCRIB SADH domain are required for tight junction assembly

586 (A) WB analysis of SCRIB and actin expression in Caco-2 cells, 3 days after

587 nucleofection with control siRNA (Ctrl) or siRNAs directed against SCRIB ORF (ORF1

588 and ORF2) or 3’ UTR (UTR1 and 2). (B) Percentage of Caco-2 cells nucleofected with

589 control or SCRIB UTR1 siRNA displaying intact of ZO-1 labeling at the cell-cell contacts.

590 (C) Representative example of Caco-2 cells displaying intact or short and disconnected 26

591 areas of ZO-1 labeling at the cell-cell contacts in the indicated conditions. (D) Caco-2

592 cells nucleofected with indicated siRNA or construct were scored 1hr after calcium

593 switch for their tight junction integrity using ZO-1 staining. Results shown are means +/-

594 SEM of 3 independent experiments, with a total of at least 100 cells. The scale bar

595 represents 10 m.

596 Legend Figure 5—figure supplement 1

597 (A) 16HBE cells were transiently nucleofected with the indicated GFP constructs and

598 analyzed by live confocal microscopy to calculate their cortical localization index. (n=50

599 for each conditions). (B) Quantitative analysis of FRAP experiment on 16HBE cells

600 nucleofected with the indicated GFP constructs (n=30 for each conditions).

601

602

603

604

605

606

607

608

609

27

Figure 1

A B GST-C + - - GST-C1 - + - Alternative GST-C2 -- + Scrib FL exon 36 (25aa) Ponceau LRRPDZ Cter Resin C C1 (1223-1424 aa) C2 short (1424-1630 aa) Spectrin β2 SPTBN1anti C long (+exon36) C2 long (+exon36) 1223 1630

C GFP-Scrib C +exon 36 + - - - - Input bound GFP-Scrib C -exon 36 - + - - - GFP-C1 - - + - - D GFP-C2 - exon 36 - - - + - GFP-C2 + exon 36 - - - - + Spectrin Binding Domain Repeats (25aa) Flag spectrin CH1 + + + + + 1 2 3 C long

anti GFP anti FLAG anti GFP anti FLAG anti GFP anti C1 C1D1 (C1 ∆1259-1284 aa) Input C1D2 (C1 ∆1313-1337 aa) C1 ∆1∆2 1223 1424

GST-C1 + - - - IP GST-C1 ∆1 - + - - GST-C1 ∆2 - - + - GST-C1 ∆1∆2 - - - + GFP-CH1 + + + + Ponceau Co IP Resin anti GFPanti E GFP-CH1

GST - + - - - bound GST-Repeat 1 - - + - - F GST-Repeat 2 - - - + - GST-Repeat 3 - - - - + SCRIB SPTBN1 SPTBN2

GFP- CH1 + + + + + Ponceau

Resin anti GFPanti

GFP-CH1

Input bound HBE cells

G HeLa cells FL C1 C2 C2+exon36 C1D1 C1D2 C1D1D2

GFP-SCRIB

GFP-SCRIB

+

RFP-CH1 spectrin

Colocalisation + + - + + + - A Spectrin b2 CH1CH2 NCH1CH2 (1-281) CH1 CH2 spectrin repeats PH domain SID (27-167) 1 2364 CH1 (56-156) Selected Interaction Domain (SID) isolated in two hybrid screen CH1CH2 (56-281)

CH2 (175-281)

B C RFP GFP RFP GFP NCH1CH2 + SCRIB C1 SID + SCRIB C1 CH1CH2 SPTBN1 GFP-NCH1CH2 + ------CH1 SPTBN1 GFP-SID - - + + ------GFP-CH1 - - - - + + - - - - RFP GFP-CH1CH2 ------+ + - - GFP-CH2 ------+ +

GST C1 Scrib - + - + - + - + - + I B I B I B I B I B Ponceau

GST C1 Phalloidin

SCRIB C1 SCRIB C1 anti GFPanti Bound GFP

1 2 3 Input Bound D GFP-NCH1CH2 - + - GFP-SID -- + anti GFP anti actin anti GFP anti actin GST C1 Scrib + ++ 1 2 3 1 2 3 1 2 3 1 2 3

GST C1 Scrib

Ponceau E F CH1 CH3 CH2 2 3 5 2 CH2 β β β β β Spectrin β2 GFP tagged

Actinin Spectrin β5 Spectrin Spectrin Spectrin Spectrin α IQGAP2 VAV3 Spectrin CH1 α actinin 1 GST-SADH3 + + ++ + + + + Spectrin β3 Input anti GFP Spectrin β2 Bound (LSB) Spectrin β CH3 Bound (HSB) Vav3

IQGAP2 GST-SADH3 Ponceau Figure 2

AB

homo GLQPLKLD YR AL AA VPSAGSVQRVP WT 1: PLSGKKFD YR AF AA LPSSRPVYDIQ 3 repeat Scrib Mus NLEPLKLD YR AL AA LPSAGSLQRGP 2: -----A------Gallus TSEPLRMD YR TL AA LPSSGSQRVNR 3: ------A------

