To examine the silencing effects of Celsr3 shrna, we co transfected 293T cells with expression

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1 Supplemental figures Supplemental Figure. 1. Silencing expression of Celsr3 by shrna. To examine the silencing effects of Celsr3 shrna, we co transfected 293T cells with expression plasmids for the shrna and Celsr3, along with an EGFP plasmid as a control. The amounts of Celsr3 protein and EGFP were analyzed by immunoblotting. The Celsr3 shrna plasmid strongly suppressed the expression of HA tagged Celsr3 (compare lane 2 with empty shrna plasmid in lane 1). To make an shrna resistant gene, we introduced a silent mutation (CGC to AGA) into the codon encoding Arg 816 of Celsr3. The shrna did not show the silencing effect on the mutant Celsr3 (shrna resistant Celsr3 HA, lane 3).

2 Supplemental Figure. 2. Celsr A431D cells have weak homophilic adhesion activity. Examples of cells in aggregation assays. A431D cells were labelled with Celltracker CM DiO (invitrogen, green) or DiI (red), then mixed at each and cultured for 8 hours on a rotatory shaker at 100 rpm. a. Aggregations of the homogenous cell lines. A431D had intrinsic binding activity (left). Aggregations of Celsr2 A431D (centre) and Celsr3 A431D (left) were larger than that of A431D. Note that green cells and red ones were well mixed in the aggregates. b. Celsr A431Ds formed selective aggregate. A431D and Celsr2 A431D (top) or Celsr3 A431D (bottom) were mixed. Aggregates mainly consisted of Celsr A431Ds (green), and A431D (red) attached peripheral of the aggregates. c. Celsr2 A431D and Celsr3 A431D selectively gathered. Compared with well mixed aggregates of homogenous cell lines (a), green cells (Celsr2 A431D) and red cells (Celsr3 A431D) appeared to form more selective aggregates. Scale bar: 50 μm.

3 Supplemental Figure 3. Tau and MAP2 staining of co culture assays. Neurons co cultured with A431D (1st and 2nd row), Celsr2 A431D (3rd and 4th row), and Celsr3 A431D (5th and 6th row) were transfected with membrane bound form of Venus (green in merged

4 images), and stained for Tau (blue) and MAP2 (red) at DIV 6. Tau positive axons are indicated by arrows. There were significant differences in axonal lengths (with A431D: 542 ± 34.2_ μm, with Celsr2 A431D: 731 ± 45.8_ μm, p < 0.05, with Celsr3 A431D: 380 ± 32.8_ μm, p < 0.05), but not in dendritic ones (with A431D: 84.3 ± 12.2_ μm, with Celsr2 A431D: 92.4 ± 15.6_ μm, with Celsr3 A431D: 70.4 ± 10.4_ μm). n = Scale bar: 50 μm. Supplemental Figure. 4. Results of co culture assays. Total neurite lengths of neurons that expressed modified Celsr molecules in co culture assays at

5 6 DIV. Schemata of the modified Celsr forms (left) and means of total neurite length with s. e. m. (error bars) are shown. Compared with EGFP expressing neurons (top), over expression of Celsr2, Chimera 2 3 or Celsr2[R2413H] moderated the effects of co culturing with Celsr3 A431D cells (see green bars with arrows at the right) on neurite growth; and over expression of Celsr3, Chimera 3 2, and Celsr3[H2573R] moderated the effects of coculturing with Celsr2 A431D (see red bars with arrowheads at the right). We hypothesized that over expression of one Celsr might interfere with the signaling of the other Celsr in this co culture assay. Neither over expression of Celsr2B (a splice variant of Celsr2 lacking a conserved region in the cytoplasmic tail) nor that of Celsr2Ex 1TM (ectodomain of Celsr2 fused to transmembrane region of N cadherin) caused such moderation. Over expression of Celsr2ΔEx (lacking the extracellular region except for the signal sequence) cancelled the growth promoting and suppressing effects of co culturing with Celsr2 (arrowhead) and Celsr3 A431D (arrow), respectively, but over expression of Celsr2BΔEx had no effects. These results suggest that the conserved region in the cytoplasmic tail was required for the moderation of the co culturing effects. Asterisks: p < 0.01 (ANOVA test) when compared with the result of co culturing with control A431D in each experiment. n = We observed that transfected Celsr2 and Celsr3 products distributed both in axons and dendrites in primary hippocampal neurons (data not shown), similar to Celsr2 protein distribution in tissue immunohistochemistry. Supplemental Figure 5. Celsr CRs increase [Ca 2+ ] i in Celsr transfected cells Time courses of normalized fluorescent ratio (340 nm/380 nm) of fura 2 in EYFP expressing control 293T cells (left), Celsr2 expressing cells (centre), and Celsr3 expressing cells (right) upon application (arrows) of 1 μg ml 1 Celsr2 CR (filled circles) and Celsr3 CR (open circles). We observed small increase in [Ca 2+ ] i upon application of 1 μg ml 1 (~ M) Celsr CRs. Celsr2 CR and Celsr3

