1
2
3 Spectrin 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 protein 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 spectrins, a molecular scaffold that
23 contributes to the cortical actin cytoskeleton 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 genes 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 gene) 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 proteins 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 human genome (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 -actinin 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
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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 sequence homology 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 calponin homology domain 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 antibodies.
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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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 antibody.
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)