Scrib Xenopus STEPL KLD YK TL AA LPAGGQAKVAR mutant 4: ------A------repeat1 Fugu STGPL KLD YK TL AA LPTASLQKINR 5: ------GG ------Homo LPANVKQA YR AF AA VPTSHPPEDAP 6: ------A------Mus LPANVKQA YR AF AA VPTVHPPENSA Gallus IPINVKQA YKTF AA VPLSHPLLETQ GST Resin 1 2 3 4 5 6

Scrib Xenopus FPANA KQA YK TY AA LPSAHPSLEQQ repeat2 GFP-CH1 ++ + + + + + Fugu GKLSP RQE YMNL AA VPRFSRPSMEL Homo PLSGKKFD YR AF AA LPSSRPVYDIQ Ponceau Mus PLSGKKFD YR AF AA LPSSRPVYDIQ Gallus SMSEKKFD YR EF AA IPSSKPVYEIQ Scrib

repeat3 Xenopus SHSDK KFD YR EF AA IPSSKPVYEIQ

Fugu SLSSK KFD YR QF AA IPSSKPVYDIQ GFPanti consensus xxxxx[+] xx Y [+] xφAA φPxxxxxxxxx GFP-CH1

Input bound

C D Y-[KR]-X-[FL]-A-A-[ILV]-P 3 SCRIB Cter VHP DMTN GST Resin BLIM Input SCRIB ABLIM1 ABLIM2 A DMTN MyoX MPP7 Afadin ABLIM1 LIM LIM LIM LIM

ABLIM2 LIM LIM LIM LIM GFP-CH1 ++ + ++ + + + + Ponceau ABLIM3 LIM LIM LIM LIM GST-25aa SCRIB (1576-1601) PLSGKKFDYR AF AA LPSSRPVYDIQ DMTN (33-339) KMDNQVLGYK DL AA IPKDKAILDIE Bound GFPanti ABLIM1 (325-121) KVDNEILDYK DL AA IPKVKAIYDIE ABLIM2 (302-326) KLGGEILDYR GL AA LPKSKAIYDID GFP-CH1 ABLIM3 (308-332) KVDNEILNYK DL AA LPKVKSIYEVQ anti anti Actin MyoX RLQYLQGD YTLH AA IPPLEEVYSLQ Actin MPP7 GSGSDTGL YELL AA LPAQLQPHVDS Afadin LTKSGPGRW KTP AA IPATPVAVSQP Figure 4

A B pre-bleach 0 S 10 S 30 S 50 S 140 S 2.5 *** 2.0 EGFP-CAAX 1.5

1.0

0.5 EGFP-SCRIB WT EGFP-SCRIB

Cortical Localisation Index Localisation Cortical 0.0

P L ) 5 6 6) 0 GF AAX 3 3 3 C on on P x x -e IB GFP- rt ( ADH1+2mut ho SCR S long (+e T EGFP-SCRIB EGFP-SCRIB SADH12mut W B SCRIB RIB WT s SC SCRI C D

** *** GFP-CAAX GFP-CAAX SCRIB SADH1+2mut SCRIB SADH1+2mut SCRIB long (+exon36) (% of initial(%of value) SCRIB long (+exon36) MobileFraction (%) FluorescenceIntensity 2.0612 cm

time (s) t 1/2 (s)

E F G Si Ctrl si SPTBN1

C anti SPTBN1 C(SADH12m) C1 C1(SADH12m) GFP anti Actin SADH3

0.6 N 90° 80 H ** ** * * GFP-Cter 60 0.4 ** *** *** 40

0.2 GFP-Cter SADH12m 20

0 % of cell%of with acorrectly oriented centrosome 0.0

% of cell% of with correctlya oriented centrosome r 3 te rib C Ctrl GFP -C1 mut) DH si GFP- FP A G -S GFP-CterGFP-C P FL Sc P si SPTBN1+ + GF GF Ctrl si-1 C(SADH12 mut) si P GF GFP-C1(SADH 12 Figure 5

A B Cancer Input GST WT R1322W Coding sequence SCRIB SADH Carcinoma GFP-Spectrin + + + + G1278R SADH1 Breast V1280M SADH1 Lung bound anti GFP A1315T SADH2 Intestine R1322W SADH2 Lung GST Ponceau P1337S SADH2 Kidney K1570N SADH3 Endometrium D C GST-SCRIB repeat 2 WT A1315T P1332L Spina bifida GFP-Spectrin + + + Coding sequence SCRIB SADH A1315T SADH2 Bound anti GFP P1332L SADH2 GST Ponceau E F P1332L GST-SCRIB repeat 2 WT 100 GFP-CAAX GFP-Spectrin + + + + + + 90 SCRIB SADH1+2mut Bound Mutants SCRIB long (+exon36) anti GFP 80 * SCRIB short (-exon36) * GST Ponceau 70 short * SCRIB R13122W WT SCRIB P1332L NaCl (mM) 200400600 200 400 600 60 long Mobile Fraction (%) Fraction Mobile

50 10 15 20 25 30 35 t=1/2 (s)