6 CR increased [Ca 2+ ] i of cells expressing Celsr2 and Ceslr3 respectively (p < by Wilcoxon test), after transient decreases in the ratios that were also seen in EYFP transfected control cells. Celsr2 CR expressing cells showed more prolonged [Ca 2+ ] i increment upon Celsr2 CR application than did Celsr3 expressing cells with Celsr3 CR, and there was a significant difference in the normalized ratios at12 minutes after ligand application (p < 0.01 by Mann Whitney test). Significant [Ca 2+ ] i increment could not be observed in control cells with either of the Celsr CRs. Celsr2 CR did not increase [Ca 2+ ] i of Celsr3 expressing cells, nor did Celsr3 CR increase that of Celsr2 expressing cells, supporting the idea that Celsrs were activated by their homophilic (Celsr2 Celsr2 and Celsr3 Celsr3) rather than heterophilic (Celsr2 Celsr3) interaction through the cadherin repeats. Error bars: s.e.m. n = One reason of the small and slow response of Celsr2 or Celsr3 expressing 293T cells could be poor presentation of Celsrs on plasma membrane. It is known that some GPCRs require accessory molecules for their proper transport and Celsrs might also have to associate those molecules. To test this idea, we co transfected Celsrs with RTP family molecules 1 and RAMPs 2 ; however, there was no significant improvement in presentation of Celsrs on plasma membrane (data not shown). Supplemental Videos Filopodial sprouting upon bath application of 1 μg ml 1 Celsr2 CR (Video 1) or 10 μg ml 1 Celsr3 CR (Video 2). Primary neurons from E18 rat hippocampus were transfected with a plasmid for the membrane bound form of Venus. We took time lapse images (one frame per 5 minutes) of hippocampal neurons in primary culture at hr. Dynamic sprouting of filopodia was observed after application of Celsr CRs (the screen drifted when it was applied).

7 Opposing roles in neurite growth control by two 7 pass transmembrane cadherins Yasuyuki Shima, Shin ya Kawaguchi, Kazuyoshi Kosaka, Manabu Nakayama, Mikio Hoshino, Yoichi Nabeshima, Tomoo Hirano, & Tadashi Uemura Supplemental notes Coculture assay In co culture assays, we seeded A431D cells at 7 80,000 cells cm 2 and hippocampal neurons from E18 Wister rat at 30,000 cells cm 2 on 0.5 mg ml 1 poly D lysine coated coverslips. For visualization of single cell morphology, we transfected neurons with an EGFP plasmid and Celsr expression plasmids by means of Nucleofector (Amaxa), and seeded them at cells cm 2. We stained cultures with rabbit anti MAP2 antibody and mouse anti Tau antibody (Chemicon). We used anti mouse Alexa 633 and anti rabbit Alexa 546 (invitrogen) as secondary antibodies. Purification of Celsr CRs We transfected 293T cells in φ15 cm culture dishes with 25 μg CR molecule plasmids in the presence of 50 μl Fugene6 (Roche) per dish and then collected the cells 48 hours after the transfection. We lysed the cells in 1 ml of cell lysis buffer (50 mm Tris ph7.6, containing 1% TritonX 100, 1% NP 40, 150 mm NaCl and protease inhibitors [Complete mini, Roche] with [for Celsr2 CR] or without [for Celsr3 CR] 1 M urea), sonicated them for 6 min, and then centrifuged them at 15, 000 g. We incubated the supernatant with 100 μl anti FLAG M2 antibody conjugated beads (Sigma) for at least 2 hours at 4 C. We washed the beads with wash buffer (50 mm Tris ph 7.6, 500 mm NaCl) for 15 minutes 3 times. For elution of FLAG tagged molecules, we incubated the beads with equal amount of PBS containing 0.4 mg ml 1 3 FLAG peptide (Sigma) for 30 minutes, and repeated this procedure once. We concentrated the elutants with Centricon (Millipore), if needed. References in supplemental materials 1. Saito, H., Kubota, M., Roberts, R.W., Chi, Q. & Matsunami, H. RTP family members induce functional expression of mammalian odorant receptors. Cell 119, (2004). 2. Christopoulos, A. et al. Novel receptor partners and function of receptor activity modifying proteins. J Biol Chem 278, (2003).